Life Sci, 1998, 62(22), 2005 - 14 The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro; Grasso G et al.; Accumulating evidence suggests that hypothalamic luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) are two hypophysiotropic factors which modulate the immune response . The aim of the present study was to determine the in vivo effects of an intravenous bolus of LHRH and TRH on plasma interferon (IFN)-gamma production in five normoprolactinemic women with irregular menstrual cycles . We also determined prolactin (PRL), thyrotropin (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels before and after intravenous administration of LHRH and TRH . The results demonstrate that intravenous bolus of LHRH/TRH increases plasma IFN-gamma levels, with the maximum response 45 min after in vivo administration of hypothalamic peptides and after peak levels of adenohypophyseal hormones (PRL: 15 min; TSH: 30 min; FSH: 30 min; LH: 30 min) . In order to investigate a possible direct action of hypothalamic hormones on immune cells, we also evaluated, in the same subjects, the influence of LHRH and TRH on IFN-gamma production by human peripheral blood mononuclear cells (PBMCs), collected before the intravenous administration of the peptides and stimulated in vitro with bacterial superantigen staphylococcal enterotoxin A (SEA) and concanavalin A (Con A) . LHRH and TRH, separately and together, significantly enhanced in vitro IFN-gamma production by SEA- and ConA-activated PBMCs . The present results suggest that hypothalamic peptides (LHRH and TRH) directly, and/or indirectly pituitary hormones (PRL, TSH, FSH, and LH) or IL-2, have stimulatory effect on IFN-gamma producing cells and are further evidence of interactions between the neuroendocrine and immune systems. FEMS Immunol Med Microbiol, 1998 Apr, 20(4), 301 - 10 Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis; Andresen LO; Exfoliative toxins of approximately 30 kDa produced by Staphylococcus hyicus strains NCTC 10350, 1289D-88 and 842A-88 were purified and specific polyclonal antisera were raised against each of the toxins . It was shown by immunoblot analysis and ELISA that three exfoliative toxins from S . hyicus were antigenically distinct . The three toxins were designated ExhA, ExhB and ExhC . From 60 diseased pigs, each representing an outbreak of exudative epidermitis, a total of 584 isolates of S . hyicus were phage typed and tested for production of exfoliative toxin . ExhA-, ExhB- and ExhC-producing S . hyicus isolates were found in 12 (20%), 20 (33%) and 11 (18%), respectively, of the 60 pig herds investigated . Production of the different types of exfoliative toxin was predominantly associated with certain phage groups . However, toxin production was found in all of the six phage groups defined by the phage typing system . Some changes in the distribution of isolates between phage groups were observed when the results of this study were compared to previous investigations . In this study two new antigenically distinct exfoliative toxins were isolated and tools for in vitro detection of toxin producing S . hyicus isolates and for further studies on the exfoliative toxins from S . hyicus have been provided. J Clin Invest, 1998 Jun 1, 101(11), 2406 - 14 In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma; Dow SW et al.; In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity . Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor . 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2 . Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times . The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors . Toxicity was minimal or absent in all dogs . Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes . Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only . Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor. Antimicrob Agents Chemother, 1998 Jun, 42(6), 1417 - 23 Pharmacokinetics of sparfloxacin in the serum and vitreous humor of rabbits: physicochemical properties that regulate penetration of quinolone antimicrobials; Liu W et al.; We have used a recently described animal model to characterize the ocular pharmacokinetics of sparfloxacin in vitreous humor of uninfected albino rabbits following systemic administration and direct intraocular injection . The relationships of lipophilicity, protein binding, and molecular weight to the penetration and elimination of sparfloxacin were compared to those of ciprofloxacin, fleroxacin, and ofloxacin . To determine whether elimination was active, elimination rates following direct injection with and without probenecid or heat-killed bacteria were compared . Sparfloxacin concentrations were measured in the serum and vitreous humor by a biological assay . Protein binding and lipophilicity were determined, respectively, by ultrafiltration and oil-water partitioning . Pharmacokinetic parameters were characterized with RSTRIP, an iterative, nonlinear, weighted, least-squares-regression program . The relationship between each independent variable and mean quinolone concentration or elimination rate in the vitreous humor was determined by multiple linear regression . The mean concentration of sparfloxacin in the vitreous humor was 59.4% +/- 12.2% of that in serum . Penetration of sparfloxacin, ciprofloxacin, fleroxacin, and ofloxacin into, and elimination from, the vitreous humor correlated with lipophilicity (r2 > 0.999) . The linear-regression equation describing this relationship was not improved by including the inverse of the square root of the molecular weight and/or the degree of protein binding . Elimination rates for each quinolone were decreased by the intraocular administration of probenecid . Heat-killed Staphylococcus epidermidis decreased the rate of elimination of fleroxacin . Penetration of sparfloxacin into the noninflamed vitreous humor was greater than that of any quinolone previously examined . There was an excellent correlation between lipophilicity and vitreous entry or elimination for sparfloxacin as well as ciprofloxacin, fleroxacin, and ofloxacin . There are two modes of quinolone translocation into and out of the vitreous humor: diffusion into the eye and both diffusion and carrier-mediated elimination out of the vitreous humor. Lik Sprava, 1998 Jan-Feb, (1), 109 - 12 {Antibody production in the blood of donors immunized with staphylococcal anatoxin}; Dyzyk HM et al.; Blood serum content was studied of specific antistaphylococcal antibodies (staphylolysins) in 576 donors immunized with staphylococcal anatoxin with the purpose of obtaining an antistaphylococcal plasma and antistaphylococcal immunoglobulin to be used in clinical settings . 292 donors had been immunized and examined prior to 1986, 284--after 1986 (before 1994) . Comparison of the immune responses in the above periods of time permitted finding out that 13.03% of immunized donors responded to the antigenic stimulus by such paradoxical reaction as disappearance of specific antibodies; the number of persons-active respondents has gotten reduced from 17.12% to 5.98% as has the number of individuals having the baseline level of staphylolysins (1-2 ME/ml) . The changes were at their greatest in donors with group A (II) blood. J Biol Chem, 1998 Jun 5, 273(23), 14085 - 9 Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine; Yamakura F et al.; Peroxynitrite has recently been implicated in the inactivation of many enzymes . However, little has been reported on the structural basis of the inactivation reaction . This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite . Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue . Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses . We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite . This residue is located near manganese and in a substrate O-2 gateway in Mn-SOD. J Mol Biol, 1998 May 15, 278(4), 879 - 94 Anion-induced folding of Staphylococcal nuclease: characterization of multiple equilibrium partially folded intermediates; Uversky VN et al.; The refolding of acid-unfolded staphylococcal nuclease (SNase) induced by anions was characterized, and revealed the existence of three different partially folded intermediates (A states) . The three intermediates lack the rigid tertiary structure characteristic of native states, and differ in their degree of folding as measured by probes of secondary structure, size, stability and globularity . The least structured conformation, A1, is stabilized by chloride (600 mM) or sulfate (100 mM) . It is about 50% folded (based on circular dichroism and small angle X-ray scattering (SAXS) data) . The next most structured intermediate, A2, is induced by trifluoroacetate (300 mM) and has approximately 70% native-like secondary structure . The most structured intermediate, A3, is stabilized by trichloroacetate (50 mM) and has native-like secondary structure content and is almost as compact as the native state . The stability toward urea denaturation increases with increasing structure of the intermediates . Moreover, ureainduced unfolding studies show that these partially folded species are separated from each other, and from the unfolded state, by significant free energy barriers, suggesting that they are distinct conformational states . Kratky plots, based on the SAXS data, indicate that the two more structured intermediates have significant globularity (i.e . a tightly packed core), whereas the less structured intermediate has very little globularity . These observations support a model of protein folding in which certain conformations are of particularly low free energy and hence populated under conditions which differentially destabilize the native state . These partially folded intermediates probably consist of ensembles of substates with a common core of native-like secondary structure, which is responsible for their stability . Consequently, it is likely that the intermediates observed here represent the equilibrium counterparts of transient kinetic intermediates. Nutrition, 1998 May, 14(5), 427 - 32 Elimination of intraluminal colonization by antibiotic lock in silicone vascular catheters; Andris DA et al.; An in vitro model was designed to evaluate the efficacy of instilled antimicrobials to reduce or eliminate intraluminal microbial colonization . Minimal inhibitory concentration and minimal bactericidal concentration activity of appropriate test anti-infectives were determined using standard methodology against clinically derived and reference test strains commonly associated with catheter-related infection . Drug activity was validated by bioassay for the test anti-infectives . Reference and clinical test strains were inoculated to the intraluminal surface of silicone catheter segments and incubated for 30 min, after which the inoculum was replaced with total parenteral nutrition (TPN) solution and reincubated for 12 h . For 7 d, instillation of antibiotic and TPN solution was alternated every 12 h to simulate clinical conditions . On days 1, 4, and 7, catheter segments were rinsed, bisected, and sonicated for quantitative plate count to determine mean microbial counts per centimeter of catheter surface . Catheter segments were also prepared for scanning electron microscopy . A significant decrease in staphylococcal intraluminal colonization after instillation of nafcillin, ceftriaxone, gentamicin, and vancomycin was demonstrated (P < 0.001) . Aztreonam, ceftriaxone, and gentamicin completely eliminated gram-negative catheter colonization (P < 0.001) . Yeast was eradicated from the internal catheter surface after treatment with amphoteracin B, and fluconazole significantly decreased intraluminal colonization (P < 0.001) . Results show a significant decrease in staphylococcal, gram-negative, and fungal intraluminal colonization after instillation of appropriate antimicrobial . In vitro results support early clinical success using this technique . Future studies are warranted to identify optimal drug concentrations and dosing intervals. Biochemistry, 1998 May 12, 37(19), 6939 - 48 Stability effects of increasing the hydrophobicity of solvent-exposed side chains in staphylococcal nuclease; Schwehm JM et al.; A total of fifty single site surface phenylalanine substitution mutants have been made in the model protein staphylococcal nuclease . The fifty residues that were replaced with phenylalanine were chosen to give a broad sampling of solvent accessibility, secondary structure, and backbone conformations . The change in the stability of each mutant protein relative to wild type was measured by guanidine hydrochloride denaturation . These results were compared to previous results obtained when these same sites were substituted with an alanine and a glycine . By this means, changes in the stability due to the loss of interactions of the wild-type side chain can be separated from the effects of introducing the bulky, hydrophobic phenylalanine in these solvent-exposed positions . In general, our results agree with the conventional wisdom that placing a hydrophobic residue in a solvent-exposed position is destabilizing in most cases, but less destabilizing than most changes in the hydrophobic core of the protein . However, the degree to which a hydrophobic surface substitution destabilizes or stabilizes a globular protein is highly context-dependent, with some mutations being as destabilizing as those in the core . This indicates that steric and packing considerations are also important on the surface of a globular protein but generally are not as important as in the interior . No evidence for the widespread occurrence of the so-called reverse hydrophobic effect at solvent-exposed sites was found . In addition, this survey of numerous sites suggests that previous measurements of alpha-helix "propensities" often seriously underestimate the importance of the environment of the side chain. J Exp Med, 1998 May 4, 187(9), 1427 - 38 A role for Fas in negative selection of thymocytes in vivo; Kishimoto H et al.; To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen . In normal mice, injection of 1-100 microg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vbeta8+ cells at the level of the semi-mature population of HSAhi CD4+ 8- cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal . SEB injection also caused marked elimination of Vbeta8+ HSAhi CD4+ 8- thymocytes in lpr/lpr mice . Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (</=1 microg) but not by high doses (100 microg) . Similar findings applied when T cell receptor transgenic mice were injected with specific peptide . These findings suggest that clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses . Previous reports documenting that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level. Z Gastroenterol, 1998 Apr, 36(4), 287 - 93 Short-term efficacy and long-term outcome of cyclosporine treatment in patients with severe ulcerative colitis; Wenzl HH et al.; Cyclosporine A (CyA) has been recommended for the treatment of severe steroid-resistant ulcerative colitis, however, long-term results are scarce . We prospectively followed a treatment plan in 14 patients with severe ulcerative colitis receiving intravenous CyA after failure to respond to at least eight days of standard therapy with prednisolone (1-1.5 mg/kg/day) . CyA was delivered in a daily dose of 5 mg/kg i.v . for a mean of 14 days (range 7-28) in addition to ongoing medical therapy . CyA whole blood levels were monitored by HPLC and maintained between 100 ng/ml and 400 ng/ml . Responders were switched to oral CyA (5-7.5 mg/kg/day) for a mean of two months, and steroids were gradually tapered . Eleven patients (79%) initially responded to i.v . CyA, three patients failed to respond and underwent urgent colectomy . Time until response averaged seven days (range 3-13) . Four of the eleven responders underwent colectomy because of severe relapse after one, eleven, twelve and 13 months of follow-up . The remaining seven patients were followed for a median of 48 months . During the first year of follow-up three out of seven had a severe relapse and responded to steroids (two patients) or to a further course of i.v . CyA (one patient) . During CyA therapy one patient developed staphylococcal sepsis, other adverse events were mild and reversible . The results confirm that CyA is effective in severe steroid-refractory ulcerative colitis . Severe relapse and colectomy are uncommon after the first year of follow-up and the colon preserving effect of CyA can be maintained in up to 50% of patients over a period of four years. Am J Physiol, 1998 Jun, 274(6 Pt 1), C1583 - 91 Endothelin-1 activates phospholipases and channels at similar concentrations in porcine coronary arteries; Jones AW et al.; Sensitivity of endothelin-1 (ET-1)-ion channel interactions has been proposed to exceed that of ET-1-phospholipase activation in vascular smooth muscle . We wanted to determine whether short-circuiting ion channels with staphylococcal alpha-toxin pores would shift the ET-1-force relation to the right as predicted from the above proposal . Medium size porcine coronary arteries (outer diameter 0.7-1.5 mm) were mounted on isometric force transducers . ET-1 concentration response curves were compared between intact rings and those subjected to alpha-toxin treatment with Ca buffered at 0.1 microM . The EC50 for treated rings (1.5 +/- 1.0 nM, n = 5 pigs) was similar to that for intact rings (1.9 +/- 0.4 nM) . The Ca sensitivity of the alpha-toxin-treated rings (EC50 = 0.43 +/- 0.08 microM) was similar to that reported by other laboratories for intact and alpha-toxin-treated arteries and was shifted eightfold to the left by a high concentration of ET-1 (10 nM) . Measurements of {32P}phosphatidic acid ({32P}PA) levels were used to evaluate phospholipase activity in intact arteries . The time courses for {32P}PA production and contraction were similar in response to high (100 nM) and to low (1 nM) ET-1 . Significant increases in both steady-state contraction and {32P}PA occurred over a wide range of ET-1 concentrations tested (0.3-100 nM) . Our findings support the concept that ET-1-phospholipase coupling is operative over the whole concentration range that induces contractile responses . It is suggested that both Ca entry and Ca sensitization processes are activated by ET-1 at low concentrations (<EC50) and that both processes contribute significantly to the integrated response. J Neuroimmunol, 1998 Mar 15, 83(1-2), 102 - 15 Marijuana, immunity and infection; Klein TW et al.; The influence of marijuana cannabinoids on immune function has been examined extensively over the last 25 yr . Various experimental models have been used employing drug-abusing human subjects, experimental animals exposed to marijuana smoke or injected with cannabinoids, and in vitro models employing immune cell cultures treated with various cannabinoids . For the most part, these studies suggest that cannabinoids modulate the function of T and B lymphocytes as well as NK cells and macrophages . In addition to studies examining cannabinoid effects on immune cell function, other reports have documented that these substances modulate host resistance to various infectious agents . Viruses such as herpes simplex virus and murine retrovirus have been studied as well as bacterial agents such as members of the genera Staphylococcus, Listeria, Treponema, and Legionella . These studies suggest that cannabinoids modulate host resistance, especially the secondary immune response . Finally, a third major area of host immunity and cannabinoids is that involving drug effects on the cytokine network . Employing in vivo and in vitro models, it has been determined that cannabinoids modulate the production and function of acute phase and immune cytokines as well as modulate the activity of network cells such as macrophages and T helper cells, Th1 and Th2 . These results are intriguing and demonstrate that under certain conditions, cannabinoids can be immunomodulatory and enhance the disease process . However, more studies are needed to determine both the health risk of marijuana abuse and the role of the cannabinoid receptor/ligand system in immune regulation and homeostasis. Z Kardiol, 1998 Apr, 87(4), 276 - 82 {Surgery of acute aortic valve endocarditis: prognosis in paravalvular abscess}; Bauernschmitt R et al.; The occurrence of paravalvular abscesses in the course of an acute endocarditis of the aortic valve indicates an advanced stadium of the disease . The infection has spread beyond the limits of the valve leaflets, and ongoing destruction of the paravalvular tissue is to be expected, if the endocarditis is continually treated by antibiotics alone . Surgery of acute endocarditis with paravalvular abscess, however, supposedly carries an increased risk of early mortality and late morbidity . The following prospective study was carried out to determine whether a radical surgical approach together with aggressive postoperative antibiotic therapy could help to improve results . Between 1988 and 1995, 138 patients were operated during the acute phase of infective endocarditis; in 102 the aortic valve was involved . Among these, 44 had paravalvular abscesses at the time of surgery . The mean age of both groups was the same, but there was a higher rate of concomitant coronary artery disease, multiple valve involvement, advanced NYHA-class, and staphylococcal disease among the patients with abscesses . All interventions were carried out with cardiopulmonary bypass and cardioplegic arrest . The aortic valve was resected, abscesses were removed, and each part of potentially infected or necrotic tissue was resected as complete as possible, irrespective of the possibility to jeopardize the conduction system or to create large tissue defects . The aortic valve was replaced with a mechanical prosthesis in each case . The postoperative antibiotic regimen was specifically directed against the microorganisms isolated preoperatively; therapy was only modified, if signs of systemic infection did not disappear three days after surgery . The operative mortality was 10% among patients without an abscess and 11% in patients with a paravalvular abscess . Early recurrent endocarditis was recorded in two patients without and in only one patient with an abscess . Late recurrent endocarditis was noted in three patients; none of them had abscesses at the time of surgery . We conclude that the operative risk of acute endocarditis of the aortic valve with a paravalvular abscess does not have to be inevitably higher compared to cases without paravalvular involvement . To achieve these results, it is necessary to use a radical surgical approach and to adjust postoperative antibiotic therapy, if infectious signs do not disappear shortly after surgery. Biochemistry (Mosc), 1998 Feb, 63(2), 212 - 8 Site-specific endonuclease SscL1 I from strain Staphylococcus species L1; Vasiljeva LY et al.; A site-specific endonuclease SscL1 I preparation has been isolated and purified to near homogeneity from the strain Staphylococcus sp . L1 without admixtures of other nuclease activity . DNA cleavage proceeds according to the scheme: 5'-G down arrow ANTC-3' 3'-CTNA up arrow G-5', and thus the isolated enzyme is an isoschizomer of restriction endonuclease HinfI and belongs to the second class of restriction endonucleases . SscL1 I works over a broad range of temperature and pH . The enzyme is characterized by high stability during storage. JAMA, 1998 May 20, 279(19), 1537 - 41 Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial . Foreign-Body Infection (FBI) Study Group; Zimmerli W et al.; CONTEXT: Rifampin-containing regimens are able to cure staphylococcal implant-related infections based on in vitro and in vivo observations . However, this evidence has not been proven by a controlled clinical trial . OBJECTIVE: To evaluate the clinical efficacy of a rifampin combination in staphylococcal infections associated with stable orthopedic devices . DESIGN: A randomized, placebo-controlled, double-blind trial conducted from 1992 through 1997 . SETTING: Two infectious disease services in tertiary care centers in collaboration with 5 orthopedic surgeons in Switzerland . PATIENTS: A total of 33 patients with culture-proven staphylococcal infection associated with stable orthopedic implants and with a short duration of symptoms of infection (exclusion limit <1 year; actual experience 0-21 days) . INTERVENTION: Initial debridement and 2-week intravenous course of flucloxacillin or vancomycin with rifampin or placebo, followed by either ciprofloxacin-rifampin or ciprofloxacin-placebo long-term therapy . MAIN OUTCOME MEASURES: Cure was defined as (1) lack of clinical signs and symptoms of infection, (2) C-reactive protein level less than 5 mg/L, and (3) absence of radiological signs of loosening or infection at the final follow-up visit at 24 months . Failure was defined as (1) persisting clinical and/or laboratory signs of infection or (2) persisting or new isolation of the initial microorganism . RESULTS: A total of 18 patients were allocated to ciprofloxacin-rifampin and 15 patients to the ciprofloxacin-placebo combination . Twenty-four patients fully completed the trial with a follow-up of 35 and 33 months . The cure rate was 12 (100%) of 12 in the ciprofloxacin-rifampin group compared with 7 (58%) of 12 in the ciprofloxacin-placebo group (P=.02) . Nine of 33 patients dropped out due to adverse events (n=6), noncompliance (n=1), or protocol violation (n=2) . Seven of the 9 patients who dropped out were subsequently treated with rifampin combinations, and 5 of them were cured without removal of the device . CONCLUSION: Among patients with stable implants, short duration of infection, and initial debridement, patients able to tolerate long-term (3-6 months) therapy with rifampin-ciprofloxacin experienced cure of the infection without removal of the implant. J Immunol, 1998 Jun 1, 160(11), 5309 - 13 Perforin and IFN-gamma are involved in the antitumor effects of antibody-targeted superantigens; Rosendahl A et al.; The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of cytokine production and cytotoxic T cell responses . To target a T cell attack against tumor cells we have genetically engineered a fusion protein of SEA and the Fab part of the tumor-reactive mAb C215 . Injection of this Fab-SEA fusion protein to mice carrying lung metastases of the poorly immunogenic B16 melanoma transfected with the C215 Ag resulted in infiltration of cytokine-producing T cells, perforin-containing CTL, and a marked tumor elimination . Fab-SEA therapy induced substantial levels of IFN-gamma and TNF-alpha in serum . In the present study we have characterized the molecular mechanisms of the antitumor effect induced by Fab-SEA treatment in vivo . Neutralization of cytokines by specific Abs demonstrated a major role for IFN-gamma in the suppression of tumor growth . In addition, a minor contribution of TNF-alpha was