Spine, 1997 Jul 1, 22(13), 1467 - 71 Freeze-dried allograft for posterior spinal fusion in patients with neuromuscular spinal deformities; Yazici M et al.; STUDY DESIGN: The effectiveness and safety of freeze-dried allograft for posterior spinal fusion in patients with neuromuscular disorders were evaluated retrospectively . SUMMARY OF BACKGROUND DATA: Because the harvest of an adequate quantity of autogenous bone graft from patients with neuromuscular deformity who have instrumentation and fusion to the pelvis is difficult at best, an alternative graft source usually is needed . Allograft bone, most commonly processed fresh-frozen or freeze-dried, has been used frequently for posterior spinal fusion in patients with neuromuscular deformity . However, a relatively high risk of infection and pseudarthrosis has been reported for this procedure . METHODS: Forty patients with neuromuscular deformity with an average age at the time of surgery of 14 years and 2 months (range, 5 years, 4 months to 23 years, 8 months) met the inclusion criteria . All of these patients underwent more than 2 years of follow-up evaluation . They were evaluated for rates of infection, pseudarthrosis, and transmissible disease . RESULTS: Thirty-eight patients had solid fusion at the most recent follow-up visit . Definite pseudarthrosis was detected in one patient (2.5% of the study group), which was treated successfully . Another patient's (2.5%) spinal curve progression of more that 10 degrees and rod breakage led the authors to diagnoses a probable pseudarthrosis . She had a stable spine that did not require revision at 68 months after surgery . For the 32 patients who underwent posterior surgery only, the pseudarthrosis rate was 3.1% . There were no acute deep wound infections . Superficial infection occurred in two patients (5%) and delayed deep sterile drainage in one patient (2.5%) . All cases of infection resolved with appropriate management . Delayed deep wound infection developed in one patient (2.5%) as a result of staphylococcus coagulase negative at 34 months after surgery . Successful treatment has consisted of implant removal, debridement, and appropriate antibiotics . Transmissible disease attributable to allograft has not been detected to date . CONCLUSION: Freeze-dried allograft fusion is a reliable and effective method for posterior spinal fusion in the patients with neuromuscular deformity. Int J Syst Bacteriol, 1997 Jul, 47(3), 724 - 6 Staphylococcus lutrae sp . nov., a new coagulase-positive species isolated from otters; Foster G et al.; Phenotypic and phylogenetic studies were performed with three strains of a catalase-positive, gram-positive, coccus-shaped bacterium isolated from otters . The results of a 16S rRNA gene sequence analysis demonstrated that these strains represent a hitherto unknown subline within the genus Staphylococcus . Based on the results of the phylogenetic analysis and phenotypic criteria, we propose that these bacteria should be classified as members of a new species, Staphylococcus lutrae . The type strain of S . lutrae is DSM 10244. Proteins, 1997 Jul, 28(3), 325 - 32 Measurement of water-amide proton exchange rates in the denatured state of staphylococcal nuclease by a magnetization transfer technique; Mori S et al.; The rates of hydrogen exchange were measured in a "physiological" denatured state of staphylococcal nuclease using a NMR magnetization transfer experiment suitable for the measurement of exchange rates faster than 0.5 s-1 . The results are compared with predicted exchange rates (kex) for the random coil state (Bai et al., Proteins 17:75-86, 1993) . No protection factors (= predicted rate/measured rate) larger than 2.4 were observed, consistent with other NMR data which strongly suggest only small amounts of residual secondary structure in this denatured state . Systematically low protection factors (0.51 +/- 0.23) were found for Asp and Glu residues, while high protection factors were observed for Gly (1.60 +/- 0.60) . We conclude that the predicted exchange rates (kex) may have an uncertainty of 2- to 3-fold . Thus, for denatured proteins only protection factors with a value of 5 or larger can be assigned structural significance . These results also demonstrate that multidimensional magnetization transfer NMR techniques are powerful tools in this research field due to its ability to measure rapidly exchanging protons (> 05 s-1) with high accuracy. J Infect Dis, 1997 Jul, 176(1), 168 - 76 Neonatal hypersusceptibility to endotoxin correlates with increased tumor necrosis factor production in mice; Cusumano V et al.; Septic shock is a major cause of mortality in neonates . The hypothesis was tested that neonatal age is associated with altered sensitivity to shock-inducing bacterial products or proinflammatory cytokines (or both) . Mice of different ages were inoculated with various doses of lipopolysaccharide (LPS), superantigenic staphylococcal enterotoxin B (SEB), or recombinant tumor necrosis factor-alpha (rTNF-alpha), alone or in combination with the sensitizing agent D-galactosamine . Neonatal mice were markedly more susceptible to LPS-induced lethality but more resistant to SEB than were adults (P < .05) . Mice of different ages did not differ, however, in their sensitivity to lethal activities of rTNF-alpha . Neonatal susceptibility to LPS and SEB correlated directly with plasma TNF-alpha but not IFN-gamma levels, which was confirmed by TNF-alpha and IFN-gamma blockade experiments . These data document marked age-related differences in the pathophysiology of septic shock and suggest that IFN-gamma is not an obligatory mediator of either LPS- or SEB-induced lethality in neonates. Gastroenterology, 1997 Jul, 113(1), 144 - 50 Mesenchymal cells stimulate human intestinal intraepithelial lymphocytes; Roberts AI et al.; BACKGROUND & AIMS: Intraepithelial lymphocytes (IELs) from human intestinal mucosa proliferate minimally to T-cell stimuli . Optimal growth may depend on factors that are missing in vitro, such as accessory cells . The aim of this study was to determine whether mesenchymal cells costimulate IELs . METHODS: IELs were isolated from human jejunum and cultured with fibroblasts or smooth muscle cells (mesenchymal cell models for mucosal myofibroblasts) and various T-cell stimuli . Proliferation was determined by {3H}thymidine incorporation, and interleukin 2 (IL-2) production was measured by enzyme-linked immunosorbent assay . Surface molecules were detected by immunofluorescence and flow cytometry . RESULTS: The proliferative responses of IELs to mitogen (phytohemagglutinin), superantigen (staphylococcal enterotoxin B), or anti-CD3 antibody were increased greatly by coculture with mesenchymal cells, while only slightly by peripheral-blood monocytes, the classical antigen-presenting cells . IL-2 production and receptor expression also increased . Mesenchymal cell costimulation of IEL growth required direct contact between the two cell types and was partly dependent on the integrin alpha4beta1 (very late activation 4{VLA-4}) and major histocompatibility complex (MHC) class I, as their respective antibodies blocked the effect . The surface molecules B7 (CD80), CD2, and MHC class II were not involved . CONCLUSIONS: Optimal IEL growth depends on their contact with mesenchymal cells, an interaction that is mediated by VLA-4 and MHC class I . In mucosal immunity, basement membrane myofibroblasts likely serve this role. Infect Immun, 1997 Jul, 65(7), 2656 - 62 Staphylococcal enterotoxin A-induced fever is associated with increased circulating levels of cytokines in rabbits; Huang WT et al.; Rabbits were injected intravenously with 10 to 100 ng of staphylococcal enterotoxin A (SEA) per kg, and colonic temperatures were monitored . The febrile responses were compared with circulating levels of interferon (IFN), tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-2, and IL-6 just before the injection of SEA . Both colonic temperatures and circulating levels of IFN, TNF, and IL-2 started to rise at 1 to 2 h and reached their peak levels at 3 to 5 h after SEA injection . Both the fever and the increased circulating levels of IFN, TNF, and IL-2 produced by SEA were decreased by pretreatment with indomethacin (a cyclo-oxygenase inhibitor) (15 mg/kg, intraperitoneally), anisomycin (a protein synthesis inhibitor) (15 mg/kg, subcutaneously), or dexamethasone (an effective anti-inflammatory and immunosuppressive agent) (4 mg/kg, intravenously) in rabbits . Rabbits were injected intravenously with 30 ng of SEA per kg on four consecutive days, and colonic temperatures were monitored . Compared to rabbits that received the single injection of SEA, rabbits that received four consecutive injections of SEA showed a lesser increase in circulating levels of IFN, TNF, and IL-2 as well as colonic temperatures in response to an intravenous dose of SEA (30 ng/kg) . The data suggest that the prevention of the febrile response elicited by SEA by indomethacin, anisomycin, or dexamethasone is due to prevention by these compounds of the increase in the circulating levels of IFN, TNF, and IL-2 . The pyrogenic hyporesponsiveness to repeated injection of SEA is associated with decreased production of these circulating cytokines. Int J Cardiol, 1997 Jun 27, 60(1), 95 - 8 Acute Staphylococcal myocarditis masquerading as an acute myocardial infarction; Raev D; It is well known that viral myocarditis can mimic acute myocardial infarction and the differentiation of both diseases presents a diagnostic challenge . This report discusses the case presentation of an 18-year-old man who developed acute Staphylococcal myocarditis in the setting of acute tonsillitis in whom clinical, enzymatic, and electrocardiographic findings have led to tentative erroneous diagnosis of acute myocardial infarction . The outcome was favorable with cephalotin and captopril therapy . This is the first reported case of bacterial myocarditis masquerading as an acute myocardial infarction. J Biol Chem, 1997 Jun 20, 272(25), 16040 - 7 Differential regulation of methionine adenosyltransferase in superantigen and mitogen stimulated human T lymphocytes; LeGros HL Jr et al.; Superantigens interact with the T cell receptor for antigen (TCR) and are, therefore, more physiological stimulators of T lymphocytes than nonspecific polyclonal T cell mitogens . The effects of these two classes of T cell stimulators on methionine adenosyltransferase (MAT) and S-adenosylmethionine (AdoMet) levels were investigated . Activation of resting human peripheral blood T lymphocytes by the mitogen phytohemagglutinin (PHA) or the superantigen staphylococcal enterotoxin B (SEB) caused a 3- to 6-fold increase in MAT II specific activity . Although the proliferative response was higher in cultures stimulated with PHA compared with SEB, MAT II activity was comparable in both cultures . Both stimuli caused down-regulation of the MAT 68-kDa lambda subunit expression and induced a comparable increase in the expression of the catalytic alpha2/alpha2' subunit mRNA and protein . However, in superantigen-stimulated cells, the expression of the noncatalytic beta subunit was down-regulated and virtually disappeared by 72 h post-stimulation; whereas, no change in the expression of this subunit was noted in PHA-stimulated cells . Thus, at 72 h following stimulation, PHA-stimulated cells expressed MAT II alpha2/alpha2' and beta subunits while SEB-stimulated cells expressed the alpha2/alpha2' subunits only; the beta subunit was no longer expressed in superantigen-stimulated cells . Kinetic analysis of MAT II in extracts of PHA- and SEB-stimulated cells using reciprocal kinetic plots revealed that in the absence of the beta subunit the Km of the enzyme for L-methionine (L-Met) was 3-fold higher than in the presence of the beta subunit . Furthermore, AdoMet levels were 5-fold higher in cell extracts lacking the beta subunit (SEB-stimulated cell extracts) compared with extracts containing MAT II alpha2/alpha2' and beta subunits . We propose that the increased levels of AdoMet in superantigen-stimulated cells may be attributed to the absence of the beta subunit, which seems to have rendered MAT II less sensitive to product feedback inhibition by (-)AdoMet . The data suggest that the beta subunit of MAT II, which has no catalytic activity, may be a regulatory subunit that imparts a lower Km for L-Met but increases the sensitivity to feedback inhibition by AdoMet . The down-regulation of the beta subunit, which occurred when T cells were stimulated via the TCR, may be an important mechanism to regulate AdoMet levels at different stages of T cell differentiation under physiological conditions. Cell Immunol, 1997 Jun 15, 178(2), 101 - 7 Treatment of murine gliomas by adoptive transfer of ex vivo activated tumor-draining lymph node cells; Plautz GE et al.; The adoptive transfer of tumor-reactive T lymphocytes has recently been demonstrated to be an effective means for mediating the regression of experimental intracranial fibrosarcomas . In this study, mice bearing syngeneic intracranial GL261 gliomas were cured by the combination of sublethal whole body irradiation followed by the intravenous transfer of tumor-draining lymph node (LN) T cells activated with anti-CD3 or staphylococcal enterotoxin C2 (SEC2) . To further identify the functional effector T cel population in the adoptive immunotherapy, LN T cells were separated into two subsets, based on the level of expression of the cell adhesion molecule CD62L (L-selectin) . As few as 5 x 10(5) CD62Llow cells could cure the