Acta Microbiol Pol, 2000, 49(3-4), 265 - 70 Plasmid occurrence and diversity in the genus Paracoccus; Baj J et al.; The results of screening for the occurrence of plasmids in several strains representing 11 out of 13 species of the genus Paracoccus are presented . We show that plasmids (ranging in size from 2.7 to above 450 kb) are widely distributed in this genus . Only one tested strain (P . alkenifer) appears to be plasmid-free . The majority of the strains harbour at least two plasmids, one of which usually fits into the class of megaplasmids. J Inorg Biochem, 2001 Feb, 83(4), 281 - 6 Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase; Kurose S et al.; The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR) . Intensities of the characteristic bands for the high spin heme b, that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (-) in the MCD spectrum decreased considerably . Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g approximately 6, of which less than 5% had been EPR detectable at pH 7 . Cyanide (x40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (x1000) was necessary to bind to the reduced NOR at an acidic pH, ca . 5 . Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed. Arch Microbiol, 2001 Feb, 175(2), 102 - 11 Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus); Kappler U et al.; The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt . Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism . Here we report the cloning and sequencing of the soxBCD genes from S . novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus . These findings suggest two distinct pathways for thiosulfate oxidation in S . novella . The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis . The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose . In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present . The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB. Braz J Med Biol Res, 2001 Apr, 34(4), 529 - 37 The low efficiency of dendritic cells and macrophages from mice susceptible to Paracoccidioides brasiliensis in inducing a Th1 response; Almeida SR et al.; In the present study we evaluated T cell proliferation and Th lymphokine patterns in response to gp43 from Paracoccidioides brasiliensis presented by isolated dendritic cells from susceptible and resistant mice . T cell proliferation assays showed that dendritic cells from susceptible mice were less efficient than those from resistant mice . The pattern of T cell lymphokines stimulated by dendritic cells was always Th1, although the levels of IL-2 and IFN-gamma were lower in T cell cultures from susceptible mice . To determine whether different antigen-presenting cells such as macrophages and dendritic cells stimulated different concentrations of Th1 lymphokines, the production of IFN-gamma and IL-2 was measured . It was observed that dendritic cells were more efficient than macrophages in stimulating lymphoproliferation in resistant mice . However, no significant difference was observed for IFN-gamma or IL-2 production . When cells from susceptible mice were used, macrophages were more efficient in stimulating lymphoproliferation than dendritic cells, but no difference was observed in the production of Th1 cytokine . Taken together, these results suggest the lower efficiency of dendritic cells and macrophages from B10.A mice in stimulating T cells that secrete Th1 lymphokines in vitro, an effect that may be involved in the progression of the disease in vivo. J Am Acad Dermatol, 2001 Apr, 44(4), 585 - 92 Chromoblastomycosis: a review of 100 cases in the state of Rio Grande do Sul, Brazil; Minotto R et al.; BACKGROUND: If not diagnosed earlier, chromoblastomycosis has a chronic evolutional course that may cause several problems, such as difficulty in managing therapy because of the recrudescent character of the disease, potential association with the growth of epidermoid carcinoma in affected regions, and poor quality of life and work incapacity to the patient . Although infrequent, new cases are reported in the state of Rio Grande do Sul every year, ratifying the necessity for further studies on this disease . OBJECTIVE: The purpose of this study was to review clinical features and response to therapy in patients with chromoblastomycosis and present data on the demography and history of this disease in the state of Rio Grande do Sul, Brazil . METHODS: We reviewed case records of 100 patients with skin lesions caused by chromoblastomycosis, who were treated between 1963 and 1998 . The cases were confirmed by the histopathologic and mycologic analyses made by the Dermatology Service of the Universidade Federal do Rio Grande do Sul at the Santa Casa de Misericordia Hospital . RESULTS: There was a predominance of male patients (4:1) and of white farmers whose ages ranged from 50 to 59 years, with lesions on their lower limbs . Most of them were from the