recorded . Injections of Fab-SEA into normal mice induced strong CTL activity but failed to promote cytotoxic function in perforin knockout mice . Also, a markedly reduced therapy was noted in perforin knockout mice, implicating a role for CTL in Fab-SEA-mediated tumor eradication . The data suggest that Fab-SEA-targeted T cells may suppress tumor growth by both perforin-dependent cytotoxicity and local release of cytokines such as IFN-gamma . The latter mechanism may have an important role in cytostatic effects against Ag-negative bystander tumor cells. J Immunol, 1998 Jun 1, 160(11), 5246 - 52 In vivo inflammatory response to a prototypic B cell superantigen: elicitation of an Arthus reaction by staphylococcal protein A; Kozlowski LM et al.; Staphylococcal protein A (SpA) is representative of a new class of Ags, the B cell superantigens (SAgs) . These SAgs, unlike conventional Ags, bind to the Fab regions of Ig molecules outside their complementarity-determining regions . In addition, B cell SAgs can react with a substantial amount of a host's serum Igs by virtue of their ability to interact with many members of an entire variable heavy chain (VH) or variable light chain gene family . For example, SpA reacts with the Fabs of most human Igs using heavy chains from the VH3 gene family (VH3+) . Members of this gene family are expressed on 30 to 60% of human peripheral B cells . We sought to determine whether the interaction of a B cell SAg with its reactive Igs can elicit immune complex-mediated tissue injury . Using the Arthus reaction in rabbits as an in vivo model of immune complex-mediated tissue inflammation, we demonstrated that untreated rabbits, which were administered SpA intradermally (i.d.), do not develop a cutaneous inflammatory response . However, when rabbits were pretreated i.v . with human IgG (hIgG), i.d . injections of SpA induced an inflammatory response with the classical histologic features of an Arthus reaction . To determine whether this Arthus-like response occurred via a B cell superantigenic mechanism, the rabbits were pretreated with VH3-depleted hIgG and then were administered SpA i.d . We found that the induction of a prominent inflammatory response by SpA was dependent upon the presence of VH3+ molecules in the hIgG pretreatment . These results provide compelling evidence that an interaction of the B cell SAg, SpA, with its reactive (VH3+) IgGs leads to an immune complex-mediated inflammatory response in vivo. J Immunol, 1998 Jun 1, 160(11), 5231 - 8 CD95 (Fas)-based, superantigen-dependent, CD4+ T cell-mediated down-regulation of human in vitro immunoglobulin responses; Stohl W et al.; Naturally occurring microbial superantigens (SAg) have been implicated in several human idiopathic disorders, and a compelling argument for the role of SAg in autoantibody-associated disorders, such as systemic lupus erythematosus, has been proposed . To test the effects of SAg on human in vitro Ig responses, CD4+ T cell + B cell cultures were stimulated with graded doses of staphylococcal enterotoxin B (SEB) . Ig-secreting cell (IgSC) responses were very weak in CD4+ T cell + B cell cultures stimulated with SEB at the optimal mitogenic concentration (high dose SEB; 100 ng/ml) but were strong in parallel cultures stimulated with low dose SEB (0.01 ng/ml) . High dose SEB actually enhanced B cell differentiation in the presence of CD4+ T cell soluble helper factors as long as the B cells were prevented from physically contacting the CD4+ T cells . However, when cell-cell contact between CD4+ T cells and B cells was permitted, high dose, but not low dose, SEB promoted increased CD4+ T cell-mediated B cell apoptosis with resulting decreases in viable CD20+ B cells and IgSC . High dose, but not low dose, SEB triggered increased levels of soluble CD95 ligand, and down-regulation of IgSC responses and incremental apoptosis of activated B cells were prevented by antagonist anti-CD95 mAb . This strongly suggests that CD4+ T cell-mediated CD95-based killing of activated B cells plays a major role in controlling SEB-driven IgSC responses . Defects in SAg-based down-regulation may contribute to autoimmune disorders such as SLE. Cornea, 1998 May, 17(3), 335 - 7 Staphylococcal endophthalmitis following cataract extraction in a patient with Darier's disease; Macsai MS et al.; PURPOSE: To report staphylococcal endophthalmitis following cataract extraction in a patient with Darier' s disease . METHODS: A 67-year-old man presented with decreased visual acuity OD and hypopyon 3 days status post-cataract extraction with intraocular lens placement . The patient was hospitalized and placed on topical and intravenous antibiotics . A diagnostic vitreous tap, pars plana vitrectomy, and intravitreal antibiotic installation were performed . RESULTS: Vitreal tap cultures indicated Staphylococcus epidermidis . His clinical status improved after vitrectomy and antibiotic therapy . The same bacteria was cultured from the patient's eyelids . CONCLUSIONS: The source of the S . epidermidis was the skin lesions on the patient's face and eyelids . Darier's disease is an exfoliative hyperkeratotic skin disease that affects all areas on the body except the buttocks . Although clean, sterile surgical techniques were followed, the risk of endophthalmitis following intraocular surgery in a patient with Darier's disease may be increased due to his or her dermatologic condition. Infect Immun, 1998 Jun, 66(6), 2778 - 81 The abilities of a Staphylococcus epidermidis wild-type strain and its slime-negative mutant to induce endocarditis in rabbits are comparable; Perdreau-Remington F et al.; The abilities of a parent and mutant pair of Staphylococcus epidermidis strains, the slime-producing parent RP62A and its slime-negative mutant, to establish endocarditis in a rabbit model of aortic valve endocarditis and to accumulate and adhere to surfaces in vitro were compared . Vegetation titer and infection rate depended on the presence or absence of a catheter (P = 0.020) and on inoculum size (P < 0.001) but not on the infecting strain . The ability of the parent strain vis-a-vis its mutant to accumulate in vitro on surfaces as demonstrated in a slime test did not correlate with any enhancement in the development of endocarditis in the rabbit model . In vitro initial adherence rates were identical . Both isolates accumulated to the same reduced extent in vitro in the presence of serum, albumin, or gelatin . Adhesion was equally promoted by addition of fibronectin . These data suggest that the in vitro phenomenon of accumulation described as slime production in the absence of serum may not be an important virulence determinant in vivo. Biochemistry, 1998 Apr 28, 37(17), 5785 - 90 Pressure denaturation of proteins: evaluation of compressibility effects; Prehoda KE et al.; One of the key pieces of information from pressure denaturation experiments is the standard volume change for unfolding (Delta V(o)) . The pressure dependence of the volume change, the standard compressibility change (Delta K(o)T), is typically assumed to be zero in the analysis of these experiments . We show here that this assumption can be incorrect and that the neglect of compressibility differences can skew the interpretation of experimental results . Analysis of experimental, variable-pressure NMR data for bovine pancreatic ribonuclease A in 2H2O at pH 2.0 and 295 K yielded the following statistically significant, non-zero values: Delta K(o) T = 0.015 +/- 0.002 mL mol-1 bar-1, Delta V(o) = -21 +/- 2 mL mol-1, and Delta G(o) = 2.8 +/- 0.3 kcal mol-1 . The experimental protein stability is in good agreement with one (Delta G(o) = 2.5 kcal mol-1) determined independently for the same protein by calorimetry at atmospheric pressure under equivalent conditions {Makhatadze, G . I., Clore, G . M., and Gronenborn, A . M . (1995) Nat . Struct . Biol . 2, 852-855} . The positive value for Delta K(o)T indicates that the denatured form of ribonuclease A is more compressible than the native form; this is explained in terms of an interplay between the intrinsic compressibility of the protein and solvation effects . When the same data were fitted to a model that assumes a zero compressibility change, the Delta G(o) value of 4 . 0 +/- 0.1 kcal mol-1 returned by the model no longer agreed with the independent measurement, and the Delta V(o) returned by the model was a very different -59 +/- 1 mL mol-1 . By contrast, it was not possible to carry out a similar thermodynamic analysis of fluorescence spectroscopic data for the denaturation of staphylococcal nuclease to yield well-defined values of Delta G(o), Delta V(o), and Delta K(o)T . This limitation was shown by evaluation of synthetic data to be intrinsic to spectroscopic data whose analysis requires fitting of the plateaus at either side of the transition . Because NMR data do not have this requirement, they can be analyzed more rigorously. Protein Eng, 1997 Dec, 10(12), 1433 - 43 Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel; Cheley S et al.; Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes . The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel . Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit . In the monomer in solution, the central domain is loosely organized and exposed to solvent . In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity . Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy . Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation . Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation . The truncated pore might make a useful framework for the construction of designed membrane active macromolecules. J Mol Biol, 1998 Apr 3, 277(3), 733 - 45 Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL; Tsurupa GP et al.; We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements . It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1 . The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution . In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme . On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1 . The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer) . The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions . Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding . The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP . No Shinkei Geka, 1998 Apr, 26(4), 357 - 62 {Multiple bacterial aneurysms: case report}; Suzuki Y et al.; A 59-year-old man presented with an internal carotid artery (ICA) bacterial aneurysm which ruptured during surgery for treatment of another bacterial aneurysm . He had been admitted to our hospital because of the recurrence of colon cancer . He had undergone aortic and mitral valve replacement because of closure incompetence due to bacterial endocarditis two months previously . Two months after treatment for colon cancer, he developed fever, and arterial blood culture demonstrated . Staphylococcus epidermidis . A few days later, he suddenly suffered severe headache and vomiting, followed by deterioration of consciousness . CT showed subarachnoid hemorrhage and angiography showed a saccular aneurysm at the opercular portion of the left middle cerebral artery (MCA) . Immediate clipping of the aneurysm was attempted . The carotid cistern was opened via a left frontotemporal craniotomy, but an ICA aneurysm, which had not been previously recognized, ruptured suddenly . The ICA aneurysm was wrapped with Vascwrap with some difficulty . The MCA aneurysm was then trapped . Postoperatively, the patient continued to be stuporous for a few days . Two weeks later, he died of complications caused by pneumonia . Bacterial aneurysm is more likely to be located in the distribution of the distal arterial tree, mainly in the distribution of the MCA . The difficulty of preoperative diagnosis and the unpredictable clinical course of bacterial aneurysms are emphasized. J Biochem (Tokyo), 1998 Apr, 123(4), 760 - 5 Presence of conserved domains in the C-terminus of MARCKS, a major in vivo substrate of protein kinase C: application of ion trap mass spectrometry to the elucidation of protein structures; Yamauchi E et al.; MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation . We have previously analyzed MARCKS purified from bovine brain using capillary liquid chromatography/electrospray mass spectrometry and found that the protein structure differed significantly from that deduced from cDNA sequences {Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K . (1994) J . Biol . Chem . 