majority of animals, whereas 2 x 10(6) CD62Lhigh cells were completely ineffective . Moreover, T cells isolated from advanced intracranial tumors were identified to be predominantly CD62Llow . In contrast, spleens contained a mixture of CD62L low and high cells similar to the transferred cell population . T cells in the glioma site were more actively proliferating than those isolated from the spleen . Mice cured of GL261 tumors demonstrated long-term immunologic memory by rejecting intracranial challenges of the original tumor but not an immunologically distinct tumor . Furthermore, despite infiltration of transferred cells into the intracranial tumors, cured mice did not exhibit any apparent neurologic abnormalities during treatment, prolonged follow-up, or after intracranial tumor rechallenge . This study demonstrates the effective treatment of an intracranial murine glioma by the systemic adoptive transfer of activated tumor-draining LN T cells and selective tumor infiltration by the therapeutically active CD62Llow T cells. J Immunol, 1997 Jun 15, 158(12), 5988 - 96 Increased acute-phase response and renal amyloidosis in aged CD2-fas-transgenic mice; Hsu HC et al.; We previously demonstrated that increased Fas expression in T cells of aged CD2-fas transgenic (Fas-Tg) CD-1 mice results in an increased immune response and T cell apoptosis . Surprisingly, despite prevention of T cell immune senescence, the average life span of Fas-Tg mice is comparable with that of nontransgenic (non-Tg) mice . Histopathologic evaluation of tissue sections showed that nearly 50% of the aged (>18-mo-old) Fas-Tg mice developed renal amyloid A amyloidosis, whereas no amyloid deposition was observed in aged non-Tg mice . The amyloid A deposition was observed primarily in glomeruli by using immunohistochemical stains and electron microscopy . The full-length amino acid coding sequence of serum amyloid A2 cDNA in CD-1 mice was identical to that of amyloid A amyloidosis-susceptible BALB/c mice . Although there was no significant difference in steady-state serum amyloid A level in the serum of aged non-Tg and Fas-Tg mice, challenging mice with staphylococcal enterotoxin B resulted in significantly higher serum levels of serum amyloid A on day 2 and IL-6 on days 1 and 2 and a higher magnitude of weight loss on day 7 in aged Fas-Tg mice compared with young mice . These parameters, at the indicated time points, were equivalent between young and aged non-Tg mice . Taken together, our data suggest that prevention of T cell senescence in Fas-Tg mice may be a factor in induction of an excessive acute-phase response triggered by T cell activation . The Fas-Tg mice are a novel model for understanding the immunologic mechanisms leading to secondary amyloidosis. J Biomed Mater Res, 1997 Jun 15, 35(4), 409 - 20 Leukocyte-biomaterial interactions in the presence of Staphylococcus epidermidis: flow cytometric evaluation of leukocyte activation; Sapatnekar S et al.; The adhesion of bacteria on a biomaterial surface is believed to be the first step in the development of biomaterial-related infection . The goal of this study was to investigate the mechanisms that permit adherent bacteria to persist on the surface of an implanted cardiovascular biomaterial . We hypothesized that circulating leukocytes are unable to adhere to the biomaterial surface under physiologic shear stress conditions, and this prevents them from interacting with adherent bacteria . To address this hypothesis, we investigated the adhesion profiles of Staphylococcus epidermidis and polymorphonuclear leukocytes (PMN), incubated under controlled shear stress conditions with the test biomaterial . We found that bacteria could adhere on the biomaterial surface, even when their concentration in the test medium was as low as 10(3) cfu/mL . At this concentration, the bacteria did not induce significant complement activation . PMN adhesion on the biomaterial surface was sensitive to shear stress and minimal at shear stress > 10 dynes/cm2 . Low concentrations of bacteria could induce a significant increase in the expression of PMN adhesion molecules CD11b and CD11c . We conclude that the presence of bacteria induces PMN activation but does not increase PMN adhesion on biomaterial surfaces under physiologic shear stress conditions . This could be a major mechanism that protects adherent bacteria from PMN antibacterial activity. J Clin Invest, 1997 Jun 15, 99(12), 3009 - 17 Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis . Implications for sustained dendritic cell activation in the skin; Pastore S et al.; Lesional skin of atopic dermatitis (AD) harbors high numbers of dendritic cells with enhanced stimulatory capacity for T lymphocytes . In this study, lesional AD skin was shown to stain heavily in both epidermal and dermal compartments for GM-CSF, a cytokine crucial to dendritic cell functions . Keratinocyte cultures established from uninvolved skin of AD patients exhibited markedly increased spontaneous and PMA-stimulated release of GM-CSF compared with keratinocytes from nonatopic controls . Correspondingly, keratinocytes from AD patients showed higher constitutive as well as PMA-induced GM-CSF gene expression . Larger amounts of GM-CSF were produced by AD keratinocytes, also in response to IL-1alpha, but not after stimulation with LPS, lipoteichoic acid, or staphylococcal enterotoxin B . Hydrocortisone reduced GM-CSF gene expression and protein release in both atopic and control keratinocytes . Supernatants from atopic keratinocytes were able to strongly stimulate PBMC proliferation in a GM-CSF-dependent manner . Moreover, conditioned medium from PMA-treated AD keratinocytes, together with exogenous IL-4, could support phenotypical and functional maturation of peripheral blood precursors into dendritic cells . Enhanced production of GM-CSF by keratinocytes may contribute relevantly to the establishment and chronicity of AD lesions, in particular to the increased number, sustained activation, and enhanced antigen-presenting functions of dendritic cells. Biochim Biophys Acta, 1997 Jun 12, 1326(2), 275 - 86 A predicted beta-sheet from class S components of staphylococcal gamma-hemolysin is essential for the secondary interaction of the class F component; Meunier O et al.; Site-directed mutagenesis was performed on genes encoding HlgA and HlgC, two of the three proteins expressed from the staphylococcal y-hemolysin locus, which originate two pore-forming toxins (HlgA + HlgB, HlgC + HlgB) . As related proteins, HlgA and HlgC were found to bind first to cell membranes . Amino acid substitutions concerned residues that would predictably disrupt a 13 amino acid conserved beta-sheet of the Chou and Fasman secondary structure prediction . The mutation of a threonin into an aspartic acid residue from HlgA (T28D) and from HlgC (T30D) that would break this predicted N-terminal structure lowered dramatically the biological activities on purely lipidic vesicles, erythrocytes and polymorphonuclear cells . The change in secondary structure was confirmed by Fourier Transformed Infrared spectroscopy . The binding of mutated and native proteins at the same kind of sites onto polymorphonuclear cells was evidenced with flow cytometry and fluorescein-labelled anti-class S antibodies or wild type HlgA or HlgC . However, the subsequent binding of fluorescein-labelled HlgB to membrane-bound mutated HlgA or HlgC complexes was inhibited . In conclusion, the first binding of class S components is essential for the subsequent binding of class F components, and a predicted beta-sheet seems to be at least one of the functional domains involved. Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6180 - 4 Three-dimensional diffuse x-ray scattering from crystals of Staphylococcal nuclease; Wall ME et al.; We have developed methods for obtaining and characterizing three-dimensional maps of the reciprocal-space distribution of diffuse x-ray scattering from protein crystals, and have used the methods to study the nature of disorder in crystals of Staphylococcal nuclease . Experimentally obtained maps are 99.5% complete in the reciprocal-space resolution range of 10 A-2.5 A, show symmetry consistent with the P41 space group of the unit cell, and are highly reproducible . Quantitative comparisons of the data with three-dimensional simulations imply liquid-like motions of the protein {Caspar, D . L . D., Clarage, J., Salunke, D . M . & Clarage, M . (1988) Nature (London) 332, 659-662}, with a correlation length of 10 A and a root-mean-square displacement of 0.36 A. J Mol Biol, 1997 Jun 6, 269(2), 270 - 80 A structural and functional comparison of staphylococcal enterotoxins A and C2 reveals remarkable similarity and dissimilarity; Schad EM et al.; Staphylococcal enterotoxins and toxic shock syndrome toxin-1 are known as superantigens due to their ability to activate a large number of T-cells by crosslinking the major histocompatibility complex class II molecules with the T-cell receptor . Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties . A structural comparison of staphylococcal enterotoxins A and C2, members of the staphylococcal superantigens, has shown large conformational differences at the putative TcR interaction site (loops between alphaN-alpha2, alpha4-beta9 and beta10-alpha5 in staphylococcal enterotoxin A) that could explain the variability in their T-cell receptor specificity . A common Zn2(+)-binding site was identified in both staphylococcal enterotoxin A and C2 that is superimposable but differs somewhat in its coordination geometry between the two molecules. J Biol Chem, 1997 Jun 6, 272(23), 14787 - 91 Lck-independent triggering of T-cell antigen receptor signal transduction by staphylococcal enterotoxins; Yamasaki S et al.; Superantigens (SAgs) activate T-cells in a manner specific to the Vbeta region of the T-cell antigen receptor . Stimulations by SAgs provoke drastic T-cell activation that leads to programmed cell death or the anergic state of responding cells . To characterize the signal transduction pathway initiated by SAgs, mutant lines derived from the human leukemic T-cell line Jurkat were tested for their reactivities against prototypic SAgs, staphylococcal enterotoxins . The J.CaM1.6 cell line, which lacks Lck expression and lost reactivity against T-cell antigen receptor-mediated stimulation, was activated by staphylococcal enterotoxins in a manner indistinguishable from the Jurkat cell line . In contrast, the J.45 . 01 cell line, which lacks expression of functional CD45, showed severely impaired reactivity . The role of Lck appears to be replaced by another Src family protein-tyrosine kinase, Fyn . In J.CaM1.6 cells, Fyn was rapidly phosphorylated and activated after staphylococcal enterotoxin treatment . The kinase-inactive mutant of Fyn significantly suppressed the reactivity against staphylococcal enterotoxin E in J.CaM1.6 cells, and the expression of the active form of Fyn reconstituted reactivity against staphylococcal enterotoxin E in J.45.01 cells . These results demonstrate that SAgs activate T-cells in an Lck-independent pathway and that Fyn plays a critical role in the process. Pharm World Sci, 1997 Jun, 19(3), 133 - 41 The treatment of staphylococcal infections with special reference to pharmacokinetic, pharmacodynamic and pharmacoeconomic considerations; Janknegt R; The choice of antibiotics for the treatment of staphylococcal infections depends to a high degree on the susceptibility patterns in the hospital in question . These may be highly variable and considerable differences between countries and hospitals exist . The insight into the pharmacodynamic aspects of antimicrobial agents has increased considerably in the last 5 years, resulting in new treatments, such as once daily administration of aminoglycosides and continuous infusion of betalactam antibiotics . The antibiotic policy in Dutch hospitals for the treatment of staphylococcal infections is discussed . In most Western countries with a relatively low incidence of MRSA, penicillin-derivatives, such as flucloxacillin (or cloxacillin, methicillin and nafcillin) will be the drug of choice, because of their good in-vitro activity, low toxicity, good clinical efficacy and relatively low cost . If the incidence of MRSA increases, drugs such as the glycopeptides will be of more importance . This will of course have a clear economic impact, as both vancomycin and teicoplanin are considerably more expensive than agents such as flucloxacillin and oral treatment is not possible . Pharmacoeconomic aspects also play a role . As a rule, intravenous antimicrobial agents are considerably more expensive than the oral formulations . Before oral administration can be recommended, a reliable oral absorption, also in seriously ill patients, must have been demonstrated . Other aspects that influence the cost of therapy are hospital stay and the possibility of outpatient treatment. J Biomol NMR, 1997 Jun, 9(4), 409 - 22 Pulse schemes for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings in 15N,13C uniformly labeled proteins; Konrat R et al.; Pulse sequences are presented for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings for all C gamma containing residues in 15N,13C uniformly labeled proteins . The methods described are based on quantitative J correlation spectroscopy pioneered by Bax and co-workers {Bax et . al . (1994) Methods Enzymol., 239, 79-105} . The combination of 3JC'C gamma and 3JNC gamma scalar coupling constants allows the assignment of discrete rotameric states about the chi 1 torsion angle in cases where such states exist or, alternatively, facilitates the establishment of noncanonical chi 1 conformations or the presence of rotameric averaging . The methods are applied to a 1.5 mM sample of staphylococcal nuclease. Med Microbiol Immunol (Berl), 1997 Jun, 186(1), 53 - 61 Pore-forming properties of proteolytically nicked staphylococcal alpha-toxin: the ion channel in planar lipid bilayer membranes; Krasilnikov OV et al.; Staphylococcal alpha-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes . Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin . Single-channel conductance in planar bilayers was decreased about threefold . The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced . At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity . In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (approximately 1.2 times) than that formed by the native toxin . This decrease was not marked enough to explain changes in the biological effects of the nicked toxin . The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin. Immunobiology, 1997 Jun, 197(1), 44 - 54 In staphylococcus enterotoxin B (SEB)-stimulated human PBMC, the LAK activity of non-T cells might have a major role in the mechanism of glomerular endothelial cells' injury; Seprenyi G et al.; In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC) . After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed . The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction . The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma . The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells . These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration. J Vet Med Sci, 1997 Jun, 59(6), 443 - 50 Pulsed-field gel electrophoresis of Staphylococcus hyicus and Staphylococcus chromogenes genomic DNA and its taxonomic, epidemiologic and ecologic applications in veterinary medicine; Shimizu A et al.; One hundred and thirty-eight strains of Staphylococcus hyicus and 21 strains of S . chromogenes isolated from animals were analyzed by pulsed-field gel electrophoresis (PFGE) after restriction endonuclease Smal digestion of chromosomal DNA . Eighty-eight strains of S . hyicus from pigs with or without exudative epidermitis (EE) generated 16 to 26 fragments in the size range of < 1 to 485 kb, and yielded 39 different patterns . With regard to the strains from pigs with EE, PFGE patterns differed according to the country of origin . Outbreaks of EE occurring on four separate pig farms in Japan involved S . hyicus with different PFGE patterns . The PFGE patterns shown by S . hyicus strains from 4 kinds of animals were compared . Strains from pigs differed from those isolated from chickens (n = 45; 18 to 24 fragments of < 1 to 425 kb), cows (n = 3; 17 to 19 fragments of < 1 to 475 kb), and goats (n = 2; 16 or 17 fragments of < 1 to 1,125 kb) . Also, each of the chicken, cow and goat strains had a host-specific fragment . The results suggest that PFGE analysis might be a useful marker for distinguishing ecovars within S . hyicus . In contrast, strains of S . chromogenes from pigs and cows generated 17 to 24 fragments ranging from < 1 to 545 kb . The PFGE patterns of S . chromogenes strains were more highly conserved than those of S . hyicus . S . chromogenes strains could be distinguished from S . hyicus strains by fragments within the range of 305 to 545 kb . The results indicate that PFGE analysis could be used to distinguish between S . hyicus and S . chromogenes . We conclude that PFGE analysis is a useful tool not only for species or strain identification but also for epidemiologic or ecologic studies of S . hyicus and S . chromogenes. J Hum Lact, 1997 Jun, 13(2), 127 - 30 Contamination in expressed breast milk following breast cleansing; Thompson N et al.; The purpose of this study was to determine if preexpression breast cleansing reduced the bacterial count in expressed breast milk . The study involved the collection of 178 breast milk samples (89 matched samples); 38 matched samples were from mothers of term infants and 51 matched samples were from mothers of preterm infants . One half of the samples were collected following breast cleansing with Phisoderm and tap water . The other half were collected following breast cleansing with tap water only . Hand and equipment washing with Phisoderm and tap water preceded all sample collections . Storage containers were sterile . Samples were cultured for pathogenic and nonpathogenic bacteria and examined at 24 and 48 hours . Data were analyzed by Wilcoxon Matched-Pairs sign test and Chi square . Breast cleansing with Phisoderm and water was not more effective than water alone at reducing nonpathogenic bacteria or eliminating pathogenic bacteria . Mothers of preterm infants had higher levels of both nonpathogenic and pathogenic bacteria than mothers of full-term infants . The most common form of bacterial contamination was coagulase-negative Staphylococcus epidermidis . This particular bacteria is found in higher levels in the stools of breast fed infants than formula fed infants and it also the most common contaminant found in the blood of preterm infants who develop sepsis . The findings of this study reveal that chemical interventions may not be effective at rendering breast milk free from pathogenic bacteria . More research is needed to determine the optimal cleansing protocol to achieve bacterial decontamination of breast milk or to determine the clinically acceptable level of contamination based on the effects on infants. Eur J Immunol, 1997 Jun, 27(6), 1413 - 21 Differential response of CD4+ V7+ and CD4+ V7- T cells to T cell receptor-dependent signals: CD4+ V7+ T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen; Soares LR et al.; V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes . This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells . In the current study, CD4+ V7+ and CD4+ V7- T cells were separated from one another and their response to various stimuli analyzed . Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4+ V7+ T cells alone produced IL-2 and proliferated vigorously . By contrast, CD4+ V7- cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28-antibody were added to the cultures . The enhancement of the V7- T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC {major histocompatibility (MHC) class II, CD86} that are known to up-regulate such signals through their interaction with counter-receptors on T cells . To assess the role of V7 in a system independent of co-stimulation, CD4+ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B . The cells responded by proliferating and then becoming anergic . Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction . These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing. Br J Pharmacol, 1997 Jun, 121(4), 743 - 50 Evidence that cyclic AMP phosphodiesterase inhibitors suppress interleukin-2 release from murine splenocytes by interacting with a 'low-affinity' phosphodiesterase 4 conformer; Souness JE et al.; 1 . We have investigated the suppressive effects of rolipram, RP 73401 (piclamilast) and other structurally diverse inhibitors of cyclic AMP-specific phosphodiesterase 4 (PDE4) on interleukin (IL)-2 generation from Balb/c mouse splenocytes exposed to the superantigen, Staphylococcocal enterotoxin-A (Staph . A) . The purpose was to determine whether their potencies are more closely correlated with inhibition of PDE4 from CTLL cells, against which rolipram displays weak potency (low-affinity PDE4), or displacement of {3H}-(+/-)-rolipram from its high-affinity binding site (HARBS) in mouse brain cytosol . 2 . RP 73401 (IC50 0.46 +/- 0.07 nM, n = 4) was a very potent inhibitor of Staph . A-induced IL-2 release from Balb/c mouse splenocytes, being > 1100 fold more potent than (+/-)-rolipram (IC50 540 +/- 67 nM, n = 3) . 3 . A close correlation (r = 0.95) was observed between suppression of IL-2 release by PDE inhibitors and inhibition of PDE4 . In contrast, little correlation (r = 0.39) was observed between suppression of IL-2 release and their affinities for the high-affinity rolipram binding site (HARBS) . 4 . RP 73401 only inhibited partially (30-40%) Staph . A-induced incorporation of {3H}-thymidine into splenocyte DNA . The PDE3 inhibitor, siguazodan (10 microM), had little or no effect on IL-2 release or DNA synthesis . This concentration of siguazodan did not enhance the inhibitory action of RP 73401 on IL-2 release but potentiated its effect on DNA synthesis, increasing potency and efficacy . 5 . Staph . A-induced DNA synthesis was only partially inhibited by anti-IL-2 neutralizing antibody, whereas dexamethazone (100 nM) and cyclosporine A (100 nM) completely blocked the response . 6 . RP 73401 (IC50 6.3 +/- 1.9 nM, n = 4) was 140 fold more potent than rolipram (IC50 900 +/- 300 nM, n = 3) in inhibiting Staph . A-induced {3H}-thymidine incorporation into splenocyte DNA . 7 . The results implicate a low-affinity form of PDE4 in the suppression of Staph . A-induced IL-2 release from murine splenocytes by PDE inhibitors . The data also indicate that mitogenic factors other than IL-2, whose elaboration or responses to which are regulated by PDE3 as well as PDE4, contribute to the superantigen-induced DNA synthesis. Melanoma Res, 1997 Jun, 7(3), 214 - 22 Superantigen-induced lysis of melanoma cells; Krull F et al.; Superantigens like the Staphylococcus enterotoxin A (SEA) can direct cytotoxic T lymphocytes expressing certain T cell receptor V beta regions to lyse MHC class II-positive target cells . This superantigen-dependent cellular cytotoxicity (SDCC) has been extended to MHC class II-negative tumour cells by targeting T cells via conjugates of a tumour-specific monoclonal antibody (moAb) and a superantigen . In the present study the MHC class II-negative human melanoma cell lines G361 and MaRI were tested for susceptibility to SDCC in vitro . Antibodies recognizing the disialoganglioside GD3 and the CD10 antigen were linked to SEA either by a recombinant protein A-SEA fusion protein or an anti-kappa moAb-SEA chemical conjugate . Specific lysis of melanoma cells was dose- and effector to target (E:T) cell ratio-dependent . Introduction of a point mutation into the SEA gene (producing SEAm9) in order to reduce MHC II affinity of the superantigen, which has already been shown to severely diminish superantigen-dependent binding and lysis of MHC class II-positive cells, did not influence antibody-targeted SDCC . Cytotoxicity was equal with both antibodies (anti-GD3 and anti-CD10) and independent of whether protein A-SEA, protein A-SEAm9 or anti-kappa-SEA were used. Proteins, 1997 Jun, 28(2), 227 - 40 Studies of the unfolding of an unstable mutant of staphylococcal nuclease: evidence for low temperature unfolding and compactness of the high temperature unfolded state; Eftink MR et al.; Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants . These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample . A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, deltaC(p), between the unfolded and native states . This analysis gives a deltaC(p) of 2.2 kcal/mol/ x K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants . These deltaC(p) values are consistent with significant population of the cold unfolded state at approximately 0 degrees C . Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography . The new chromatographic peak is seen near 0 degrees C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein . Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact . Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms . Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements). Am J Forensic Med Pathol, 1997 Jun, 18(2), 140 - 3 Complications following butane inhalation and flash fire; Huston BM et al.; Solvent inhalation is a well-documented form of drug abuse that can cause euphoria and hallucinations . Sudden death involving a volatile substance is most commonly caused by cardiac arrythmias, asphyxia, direct drug effects, and trauma . The victim in this paper suffered superficial partial thickness (12% total body surface area) burns from a flash fire that occurred when lighting a match after inhaling butane in an enclosed vehicle . The victim was admitted to the hospital for 2 days of observation but did not develop any respiratory symptoms under 2 days following her release . The victim died during her readmission, 9 days after the flash fire . Postmortem examination showed extensive epithelial injury from the upper airway and trachea to the terminal bronchioles, most likely due in part to both the initial inhalation injury and the resulting adult respiratory distress syndrome (ARDS) and staphylococcal infection . Many victims with superficial burn injuries may not seek medical attention owing to either embarrassment or fear of legal prosecution . Even those who do seek medical assistance may not reveal solvent abuse as the cause of their injuries . It is possible that delayed death may occur at home following volatile substance abuse but may remain unrecognized even with a thorough scene investigation. Clin Cardiol, 1997 Jun, 20(6), 579 - 80 Eustachian valve endocarditis: detection with multiplane transesophageal echocardiography; Palakodeti V et al.; Right-sided involvement is fairly common in infective endocarditis, but involvement of the eustachian valve is distinctly rare . We present the case of a 36-year-old intravenous drug user with staphylococcal bacteremia and septic pulmonary emboli . Transthoracic echocardiography was normal, but transesophageal echocardiography revealed a large eustachian valve vegetation . This case illustrates the utility of multiplane transesophageal echocardiography in the evaluation of eustachian valve pathology. Gastroenterology, 1997 Jun, 112(6), 1876 - 86 CD4+ T-cell population mediates development of inflammatory bowel disease in T-cell receptor alpha chain-deficient mice; Takahashi I et al.; BACKGROUND & AIMS: Increase of T cells expressing CD4 and T-cell receptor (TCR) alpha- beta+ (beta{dim}) was observed in the mucosal and peripheral lymphoid tissues of TCR alpha-/- mice with inflammatory bowel disease (IBD) . The aim of this study was to characterize the CD4+ TCR alpha-beta+ T cells . METHODS: Cytokine production, TCR V beta usage, and helper function for Peyer's patch B cells by the CD4+ TCR alpha-beta+ T cells were assessed . RESULTS: The CD4+ TCR alpha-beta+ T cells purified from mesenteric lymph nodes and lamina propria of the intestine of IBD mice exclusively produced interleukin 4, used selected subsets (V beta6, V beta8, V beta14, and V beta15) of TCR, and massively proliferated after stimulation with staphylococcal enterotoxin B . Addition of the CD4+ TCR alpha-beta+ T cells to Peyer's patch B-cell cultures markedly enhanced immunoglobulin (Ig) A, IgG, and IgM antibody responses . Furthermore, depletion of the TCR alpha-beta+ T cells with monoclonal antibody against TCR beta chain completely suppressed the onset of IBD and polyclonal B-cell activation in the TCR alpha-/- mice . CONCLUSIONS: These findings suggest the CD4+ TCR alpha-beta+ T cells-mediated development of IBD in TCR alpha-/- mice. Infect Immun, 1997 Jun, 65(6), 2000 - 5 Functional analysis of Mycoplasma arthritidis-derived mitogen interactions with class II molecules; Bernatchez C et al.; The ability of superantigens (SAGs) to trigger various cellular events via major histocompatibility complex (MHC) class II molecules is largely mediated by their mode of interaction . Having two MHC class II binding sites, staphylococcal enterotoxin A (SEA) is able to dimerize MHC class II molecules on the cell surface and consequently induces cytokine gene expression in human monocytes . In contrast, cross-linking with specific monoclonal antibodies or T-cell receptor is required for staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST-1) to induce similar responses . In the present study, we report how Mycoplasma arthritidis-derived mitogen (MAM) may interact with MHC class II molecules to induce cytokine gene expression in human monocytes . The data presented indicate that MAM-induced cytokine gene expression in human monocytes is Zn2+ dependent . The MAM-induced response is completely abolished by pretreatment with SEA mutants that have lost their capacity to bind either the MHC class II alpha or beta chain, with wild-type SEB, or with wild-type TSST-1, suggesting that MAM induces cytokine gene expression most probably by inducing dimerization of class II molecules . In addition, it seems that SEA and MAM interact with the same or overlapping binding sites on the MHC class II beta chain and, on the other hand, that they bind to the alpha chain most probably through the regions that are involved in SEB and TSST-1 binding. J Immunol, 1997 Jun 1, 158(11), 5229 - 36 Gene transfer directly demonstrates a role for TCR V alpha elements in superantigen recognition; Donson D et al.; Recent structure-function studies of ours and others indicating that regions of the TCR other than V beta are involved in the TCR-superantigen (SAg)-MHC class II trimolecular interaction were correlative; thus, while the conclusions were persuasive, they were not unequivocal . The transfection experiments described in this report show that 1) responsiveness to staphylococcal enterotoxin B in V beta6 T cells was transferred by a V alpha4- but not by V alpha8- and V alpha10-containing alpha-chain cDNA constructs, 2) responsiveness was not transferred by a chimeric alpha-chain construct containing the N and J regions from a responsive T hybrid clone and the V alpha10 V alpha region from a nonresponsive clone, and 3) responsiveness was transferred by a chimeric alpha-chain construct in which most of the V alpha region (from the N terminus to the C-terminal end of the complementarity-determining region 2) was derived from the V alpha4 alpha-chain of a responsive T hybrid and the rest (framework 3, N, and J) from the V alpha8 alpha-chain of a nonresponsive T hybrid . Thus, these data provide the first direct evidence for a specific SAg response facilitating activity in a defined V alpha segment and map this activity N-terminal of framework region 3 . Furthermore, the diversity in the alpha- and beta-chain junctional regions of a panel of staphylococcal enterotoxin B-responsive V beta6 T hybrid clones excludes a stringent corequirement for a particular junctional region for the V alpha4 segment to mediate its facilitating activity . Finally, a model postulating a universal role for V alpha elements in TCR recognition of SAg is presented. Biochem Biophys Res Commun, 1997 May 29, 234(3), 660 - 5 The solution structure of a class II major histocompatibility complex superantigen binding domain; Jablonsky MJ et al.; We have used 600 MHz 1H NMR spectroscopy data to determine the solution structure of a 31-residue domain of a murine class II major histocompatibility (MHC) protein . This domain, I-Ab(beta)-(60-90), binds to the superantigen staphylococcal enterotoxin A . Distance geometry and dynamical simulated annealing calculations were performed using NOESY- and COSY-deduced constraints . I-Ab(beta)-(60-90), which is mostly alpha-helical, is more similar to the corresponding region of the class II MHC protein HLA-DR1 than to the class I MHC protein HLA-A2 . Arg-72 and Arg-80 lie on the same side of the helix and face away from the antigenic peptide binding groove . His-81, implicated in both superantigen and peptide binding, is located midway between the surface defined by Arg-72/Arg-80 and residues that define the inside of the peptide binding groove, allowing for its participation in both types of binding. Biochemistry, 1997 May 27, 36(21), 6529 - 38 Kinetic folding and cis/trans prolyl isomerization of staphylococcal nuclease . A study by stopped-flow absorption, stopped-flow circular dichroism, and molecular dynamics simulations; Ikura T et al.; We studied the urea-induced unfolding transition of staphylococcal nuclease (SNase) and its five proline mutants (P47A, P47T, P117G, P47T/P117G, and P47A/P117G) {corrected} by peptide and aromatic circular dichroism and aromatic absorption spectroscopy at equilibrium and the refolding-unfolding kinetics of the proteins by stopped-flow circular dichroism and stopped-flow absorption techniques . Recent studies have revealed that the cis/trans isomerizations about the Pro47 and Pro117 peptide bonds of SNase occur not only in the unfolded state but also in the native state . The mutational effects on the stability and the refolding-unfolding kinetics of SNase were, however, remarkably different between the two sites . The substitution of Ala or Thr for Pro47 neither changed the stability nor affected the refolding-unfolding kinetics of SNase, whereas the substitution of Gly for Pro117 increased the protein stability by 1.2 kcal/mol (pH 7.0 and 20 degrees C) and affected the kinetics . These results have been attributed to the high flexibility of the loop around Pro47, which has been revealed by molecular dynamics simulations of native SNase . Under every condition studied, cooperative refolding-unfolding kinetics of SNase were observed . Refolding of wild-type SNase was represented by two urea concentration-dependent fast phases and a urea concentration-independent slow phase . The double mutant (P47T/P117G) {corrected} of SNase still showed multiphasic refolding kinetics that involved two urea concentration-independent slow phases, suggesting that isomerization of proline residues other than Pro47 and Pro117 may occur in the unfolded state of the mutant . Two phases were observed in the unfolding of the wild-type and mutant proteins that contained Pro117, a fast phase corresponding to the unfolding of the trans isomer and a slow phase corresponding to that of the cis isomer . On the basis of these results, the folding scheme of SNase is discussed. Biochemistry, 1997 May 13, 36(19), 5795 - 805 Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease; Walkenhorst WF et al.; The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form . A central issue in protein folding is whether any of these kinetic events represent conformational steps important for efficient folding rather than side reactions caused by slow steps such as proline isomerization or misfolding of the polypeptide chain . In order to address this question, we used stopped-flow fluorescence techniques to characterize the kinetic mechanism of folding and unfolding for a Pro- variant of SNase in which all six proline residues were replaced by glycines or alanines . Compared to the wild-type protein, which exhibits a series of proline-dependent slow folding phases, the folding kinetics of Pro- SNase were much simpler, which made quantitative kinetic analysis possible . Despite the absence of prolines or other complicating factors, the folding kinetics still contain several phases and exhibit a complex denaturant dependence . The GuHCl dependence of the major observable folding phase and a distinct lag in the appearance of the native state provide clear evidence for an early folding intermediate . The fluorescence of Trp140 in the alpha-helical domain is insensitive to the formation of this early intermediate, which is consistent with a partially folded state with a stable beta-domain and a largely disordered alpha-helical region . A second intermediate is required to model the kinetics of unfolding for the Pro- variant, which shows evidence for a denaturant-induced change in the rate-limiting unfolding step . With the inclusion of these two intermediates, we are able to completely model the major phase(s) in both folding and unfolding across a wide range of denaturant concentrations using a sequential four-state folding mechanism . In order to model the minor slow phase observed for the Pro- mutant, a six-state scheme containing a parallel pathway originating from a distinct unfolded state was required . The properties of this alternate unfolded conformation are consistent with those expected due to the presence of a non-prolyl cis peptide bond . To test the kinetic model, we used simulations based on the six-state scheme and were able to completely reproduce the folding kinetics for Pro- SNase across a range of denaturant concentrations. J Immunol Methods, 1997 May 12, 204(1), 33 - 41 Selection of phage-displayed superantigen by binding to cell-surface MHC class II; Wung JL et al.; We have expressed the superantigen staphylococcal enterotoxin A (SEA) on the surface of bacteriophage as a fusion with the gene VIII protein (gVIIIp) . This phage-displayed superantigen retains the properties inherent in the natural protein . It binds to MHC class II and activates T-cells bearing appropriate V beta regions . A flexible 5-amino acid linker sequence between the SEA molecule and the phage coat protein improved the production of functional phage-displayed SEA . Binding to MHC class II-expressing cells effectively selected SEA-phage from non-SEA-phage background . This indicates that this will be an effective method for selecting new specificities of superantigen from libraries of SEA mutants and for cloning of novel superantigens. Aust N Z J Ophthalmol, 1997 May, 25 Suppl 1, S20 - 2 Interactions between the constitutive host defences of tears and Staphylococcus epidermidis; Leitch EC et al.; As part of the normal microbiota of the external eye, Staphylococcus epidermidis has probably developed strategies to overcome tear defences . The interaction of this bacteria with constitutive tear proteins was examined . Isolates of S . epidermidis grown in 10% serum or artificial tear fluid containing lysozyme or lactoferrin were resistant to the action of these proteins . Using ELISA, cell wall binding of C3, vitronectin and lactoferrin differed quantitatively between strains and in closed-eye compared to open-eye conditions . No differences were observed between ocular and non-ocular strains . This suggests that ocular isolates originate from the general host microbiota and S . epidermidis isolates are resistant to individual constitutive tear proteins. Dev Comp Immunol, 1997 May-Jun, 21(3), 311 - 22 Characterization of five monoclonal antibodies specific for swine class II major histocompatibility antigens and crossreactivity studies with leukocytes of domestic animals; Bullido R et al.; A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized . These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation . By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules . mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattle but not those of human, dog and horse . These mAbs effectively blocked the mixed lymphocyte reaction and the proliferative response to viral antigens (African swine fever virus) and to staphylococcal enterotoxin B . Therefore, these mAbs can be useful reagents for studying MHC class II molecules of pig and crossreactive species, and the immunological processes where they are involved. J Surg Res, 1997 May, 69(2), 372 - 8 Prostaglandin E2 modulates monocyte MHC-II (Ia) suppression in biomaterial infection; Henke PK et al.; Staphylococcus epidermidis biomaterial