northern regions of the state . The average time between the appearance of the disease and medical diagnosis was 14 years . The verrucous type proved to be the most frequently reported lesion (53%) . Thorn wounds were associated with the disease in 16% of the cases . Lesions uncommon to some parts of the body were also reported . In two of the cases, cutaneous lesions caused by paracoccidioidomycosis and chromoblastomycosis were found in the same patient . Epidermoid carcinoma was found in the same parts of the body affected by chromoblastomycosis . Eumycotic mycetoma and chromoblastomycosis were associated . Fonsecaea pedrosoi was found in 96% of the cases, and Phialophora verrucosa in 4% of the cases . CONCLUSION: In our study, we observed a predominance of cases in the regions of Missoes and Alto Uruguay, followed by the upper and lower northeastern slopes and the lowlands . Severe cases of chromoblastomycosis with intense skin involvement (eg, lesions with carcinoma) were observed . Statistical analysis showed recrudescence of the disease in 43% of cases despite the treatment used. Med Trop (Mars), 2000, 60(3), 281 - 90 {Systemic tropical mycoses}; Marty P et al.; Systemic tropical mycoses have been emerging since the beginning of the AIDS epidemic . The incidence of these infections is probably even underestimated since most cases occur in populations with poor access to medical care in regions where modern diagnostic methods are unavailable . In Europe, this pathology is sometimes observed in returning travelers, aid workers, and immigrants . Differential diagnosis of imported systemic tropical mycosis may be difficult for uniformed physicians . The purpose of this review of the literature is to provide up-to-date epidemiological, clinical, diagnostic, and therapeutic data on the six main systemic tropical mycoses with disseminated forms . The six mycoses described in this study are histoplasmosis, coccidioidomycosis, paracoccidioidomycosis, blastomycosis, sporotrichosis, and penicilliosis due to Penicillium marneffei . Strictly superficial and subcutaneous mycoses are not covered. Expert Opin Pharmacother, 2000 Jan, 1(2), 287 - 304 Itraconazole; Pierard GE et al.; Itraconazole is a broad spectrum triazole antifungal agent . It has favourable pharmacodynamic and pharmacokinetic profiles and is available as both oral and i.v . formulations . Over the last two decades, clinical and animal infection studies have demonstrated the efficacy of itraconazole in a wide range of superficial fungal infections including difficult-to-treat dermatophytoses and onychomycoses . Furthermore, shortened treatment regimens have proven to be effective, ranging from 1-day treatment for vaginal candidosis to 1-week pulse therapy per month, for 2-4 months, in onychomycosis and follicular dermatophytosis . Clinical experience with itraconazole in the treatment of deep mycoses is less comprehensive . However, results in systemic candidosis, sporotrichosis, blastomycosis, paracoccidioiodomycosis, certain types of histoplasmosis and aspergillosis are extremely encouraging . Itraconazole is less effective in the treatment of chromomycosis and coccidioidomycosis . Nevertheless, considering the refractory nature of these diseases, itraconazole has proven to be a valuable addition to the antifungal drugs currently available for treatment . Itraconazole has been well-tolerated with doses of up to 400 mg/day being generally free of serious adverse effects . However, a potential for drug interactions exists, mediated through the cytochrome P450 enzyme 3A4 system, which should be considered when itraconazole is used as part of a multi-drug regimen. Biochem Biophys Res Commun, 2001 Mar 2, 281(3), 788 - 94 The cytochrome c domain of dimeric cytochrome cd(1) of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli; Gordon EH et al.; Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre . The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69 . Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here . The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1) . Cytokine, 2001 Feb 21, 13(4), 248 - 52 Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis; Benard G et al.; Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43) . To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P . brasiliensis-sensitized subjects . gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10 . Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM . gp43-pulsed adherent cells, but not non-adherent cells, were the main source of IL-10 . Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects . Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients . J Med Microbiol, 2001 Feb, 50(2), 127 - 34 Antibody isotypes to a Paracoccidioides brasiliensis somatic antigen in sub-acute and chronic form paracoccidioidomycosis; Juvenale M et al.; This report describes the differences in isotype antibody reactivity against a crude Paracoccidioides brasiliensis antigenic preparation in the sub-acute (SAF) and chronic (CF) forms of paracoccidioidomycosis before treatment . IgG antibodies were detected in all patients, with a slightly but not significantly higher reactivity in the SAF . IgG1 antibodies were present, frequently at high levels, in both forms, whereas IgG3 was always low or absent . IgG2 antibodies were detectable in most patients, but at high levels in only a few CF patients . IgG4 was found mainly in SAF patients, whereas IgA was detected almost only in CF patients, probably due to a Th2 pattern of immune response in the more severe SAF, and the characteristic mucosal involvement of the CF, respectively . Immunoblot analysis showed that, in addition to the 43-kDa immunodominant fraction, other less well-characterised fractions were also recognised differentially by the isotypes and deserve further investigation. Med Mycol, 2000 Dec, 38(6), 437 - 41 RFLP analysis reveals marked geographical isolation between strains of Paracoccidioides brasiliensis; Nino-Vega GA et al.; Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America . The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated . Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders . These results underline the belief that P . brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment. Mycoses, 2000, 43(11-12), 403 - 7 In vitro susceptibilities of Paracoccidioides brasiliensis yeast form to antifungal drugs; Hahn RC et al.; A total of 12 Paracoccidioides brasiliensis yeast form strains, including clinical and environmental isolates, were tested for their susceptibilities to amphotericin B, ketoconazole, fluconazole, and itraconazole . The tests were determined using a broth macrodilution procedure . In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates. Med Mycol, 2000, 38 Suppl 1, 189 - 97 Molecular typing of pathogenic fungi; McEwen JG et al.; In this Round Table, the application of several methods of molecular typing were discussed in reference to four important pathogenic fungi: Coccidioides immitis, Histoplasma capsulatum, Candida albicans and Paracoccidioides brasiliensis . Among the different methods the following were discussed: restriction fragment length polymorphisms (RFLP), single nucleotide polymorphisms, random amplified polymorphic DNA (RAPD), polymerase chain reaction (PCR)-RFLP and microsatellites . By means of these methods, several important biological questions related to speciation, mode of reproduction and population genetics could be approached . The basic information obtained from this approach has implications in the understanding of these pathogenic fungi in relation to their behavior and the development of pathogenic features, such as resistance to antimicrobials and virulence factors used for colonization of mammalian hosts . The knowledge obtained from these studies could also be used for the development of innovative diagnostic methods, as well as for novel therapeutic approaches and production of vaccines. Med Mycol, 2000, 38 Suppl 1, 139 - 45 Conventional versus molecular diagnostic tests; Elias Costa MR et al.; In this session, emphasis was placed on the diagnosis of various mycoses through the identification of antibodies and antigens in sera, as well as on new techniques to properly identify medically important fungi through molecular biological procedures . The use of restriction fragment length polymorphism (RFLP) on fungal mitochondrial DNA (mtDNA) has enabled the identification of different strains of Sporothrix schenkii, several dermatophytes, Candida spp . and black fungi according to their species-specific mtDNA-RFLP patterns . In some species, distinct specific types where found in relation to the geographic origin of the patients . These particular molecular diagnostic tests are useful in the identification of strains and in epidemiological studies . An account of the applications of serological methods in the diagnosis of paracoccidioidomycosis was presented . Serology has been used in the identification of paracoccidioidomycosis using a specific, sensitive and rapid antibody-based immunodiagnosis method . Using the gp43 antigen, the diagnostic coverage of inmunodifussion has been improved from the 93-95% achieved with crude antigens, to 100% in an enzyme-linked immunodiffusion assay capture test . Cross-reactions were eliminated by treatment of the antigen with sodium metaperiodate . Antibody detection is useful, especially in paracoccidioidomycosis and histoplasmosis. Med Mycol, 2000, 38 Suppl 1, 113 - 23 Pathogenesis II: fungal responses to host responses: interaction of host cells with fungi; Mendes-Giannini MJ et al.; Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures . The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules . Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival . Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity . Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules . The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization . Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic . It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation . For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence . Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell . These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface . The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper. Med Mycol, 2000, 38 Suppl 1, 1 - 8 From magic to science: a journey throughout Latin American medical mycology; San-Blas G; The start of Latin America's love story with fungi may be placed in pre-Hispanic times when the use of fungi in both ritual ceremonies and daily life were common to the native civilizations . But the medical mycology discipline in Latin America started at the end of the 19th Century . At that time, scholars such as A . Posadas, R . Seeber, A . Lutz and P . Almeida, discovered agents of fungal diseases, the study of which has influenced the regional research ever since . Heirs to them are the researchers that today thrive in regional Universities and Research Institutes . Two current initiatives improve cooperation among Latin American medical mycologists . First, the periodical organization of International Paracoccidioidomycosis Meetings (seven so far, from 1979 to 1999); second, the creation of the Latin American Association for Mycology in 1991 (three Congresses, from 1993 to 1999) . Latin American publications have increased in international specialized journals such as that from our Society (ISHAM) (from 8% in 1967 to 19% in 1999), and the Iberoamerican Journal of Mycology (Revista Iberoamericana de Micologia; > 40% from 1997 to 1999) . In addition, Latin American participation at ISHAM International Congresses has risen from 6.9% in 1975 to 21.3% in 1997, and 43.2% at the 14th ISHAM Congress, held for the first time in a Latin American country, Argentina . A significant contribution of women to the scientific establishment of Latin American medical mycology (e.g., 45% of Latin American papers vs . 18% of other regions published in Journal of Medical and Veterinary Mycology in 1987, had women as authors or coauthors) suggests a better academic consideration of Latin American women against their counterparts in the developed world . Taken together, all these figures reflect the enthusiasm of our Latin American colleagues in the field, despite the difficulties that afflict our region, and affect our work. Analyst, 2000 Nov, 125(11), 1993 - 9 Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol-gel matrix; Ferretti S et al.; Nitrite is an important human health and environmental analyte . As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM) . In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus . The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined . The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9 . Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection . The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity . The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM . In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1 . This structure enabled the determination of nitrite concentrations within ca . 5 min . The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C. J Biol Inorg Chem, 2001 Jan, 6(1), 23 - 6 An amicyanin C-terminal loop mutant where the active-site histidine donor cannot be protonated; Remenyi R et al.; A novel blue copper protein was constructed by replacing the C-terminal loop of amicyanin (Paracoccus versutus) by the homologous loop of rusticyanin . The C-terminal loop of both amicyanin and rusticyanin contains three (His, Cys, Met) of the four copper ligands . The amicyanin mutant exhibits all spectroscopic properties normally encountered for blue copper sites . The midpoint potential (369 mV) is the highest reported value for an amicyanin mutant . Cyclic voltammetry and NMR studies of the reduced form indicate that, in contrast to wild-type amicyanin and all amicyanin mutants described so far, the C-terminal histidine ligand does not protonate in the accessible pH range (pKa<4.5). Mycopathologia, 1999 Dec, 148(3), 123 - 30 Paracoccidioides brasiliensis isolated from armadillos is virulent to Syrian hamsters; Peracoli MT et al.; Isolates of Paracoccidioides brasiliensis may vary in virulence according to time of in vitro subcultivation . The present study compared the morphology and pathogenicity to hamsters of two P . brasiliensis isolates: one obtained from human lesions and maintained in the laboratory for several years (Pb-18) and the other isolate recovered from hamsters inoculated with organ homogenates from armadillos (Pb-T) . The microscopic morphology of Pb-18 and Pb-T showed yeast cells with similar