269, 18299-18302} . Moreover, the alignment of the protein from various species showed a lack of any conserved sequences in the C-terminal half of the molecule . This prompted us to reexamine the C-terminal amino-acid sequence of bovine MARCKS . The purified protein was digested with lysyl endoprotease, and the obtained C-terminal peptide was further digested with either Staphylococcus V8 protease or NTCB . The small peptides thus obtained were analyzed by liquid chromatography/electrospray/tandem mass spectrometry . This combined with gas-phase Edman sequencing allowed us to determine the C-terminal primary structure . The sequence obtained differed significantly from that reported previously, and the comparison with other species revealed the presence of a novel conserved domain in the C-terminal region of MARCKS. J Immunol, 1998 May 15, 160(10), 5170 - 80 In vivo effects of a bacterial superantigen on macaque TCR repertoires; Kou ZC et al.; A macaque model was employed to explore staphylococcal enterotoxin B (SEB) superantigen-driven T lymphocyte responses . The SEB-reactive Vbeta+ cell subpopulations demonstrated a striking tri-phase response in rhesus monkeys following an SEB challenge in vivo . The hyperacute down-regulation, seen as early as 2 h through 2 days after SEB injection, was characterized by a disappearance of the reactive Vbeta-restricted PBL subpopulations from the circulation and decreased expression of these cell subpopulations in lymphoid tissues . Following this, a dominant expansion of reactive Vbeta-expressing CD4+ cell subpopulations occurred in lymph nodes and spleens, whereas in the peripheral blood a preferential expansion of reactive Vbeta-expressing CD8+ cell subpopulations was seen . An exhaustion of this response was then seen, with a prolonged decrease in the number of the reactive Vbeta+ CD4+ lymphocyte subpopulations . Interestingly, monoclonal or oligoclonal dominance was seen in the reactive Vbeta+ cell subpopulations in the period of the transition from the polyclonal cellular expansion to the exhaustion of the response, suggesting that some Vbeta+ cell clones may be more resistant than others to superantigen-mediated depletion . These results indicate that in vivo SEB superantigen-mediated effect on lymphocyte subpopulations in macaques is complex, suggesting that profound dynamics in the TCR repertoires may in part account for the susceptibility of higher primates to SEB-induced diseases. J Heart Valve Dis, 1998 Mar, 7(2), 235 - 9 Repair of fungal aortic prosthetic valve endocarditis associated with periannular abscess; Remsey ES et al.; The incidence of prosthetic valve endocarditis is 2-4%; in most cases the involved organisms are Staphylococcus epidermidis and Staph . aureus . Fungal endocarditis is much less common (incidence < 0.1%), but it is often fatal, with a long-term mortality rate of 90-100% . Most fungal endocarditis cases occur after aortic valvular surgery, due to Candida sp . Late-onset symptoms, long-term development and aggressive nature of the fungus makes its eradication complicated and treatment difficult . Fungal valvular mycoses produce systemic embolization and cause serious perioperative bleeding on resection of infected tissue . Usually surgery includes aortic root replacement with an aortic homograft conduit after radical debridement, to attain local sterilization . This report describes a patient with complex infection, requiring replacement of an infected prosthetic valve with an aortic homograft conduit, aggressive and radical debridement of infected tissue, and reconstruction using biologic tissues . The case demonstrates the importance of perioperative and long-term antifungal treatment and presents a modified 'Cabrol procedure' to prevent critical intraoperative hemorrhage. Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5480 - 3 Association-induced folding of globular proteins; Uversky VN et al.; It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding . We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties . The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations . In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states. Eur J Immunol, 1998 Apr, 28(4), 1417 - 25 The crucial role of IL-10 in the suppression of the immunological response in mice exposed to staphylococcal enterotoxin B; Hasko G et al.; Staphylococcal enterotoxin B (SEB), a bacterial superantigen, activates the immune system resulting in a burst of pro- and anti-inflammatory cytokines . A central anti-inflammatory mediator in this process is IL-10 . Using IL-10-deficient C57BL/6 (IL-10 KO) mice, we studied the role of endogenous IL-10 in the regulation of the immune response to SEB . SEB (100 microg) induced the release of IL-10 in control C57BL/6 {IL-10 wild type (WT)} mice, but not in their IL-10 KO counterparts . SEB-evoked plasma levels of TNF-alpha, IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma were significantly higher in the IL-10 KO mice than in the WT animals . The release of macrophage inflammatory proteins-1alpha and -2 was also enhanced in the IL-10 KO mice . Further, upon SEB challenge, mice deficient in IL-10 produced higher levels of nitric oxide than the WT animals . IL-10 deficiency resulted in a marked enhancement of the SEB-induced apoptosis of thymocytes . Finally, IL-10 KO mice were more susceptible to SEB-induced lethal shock than their WT controls . These results show that IL-10 plays an important immunoregulatory role in the response to a superantigenic stimulus, by dampening of the shock-inducing inflammatory response and early activation-induced cell death elicited by SEB. Eur J Haematol, 1998 Apr, 60(4), 233 - 9 Antibody-directed superantigen-mediated T-cell killing of myeloid leukaemic cell line cells; Gidlof C et al.; Bacterial superantigens (SAgs) bound to MHC class II molecules on target cells are efficient activators of cytotoxic T cells expressing certain T cell receptor (TCR) Vbeta regions We described earlier that the specificity of the SAg Staphylococcus enterotoxin A (SEA) can be changed by introducing a D227A point mutation in the major MHC class II binding site and by genetically fusing the SEA mutant (SEAm) to protein A (PA) . This SEAm-PA fusion protein can then be used to direct cytotoxic T cells to tumour cells coated with monoclonal antibodies (mAbs) . In this communication, we tested the PA-SEAm fusion protein together with mAbs against the myeloid cell surface antigens CD13, CD15 and CD33 . A SEA-reactive T cell line was used as effector cells against 10 different myeloid leukaemic cell lines . Optimal lysis of antigen positive leukaemic cells was obtained at a PA-SEAm concentration of 1 ng/ml and effector : target cell ratios of 15 : 1 . No correlation between target cell sensitivity and the level of surface antigen expression could be seen . The 6 acute myeloid leukaemia (AML) cell lines tested appeared to be more sensitive than the 4 chronic myeloid leukaemia (CML) cell lines . The sensitivity of the AML cell line HL-60 could be improved further by stimulation with TNFalpha . This was accompanied by increased surface ICAM-1 expression whereas specific target molecule expression (CD13, CD33) was unchanged . This suggests that sensitivity to lysis is related to the leukaemic subtype and ICAM-1 expression but not to the tumour antigen density . Our results show that it is possible to direct cytotoxic T cells to myeloid leukaemia cells by using SAgs linked to mAbs, and encourage the construction and testing of a recombinant direct SAg-mAb fusion protein as a candidate drug for therapy of myeloid leukaemias. Cell Immunol, 1998 Jan 10, 183(1), 42 - 51 Quantitative defect in staphylococcal enterotoxin A binding and presentation by HLA-DM-deficient T2.Ak cells corrected by transfection of HLA-DM genes; Albert LJ et al.; HLA-DM facilitates peptide acquisition by MHC class II proteins within the endosomes of APC by facilitating release of invariant chain peptide intermediates (CLIP) from the class II molecules . T2 cells have a deletion in the MHC II region which deletes HLA-DM and MHC II genes . T2 cells transfected with MHC class II proteins are defective in protein presentation, a defect that is corrected by HLA-DM transfection . Here we show that T2 cells transfected with Ak are also impaired in binding and presentation of the superantistaphylococcal enterotoxin A and that HLA-DM transfection corrects this defect . The poor ability of SEA to bind to Ak on DM-deficient cells is somewhat surprising since Ak has a low affinity for CLIP and is not predominantly occupied with CLIP on T2 cells compared to wide-type APC . These data suggest an influence of HLA-DM on the structure or composition of the Ak/peptide complex beyond its role in the release of invariant chain peptides. J Clin Pathol, 1998 Jan, 51(1), 62 - 7 Identification of multiresistant Staphylococcus epidermidis in neonates of a secondary care hospital using pulsed field gel electrophoresis and quantitative antibiogram typing; Sloos JH et al.; AIMS: To determine the diversity of types of Staphylococcus epidermidis in a neonatal care unit of a secondary care hospital in the Netherlands . METHODS: In a prospective study, specimens from nose, ear, axilla, umbilicus, and groin were taken from patients twice a week during a period of up to two weeks . All isolates were typed by both pulsed field gel electrophoresis (PFGE) and antibiogram analysis . RESULTS: Fifty three S epidermidis isolates from 15 of 24 patients were obtained in one to four surveys . Fourteen isolates from six patients had a common PFGE pattern and were of one multiresistant antibiogram type . The remaining 39 isolates were allocated to 24 sporadic PFGE types and were more susceptible to antibiotics . Colonisation with the multiresistant strain correlated with a long period of stay and with the use of specific antibiotics . The multiresistant isolates were related closely to isolates of S epidermidis found in a recent study in a teaching hospital in the vicinity of the secondary care hospital . CONCLUSION: Repeated sampling and the use of two typing methods allowed the identification of two closely related multiresistant S epidermidis strains in two hospitals in the same area. J Vasc Surg, 1998 Apr, 27(4), 689 - 98 Treatment of vascular graft infection by in situ replacement with cryopreserved aortic allografts: an experimental study; Knosalla C et al.; PURPOSE: The purposes of this study were to prove the efficacy of cryopreserved aortic allografts to treat an established vascular graft infection by in situ replacement in an animal model and to evaluate the role of the antibiotics normally used to decontaminate the allografts . METHODS: Twenty-three dogs underwent infrarenal aortic replacement with a gelatin-sealed knitted polyester graft contaminated in vitro by Staphylococcus epidermidis RP-62 . One week later, the 18 surviving animals underwent reoperation for graft removal and were randomized into three groups for in situ replacement: group I (control, n = 6) received a new gelatin-sealed graft; group II (n = 6) received a non-antibiotic-treated cryopreserved allograft; and group III (n = 6) received an antibiotic-treated cryopreserved allograft . Control grafts and allografts were removed 4 weeks after the initial intervention for quantitative bacteriologic analysis and histologic analysis . Bacteriologic results were expressed as colony-forming units per square centimeter of graft material . Qualitative bacteriologic analysis was also obtained from perigraft fluid and tissue . RESULTS: All of the initially implanted grafts and all of the control grafts (group I) were infected at the time of removal . In group II, three out of six allografts were not totally incorporated, whereas in group III incorporation was always complete, with a significantly decreased inflammatory reaction . All of the antibiotic-treated allografts were sterile, whereas three untreated allografts grew bacteria . CONCLUSIONS: In this model, cryopreserved aortic allografts were more resistant to reinfection than synthetic grafts after in situ replacement of an infected prosthetic graft . However, the antibiotic loading of the cryopreserved aortic allograft appears to be essential to obtain optimal therapeutic effects. Klin Lab Diagn, 1998 Mar, (3), 36 - 8 {Method of quantitative determination of staphylococcal hyaluronidase activity}; Generalov II; The proposed method for measuring hyaluronidase activity of microorganism is based on prevention of hyaluronic acid clot formation with rivanol under the effect of hyaluronidase . This made possible the quantitative and qualitative detection of hyaluronidase activities of different staphylococcus species and strains . The maximum level of the enzyme and highest rate of its detection were typical of St . aureus . Its strains producing hyaluronidase in quantities of at least 0.5 IU are significantly (p < 0.01) more often isolated from medical staff. J Bacteriol, 1998 May, 180(9), 2273 - 9 Regulation of lactose utilization genes in Staphylococcus xylosus; Bassias J et al.; The lactose utilization genes of Staphylococcus xylosus have been isolated and characterized . The system is comprised of two structural genes, lacP and lacH, encoding the lactose permease and the beta-galactosidase proteins, respectively, and a regulatory gene, lacR, coding for an activator of the AraC/XylS family . The lactose utilization genes are divergently arranged, the lacPH genes being opposite to lacR . The lacPH genes are cotranscribed from one promoter in front of lacP, whereas lacR is transcribed from two promoters of different strengths . Lactose transport as well as beta-galactosidase activity are inducible by the addition of lactose to the growth medium . Primer extension experiments demonstrated that regulation is achieved at the level of lacPH transcription initiation . Inducibility and efficient lacPH transcription are dependent on a functional lacR gene . Inactivation of lacR resulted in low and constitutive lacPH expression . Expression of lacR itself is practically constitutive, since transcription initiated at the major lacR promoter does not respond to the availability of lactose . Only the minor lacR promoter is lactose inducible . Apart from lactose-specific, LacR-dependent control, the lacPH promoter is also subject to carbon catabolite repression mediated by the catabolite control protein CcpA . When glucose is present in the growth