infection is associated with local cellular immune suppression measured by a depressed monocyte (M phi) Ia expression . The purpose of this study was to define the effect of proinflammatory mediators on Ia expression and bacterial clearance in experimental biomaterial infection . A 1-cm-long Dacron tube graft, sterile or colonized with Staphylococcus epidermidis (1 x 10(7) cfu/ml2), was implanted in Swiss-Webster mice . Perigraft fluid was collected at 7, 10, 14, and 28 days and assayed by enzyme-linked immunoassays for tumor necrosis factor alpha (TNF alpha), interleukin (IL)-I alpha, IL-4, IL-10, and prostaglandin E2 (PGE2) . Grafts were sonicated and plated for quantitative growth . In vivo effector inhibitions was accomplished with anti-TNF alpha, anti-IL-1 alpha antibodies (7 micrograms/24 hr), or indomethacin (50 micrograms/24 hr) via an Alzet 7-day microinfusion pump . M phi Ia expression was determined by flow cytometry . A significant elevation of TNF alpha, IL-1 alpha, and PGE2 was found during the first 10 days in the infected compared with sterile (P < or = 0.05) grafts and correlated with maximal Ia suppression . Neither IL-4 nor IL-10 was significantly different in the sterile or infected perigraft fluid . Indomethacin completely prevented M phi Ia suppression, while anti-IL-1 alpha only partially (94%) prevented M phi Ia suppression with a corresponding decrease in PGE2 production in infected grafts . Anti-TNF alpha increased PGE2 production by 189% and was associated with depressed M phi Ia expression . Indomethacin treatment improved mean graft-adherent bacterial clearance by 54% at 7 days and 75% at 28 days compared with control (not significant) . Interleukin-1 alpha but not TNF alpha increases PGE2 production which modulates M phi Ia suppression . To improve treatment of biomaterial infections, local immunomodulation of PGE2 and IL-1 alpha is promising. Optom Vis Sci, 1997 May, 74(5), 288 - 92 Efficacy of hand washing procedures on bacterial contamination of hydrogel contact lenses; Ly VT et al.; The effect of various hand washing regimens on transfer of bacterial contaminants from the hands to a hydrogel contact lenses was evaluated . Each of 47 subjects performed 5 different hand washing procedures, and then handled a new, sterile hydrogel contact lens . The lenses were cultured to determine colony-forming units (CFUs) and microbial identity . Median CFUs on lenses handled after washing with water, soap and water, or soap and water followed by towel drying were higher than the median CFU for lenses handled after no hand washing . The median CFU for lenses handled after soap and water washing followed by an alcohol wipe was not different from the no washing group . The majority of the contaminants were identified as Staphylococcus epidermidis . These results show that ordinary hand washing alone does not decrease, and may even increase, the amount of contaminants transferred from the hands to a hydrogel lens . Use of an alcoholic wipe after hand washing reverses this effect . Hand washing is still recommended in contact lens hygiene for removal of more pathogenic contaminants. Br J Dermatol, 1997 May, 136(5), 762 - 7 Nikolsky's sign: is it 'dry' or is it 'wet'? Salopek TG. Nikolsky's signs refers to the ability to induce peripheral extension of a blister as a consequence of applying lateral pressure to the border of an intact blister . Although initially used in reference to the pemphigus group of blistering dermatoses, a positive Nikolsky's sign can be seen in other bullous diseases such as toxic epidermal necrolysis and staphylococcus scalded skin syndrome . Appreciating whether the blister is 'wet' or 'dry' at the site of a positive Nikolsky's signs may have both diagnostic and prognostic significance which I illustrate with several clinical cases . Lastly, I review the significance of a positive Nikolsky's sign. Br J Dermatol, 1997 May, 136(5), 730 - 3 IgG subclasses specific to Staphylococcus epidermidis and Propionibacterium acnes in patients with acne vulgaris; Ashbee HR et al.; IgG subclasses specific to Staphylococcus epidermidis and Propionibacterium acnes were determined in sera from patients with mild, moderate or severe acne and from a control group . Titres specific to S . epidermidis were all within the same range and did not differ between groups . The titres of IgG subclasses to specific to P . acnes did vary between groups . IgG1 and IgG3 were significantly higher in severe acne patients compared with moderate acne patients, while IgG2 was significantly higher in moderate and severe patients compared with controls . Titres of IgG4 did not differ between groups . The pattern of titres observed suggests that, while the antibody response to S . epidermidis is relatively harmless, antibodies to P . acnes may be involved in the pathogenesis of acne vulgaris. Cytokine, 1997 May, 9(5), 295 - 9 Biphasic control of NF-kappa B activation induced by the triggering of HLA-DR antigens expressed on B cells; Leonardi A et al.; The regulation of NF-kappa B activation following the triggering of HLA-DR antigens by mAb L243 has been studied at various times in Raji cells . Electrophoretic mobility shift assays demonstrated a strong increase of NF-kappa B DNA binding after triggering of HLA-DR antigens . Using TNF-alpha-activity neutralizing antibodies, the authors demonstrated that the upregulation of NF-kappa B was found to depend, at later time point, on an autocrine effect of TNF-alpha secreted following triggering of HLA-DR antigens . In contrast, it was found to be TNF-alpha independent in the early time point . Moreover, the upregulation of NF-kappa B binding activity is regulated by the triggering of selected epitopes of HLA-DR antigens . In fact, mAb L243 but not the staphylococcal superantigens, staphylococcal exotoxin toxic shock syndrome toxin-I or staphylococcal enterotoxin B, regulate the NF-kappa B binding activity. Biosci Biotechnol Biochem, 1997 May, 61(5), 846 - 51 Sequential binding of Staphylococcal gamma-hemolysin to human erythrocytes and complex formation of the hemolysin on the cell surface; Kaneko J et al.; Staphylococcal gamma-hemolysin consists of two protein components, F (or H gamma I) and H gamma II . To elucidate the mode of action of gamma-hemolysin, we studied the binding order of F and H gamma II to human erythrocytes and the cell-bound state of the two components . The binding of F to human erythrocytes preceded the binding of H gamma II to the cells, and thereafter hemolysis occurred . Western immunoblot analysis of the cell-bound gamma-hemolysin indicated that F and H gamma II components form high-molecular-mass (150-250 kDa) complexes on the erythrocytes . The toxin complexes were recovered in a Triton X-100-insoluble fraction of the erythrocytes, which contains cytoskeleton proteins . Neither the formation of the toxin complex(es) nor hemolysis occurred when the erythrocytes were treated with proteinase K . Abortion of the complex formation on the proteinase K-treated erythrocytes may be due to the failure of the binding of H gamma II to the cells, because F bound to the proteinase K-treated erythrocytes to the same extent as to the non-treated erythrocytes. Anal Biochem, 1997 May 1, 247(2), 223 - 7 Bacterial typing: storing and processing of stabilized reference bacteria for polymerase chain reaction without preparing DNA--an example of an automatable procedure; Rogers C et al.; This paper examines the use of bacteria killed on blood-storage paper as templates (ghosts) and takes, as an example, the PCR ribotyping (amplification of the intergenic spacer regions between the 16S and 23S ribosomal RNA genes) of bacteria as described by Kostman et al . (J . Infect . Dis . 171, 204-208, 1995) . All procedures have been particularly designed to be compatible with automation . DNA preparation is inappropriate for routine, high-volume sequence amplification from a diversity of microorganism cultures . Blood-storage/processing media provide another way of processing samples for PCR with distinctive aspects of increased safety and ease of automatibility . Blood-storage paper can be used for killing and processing bacteria to DNA-containing ghosts for reliable PCR . From as little as a few microliters of an overnight culture or a reasonably sized, single colony, rapid processing of large sample numbers is possible . FTA blood-storage medium has additional utility in that long-term cataloguing, storage, and processing of paper, loaded with culture, for PCR, is possible via a variety of sample wash sequences . It can be performed at convenience, after collection, and can be delayed indefinitely . using this approach, the repeatability of some PCR-ribotyping methodology of the type used by Kostman et al . (J . Clin . Microbiol . 30, 2084-2087, 1992) was examined as an exercise to demonstrate the practicality of FTA blood-paper usage and to check some basic features of PCR ribotyping . Five strains of Staphylococcus and one strain Escherichia coli were stored and processed on FTA blood paper, the PCR-ribotype patterns were analyzed and found to be the equal of patterns previously seen via additional DNA preparations. J Clin Oncol, 1997 May, 15(5), 1994 - 2007 Superantigen-based immunotherapy: a phase I trial of PNU-214565, a monoclonal antibody-staphylococcal enterotoxin A recombinant fusion protein, in advanced pancreatic and colorectal cancer; Giantonio BJ et al.; PURPOSE: To establish the maximum-tolerated dose (MTD) and define the toxicities of a single-dose infusion of PNU-214565, a recombinant Escherichia coli-derived fusion protein of Staphylococcal enterotoxin A (SEA) and the Fab-fragment of the C242 monoclonal antibody in patients with advanced colorectal and pancreatic carcinomas . To investigate the capability of PNU-214565 to induce a superantigen (SAg) response resulting in cytokine production and tumor regression . PATIENTS AND METHODS: Twenty-one patients (age range, 39 to 76 years; median, 64; 12 men, nine women; 18 colorectal, three pancreatic cancers) were treated with a single 3-hour infusion of PNU-214565, with doses ranging from 0.01 to 1.5 ng/kg . All patients had prior chemotherapy and a good performance status Eastern Cooperative Oncology Group {ECOG} performance status {PS} = 0 {n = 10}; PS = 1 {n = 11}), 10 had prior radiation, and 18 had prior surgery . RESULTS: Fever and hypotension were the most common toxicities . Fever of any grade occurred in 16 of 21 patients (76%): four of 21 (19%) with grade 2 and two of 21 (9.5%) with grade 3 . Hypotension of any grade occurred in 13 of 21 (62%): four of 21 with grade 2 and one of 21 (5%) with grade 3 . Interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF alpha) induction correlated with toxicity . In the two patients with grade 3 fever, peak IL-2 and TNF alpha levels were 2.9 IU/mL and 165 pg/mL, and 8.3 IU/mL and 245 pg/mL, respectively . Transient, > or = 50% decreases in circulating monocytes were observed in 17 of 21 patients as early as 0.5 hours (median time, 2 hours) from the start of infusion . Decreases (mean 33%) in circulating lymphocytes were observed in seven of 21 patients . All three patients with grade 3 toxicity were treated at the 0.5-ng/kg dose . The significance of baseline anti-SEA, human antimouse antibody (HAMA), CA242-soluble antigen levels, and T-cell receptor variable beta region (TCR V beta) subsets and histocompatibility leukocyte antigen-DR (HLA-DR) genotypes was assessed as possible predictors of toxicity . All toxicities were transient and easily managed . No grade 3 toxicity occurred at the higher dose levels . CONCLUSION: PNU-214565, a SAg-based tumor targeted therapy, is safe when given as a single 3-hour infusion at doses up to 1.5 ng/kg . The MTD for a single dose was not determined . The safety of a repeated dose schedule is currently under investigation, beginning with doses determined to be safe in this trial. Scand J Immunol, 1997 May, 45(5), 551 - 6 The amino acid sequence of the glycosylated amyloid immunoglobulin light chain protein AL MS; Omtvedt LA et al.; The authors report on the amino acid sequence of the glycosylated amyloid protein AL MS . The amyloid fibrils were extracted from the spleen of a patient (MS.) with amyloidosis . The protein AL MS was purified from the amyloid fibrils by gel filtration . SDS-PAGE performed on the purified protein material showed glycosylated protein bands in the range of 22 to 32 kDa, corresponding to polymerization of N-terminal fragments . The protein was characterized by amino acid analysis and Edman degradation . Tryptic digest combined with Staphylococcal V8 protease, chymotrypsin and pyroglutamate aminopeptidase digestion, as well as cleavage with BNPS-skatole, established the complete amino acid sequence of 168 residues . The protein was compared to other proteins in the SWISSPROT databank, showing homology with the immunoglobulin light chain variable subgroup lamda I . AL MS showed some unique amino acid substitutions . Highly conserved residues Gly(57) and Arg(61), were exchanged to arginine and glutamine, respectively, possibly altering the three- dimensional structure of the protein. Br J Urol, 1997 May, 79(5), 781 - 4 A comparative study of the distribution of ofloxacin and ciprofloxacin in prostatic tissues after simultaneous oral ingestion; Png JC et al.; OBJECTIVES: To determine the levels of two quinolones, ofloxacin and ciprofloxacin, potent broad-spectrum antibiotics with very good oral bioavailability and low minimum inhibitory concentrations (MICs) for most pathogens, in