diameter . However, Pb-T produced a significantly higher number of buds per mother cell than Pb-18 . Besides, the mycelial form of Pb-T developed abundant sporulation during 8 weeks of culture which was absent in the Pb-18 isolate . Virulence studies demonstrated that mortality rates, antibody levels, fungal load and extent of lesions in the organs were significantly higher in animals infected with Pb-T . The results demonstrated that Pb-T recently isolated from an animal was more virulent than Pb-18 . These differences between the two P . brasiliensis isolates may be indicators of virulence attenuation in this fungal species. Nippon Ishinkin Gakkai Zasshi, 2001, 42(1), 23 - 7 Detection of gp43 and ITS1-5.8S-ITS2 ribosomal RNA genes of Paracoccidioides brasiliensis in paraffin-embedded tissue; Sano A et al.; Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries . An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan . To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study . We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P . brasiliensis genes from paraffin-embedded tissue specimens . Successfully amplified were: a 473 base pairs fragment of gp43 gene of P . brasiliensis (located from 741st to 1,213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P . brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P . brasiliensis yeast cells . The authenticity of the PCR products was confirmed by nucleotide sequence analysis . These results indicate that the two nested PCR methods may be useful for diagnosis of PCM. Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 19 - 24 Structure elucidation of sphingolipids from the mycopathogen Sporothrix schenckii: identification of novel glycosylinositol phosphorylceramides with core manalpha1-->6Ins linkage; Toledo MS et al.; Acidic glycosphingolipid components were extracted from the mycelium form of the thermally dimorphic mycopathogen Sporothrix schenckii . Two fractions from the mycelium form (Ss-M1 and Ss-M2), having the highest Rf values on HPTLC analysis, were isolated and their structures elucidated by 1- and 2-D 13C- and 1H-nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry with lithium adduction of molecular ions . The structures of Ss-M1 and Ss-M2 were determined to be Manalpha1-->Ins1-P-1Cer and Manalpha1--> 3Manalpha1-->Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester) . The Manalpha1-->6Ins motif is found normally in diacylglycerol-based glycophosphatidylinositols of Mycobacteria, but this is the first unambiguous identification of the same linkage making up the core structure of fungal glycosylinositol phosphorylceramides (GIPCs) . These results are discussed in relation to the structures of GIPCs of other mycopathogens, including Histoplasma capsulatum and Paracoccidioides brasiliensis . J Clin Microbiol, 2001 Jan, 39(1), 309 - 14 Phylogenetic analysis of Lacazia loboi places this previously uncharacterized pathogen within the dimorphic Onygenales; Herr RA et al.; Lacazia loboi is the last of the classical fungal pathogens to remain a taxonomic enigma, primarily because it has resisted cultivation and only causes cutaneous and subcutaneous infections in humans and dolphins in the New World tropics . To place it in the evolutionary tree of life, as has been done for the other enigmatic human pathogens Pneumocystis carinii and Rhinosporidium seeberi, we amplified its 18S small-subunit ribosomal DNA (SSU rDNA) and 600 bp of its chitin synthase-2 gene . Our phylogenetic analysis indicated that L . loboi is the sister taxon of the human dimorphic fungal pathogen Paracoccidioides brasiliensis and that both species belong with the other dimorphic fungal pathogens in the order Onygenales . The low nucleotide variation among three P . brasiliensis 18S SSU rDNA sequences contrasts with the surprising amount of nucleotide differences between the two sequences of L . loboi used in this study, suggesting that the nucleic acid epidemiology of this hydrophilic pathogen will be rewarding. Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 674 - 7 A novel conformer of oxidized Paracoccus pantotrophus cytochrome cd(1) observed by freeze-quench NIR-MCD spectroscopy; Allen JW et al.; Paracoccus pantotrophus cytochrome cd(1) is a physiological nitrite reductase and an in vitro hydroxylamine reductase . The oxidised "as isolated" form of the enzyme has bis-histidinyl coordinated c-heme and upon reduction its coordination changes to histidine/methionine . Following treatment of reduced enzyme with hydroxylamine, a novel, oxidised, conformer of the enzyme is obtained . We have devised protocols for freeze-quench near-ir-MCD spectroscopy that have allowed us to establish unequivocally the c-heme coordination of this species as His/Met . Thus it is shown that the catalytically competent, hydroxylamine reoxidised, form of P . pantotrophus cytochrome cd(1) has different axial ligands to the c-heme than "as isolated" enzyme . J Bacteriol, 2001 Jan, 183(1), 257 - 63 Identification of ccdA in Paracoccus pantotrophus GB17: disruption of ccdA causes complete deficiency in c-type cytochromes; Bardischewsky F et al.; A transposon Tn5-mob insertional mutant of Paracoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20% . Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining . However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue . The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43 . With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15 . Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently . ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis . In strain TP43 Tn5-mob was inserted into ccdA . The deduced ORFA product showed no similarity to any protein in databases . However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis . For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines . This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins . CcdA of P . pantotrophus is 42% identical to a protein predicted by ORF2, and its location within the sox gene cluster coding for lithotrophic sulfur oxidation suggested a different function. Biochem J, 2000 Dec 15, 352 Pt 3, 859 - 64 Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus; Butler CS et al.; The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a {4Fe-4S} cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite . In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated . Results are presented that show NAP is sensitive to SCN(-) (thiocyanate) inhibition, with SCN(-) acting as a competitive inhibitor of nitrate (K(i) approximately 4.0 mM) . The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) approximately 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN(-) . Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three Mo-S ligands at 2.35 A (1 A=0.1 nm) and a Mo-O ligand at 2.14 A . The addition of SCN(-) to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 A) Mo(IV) species with four Mo-S ligands at 2.34 A . Taken together, the competitive nature of SCN(-) inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR g(av) value following redox cycling in the presence of SCN(-) and the increase in sulphur co-ordination of Mo(IV) upon SCN(-) binding, provide strong evidence for the direct binding of SCN(-) via a sulphur atom to Mo. Braz J Infect Dis, 1998 Jun, 2(3), 118 - 127 Management of Opportunistic Infections in HIV(+) Patients: Contrasts Between Europe and South America; Levi GC; The author discusses the management of some opportunistic diseases more commonly observed in South American AIDS patients than in European ones . Characteristics of coinfection with HIV and leprosy, paracoccidioidomycosis, Chagas' disease, mucocutaneous leishmaniasis, malaria, disseminated BCG and strongyloidiasis are reviewed, with special emphasis on preferred therapeutic schedules for these conditions. Med Mycol, 2000 Oct, 38(5), 371 - 7 Correlation among immune response, morphogenesis of the granulomatous reaction and spleen lymphoid structure in murine experimental paracoccidioidomycosis; Soares AM et al.; We studied the correlation among cellular immune response, the pattern of lung granulomatous lesions and alterations in spleen lymphoid structure in Swiss mice inoculated intravenously with Paracoccidioides brasiliensis strain 18 . The animals were evaluated at 24, 48 and 96 h after infection and further studied weekly for 18 weeks by: (i) the macrophage migration inhibition test with phytohemagglutinin (PHA) and P . brasiliensis antigen (PbAg); and (ii) histopathology of the lung and spleen lesions . One group of animals was gamma-irradiated (8 Gy), infected under the same conditions and evaluated for the pattern of lung granulomatous lesions and spleen lymphoid structure at 24, 48 and 96 h after infection . During the first week of infection, the non-irradiated animals presented a positive response to PHA and PbAg, compact granulomas in the lungs and a typical hyperplasia of the spleen white pulp . However, from weeks 2 to 5, a depression of the cell-mediated immunity (CMI) response to PHA and PbAg was observed in association with granulomas presenting only large mononuclear cells and lacking both giant cells and a peripheral halo of small mononuclear cells . This pattern of granuloma formation was similar to that seen in gamma-irradiated animals, whose cells involved in CMI were absent . After week 7, the non-irradiated animals showed granulomas characterized by the presence of giant cells and a peripheral halo of small mononuclear cells . This type of granuloma was formed concomitantly with recovery of the CMI and of the lymphoid structure of the spleen . The results showed a correlation among granulomas composed of large mononuclear cells, hypoplasia of the splenic tissue and impaired CMI . This correlation indicated