medium, lacPH transcription initiation is reduced . Upon ccpA inactivation, repression at the lacPH promoter is relieved . Despite this loss of transcriptional regulation in the ccpA mutant strain, beta-galactosidase activity is still reduced by glucose, suggesting another level of control. Intensive Care Med, 1998 Mar, 24(3), 258 - 61 Fibrinolytic changes in a patient with toxic shock syndrome; release of active u-PA; Haj MA et al.; OBJECTIVE: Definition of the changes in the activators of plasminogen, u-PA and t-PA, and examination of the possible generation of plasmin in the circulation in overwhelming sepsis . DESIGN: Serial blood analysis starting 4 h after development of symptoms of toxic shock syndrome . SETTING: Intensive care unit . PATIENT: A previously healthy woman underwent endometrial ablation and rapidly thereafter developed staphylococcal toxic shock syndrome with organ failure . MEASUREMENT AND RESULT: t-PA, PAI-1, t-PA-PAI-1 complexes, plasminogen, fibrinogen and plasmin-alpha 2-antiplasmin complexes were measured serially by ELISA and free u-PA by SDS-PAGE with zymography . The onset of symptoms was accompanied by a rise of t-PA antigen-followed rapidly by PAI-1 antigen . Plasmin-alpha 2-antiplasmin complex was generated in large amounts but disappeared within hours . From day 3, free u-PA was detectable in the circulation without plasmin generation . CONCLUSION: Despite the sustained presence of active u-PA in the circulation and of t-PA antigen at the onset of symptoms, plasmin-alpha 2-antiplasmin generation was largely suppressed by high levels of PAI-1 . The suppression of plasmin generation by u-PA and t-PA may ensure the persistence of fibrin in the microcirculation and so contribute to organ failure. J Bone Joint Surg Am, 1998 Apr, 80(4), 481 - 91 Infection after total elbow arthroplasty; Yamaguchi K et al.; The purpose of this study was to review our experience with the treatment of twenty-five infections (in twenty-five patients) after total elbow arthroplasty and to examine indications for salvage of the prosthesis compared with those for resection arthroplasty . The patients were divided into three groups on the basis of treatment . Group I comprised fourteen patients who were managed with multiple, extensive irrigation and debridement procedures with retention of the original components . The primary indication for retention of the prosthesis was evidence that it was well fixed as determined both radiographically and intraoperatively . Group II comprised six patients who had removal of the prosthesis and debridement followed by immediate or staged reimplantation . Group III comprised five patients who were managed with resection arthroplasty . The infection was successfully eradicated in seven of the fourteen elbows that had salvage of the prosthesis with irrigation and debridement . The results were strongly dependent on the causative organism; attempts at debridement failed in the four elbows that were infected with Staphylococcus epidermidis compared with three of the ten that were infected with another organism . Four of the six patients in Group II had successful reimplantation of a prosthesis; in three, the infection had been caused by an organism other than Staphylococcus epidermidis . Only one of the three patients who had a Staphylococcus epidermidis infection had a successful reimplantation . None of the five patients who had a resection arthroplasty had signs of infection at the latest follow-up examination . We concluded that salvage of the prosthesis with extensive irrigation and debridement in the presence of an infection about the elbow can be reasonably successful if the infecting organism is not Staphylococcus epidermidis and if the components are well fixed . When removal of the components is warranted, staged reimplantation can also be highly successful when the infecting organism is not Staphylococcus epidermidis . However, the repeated operations necessary to retain a prosthesis and the high rates of complications seen with this approach--and the relatively good rates of satisfaction obtained with resection arthroplasty--suggest that resection arthroplasty remains the procedure of choice in medically frail patients or in patients for whom function of the elbow is less of a concern. Ophthalmologica, 1998, 212(3), 184 - 7 In vitro Staphylococcus epidermidis growth in some viscoelastic substances containing sodium hyaluronate; Gallenga PE et al.; The aim of our study was to verify the in vitro growth of Staphylococcus epidermidis in various dilutions of some viscoelastic substances containing hyaluronic acid (Healon and Healon GV, IAL, Biolon) . Serial twofold dilutions of each sterile viscoelastic substance, prepared so as to obtain a final concentration ranging from 50 to 0.78% of the product in sterile saline solution (0.85% NaCl), were taken out with a pipette that delivered 1.0 ml/tube . One hundred microliters of the S . epidermidis inocula, used for the evaluation of the positive control of the test organism, was dispensed into each tube . After 24 h of aerobic incubation at 37 degrees C, 100 microl of sample was taken out from each tube and plated into the specific medium for the growth of the test organism . After 24 h of incubation at 37 degrees C, these agar plates were examined and the colony-forming unit count of the test organism was compared to the corresponding total colony count, acting as a positive control, in order to determine the quantitative variation of the test organism grown in the presence of the viscoelastic compounds . For the lowest dilutions (from 1:2 to 1:8) statistically significant bacterial growth was detected in all tested viscoelastic substances . For the highest dilutions (1:64 and 1:128) Biolon and Healon GV showed a significant inhibition of S . epidermidis growth . A significant inhibition was also observed in the highest dilution (1:128) of Healon . In every dilution of IAL a statistically significant increase in bacterial growth was observed . It remains to be carefully considered whether S . epidermidis, accidentally penetrating the eye via the intraocular lens, could find a culture medium in a small amount of sodium hyaluronate left in the capsular bag behind the optic. Immunol Invest, 1998 Jan-Feb, 27(1-2), 73 - 88 T cell dependent B cell activation occurs during the induction of T cell anergy by staphylococcal enterotoxin B in mice; Abdul-Majid KB et al.; Staphylococcal enterotoxin B (SEB) can activate specific T cell clones bearing specific TcR V beta domains together with MHC class II ligands on accessory cells . The release of proinflammatory cytokines is the consequence of this activation as well as the main pathological aspect involved in SEB infection . This current study looked at the active role of both T and B cells during the induction of anergy by SEB in vivo . Euthymic and nude BALB/c mice were injected with SEB and over a period of 8 days, cells from the spleen and sera from the blood were collected . After a single injection with SEB (50 micrograms/mouse), a transient increase of CD4+V beta 8+ T cells were detected after 2 days followed by a decrease after 4 days, which persisted until day 8 . These clones were rendered anergic upon restimulation in vitro with SEB . Interestingly, cells taken out 2 days after SEB injection, exhibited reduced proliferation in response to Con A . However, this response gradually recovered on days 4, 6 and 8 . Furthermore, early IgM antibody production (day 2) was observed after SEB injection . SEB-induced IgM antibody production in euthymic BALB/c was found to have specificity against SEB, cardiolipin (CL) and phosphatidylethanolamine (PE) . SEB-treated nude mice did not produce antibody secreting cells in response to SEB, indicating that this process is T cell dependent. Eur J Immunol, 1998 Mar, 28(3), 874 - 80 Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice; Stoffel B et al.; Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways . We used an aSMase-deficient mouse line (asmase-/-) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B . All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor . However, in asmase-/- mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated . Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50% after 72 h in comparison to wild-type cells . We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes. Acta Paediatr, 1998 Mar, 87(3), 349 - 50 Coexistence of acquired protein S and protein C deficiency and the Arg506Gln mutation in factor Va in a child with severe thromboembolic disease; Shavit I et al.; An 11-y-old girl who presented with cellulitis and clinical signs of deep vein thrombosis (DVT) is reported here . She developed staphylococcal sepsis, recurrent septic emboli and a large vegetation on the tricuspid valve . The patient was found to be heterozygous for the Arg506Gln mutation in factor Va and had low levels of protein C and protein S during the sepsis . The coexistence of the two thrombophilic states may explain the severe thromboembolic manifestations. Gen Pharmacol, 1998 May, 30(5), 777 - 82 Cyclosporin A and FK-506 inhibit development of superantigen-potentiated collagen-induced arthritis in mice; Takaoka Y et al.; 1 . Staphylococcal enterotoxine B (SEB; superantigen) accelerated the onset of arthritis in mice preimmunized with type II collagen (SEB-potentiated collagen-induced arthritis) . Cyclosporin A and FK-506 inhibited the induction and development of clinical signs and histopathological changes of SEB-potentiated collagen-induced arthritis in mice . 2 . Simultaneously, both cyclosporin A and FK-506 inhibited the development of humoral and cellular immunity to type II collagen . 3 . The expression of IL-2 receptor (CD25) by SEB on splenocyte T cells from collagen-preimmunized mice was inhibited by both agents in ex vivo experimentation. Bull Soc Pathol Exot, 1998, 91(1), 104 - 8 {A great Franco-Mauritian epidemiologist: Joseph Désiré Tholozan (1820-18970}; Theodorides J; Born in 1820 from French parents in Diego Garcia, an islet then linked to Mauritius where he started in Port-Louis his school years, Joseph Desire Tholozan was an original personality . He undertook medical studies in France (M . D . thesis, Paris, 1843) after having joined the military Health Service (1841) as a surgeon serving in various garrisons in the country and later at the Hospital of the Valde-Grace in Paris (1849) . Successful at the "agregation" of Medicine in 1853, he later participated to the Crimean War (1854-1855) where he performed-interesting medical observations . In 1858, he was appointed personal physician to Nasreddin Shah and remained in Persia until his death in Teheran (1897) where he is buried . Tholozan published between 1847 and 1892 over fifty articles and books dealing chiefly with infectious pathology and epidemiology, written at a time when microbial etiology and specificity of such diseases were wholly unknown . He considered chiefly bubonic plague, studying as soon as 1871 the focus of the Iranian Kurdistan, a research which will be resumed by M . Baltazard and his collaborators between 1947 and 1971, i.e . a century later . He was also deeply interested by the "oriental" cholera of which he recalled masterly the history and geography in the Near and Middle East . He also performed, while in Crimea and Persia, personal observations on tuberculosis, diptheria, remittent fever, acrodynia and had studied in France in his early years various other diseases such as cutaneous staphylococcic infections, glanders, pulmonary haemorrhagies, etc . In Persia, he reorganized Public Health and medical teaching and educated many local physicians and surgeons . Being assured of the unlimited confidence of the Shah, he played an important cultural role, promoting French influence in Persia . Holder of many French and foreign decorations, Tholozan was Fellow of the French Academies of Sciences and Medicine . His name was given by Laboulbene to Ornithodoros tholozani, a tick vector of a recurrent fever (spirochetosis due to Borrelia persica), of which he had described both the symptoms and the vector in 1882. Jpn J Thorac Cardiovasc Surg, 1998 Feb, 46(2), 170 - 4 {Postoperative infections related to pacing wires, pulmonary arterial catheters, and drainage tubes temporarily inserted during open-heart surgery}; Kanoh M et al.; Bacterial examinations of temporary pacing wires (P-wires), pulmonary arterial (P-A) catheters, and drainage tubes temporarily inserted during open-heart surgery were performed in 213 patients . Bacteria were detected in 19 (2.8%) of 672 specimens gathered from the subject patients, with coagulase-negative Staphylococcus (CNS) being most frequently observed . P-wires accounted for 17 out of 19 of the culture-positive specimens, and 7 of the P-wires remained in place for more than two weeks . The frequency of infection with the P-wires was significantly higher than with the P-A catheters or drainage tubes . The period of time that the P-wire was left in place significantly longer than for P-A catheter or drainage tube . There was, however, no statistically significant difference between the culture-positive and negative groups in respect to age, detention periods, operation times, CPB times, or length of ICU stay . As a result of these findings, we have concluded that P-wires should be removed as soon as possible following surgery, and in any case, a meticulous care should be taken to prevent transcutaneous