the prostates of patients who underwent transurethral resection of the prostate (TURP) after oral ingestion for surgical prophylaxis . PATIENTS AND METHODS: Twenty-eight patients with BPH requiring a TURP ingested 250 mg of both drugs 2-4 h before operation . The levels of the drugs in the serum and prostate were measured using high-performance liquid chromatography and the levels of both drugs determined at the 6 and 9 o'clock positions in the prostate to examine any local variations in drug concentration . RESULTS: Ofloxacin concentrations were significantly higher in the serum and prostatic tissues compared with ciprofloxacin for the same dose, but its penetrance into the prostate was lower . This mainly reflected its higher oral bioavailability . Both drugs were present in concentrations 50% higher at the 6 o'clock than at the 9 o'clock position but both exceeded the MICs for most Gram-negative organisms except Pseudomonas . CONCLUSION: Ofloxacin has the advantage against ciprofloxacin of exceeding the MICs for Staphylococcus and Chlamydia . However, ciprofloxacin has the advantage of having prostate-to-serum ratios of unity, but for the same dose the prostatic concentrations of ofloxacin is significantly higher. Am J Respir Crit Care Med, 1997 May, 155(5), 1723 - 8 Lack of clinical utility of bronchoalveolar lavage cultures for cytomegalovirus in HIV infection; Mann M et al.; This study assessed the presence of cytomegalovirus (CMV) in bronchoalveolar lavage (BAL) in three subpopulations of HIV-infected patients and correlated its presence with clinical status during 3 mo of follow-up . Nineteen asymptomatic volunteers, six patients with CMV retinitis, and 46 patients with acute pulmonary symptoms underwent BAL and were assessed for CMV by cytopathology, conventional shell vial cultures, and antigen detection . Transbronchial biopsies were also obtained when possible and evaluated for histopathologic changes of CMV . All patients were followed for approximately 3 mo . Cytomegalovirus was detected in BAL in nine of 19 (47%) asymptomatic volunteers, in all six patients with CMV retinitis, and in 33 of 46 (72%) patients with pulmonary symptoms . Only one symptomatic patient with a positive CMV BAL culture developed clinically significant CMV pulmonary disease; this patient developed disseminated CMV and died . The only other death occurred in a patient with CMV retinitis who developed staphylococcal bacteremia . None of the asymptomatic volunteers or patients with CMV retinitis developed evidence of CMV pneumonia or any other organ disease with CMV . Cytomegalovirus is frequently detected in BAL from HIV-infected patients regardless of their pulmonary symptoms and its presence does not clinically predict significant pulmonary morbidity or mortality in 3 mo of follow-up. J Med Microbiol, 1997 May, 46(5), 430 - 2 Microbiology of chronic maxillary sinusitis: comparison between specimens obtained by sinus endoscopy and by surgical drainage; Brook I et al.; The aerobic and anaerobic microbiology of sinus aspirates obtained during surgery was compared with culture of samples obtained by endoscopy . Six patients with chronic maxillary sinusitis were evaluated . Polymicrobial flora was found in all specimens (two-to-five isolates/sample) . A total of 24 isolates (18 anaerobic, five aerobic and one micro-aerophilic) was obtained from sinus aspirates, and 25 isolates (16 anaerobic and nine aerobic) were found in endoscopic specimens . The predominant organisms were Prevotella spp., Fusobacterium nucleatum, Peptostreptococcus spp . and Staphylococcus spp . Concordance in the type and concentration of organisms was found in all cases . Sixteen of the 18 anaerobes isolated from sinus aspirates were also found in the concomitant endoscopic sample . Five aerobic isolates were found in both sinus aspirates and endoscopic samples and their concentration was similar . However, four aerobic gram-positive bacteria (<10(4) cfu/sample) were found only in endoscopy samples . This pilot study demonstrates the usefulness of endoscopic aspiration in the isolation of bacteria from chronically infected maxillary sinuses. J Neuroimmunol, 1997 May, 75(1-2), 163 - 8 Inhibition of T cell superantigen responses following treatment with the kappa-opioid agonist U50,488H; Guan L et al.; Previous work in our laboratory has shown that cytokine production by primary murine macrophages, and macrophage cell lines, is inhibited following treatment with the kappa-opioid agonist U50,488H . Furthermore, we have found that the participation of both accessory cells and T cells in an antibody response is suppressed by this compound . We have utilized the superantigen staphylococcal enterotoxin B (SEB) to further examine the effects of U50,488H on accessory and T cell function . The results showed that the proliferative response of lymph node T cells to SEB presented by activated macrophages was significantly inhibited by the kappa-opioid agonist at concentrations as low as 100 nM . However, suppression of the T cell response to SEB presented by resting macrophages required 100 times the concentration of U50,488H . On the other hand, the production of IL-2 in response to lymph node T cell stimulation with SEB was not altered by the opioid treatment . Additional experiments utilizing the opiate antagonist naloxone and the kappa-selective antagonist nor-binaltorphimine (norBNI) were performed in order to further characterize the opioid receptor involved in the suppressive activity of U50,488H . Results showed that both naloxone and norBNI were able to block the inhibitory activity of U50,488H . Further analysis showed that the proliferative response of thymic T cells was more sensitive to the effects of U50,488H, and the response with both activated and resting macrophages was suppressed . In addition, the production of IL-2 by the thymic T cells was also inhibited by the opioid treatment . The mechanism of suppression of superantigen-induced T cell responses is discussed. Am J Pathol, 1997 May, 150(5), 1607 - 18 Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses; Litton MJ et al.; Repeated injections of a fusion protein containing the superantigen staphylococcal enterotoxin A (SEA) combined with a Fab fragment of a tumor-specific antibody is a highly efficient immunotherapy for mice expressing lung melanoma micrometastasis . In the present study, the systemic and local immune responses generated by this therapy were analyzed at a cellular level . Two distinct but coupled immune reactions occurred after repeated therapy . Tumor necrosis factor and macrophage inflammatory protein-1 alpha and -1 beta were immediately synthesized, in the absence of T lymphocytes, at the local tumor site in the lung . This was followed by the induction of VCAM-1 adhesion molecule expression on pulmonary vascular endothelial cells . Concurrently, the early response in the spleen was characterized by the induction of selective T cells producing interleukin (IL)-2 . The primed and expanded SEA-reactive V beta 3- and V beta 11-expressing T lymphocytes accumulated to the tumor area only after Fab-SEA therapy and were not present in the lung when SEA, Fab fragment, or recombinant IL-2 was injected . The tumor-infiltrating T cells produced large amounts of interferon-gamma, but no IL-2 or Th2 type of lymphokines were detected at the tumor site in the Fab-SEA-targeted antitumor immune response . These results emphasize the necessity to investigate several sites of antigen presentation to elucidate the effects of immunotherapy. J Immunol, 1997 May 1, 158(9), 4245 - 51 Functional characterization of the interaction between the superantigen staphylococcal enterotoxin A and the TCR; Antonsson P et al.; In this report, we show that despite an overall amino acid residue identity of more than 80% between the staphylococcal enterotoxins (SE) A and E, these proteins markedly differ in their absolute requirement for the MHC class II during T cell activation . The superantigens were produced as C215Fab-SE fusion proteins and analyzed for their ability to activate T cells in a MHC class II-independent manner, using C215 Ag expressing cell lines as pseudo super-APCs . C215Fab-SEA, but not C215Fab-SEE, induced T cell cytotoxicity and proliferation in these MHC class II-independent systems . Introduction of a region from SEA, comprising amino acids 20-27, to SEE transferred the ability to engage T cells in the absence of MHC class II . Analysis of the Vbeta specificity of the chimeric SEA/SEE molecules and a panel of SEA mutants demonstrated that the site for TCR interaction covers the edge surrounding the shallow cavity on top of the SEA molecule. J Mol Biol, 1997 Apr 25, 268(1), 170 - 84 Characterization of long-range structure in the denatured state of staphylococcal nuclease . II . Distance restraints from paramagnetic relaxation and calculation of an ensemble of structures; Gillespie JR et al.; Structural analysis of delta131delta, a fragment model of the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between chain segments by paramagnetic relaxation enhancement . Fourteen unique PROXYL spin labels were introduced at sites that are solvent-exposed in the native state, and the resulting enhancements of T2 for the amide protons were measured by NMR spectroscopy . When these data were combined with either measured or estimated correlation times tau(c), the r(-6)-weighted, time and ensemble-averaged distance between the spin label and 30 to 60 amide protons could be calculated for each spin-labeled protein . On the basis of approximately 700 such loose distance restraints, ensembles of compatible structures were generated by a combined distance geometry/molecular dynamics approach . Because of the large uncertainty in the physical basis of these distance restraints, a number of calculations were carried out to establish the sensitivity of the calculated structures to systematic errors in these restraints . Overall, the structural features reflected in the paramagnetic relaxation data were robust; large variations in tau(c), in the bounds window of allowed distances, or in the number of restraint distances used had small effects on the general features common to all calculated structures . The global topology of this denatured form of staphylococcal nuclease, as described by an ensemble of conformations consistent with the data, is strikingly similar to that of the native state, the major difference being the segregation of two hydrophobic segments that form a beta hairpin in the native state . These findings suggest that the topology of a protein's fold is established in the denatured state in the absence of cooperative interactions involving tight packing or stable hydrogen bonding . Hydrophobic interactions alone may encode global topology. J Mol Biol, 1997 Apr 25, 268(1), 158 - 69 Characterization of long-range structure in the denatured state of staphylococcal nuclease . I . Paramagnetic relaxation enhancement by nitroxide spin labels; Gillespie JR et al.; Structural analysis of delta131delta, a fragment model of the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between protein chain segments based on paramagnetic relaxation enhancement methods . PROXYL spin labels were attached at unique cysteine residues introduced at 14 different sites along the polypeptide chain, and the resulting enhancements of amide proton relaxation were measured by NMR spectroscopy . To minimize perturbation of denatured state structure, these labeling sites were chosen on the basis of a high solvent exposure in the native state and a small change in stability and m-value upon mutation of the wild-type residue to cysteine or alanine . EPR spectroscopy confirmed that in all cases the PROXYL label of the modified protein was solvent-exposed and undergoing free isotropic rotation . By quantifying at 500 MHz and 600 MHz the enhancement of both T1 and T2 relaxation for amide protons resolved in a 1H-15N correlation spectrum, the apparent correlation time for the free electron-proton vectors for six PROXYL-labeled proteins could be estimated . With these data plus the enhancements in transverse relaxation rate (R2) for the other eight proteins, the time-averaged, r(-6) weighted distance between the free electron on the unique nitroxide and 30 to 60 amide protons in each protein could be approximated . Inspection of the pattern of R2 enhancements reveals a significant amount of long-range structure in this denatured state, a clear indication that it is not a random coil. J Exp Med, 1997 Apr 21, 185(8), 1435 - 45 Lack of allelic exclusion in B cell chronic lymphocytic leukemia; Rassenti LZ et al.; We determined the immunoglobulin (Ig) V(H) subgroup expressed by the leukemia cells of 108 patients with B cell chronic lymphocytic leukemia (CLL) . Surprisingly, we found that six samples (5%) each expressed Ig of more than one V(H) subgroup . Southern blot analysis demonstrated that these samples each had rearrangements involving both Ig heavy chain alleles . Nucleic acid sequence analyses of the Ig cDNA revealed each to express two functional Ig V(H) genes: V(H)3-33 and V(H)4-39; V(H)3-7 and V(H)4-39; V(H)3-23 and V(H)4-61; V(H)2-70 and V(H)3-30.3; or V(H)3-30 and V(H)4-b (DP67) . One sample expressed three Ig V(H) genes: V(H)2-70, V(H)3-7, and V(H)4-59 . Despite having more than one Ig heavy chain transcript, each sample was found to express only one functional Ig light chain . From the primary sequence, we deduced that the Ig of some of these CLL samples should react with Lc1, a monoclonal antibody (mAb) reactive with a supratypic cross-reactive idiotype present on Ig encoded by a subgroup of Ig V(H)4 genes (namely, V(H)4-39, V(H)4-b {DP-67}, V(H)4-59, or V(H)4-61), and B6, an mAb that reacts with Ig encoded by certain Ig V(H)3 genes (namely, V(H)3-23, V(H)3-30, or V(H)3-30.3), and/or modified staphylococcal protein A (SpA), a 45-kilodalton bacterial "superantigen" that reacts with most Ig of the V(H)3 subgroup . Flow cytometric analyses revealed that such samples did in fact react with Lc1 and B6 and/or SpA, but not with control mAbs of irrelevant specificity . This study demonstrates that a subset of CLL patients have leukemic B cells that express more than one functional Ig heavy chain. Int J Cardiol, 1997 Apr 18, 59(2), 113 - 8 Atrio-ventricular valve dysplasia in 22 newborn infants; Bonnet D et al.; We retrospectively studied the experience of our institution with isolated dysplasia of one or both atrio-ventricular valves in 22 newborn infants . All patients with associated cardiac malformations were excluded . Ten patients exhibited isolated tricuspid valve dysplasia . One patient had tricuspid valve dysplasia and a dysplastic pulmonary valve . In 10 patients, both atrio-ventricular valves were affected . Finally, mitral valve dysplasia was associated with pulmonary valve stenosis in 1 case . Associated syndromes and/or chromosomal anomalies were: Down syndrome (n=2), trisomy 18 (n=1), Noonan syndrome (n=1), Marfan syndrome (n=3), Ehlers-Danlos and Cutis laxa (n=2) . Mortality was 27.2% during follow-up (mean 51 months): 3 patients with chromosomal aneuploidies, 2 patients with severe neonatal Marfan syndrome and 1 with Ehlers-Danlos . Complications were: sustained supra-ventricular tachycardia in 3, neonatal staphylococcal tricuspid valve endocarditis in 1, persistent significant valvular disease in 8 . In the remaining 9 survivors, the dysplasia of the atrio-ventricular valves persists with absent or mild incompetence . Beside obvious chromosomal anomalies, newborn infants with dysplastic valves should be investigated for manifestations of connective tissue disorders . This may help to identify new pleiotropic syndromes which include valvular dysplasia as one manifestation. Mol Gen Genet, 1997 Apr 16, 254(3), 312 - 8 Secretion of the lantibiotics epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on epidermin production and their regulation by EpiQ; Peschel A et al.; The closely related lantibiotics epidermin and gallidermin are produced by Staphylococcus epidermidis Tu3298 and S . gallinarum Tu3928, respectively . The epidermin biosynthetic genes involved in maturation, regulation, and immunity have been identified previously . How epidermin or gallidermin is secreted, however, has remained unclear . Here, we characterize two additional genes, epiH and epiT, as well as the homologous gallidermin genes gdmH and gdmT . EpiT and GdmT are similar to one-component ABC transporters that are involved in the secretion of proteins or peptides . EpiH and GdmH are hydrophobic proteins without conspicuous similarities to other proteins . Comparison of the gene sequences revealed that epiT is incomplete, having an internal deletion that causes a frame shift and a second deletion at the 3'-end, while gdmT is intact . Introduction of epiT and epiH into the heterologous host S . carnosus (pTepi14) bearing the maturation and regulation genes had no significant effect on the rather low level of epidermin production . The presence of the homologous gdmT and gdmH, however, resulted in a strong increase (seven- to tenfold) in the production level, which is very likely to be due to increased efficiency of epidermin secretion . Both gdmT and gdmH were necessary for this effect, indicating that the two gene products cooperate in some way . In the epidermin-producing wild-type strain Tu3298, which contains epiH and the disrupted epiT, the addition of gdmT alone led to a two-fold increase in epidermin production . Both gdmT and gdmH and the corresponding epi genes were activated by the transcriptional regulator EpiQ; this is in accordance with the presence of putative EpiQ operator sites in the promoter regions. J Immunol, 1997 Apr 15, 158(8), 3936 - 46 Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit; Agren LC et al.; Cholera toxin (CT) is an exceptionally potent adjuvant but, unfortunately, also very toxic . Here we present a powerful new approach to separate toxicity from adjuvanticity by constructing a fusion protein that combines the enzymatically active cholera toxin A1 subunit (CTA1) with targeting to B cells . The CTA1 was genetically linked at its C-terminal end to two Ig-binding domains, DD, of staphylococcal protein A and produced in Escherichia coli . The highly purified, monomeric CTA1-DD fusion protein, with a molecular mass of 37 kDa, was found to exhibit strong ADP-ribosyltransferase activity and bound, via the DD moiety, to both Fc and Fab fragments and to all IgG subclasses--IgE, IgA, and IgM . After i.v . injection of the fusion protein, FACS analysis revealed binding of CTA1-DD to splenic IgM+ B cells, but not CD3+ T cells, indicating cell-specific targeting in vivo . Strikingly, we found that the adjuvant ability of CTA1-DD to enhance systemic IgG as well as mucosal IgA responses to the unrelated Ags, OVA, or keyhole limpet hemocyanin, administered i.v or intranasally, was comparable to that of intact CT . In addition, the enhancing effect on specific IgG1, IgG2a, and IgG2b responses mimicked that of CT and suggested involvement of both Th1 and Th2 CD4+ T cell activity . The CTA1-DD, as well as CT, up-regulated expression of the CD80 and CD86 molecules on the targeted B cells, indicating that enhanced T cell costimulation may be responsible for the adjuvant effect . Contrary to CT, however, CTA1-DD was completely nontoxic . Thus, the CTA1-DD adjuvant should find general applicability in systemic and mucosal vaccines, and the strategy used may also be explored for other regimens requiring targeted immunomodulation. J Immunol, 1997 Apr 15, 158(8), 3698 - 704 Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain; Thibodeau J et al.; Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR . Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules . Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain . Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR . Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA . Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR . These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosine phosphorylation through the TCR. Acta Gastroenterol Belg, 1997 Apr-Jun, 60(2), 176 - 9 Experimental hepatitis and role of cytokines; Tiegs G; Activated T lymphocytes appear to be responsible for liver damage in chronic active hepatitis and autoimmune liver disease . We described three experimental mouse models of T cell dependent liver injury . D-galactosamine (GalN)-sensitized mice challenged with either T cell activating anti-CD3 monoclonal antibody (mAb) or with the superantigen staphylococcal enterotoxin B (SEB) developed severe liver injury characterized by internucleosomal DNA fragmentation as well as by histological hallmarks of hepatocyte apoptosis, both preceding the increase of plasma transaminases . Administration of the T cell mitogen concanavalin A (Con A) to unsensitized mice also resulted in hepatic apoptosis and the ensuing necrosis . Anti-CD3 mAb as well as SEB or Con A induced the release of systemic tumor necrosis factor (TNF), interferon gamma (IFN gamma), and various other cytokines . Passive immunization against TNF or pretreatment with immunosuppressive drugs such as cyclosporin A, FK 506 or dexamethasone protected mice from liver injury . T lymphocytes were identified as effector cells of Con A in vivo i) by proof of resistance of athyrnic nude mice against Con A and ii) by restoration of susceptibility in nude by lymphocyte transfer from control mice . Moreover, antibody-dependent depletion of CD4+ T cells fully protected against Con A, whereas depletion of CD8+ T cells failed to prevent liver injury . These results indicated that cytokines released following T helper cell activation rather than cytotoxic T cells mediated liver injury . We recently found that IFN gamma is also a critical mediator of Con A-induced hepatic damage . In conclusion, these T cell-dependent models of inflammatory liver injury allow the investigation of basic principles of hepatic disorders associated with T cell activation and infiltration as well as pharmacological in vivo studies for the development of hepatoprotective drugs. Acta Virol, 1997 Apr, 41(2), 97 - 9 Summing up, weak immunomodulating signals of Tahyna virus and immunomodulating drugs induce immunosuppression in mice; Semenova IB et al.; The successive injection of non-immunomodulating doses of Tahyna virus (100 LD50) and non-immunomodulating doses of immunomodulating drugs, such as purified staphylococcal toxoid or glucosaminylmuramyldipeptide (Likopid), to mice were accompanied by a decrease in the IgM plaque-forming cell response to sheep red blood cells. Protein Eng, 1997 Apr, 10(4), 455 - 62 Production of correctly folded recombinant {13C, 15N}-enriched guinea pig {Val90}-alpha-lactalbumin; Kim S et al.; The M90V mutant of guinea pig alpha-lactalbumin (gpLA) was expressed intracellularly in Escherichia coli using a gpLA gene fusion to the IgG-binding ('Z') domain coding sequences of staphylococcal protein A . The fusion protein was expressed as an inclusion body, then purified and refolded in vitro; CNBr cleavage of the fusion polypeptide yielded native alpha-lactalbumin . The recombinant M90V gpLA was virtually identical with natural gpLA with respect to its ability to stimulate lactose synthesis by galactosyl transferase and the recombinant and natural molecules also exhibited similar circular dichroism spectra, thermal melting profiles and NMR spectra . However, modest perturbations in the chemical shifts of amide protons in the C-helix residues, attributable to the Met-->Val mutation, were observed . In defined media, this expression system enabled the production of highly-enriched 15N- and 13C, 15N-labeled gpLA . Use of this material will allow the solution conformations of the native and molten globule states of gpLA to be characterized by high-resolution multidimensional NMR. Cancer Immunol Immunother, 1997 Apr, 44(2), 77 - 82 Prevention of superantigen-induced tolerance in vivo by interleukin-2 treatment; Belfrage H et al.; Injection of the superantigen staphylococcal enterotoxin A (SEA) activates both CD4+ and CD8+ T cells expressing certain families of T cell receptor (TCR) variable-region beta (V beta) chain . T cells respond with profound cytokine production and induction of cytotoxicity . Repeated injections, however, cause deletion and anergy of both CD4+ and CD8+ T cells, resulting in reduced frequency of SEA-responsive cells TCR-V beta11+ as well as reduced cytokine levels in serum upon challenge with SEA . Exogenous interleukin-2 (IL-2) in vivo rescued SEA-responsive CD4+ and CD8+ cells from SEA-induced deletion and/or increase expansion of SEA-primed cells as well as preventing downregulation of endogenous IL-2 production in vivo . Combined treatment with SEA and IL-2 also superinduced production of important cytokines for the cytotoxic function of T cells, tumour necrosis factor alpha, interferon gamma and IL-6, on a cellular level . These studies show that continuous stimulation with IL-2 in vivo could be useful for superantigen-based immunotherapy by induction of excessive T cell activation and by prevention of the development of T cell deletion and anergy. Jpn J Clin Oncol, 1997 Apr, 27(2), 115 - 8 Mitomycin-C induced hemolytic uremic syndrome: a case report and literature review; Wu DC et al.; Hemolytic uremic syndrome spontaneously arises in a few patients with advanced cancer, but it is more commonly related to the use of certain chemotherapeutic agents . Mitomycin-C is, etiologically, the most common causative agent inducing hemolytic uremic syndrome, in a dose dependent manner . We report this syndrome, attributable to mitomycin-C at a cumulative dose of 40 mg/m2, in a gastric cancer patient . A 42-year-old female with stage III gastric cancer underwent radical gastrectomy and was given mitomycin-C at 10 mg/m2 intravenously every four weeks as adjuvant therapy . Hemolytic uremic syndrome was diagnosed three months after the last dose of mitomycin-C administration . The most prominent symptoms included pallor, hypertension and anasarca, with laboratory evidence of microangiopathic hemolytic anemia, azotemia and hyperkalemia . Her disease was progressive, but fortunately stabilized after staphylococcus column A dialysis . Her disease remained in remission for 24 months from the time of diagnosis, and then relapsed in the form of peritoneal carcinomatosis with partial intestinal obstruction. Eur J Immunol, 1997 Apr, 27(4), 825 - 33 Superantigen and endotoxin synergize in the induction of lethal shock; Blank C et al.; Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock . While endotoxin primarily interacts with CD14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells . Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock . We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock . We report that LPS and SEB operate synergistically . Lethal doses of both inducers were reduced 100-fold when given in combination . The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-gamma (IFN-gamma) were elevated and remained high for a prolonged period . Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D-galactosamine (D-GalN) . Opposed to D-GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel . Cyclosporin A and treatment with anti-IFN-gamma monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-gamma is the mediator of the observed synergism . Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction . Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia. Biotechnol Appl Biochem, 1997 Apr, 25 ( Pt 2), 173 - 80 In vitro complex-formation between the molecular chaperone DnaK and staphylococcal protein A derivatives produced in Escherichia coli and its use in the purification of DnaK; Gustavsson K et al.; Complex-formation between a truncated staphylococcal Protein A produced in Escherichia coli and a native E coli molecular chaperone, DnaK, can be used for the purification of DnaK by IgG-affinity chromatography . The half-time constant for in vitro formation of the Protein A-DnaK complex is about 14 min . Complex-formation in the presence of ATP is faster, but pre-incubation of DnaK with ATP decreases the final amount of the complex . A second complex with a slower migration on native PAGE is formed when the ratio of DnaK to Protein A is increased . A derivative of Protein A, ZZ, which essentially contains only two modified domains of Protein A, did not bind DnaK . After insertion of a tryptophan-rich peptide close to the C-terminus, the resulting protein, ZZT3, became able to bind DnaK . The binding of these three proteins to DnaK correlates with proteolysis in E coli, indicating a possible role for the binding of DnaK in the control of proteolysis. Infect Immun, 1997 Apr, 65(4), 1522 - 6 Expression of slime interferes with in vitro detection of host protein receptors of Staphylococcus epidermidis; Baldassarri L et al.; We hypothesized that slime may mask bacterial molecules important in the attachment of Staphylococcus epidermidis to inanimate surfaces . In support of this hypothesis, we found that slime-negative strains attached significantly better to fibrinogen or fibronectin than the parent strains and exhibited greater surface hydrophobicity . Comparable results were obtained with 53 clinical isolates. Transfusion, 1997 Apr, 37(4), 423 - 35 Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light; Lin L et al.; BACKGROUND: A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates . This process is based on the photochemical reaction of a novel psoralen, S-59, with nucleic acids upon illumination with long-wavelength ultraviolet light (UVA, 320-400 nm) . STUDY DESIGN AND METHODS: High levels of pathogens were added to single-donor platelet concentrates containing 3 to 5 x 10(11) platelets in 300 mL of 35-percent autologous plasma and 65-percent platelet additive solution . After treatment with S-59 (150 microM) and UVA (0-3 J/cm2), the infectivity of each pathogen was measured with established biologic assays . In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays . The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model . RESULTS: The following levels of pathogen inactivation were achieved: >10(6.7) plaque-forming units (PFU) per mL of cell-free human immunodeficiency virus (HIV), >10(6.6) PFU per mL of cell-associated HIV, >10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus), >10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus), >10(6.6) colony-forming units of Staphylococcus epidermidis, and >10(5.6) colony-forming units of Klebsiella pneumoniae . Expression of integrated HIV was inhibited by 0.1 microM S-59 and 1 J per cm2 of UVA . In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment . CONCLUSION: Photochemical treatment of platelet concentrates offers the potential for reducing transfusion-related viral and bacterial diseases. Int J Syst Bacteriol, 1997 Apr, 47(2), 313 - 23 Ribotype delineation and description of Staphylococcus sciuri subspecies and their potential as reservoirs of methicillin resistance and staphylolytic enzyme genes; Kloos WE et al.; Three subspecies of Staphylococcus sciuri, S . sciuri subsp . sciuri Kloos, Schleifer, and Smith 1976, 23AL emend . Kloos et al . 1997 {corrected}, S . sciuri subsp . carnaticus subsp . nov., and S . sciuri subsp . rodentium subsp . nov., are described on the basis of their ribotype patterns, DNA-DNA liquid hybridization data, and phenotypic characteristics . Normalized ribotyping subdivided the S . sciuri patterns into three blocks of patterns, each corresponding to a subspecies . Each subspecies formed a separate, well-defined DNA similarity group when DNA-DNA hybridizations were conducted under stringent (70 degrees C) reassociation conditions . S . sciuri subsp . sciuri could be distinguished from the other subspecies on the basis of its ability to produce acid from D-cellobiose, alkaline phosphatase activity, and inability to produce either clumping factor or protein A . S . sciuri subsp . carnaticus could be distinguished by its ability to produce acid aerobically from D-xylose and maltose, inability to produce acid from D-melezitose, and smaller colony size on P agar . S . sciuri subsp . rodentium could be distinguished by its positive reaction in the latex agglutination test for clumping factor and/or protein A and generally higher frequencies and levels of oxacillin and methicillin resistance . All 40 strains of S . sciuri tested (including representatives of all three subspecies) hybridized with the mecA gene probe . All strains of S . sciuri subsp . sciuri, 79% of the strains of S . sciuri subsp . carnaticus and 89% of the strains of S . sciuri subsp . rodentium exhibited extracellular, staphylolytic enzyme activity . This activity was associated with an enzyme(s) that immunoblotted with a lysostaphin-specific monoclonal antibody; however, only three strains hybridized with a lysostaphin (end) gene probe . The type strain of S . sciuri subsp . carnaticus is DD 791 (= ATCC 700058), and the type strain of S . sciuri subsp . rodentium is DD 4761 (= ATCC 700061). Protein Sci, 1997 Apr, 6(4), 789 - 93 The kinetic basis for the stabilization of staphylococcal nuclease by xylose; Frye KJ et al.; The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence-detected pressure-jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234-10239) . The activation volumes for both folding and unfolding and the equilibrium volume change for folding were all positive . Their values were within experimental error of those reported previously (Vidugiris GJA, Markley JL, Royer CA, 1995, Biochemistry 34:4909-4912) and were independent of xylose concentration . The major effect of xylose concentration was to increase significantly the rate of folding . The large positive activation volume for folding was interpreted previously as indicating that the rate-limiting step in nuclease folding involves dehydration of a significant amount of surface area . A large effect of xylose on the rate constant for folding provides strong support for this interpretation, because xylose, an osmolyte, stabilizes the folded state of proteins through surface tension effects . These studies further characterize the transition state in nuclease folding as lying closer to the folded, rather than the unfolded state along the folding coordinate in terms of the degree of burial of surface area . The image of the transition state that emerges is consistent with a dry molten globule. Gastroenterology, 1997 Apr, 112(4), 1169 - 78 Interleukin 12 is expressed and actively released by Crohn's disease intestinal lamina propria mononuclear cells; Monteleone G et al.; BACKGROUND & AIMS: Cell-mediated immunity is a feature of Crohn's disease (CD) . The heterodimer interleukin (IL)-12, produced by phagocytes, induces T-cell cytokines, primarily interferon (IFN)-gamma . This study examined whether CD lamina propria mononuclear cells (LPMCs) express and release bioactive IL-12 . METHODS: LPMCs were isolated from 13 patients with CD, 9 with ulcerative colitis (UC), and 13 controls . Messenger RNA for p40 and p35 IL-12 subunits was evaluated by reverse-transcription polymerase chain reaction . IL-12 was measured by enzyme-linked immunosorbent assay in LPMC culture supernatants . The INF-gamma-inducing effect of unstimulated LPMC supernatants was evaluated . RESULTS: Messenger RNA for both IL-12 subunits was detected in LPMCs of 11 of 13 patients with CD, 1 of 9 patients with UC, and 1 of 13 controls (P < 0.001) . IL-12 was measured (10.5 +/- 2 pg/mL at 24 hours) in unstimulated CD LPMCs and was enhanced by pokeweed mitogen, lipopolysaccharide, and staphylococcal enterotoxin B . No IL-12 was detectable in 8 of 9 patients with UC and 12 of 13 control-unstimulated LPMCs . IL-12 induced by pokeweed mitogen and staphylococcal enterotoxin B in UC was lower than in CD and did not differ from controls . An IFN-gamma-inducing effect was restricted to unstimulated CD LPMC supernatants and was inhibited by an anti-IL-12 antibody in a dose-dependent fashion . CONCLUSIONS: IL-12 transcripts are expressed in CD intestinal tissues . CD LPMCs are up-regulated in their capability of releasing bioactive IL-12 . Expression and release of bioactive IL-12 seem to differentiate CD from UC. Proc Natl Acad Sci U S A, 1997 Mar 18, 94(6), 2489 - 94 Genetically engineered superantigens as tolerable antitumor agents; Hansson J et al.; Superantigens (SAg) are a family of bacterial and viral proteins with strong immunostimulatory properties . SAg bound to major histocompatibility complex (MHC) class II molecules activate a high frequency of T cells and represent the most potent known activators of T cells to date . To explore the use of SAg for T cell-based tumor therapy we have created a tumor-reactive SAg by engineering a fusion protein composed of a tumor-reactive mAb (C215Fab) and the bacterial SAg staphylococcal enterotoxin A (SEA) . A point mutation D227A was introduced at the major MHC class II binding site in SEA to reduce systemic toxicity . Treatment of tumor bearing mice with the Fab-SEA D227A fusion protein resulted in profound antitumor effects with a markedly reduced toxicity as compared with the wild-type Fab-SEA fusion protein . The reduced toxicity was probably due to a weak distribution of the SEA D227A fusion protein in tissues with a high MHC class II expression and low systemic cytokine levels as exhibited in mice and rabbits . The data presented demonstrate the efficacy of immunoconjugates containing a mutated SAg in directing a T cell attack against tumor cells with minimal systemic immune activation. EMBO J, 1997 Mar 17, 16(6), 1436 - 43 The structure of the translational initiation factor IF1 from E.coli contains an oligomer-binding motif; Sette M et al.; The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy . Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics . From this set, 19 structures were selected, having low constraint energy and few constraint violations . The ensemble of 19 structures displays a root-mean-square deviation versus the average of 0.49 A for the backbone atoms and 1.12 A for all atoms for residues 6-36 and 46-67 . The structure of IF1 is characterized by a five-stranded beta-barrel . The loop connecting strands three and four contains a short 3(10) helix but this region shows considerably higher flexibility than the beta-barrel . The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N-terminal domain of aspartyl-tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer-binding motif . Several proteins of this family are nucleic acid-binding proteins . This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions . Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes. Cell Immunol, 1997 Mar 15, 176(2), 166 - 72 The conventional CD4+ T cell response to staphylococcal enterotoxin B is modified by its superantigenic activity; Wen R et al.; Because of the massive cytokine response elicited by superantigen exposure, it has been suggested that superantigens may act as adjuvants to boost conventional antigen responses . However, most previous studies have shown that in vivo exposure to superantigen suppressed subsequent T cell responses . Here we analyzed the effect of the superantigen Staphylococcal enterotoxin B (SEB) on a concurrent CD4(+) immune response to a conventional antigen, an I-Ab-restricted epitope derived from the same protein (SEB127-142) . Heat-inactivated SEB, which had lost all superantigenic activity, was capable of eliciting a strong CD4(+) proliferative T cell response to SEB127-142 . In contrast, native SEB was relatively nonimmunogenic, even when administered in association with complete Freund's adjuvant . High doses of native SEB coadministered with heat-inactivated SEB had no effect on the peptide response . However, low doses of native SEB were able to strongly enhance the ability of inactive SEB to prime CD4(+) T cells to SEB127-142 . Thus, SEB is not always immunosuppressive, and low doses may actually enhance a concurrent immune response . Also, the contribution of Vbeta8(+)/CD4(+) T cells to peptide reactivity was not affected by the presence of low doses of native SEB, suggesting that the enhanced reactivity was not a Vbeta-specific effect of SEB, but was cytokine-mediated. J Immunol, 1997 Mar 15, 158(6), 2856 - 61 CD28 ligation prevents bacterial toxin-induced septic shock in mice by inducing IL-10 expression; Wang R et al.; The pathogenesis of septic shock is due mainly to bacterial toxin stimulation of the immune system, resulting in an excessive production of proinflammatory cytokines . TNF-alpha has been implicated as a major mediato