infection. J Immunol, 1998 Apr 15, 160(8), 3855 - 60 CTLA-4 regulates tolerance induction and T cell differentiation in vivo; Walunas TL et al.; Cytotoxic T lymphocyte Ag-4 (CTLA-4; CD152) is an important T cell regulatory molecule . In vitro experiments have shown that the blockade of signals through CTLA-4 augments T cell expansion, while CTLA-4 cross-linking results in decreased T cell proliferation due to decreased IL-2 production . However, less is known about the role of CTLA-4 in regulating an ongoing immune response . In this study, we examined the role of CTLA-4 in the expansion, decline, tolerization, and differentiation of T cells following treatment with staphylococcal enterotoxin B (SEB) . Anti-CTLA-4 treatment resulted in increased numbers of SEB-reactive T cells and blockade of subsequent tolerance induction . Further examination of the SEB-reactive cells from anti-CTLA-4-treated mice demonstrated that both the CD4+ and CD8+ Vbeta8+ T cells produced IL-4, providing evidence that not only do signals through CTLA-4 regulate T cell-tolerizing events, but they also play an important role in the differentiation of T cells in vivo. Biochemistry (Mosc), 1998 Apr, 63(4), 470 - 5 Equilibrium unfolding of partially folded staphylococcal nuclease A2- and A3-forms is accompanied by the formation of an intermediate state; Uversky VN; Structural properties of an equilibrium intermediate formed upon urea-induced unfolding of more ordered staphylococcal nuclease A-forms (A2 and A3) are studied . The effect of association on the structural properties and conformational stability of this unfolding intermediate is also considered . A close structural similarity (including tendency for association) is shown between this intermediate and the least ordered A1-form, induced in the acid-unfolded nuclease by moderate sulfate or chloride concentrations. Biochemistry (Mosc), 1998 Apr, 63(4), 463 - 9 Structural properties of staphylococcal nuclease in oligomeric A-forms; Uversky VN et al.; Association affects the structural properties of different partially folded conformations of staphylococcal nuclease induced by anions of different nature . It is shown that oligomerization induces new structural levels in non-native A-forms . A close structural similarity between the monomeric A2 and the dimeric (A1)2 forms as well as between the monomeric A3 and oligomeric {(A1)2}M and {A2}M forms is established . This suggests that association of a protein molecule in partially folded conformations can be an additional structure forming factor. Biochemistry (Mosc), 1998 Apr, 63(4), 456 - 62 Structural effect of association on protein molecules in partially folded intermediates; Uversky VN et al.; Fluorescence and circular dichroism spectroscopy, small angle X-ray scattering and high performance liquid gel filtration have shown that oligomerization considerably affects the structural properties and conformational stability of partially folded intermediates of staphylococcal nuclease . Conformational transitions induced by different anions and association in the acid-unfolded protein are described . It is shown that association of non-native conformations of the protein molecule can be an additional structuring factor . The corresponding folding schemes and phase diagrams are suggested. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1998 Jan-Feb, 39(1), 55 - 7 Pneumonia with pneumatocele formation caused by Mycobacterium tuberculosis: report of one case; Kao HC et al.; Pneumatoceles are usually characteristic of staphylococcal pneumonia . They are rarely formed as one of the complications of Mycobacterium tuberculosis pneumonia . We report a 1-year-and-5-month old male child with pneumonia who was confirmed to have the rare complication, pneumatocele formation, by chest radiography and computed tomography . Since the patient did not respond to empiric antibiotic therapy, gastric larvage through a nasogastric tube was performed on three consecutive mornings and, as a result, acid fast rods were found on the three specimens . The cultures subsequently yielded M . tuberculosis . He was finally cured with a 6-month course of antituberculous chemotherapy . We conclude that tuberculosis should be considered in infants or young children with pneumonia that presents radiologically as pneumatocele formation, especially in whom there has been no response to empiric antibiotic therapy. J Immunol, 1998 Jan 1, 160(1), 225 - 32 Staphylococcal enterotoxin D is a promiscuous superantigen offering multiple modes of interactions with the MHC class II receptors; Al-Daccak R et al.; Dimerization of MHC class II molecules on the cell surface of human THP-1 monocytic cell line is a requirement for staphylococcal superantigen (SAG)-induced cytokine gene expression . The capacities of various SAG to induce this response are governed by their modes of interaction with MHC class II molecules . Staphylococcal enterotoxin A (SEA), with its two binding sites, dimerizes MHC class II molecules and subsequently induces cytokine gene expression in THP-1 cells . Here, we demonstrate that staphylococcal enterotoxin D (SED) and staphylococcal enterotoxin E (SEE) induce, similarly, IL-1beta and TNF-alpha gene expression in these cells . Using mutated toxins that lost their binding site with the MHC class II alpha- or beta-chain, we demonstrate that this response is also mediated by the dimerization of MHC class II molecules through two binding sites . Furthermore, SED forms Zn2+-dependent homodimers that allow multiple modes of MHC class II clustering, including ligation of alpha-chains (alpha/alpha), beta-chains (beta/beta), or the alpha- and beta-chains of two different class II molecules . The beta/beta interaction following Zn2+-dependent SED/SED homodimer formation seems to be mediated by the appearance of a novel binding site on SED that interacts with histidine 81 of the MHC class II beta-chain . The different modes of SED interactions also influence SED-induced T cell activation where simultaneous ligation of the alpha- and beta-chains is essential for optimal response . These various modes of SED binding may be used to preserve bivalency regardless of variability in the MHC class II alpha/beta/peptide complexes. J Immunol, 1998 Jan 15, 160(2), 615 - 23 Intercellular adhesion molecule-1 and leukocyte function-associated antigen-3 provide costimulation for superantigen-induced T lymphocyte proliferation in the absence of a specific presenting molecule; Lamphear JG et al.; Bacterial superantigens can bind TCR in the absence of MHC class II molecules and activate T lymphocytes when cocultured with certain class II-deficient accessory cells . It has not been determined, however, whether these accessory cells provide direct costimulation to the T cell or serve to present superantigens via a nonconventional ligand . We have identified a human adenocarcinoma cell line, SW480, that assists in the activation of human T cells by the staphylococcal enterotoxins B (SEB), C1 (SEC1), and D (SED), but not SEA, SEC2, SEC3, or SEE . SW480 cells did not express class II molecules, and anti-class II mAbs did not inhibit T cell proliferation, supporting the hypothesis that class II is not absolutely required for enterotoxin-mediated T cell activation . The TCR Vbeta profile of T cells stimulated by SEB plus SW480 cells was similar to that of T cells stimulated by SEB plus class II+ APC, indicating that TCR-SEB interactions were preserved in the absence of class II molecules . Binding studies failed to detect specific association of SEB with SW480 cells, suggesting that SW480 cells do not express receptors for enterotoxin . SEB coupled to beads, however, stimulated T cell proliferation, but only in the presence of SW480 cells . SW480 cells express both ICAM-1 and LFA-3 molecules, and the addition of Abs to these receptors inhibited T cell proliferation . These findings support a model in which certain enterotoxins engage the TCR independent of MHC class II or other specific presenting molecules and induce T cell proliferation with signals provided by nonconventional accessory cells. J Immunol, 1998 Jan 15, 160(2), 559 - 65 Suppression of immune responses by CD8 cells . I . Superantigen-activated CD8 cells induce unidirectional Fas-mediated apoptosis of antigen-activated CD4 cells; Noble A et al.; Stimulation of mature CD4 cells through the TCR induces cellular activation and expansion that are often followed by clonal elimination by a form of apoptosis termed activation-induced cell death . This process of CD4 cell apoptosis is generally thought to reflect clonal suicide and to be independent of other cell types . Here we show that during the response to the superantigen Staphylococcal enterotoxin A, activated CD8 cells, but not activated CD4 cells, suppress the CD4 proliferative response . Suppression by CD8 cells reflects their ability to induce CD4 cell apoptosis via ligation of Fas . Moreover, although activated CD8 cells that express Fas ligand and Fas eliminate CD4 cells through a Fas-dependent mechanism, they are themselves resistant to Fas-dependent apoptosis . These findings indicate a fundamental difference between the two major T cell subsets with regard to sensitivity to Fas-dependent apoptosis, expression of Fas ligand, and mediation of suppressive activity following immunization with superantigen. J Immunol, 1997 Dec 15, 159(12), 5936 - 45 Maternal immunization with a soluble TCR-Ig chimeric protein: long term, V beta-8 family-specific suppression of T cells by maternally transferred antibodies; McKeever U et al.; Maternal transfer of TCR clonotypic Ab protected young NOD mice against the adoptive transfer of diabetes by the BDC 2.5 T cell clone . The effect of maternal anti-TCR Vbeta-8 Ab on T cell development and function has now been investigated . SJL/J mice, which lack TCR Vbeta-8, were immunized with soluble, chimeric D10 TCR-IgG1 containing Vbeta-8.2 . The (SJL/J x AKR/J) F1 offspring of immunized female SJL/J mice were severely depleted of peripheral T cells bearing Vbeta-8 until 11 to 17 wk of age . The loss of Vbeta-8 expression did not appear to be due to modulation of cell surface TCR . Since the Vbeta-8+ T cell population was unperturbed in the (AKR/J x SJL/J) F1 offspring of D10 TCR-IgG1-immunized AKR/J mothers making D10 clonotypic Ab, the effect was immunologically specific . The deletion of Vbeta-8+ T cells had functional consequences . In the in vitro response to the superantigen, staphylococcal enterotoxin B, the usually observed participation of Vbeta-8.2+ T cells was largely suppressed, whereas the recruitment of Vbeta-3+ T cells remained unaltered . In control mice, T cell responses to the 134- to 146-residue peptide of conalbumin (pCA(134-146)) were biased toward use of Valpha-2/Vbeta-8.2 TCR . In D10 TCR-IgG1 maternally immunized (SJL x AKR/J) F1 mice, the T cell responses to pCA(134-146) were suppressed, and T cell lines derived from these in vitro were devoid of Vbeta-8.2 expression . With an increased understanding of TCR V gene usage in autoimmune diseases, similar strategies for the depletion of autoreactive T cells may become feasible in humans. J Biol Chem, 1998 Mar 27, 273(13), 7657 - 67 Construction and binding kinetics of a soluble granulocyte-macrophage colony-stimulating factor receptor alpha-chain-Fc fusion protein; Monfardini C et al.; Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha-chain (GM-CSFRalpha), which specifically binds GM-CSF, and a beta-chain (betac), shared with the interleukin-3 and interleukin-5 receptors . GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha) and a soluble form (sGM-CSFRalpha) . We designed an sGM-CSFRalpha-Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha . The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a . Purified sGM-CSFRalpha-Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions . The sGM-CSFRalpha-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF . The sGM-CSFRalpha-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist . Kinetics of sGM-CSFRalpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems . In the first, the sGM-CSFRalpha-Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined . This revealed a rapid koff of 2.43 x 10(-2)/s . A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha-Fc in solution . The dissociation rate constant (koff) for the sGM-CSFRalpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of binding in this assay . These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha-Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity. J Mol Biol, 1998 Mar 20, 277(1), 61 - 79 Crystal structure of microbial superantigen staphylococcal enterotoxin B at 1.5 A resolution: implications for superantigen recognition by MHC class II molecules and T-cell receptors; Papageorgiou AC et al.; Staphylococcal enterotoxin B is a member of a family of toxins known as superantigens that activate a large number of T-cells (up to 20%) by cross-linking MHC class II molecules with T-cell receptors in a Vbeta-restricted fashion . The crystal structure of staphylococcal enterotoxin B presented here has been determined at 1.5 A resolution, the highest resolution so far for a superantigen . The final model contains 1948 protein atoms and 177 water molecules and has excellent geometry with root-mean-square (rms) deviation of 0.007 A and 1.73 degrees in bond lengths and bond angles, respectively . The overall fold is similar to that of other microbial superantigens, but as it lacks the zinc-binding site found in other members of this family, such as staphylococcal enterotoxin A, C2 and D, this enterotoxin possesses only one MHC class II binding site . Comparison of the crystal structure of free SEB and in complex with an MHC class II molecule revealed no major changes in the MHC-binding site upon complex formation . However, a number of water molecules found in the free SEB may be displaced in the complex or contribute further to its stability . Detailed analysis of the TcR-binding site of SEB, SEA and SEC2 shows significant differences which may account for the ability of each superantigen to bind specific Vbeta sequences . J Immunol, 1997 Dec 1, 159(11), 5293 - 300 Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense; Berthou C et al.; In vertebrate tissues, cell integrity is maintained by at least three mechanisms . During an immune response, injured cells are eliminated by cytotoxic lymphoid cells that produce perforin, granzyme B, and Fas ligand (FasL) . Second, epithelial cells can produce FasL as an immunosuppressive protein, probably to protect the tissue against immune-mediated damage . Third, locally secreted antimicrobial peptides can be operative in the protection of animal and human epithelia . In this work, as another contribution to local mechanisms of host defense, the ability of human epidermal keratinocytes to produce cytotoxic proteins was investigated . To address this question, freshly isolated human epidermal cells and keratinocytes grown in vitro were studied . Freshly isolated epidermal cells did not express the cytolytic proteins . In contrast, keratinocyte growth to confluence was associated with granzyme B, perforin, and FasL mRNA and protein synthesis . These proteins were secreted in the culture medium . Further analysis showed that they were identical with the ones used by cytotoxic lymphocytes . Their function was then investigated with a view to a potential role in epidermal cell integrity . The data showed that activated human keratinocytes were able to protect against invading pathogens through granzyme B expression . This was demonstrated by the ability of granzyme B to greatly decrease the bacterial growth of Staphylococcus epidermidis . In addition, keratinocytes expressing FasL were found to prevent immune epidermal cell damage . Apoptosis of Fas-sensitive T cells occurred during coculture with confluent epidermal keratinocytes and was largely reduced by the addition of a FasL inhibitor . The data favor keratinocyte involvement in the regulation of dermal inflammatory responses. Surg Today, 1998, 28(3), 325 - 7 Infected atherosclerotic ulcer of the abdominal aorta as a cause of mycotic aneurysm treated by in-situ prosthetic graft reconstruction: report of a case; Moriyama Y et al.; A 68-year-old man with an infrarenal mycotic aneurysm underwent successful in-situ graft reconstruction using a woven Dacron graft . A tissue culture taken from the excised aortic wall revealed Staphylococcus epidermidis, and histological study subsequently showed penetrating atherosclerotic ulcers (PAU) involving all layers of the aortic wall and marked neutrophilic infiltration with abscess formation inside the ulcer . Atherosclerotic aortic lesions such as PAU are considered susceptible to bacterial infection, which may lead to the formation of an aneurysm after destruction of the vessel wall . Hence, elderly hypertensive patients, being at high risk for such aortic pathology, require careful studies performed to assess the aorta . The usefulness of computed tomographic (CT) scans to determine the presence of PAU or surrounding inflammation should be borne in mind even when a small mycotic aneurysm exists. J Pediatr, 1998 Mar, 132(3 Pt 1), 535 - 7 Successful treatment of a staphylococcal endocarditis vegetation with tissue plasminogen activator; Fleming RE et al.; A 930 gm premature infant had Staphylococcal endocarditis with a tricuspid valvular vegetation that was unresponsive to antibiotics and not amenable to resection . Infusion of tissue plasminogen activator over a 3-day period completely lysed the vegetation . The infection cleared with continued antibiotics, and the infant recovered without sequelae. Arch Pediatr, 1997 Dec, 4(12), 1204 - 6 {Postoperative mediastinitis due to methicillin resistant Staphylococcus epidermidis with low sensitivity to vancomycin}; Hulin S et al.; BACKGROUND: Vancomycin is the drug of choice for methicillin-resistant Staphylococcus . Antibiotherapy failure is rarely clinically related to Staphylococcus with vancomycin low susceptibility . CASE REPORT: A surgical cure of an aortic stenosis in a neonate was complicated by a Staphylococcus mediastinitis . After initiation of antibiotherapy with vancomycin and rifampin and surgical debridement, there was a rapid improvement . Few days later, failure of therapy was obvious . Despite continuous infusion of vancomycin, with a serum level of 29 mg/L, blood cultures were positive again to Staphylococcus . There was no endocarditis or inadequate surgical drainage . Susceptibility of the Staphylococcus was tested, looking for a tolerant strain . The vancomycin minimum bactericidal concentration was 30 mg/L (above usual value 2 to 8 mg/L), while the minimum inhibitory concentration was 3.75 mg/L . A higher dosage of vancomycin associated with fusidic acid was rapidly efficient, and total recovery was achieved . CONCLUSION: In case of failure of vancomycin therapy, despite correct serum levels, the susceptibility of the Staphylococcus strain has to be determined . A low susceptibility strain prescribes more prolonged combination of two antibiotics. Int J Cancer, 1998 Apr 13, 76(2), 274 - 83 Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth; Rosendahl A et al.; Superantigens (SAg) are microbial proteins with the capacity to activate a large proportion of T cells . We have developed a novel approach for cancer immunotherapy by genetically fusing the SAg staphylococcal enterotoxin A (SEA) to a Fab-fragment of a tumor-specific antibody . Repeated exposure to SEA induces a state of unresponsiveness including cell deletion and functional hyporesponsiveness, i.e., anergy . In this study we have developed improved therapeutic schedules to allow repeated injections of Fab-SEA, limit development of immunological unresponsiveness and promote maximal anti-tumor response . Four daily injections of Fab-SEA to mice carrying B 16-C215 lung metastases resulted in 90-95% reduction in the number of metastases . However, the animals did retain a minimal residual tumor disease . The immune system was in a hyporesponsive state after 4 daily Fab-SEA injections, and further injections did not improve therapy . Two repeated cycles, each comprising 4 daily injections of Fab-SEA, significantly prolonged the survival and resulted in complete cure of a fraction of the animals . A rest period of 10 days between the cycles was required to mount an efficient secondary anti-tumor response . This secondary immune response was characterized by partial recovery of cytokine production i.e., interleukin-2, interferon-gamma and tumor necrosis factor-alpha . Strong CTL activity was detected in animals that had rested for 8 weeks between the 2 cycles . Interestingly, irrespective of the resting period, the CD4+ SEA-reactive T cells expanded in response to all 4 additional Fab-SEA injections both locally and in spleen . In contrast, only marginal expansion of CD8+ T cells was seen if restimulation was given within 1 month . Our data show that potent anti-tumor effector functions can be induced after repeated stimulation cycles with a SAg-monoclonal antibody fusion protein resulting in a CD4+ T cell-dependent cytokine release, prolonged survival and induction of complete cures. Pediatr Infect Dis J, 1998 Mar, 17(3), 179 - 83 Selective use of vancomycin to prevent coagulase-negative staphylococcal nosocomial bacteremia in high risk very low birth weight infants; Baier RJ et al.; OBJECTIVE: To determine whether vancomycin added to parental nutrition (PN) fluids could prevent nosocomial infections in very low birth weight newborns and which infants would benefit most from prophylaxis . DESIGN: Double blind, randomized controlled study . SETTING AND STUDY POPULATION: Very low birth weight infants receiving PN in a tertiary neonatal intensive care unit . METHODS: Thirty-eight infants with and without central vascular catheters were randomized to receive no medication or 25 microg/ml vancomycin added to PN for the duration of the infant's PN requirements . RESULTS: The addition of 25 microg/ml vancomycin to PN prevented bacteremia in very low birth weight infants receiving PN . There was a significant reduction in the number of coagulase-negative staphylococcal (CONS) bacteremias (defined as isolation of the same organism from two positive blood cultures) during PN (5 vs . 0; P = 0.037) as well as the total number of bacteremias and fungemias (9 vs . 1; P = 0.036) . The total number of hospital days (108 +/- 13 vs . 76 +/- 6; P = 0.039) were reduced in infants receiving vancomycin . Infants with birth weights of < 1000 g who received corticosteroids for treatment of chronic lung disease benefitted most from treatment . No vancomycin-resistant strains of CONS or enterococci were detected during the study period . CONCLUSIONS: Prophylactic treatment with vancomycin effectively prevented CONS bacteremia under the conditions of the study . Its use was most effective in infants with birth weights of <1000 g. Intensive Crit Care Nurs, 1997 Oct, 13(5), 308 - 9 Teicoplanin (Targocid, Hoechst Marion Roussel): a new glycopeptide antibiotic; MacConnachie AM; The glycopeptides are important antibiotics in the management of Staphylococcal infections . Teicoplanin, the latest member of the group, may offer some advantages over vancomycin, the workhorse drug, which has retained its importance in the presence of increasing major resistance to other antistaphylococcal agents. Scand J Immunol, 1980, 11(3), 253 - 60 Determination of the immunoglobulin class of complement-dependent cytotoxic antibodies in serum of D23 hepatoma-bearing rats; Lando P et al.; The immunoglobulin class of the complement-dependent cytotoxic antibodies in serum from D23 hepatoma-bearing rats (D23 TBS) for D23 hepatoma cells was analysed . When studied by affinity chromatography with concanavalin A, protamine, or staphylococcal protein A conjugated to Sepharose, the cytotoxic activity bound to the former two but not protein A . The binding fractions were further characterized by column chromatography on Sepharose CL-4B . The cytotoxic activity was recovered exclusively in the high molecular weight fractions corresponding to human IgM . Monitoring with IgG- or IgM-specific rabbit antibodies indicated that these high molecular weight cytotoxic fractions contained both IgG and IgM . However, fractionation of D23 TBS at low pH suggested that cytotoxicity was due to IgM antibodies rather than to immune-complexed IgG antibodies . This was supported by the findings that rabbit antirat IgM antibodies inhibited the cytotoxicity of TBS completely when added at high dilutions. J Surg Res, 1998 Jan, 74(1), 17 - 22 Bacterial products primarily mediate fibroblast inhibition in biomaterial infection; Henke PK et al.; PURPOSE: The stimulation of fibroblast growth is essential for the normal healing and tissue integration of biomaterials . The local elevation of proinflammatory mediators in infected perigraft fluid (PGF) may inhibit this growth . We sought to determine whether infected PGF inhibited fibroblast growth, and, if so, whether this was primarily dependent on the biomaterial, bacteria, or host . METHODS: In vivo Dacron or expandable polytetra-fluoroethylene (ePTFE) grafts, sterile or colonized with slime-producing (RP-62A, viable or formalin-killed) or nonslime-producing (RP-62NA) Staphylococcus epidermidis (1 x 10(7) CFU/cm2), were implanted in Swiss Webster mice, and the PGF was harvested at 7 and 28 days . Antibodies to tumor necrosis factor alpha, interleukin 1 alpha, interferon gamma (7 micrograms/day), and indomethacin (50 micrograms/day) were administered by microinfusion pumps for 7 days and the PGF was harvested . Inhibition of the proinflammatory mediators was confirmed by enzyme-linked immunosorbant assay . The nontreated, heat-treated, or trypsin-digested in vivo PGF was incubated with an in vitro {3H}thymidine murine fibroblast (ATCC CCL-12) proliferation assay . RESULTS: Fibroblast inhibition was significant at 7 and 28 days with infected PGF incubation compared with sterile and was not dependent on bacterial slime production or viability . Dacron sterile PGF did not significantly inhibit fibroblasts compared with control, whereas sterile ePTFE stimulated (P < 0.05) fibroblasts . Treatment of the PGF with proinflammatory cytokines, heat, and trypsin failed to reverse fibroblast inhibition in the infected state . CONCLUSION: Biomaterial infection is associated with fibroblast inhibition that is dependent primarily on bacterial products and not the host or biomaterial . Conservative intervention strategies for graft infection need to address the problem of poor healing as well as bacterial clearance. Immunology, 1998 Jan, 93(1), 20 - 5 Synovial fibroblasts as target cells for staphylococcal enterotoxin-induced T-cell cytotoxicity; Kraft M et al.; Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown aetiology . Recently, superantigens have been implied in the pathogenesis of RA . Superantigens activate a large fraction of T cells leading to the production of cytokines and proliferation . In addition, superantigens direct cellular cytotoxicity towards major histocompatibility complex (MHC) class II-expressing cells . There is now increasing evidence that cytotoxic T cells may be involved in the pathogenesis of RA . In the inflamed synovia class II-positive synovial fibroblasts (SFC) are found . In the present study it was tested whether MHC class II-positive SFC serve as target cells for superantigen-induced cellular cytotoxicity . SFC were stimulated with interferon-gamma to express class II antigens, then they were cultivated in the presence of CD4-positive T cells with or without staphylococcal enterotoxins (SE) . Cytotoxicity of T cells was measured as release of lactate dehydrogenase from SFC . Specific cytotoxicity was only found in the presence of class II-positive SFC depending on the dose of SE . Maximum lysis was seen after 20 hr . T-cell cytotoxicity was inhibited by antibodies to MHC class II antigens . The data suggest that class II-positive SFC not only function as accessory cells for SE-mediated T-cell proliferation and interleukin-2 production but may also be the targets of superantigen-mediated cellular cytotoxicity. Mol Microbiol, 1998 Mar, 27(5), 967 - 76 Identification of novel staphylococcal virulence genes by in vivo expression technology; Lowe AM et al.; We have applied in vivo expression technology (IVET) to the study of staphylococcal virulence . Using a promoter trap that relies on genetic recombination as a reporter of gene expression, we identified 45 staphylococcal genes that are induced during infection in a murine renal abscess model . Of these, only six were known previously; 11 others have homology to known non-staphylococcal genes . The known staphylococcal genes include agrA, part of a key locus regulating numerous virulence products, and a glycerol ester hydrolase, which may enhance staphylococcal survival in abscesses . We constructed 11 strains containing mutations in previously unknown ivi genes . Of these strains, seven were significantly attenuated in virulence compared with the wild-type parent . The mutagenized ivi genes may encode novel staphylococcal virulence factors. J Infect Dis, 1998 Apr, 177(4), 1013 - 22 Differential immune responses to staphylococcal enterotoxin B mutations in a hydrophobic loop dominating the interface with major histocompatibility complex class II receptors; Woody MA et al.; Bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can trigger acute pathologic effects in humans . A hydrophobic loop on the surface of SEB and other bacterial superantigens, centered around a conserved leucine (L45) residue, is essential for binding to class II major histocompatibility complex molecules . Single residue changes of wild type SEB, designated Q43P, F44P, or L45R, resulted in nonlethal proteins at a dose equivalent to 30 murine LD50 of SEB . Relative to SEB, the mutant proteins did not elevate serum concentrations of proinflammatory cytokines in mice and caused minimal proliferation of human lymphocytes . Anti-SEB titers of mice immunized with Q43P, F44P, L45R, or SEB were similar and protected 77%-100% of animals against a lethal SEB challenge . Levels of toxin-specific IgG1, IgG2a, IgG2b, and IgG3 in mice immunized with SEB, Q43P, or F44P were equivalent, but animals immunized with L45R had significantly elevated levels of IgG2a and IgG2b . Vaccines against staphylococcal superantigens should focus on this critical leucine residue. J Infect Dis, 1998 Apr, 177(4), 905 - 13 gp41 envelope protein of human immunodeficiency virus induces interleukin (IL)-10 in monocytes, but not in B, T, or NK cells, leading to reduced IL-2 and interferon-gamma production; Barcova M et al.; The effect of extracellular domain of human immunodeficiency virus (HIV-1) transmembrane glycoprotein gp41 on interleukin (IL)-10, IL-2, interferon (IFN)-y, IL-4, and tumor necrosis factor-alpha production by human peripheral blood mononuclear cells (PBMC) was assessed by ELISA . Rapid gp41-induced increase of IL-10 production was detected in resting PBMC and isolated monocytes but not in B, T, or NK cells . Furthermore, gp41 also enhanced IL-10 production in staphylococcal enterotoxin B-stimulated PBMC, while synthesis of IL-2, IFN-gamma, and IL-4 in these cells was down-modulated . Kinetic studies revealed that increased IL-10 production preceded reduction of IL-2, indicating the possible IL-10 regulatory role in the gp41-induced down-modulation of this cytokine . Anti-IL-10 antibody reversed almost completely the gp41 inhibitory effect on IL-2 production . In this study, HIV-1 gp41 was a potent modulator of cytokine production by PBMC, in particular by increasing IL-10 secretion from normal monocytes/macrophages and consequently down-regulating IL-2 and IFN-gamma. Proteins, 1998 Mar 1, 30(4), 381 - 7 A fusion protein between serum amyloid A and staphylococcal nuclease--synthesis, purification, and structural studies; Meeker AK et al.; We developed a recombinant DNA system to overexpress a fusion protein between the small, minimally soluble acute phase serum protein, serum amyloid A (SAA), and the bacterial enzyme staphylococcal nuclease (SN) . This fusion protein is very soluble and is immunoreactive to polyclonal anti-SAA antibodies . Tryptophan fluorescence shows smooth denaturation curves for the fusion protein in guanidinium HCl or potassium thiocyanate . Fluorescence also indicates that only a single tryptophan residue (of the four present) is accessible to iodide quenching and, presumably, is exposed on the surface of the fusion protein . Circular dichroism (CD) shows a significant signal indicating alpha-helix, which can be attributed to the SAA portion of the molecule; these are the first CD spectral data available for SAA . pH titration shows persistence of helix domains for the fusion protein at pH 3.0, in contrast to the denaturation of SN under the same conditions . (The entire fusion protein shows a random coil pattern below pH 3.0.) By exploiting the structural and solubility properties of SN, this fusion protein has provided the first structural data about SAA-the precursor of the amyloid deposits in secondary amyloidosis . This fusion protein should be useful for further physical and physiologic studies of SAA. Zentralbl Bakteriol, 1998 Jan, 287(1-2), 135 - 45 Effect of surface modifications of intraocular lenses on the adherence of Staphylococcus epidermidis; Schmidt H et al.; The development of polymers with different surface properties and surface modifications of intraocular lenses (IOL) should reduce foreign body reactions after implantation by reducing the surface hydrophobicity of the lenses . It was examined how far such surface variations influenced the adhesiveness of bacteria . The most common organism isolated from cases of postoperative endophthalmitis is Staphylococcus epidermidis . For this reason, three strains of this species, the type strain ATCC 14990 and two clinical isolates (8687, 6579 I), with different hydrophobic surfaces, were studied . IOL made of PMMA, silicone, and a copolymer as well as PMMA lenses with modified surfaces (unpolished, polished, silanized, and heparinized) were used . Bacteria were radiolabelled with 3H-thymidine and the adherent bacteria were calculated per mm2 of lens surface . The three strains adhered better to the unpolished surface of silicone than to PMMA . Treatment of PMMA surface by polishing diminished the differences between the strains . An influence of hydrophobic interactions on the adherence of S . epidermidis ATCC 14990 was demonstrated . The adherence of this hydrophobic type strain was clearly reduced by heparinization of the PMMA surface . In contrast, the hydrophilic catheter isolate 6579 I adhered better to modified surfaces . This strain differed clearly in its PFGE pattern from both hydrophobic strains . Hydrophobic interactions play a role in the bacterial adherence to intraocular lenses in vitro and in vivo . Modifications of polymer surfaces, however, can result in rather different effects depending on the bacterial surface composition and properties. Zentralbl Bakteriol, 1998 Jan, 287(1-2), 19 - 31 In vitro anti-staphylococcal activity of heparinized biomaterials bonded with combinations of rifampicin; Fallgren C et al.; Biomaterial implants in various human body tissues are highly susceptible to bacterial colonization . We report here on the coating of heparinized biomaterials with heparin binding extracellular matrix proteins giving special regard to the efficient adsorption and slow release of antibiotics . Heparin was partially degraded and the resulting fragments were covalently end-point attached to 0.5 cm long silicone biomaterial surface . Collagen type I was immobilized on the heparinized biomaterials and then cross-linked with acyl-azide or carbodiimide . Finally, the resulting biosurfaces were exposed to antibiotics, i.e . rifampicin in combination with cefuroxime, fusidic acid, ofloxacin or vancomycin, respectively . The antibiotic bonded biomaterials were evaluated for their anti-staphylococcal activity after elution in NaCl, serum or blood by measuring the zones of inhibition for S . epidermidis strain RP12 . Furthermore, we examined the in-vitro colonization resistance to S . epidermidis RP12 for these combinations of rifampicin-bonded biomaterials by an ATP bioluminescence assay . The ATP measurements showed that initially adherent bacteria were eradicated from the polymer surface, for at least 24 or 48 h (fusidic acid > cefuroxime > vancomycin > ofloxacin) . The anti-staphylococcal activity of rifampicin-fusidic acid bonded heparinized biomaterials seems of sufficient duration and efficacy to merit testing in an animal model. Zentralbl Bakteriol, 1998 Jan, 287(1-2), 7 - 18 Quantitation of bacterial adhesion to polymer surfaces by bioluminescence; Stollenwerk M et al.; Quantitation of microbes adhering to a surface is commonly used in studies of microbial adhesion to different surfaces . We have quantified different staphylococcal strains adhering to polymer surfaces by measuring bacterial ATP (adenosine triphosphate) by bioluminescence . The method is sensitive, having a detection limit of 10(4) bacterial cells . Viable counting of bacterial cells may yield falsely low results due to the presence of "dormant" and adherent bacteria . By using bioluminescence, this can be avoided . Cells of different bacterial species and cells of strains of the same species were shown to differ significantly in their basal ATP content (8.7 x 10(-13) - 5.2 x 10(-22) MATP) . The size of adherent and planktonic bacteria decreased with time (0.7 micron-->0.3 micron, 20 days) . During incubation in nutrient-poor buffer ("starvation"), the ATP content of adherent bacteria decreased after 24-96 h whereas that of planktonic bacteria was stable over 20 days . The presence of human serum or plasma did not interfere significantly with the test results . Since the ATP concentration of bacterial strains of different species varies and is also influenced by the growth conditions of bacteria (solid or liquid culture medium), a species-specific standard curve has to be established for bacteria grown under the same culture conditions . We conclude that the method is a sensitive tool to quantify adherent bacteria during experiments lasting for less than 6 h and constitutes a valuable method to be used in conjunction with different microscopical techniques. Int Immunol, 1998 Feb, 10(2), 175 - 83 Regulation of programmed cell death following T cell activation in vivo; Yang Y et al.; Activation of T cell hybridomas in vitro induces rapid Fas-Fas ligand (FasL)-mediated programmed cell death (apoptosis) . In contrast, T cells activated by antigen or superantigen in vivo undergo a population expansion and then decline due to Fas-FasL-mediated activation-induced apoptosis (AIA) . We asked how T cells activated by antigen in vivo proliferated before undergoing apoptosis . Two possibilities were analyzed: either (i) the apoptosis program was not 'turned on' or (ii) was 'blocked' during the period of cellular proliferation in vivo . Data presented in this manuscript support the second of these possibilities . CD4+ T cells activated in vivo were resistant to anti-fas-mediated apoptosis until 48 h following staphylococcal enterotoxin B (SEB) administration, despite the fact that activated proliferating T cells expressed high levels of Fas (CD95) antigen and many 'apoptosis genes' were induced within 24 h of SEB administration . The analysis of the expression patterns of 'apoptosis genes' during the T cell activation further suggested that temporal blockade of AIA may be due to the induction of apoptosis-preventing genes, such as bag-1. Infect Immun, 1998 Apr, 66(4), 1579 - 87 Characterization of leptospiral outer membrane lipoprotein LipL36: downregulation associated with late-log-phase growth and mammalian infection; Haake DA et al.; We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36 . We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe . A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified . Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E . S . Shang, T . A . Summers, and D . A . Haake, Infect . Immun . 64:2322-2330, 1996) . The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site . LipL36 is solubilized by Triton X-114 extracti