that although granuloma morphogenesis per se does not depend on the activation of CMI, this response is important at later stages during modulation of the cellular composition of the granulomas. Plasmid, 2000 Nov, 44(3), 209 - 19 Sequence diversity of the plasmid replication gene repC in the Rhizobiaceae; Palmer KM et al.; The repABC operon is essential for stable maintenance of some Rhizobiaceae plasmids and of pTAV320 from Paracoccus versutus . These plasmids are the largest described family of homologous, yet compatible replicons . The repC gene is essential for plasmid replication, and previous work identified four distinct sequence groups (repC1, repC2, repC3, and repC4) that appear to define different compatibility classes . Probes for these different groups were used to characterize plasmids in Rhizobium leguminosarum population studies and three new repC sequence groups, repC5, repC6, and repC7 were identified . The general repC primers were modified to amplify a wider range of repC sequences and repC sequences were identified in Sinorhizobium and Mesorhizobium type strains . We also showed that the repC3 group-specific primers described previously do not amplify all repC3 sequences and developed a new repC3 amplification strategy . J Clin Microbiol, 2000 Nov, 38(11), 3960 - 6 Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis; Morais FV et al.; The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM) . It is protective against murine PCM and is a putative virulence factor . The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp . Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points . In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P . brasiliensis isolates after sequencing two PCR products from each fungal sample . The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp . Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein . The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms . The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots . The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine. Rev Inst Med Trop Sao Paulo, 2000 Sep-Oct, 42(5), 239 - 43 Inoculation of BALB/c mice with Lacazia loboi; Madeira S et al.; In a previous study, the authors inoculated Swiss mice with Lacazia loboi (L . loboi) and succeeded in maintaining a granulomatous infiltrate and viable fungal cells up to one year and six months after inoculation . Considering the experimental work on paracoccidioidomycosis, 0.03 ml of a fungal suspension obtained from a biopsy of a Jorge Lobo's Disease patient were inoculated into both hind foot pads of 32 six week-old BALB/c mice of both sexes . The animals were sacrificed 1, 4, 7 and 10 months post inoculation . The suspension contained 1.3 x 10(6) fungi/ml and presented 38% viability . Seven months after inoculation, most of the animals presented profuse infiltrates consisting of isolated histiocytes, foreign body and Langhans' giant cells and a large number of fungi, most of them viable . Emergence of macroscopic lesions was observed during the 8th month . Based on fungal count, viability index before and after inoculation, presence of macroscopic lesions and histopathological findings similar to the findings in humans, the authors believe that BALB/c mice may be a good experimental model to study Jorge Lobo's Disease, mainly regarding therapeutic evaluation. Clin Infect Dis, 2000 Oct, 31(4), 1032 - 9 Epub 2000 Oct 20. Paracoccidioidomycosis: a model for evaluation of the effects of human immunodeficiency virus infection on the natural history of endemic tropical diseases; Benard G et al.; The interaction of human immunodeficiency virus (HIV) infection with endemic tropical diseases has become a major concern, but its mechanisms are still poorly understood . Paracoccidioidomycosis (PCM), a South America endemic deep mycosis, may provide an interesting model to investigate this interaction, as clinical-epidemiological features of most HIV-PCM-coinfected patients are difficult to classify into the standard acute and chronic forms of PCM . Such patients have presented clinical features indicative of an uncontrolled infection with lymphohematogenous dissemination, similar to the more severe, acute form . However, this infection probably resulted from reactivated latent foci that, in nonimmunocompromised hosts, leads to the less severe chronic form, characterized by mucosal lesions . We propose that a new outcome of the Paracoccidioides brasiliensis-host interaction is induced by concomitant HIV infection . This outcome probably reflects an impaired anti-P . brasiliensis immune response during coinfection that is similar to that seen in the acute form, although the patients have a chronic P . brasiliensis infection. Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1797 - 802 Proposal for the reclassification of Thiobacillus novellus as Starkeya novella gen . nov., comb . nov., in the alpha-subclass of the Proteobacteria; Kelly DP et al.; Thiobacillus novellus is a facultatively chemolithoautotrophic and methylotrophic, Gram-negative, rod-shaped sulfur bacterium, shown by 16S rRNA gene sequence analysis to be a member of the alpha-2 subclass of the Proteobacteria . As such, it must be excluded from the genus Thiobacillus, whose species are members of the beta-Proteobacteria . It closest phylogenetic neighbour appears to be Ancylobacter, from which it is distinct morphologically and in some physiological characteristics . It is distinct physiologically and biochemically in a number of diagnostic features from Paracoccus versutus, in the alpha-3 subclass of the Proteobacteria and does not appear to be sufficiently closely related to any other genus of the alpha-Proteobacteria to be reassigned to a known genus . The new genus and species name Starkeya novella is proposed for T . novellus . The type strain is ATCC 8093T (= NCIMB 10456T = NCIMB 9113T = DSM 506T = IAM 12100T = IFO 12443T = CCM 1077T). J Biol Chem, 2001 Feb 23, 276(8), 5846 - 55 Epub 2000 Oct 16. Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase; Steensma E et al.; The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy . In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase . In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17 . An equilibrium mixture of heme ligands is present in isolated oxidized c domain . Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains . This form is in equilibrium with a high-spin form in which Met106 has left the heme iron . Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form . The thermally denatured forms are high-spin in both oxidation states . The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme . This process is not solely a function of the properties of the c domain . The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed. J Eur Acad Dermatol Venereol, 2000 May, 14(3), 166 - 71 The role of HLA antigens in the development of paracoccidioidomycosis; Dias MF et al.; BACKGROUND: Paracoccidioidomycosis is a systemic granulomatous disease that involves primarily the lungs and may disseminate to other organs and systems . It is caused by Paracoccidioides brasiliensis, a fungus that exhibits reversible thermal dimorphism and whose natural habitat is presently unknown . There are two main clinical forms: the acute (subacute) juvenile form and the chronic adult form . The former runs a more rapid course and is more severe than the latter . This mycosis is found throughout Latin America . Brazil accounts for 80% of reported cases . Presumably P . brasiliensis thrives in humid and hot places, especially near forests or farms . The infection is endemic in certain areas, especially in Brazil, Colombia and Venezuela, where nearly 100% of the population show cutaneous paracoccidioidina positive skin tests, indicating previous contact with the fungus, although a small percentage show clinical manifestations of the disease . METHODS: We compared the expression of HLA class I antigens in a healthy group (control) and in a group of patients with paracoccidioidomycosis (chronic adult form) using the Terasaki lymphocytotoxicity test modified by Amos for HLA antigen analysis . AIMS: To discover indications of whether or not individual susceptibility to P . brasiliensis might depend on some specific immunological defect . RESULTS: There is no evidence of association between a specific HLA antigen and paracoccidioidomycosis in the subjects studied . Further investigations are recommended. Nat Struct Biol, 2000 Oct, 7(10), 885 - 8 A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis; Allen JW et al.; Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates . Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle . Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis . A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with 'switched' axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine . This activated conformer returns over 20 min at 25 degrees C to the state previously observed for oxidized 'as isolated' cd1, which is catalytically inactive towards the same substrates. Rev Argent Microbiol, 2000 Jul-Sep, 32(3), 116 - 22 {In vitro infection by different strains of Paracoccidioides brasiliensis}; Canteros CE et al.; We analyzed the in vitro infection process by P . brasiliensis and the effect of extracellular factor(s) produced on monolayers of mammalian Vero cell lines . The yeast phase of four strains was studied: B339 (avirulent or slightly virulent), U, (intermediate virulence), 93745 and 63265 (both highly virulent) . Strains of intermediate and high virulence had higher adherence at first contact (about 16%) . Strain B339 had a slower adherence at first contact (8%) than the others during the same period . The production of extracellular proteases, soluble extracellular factor(s) and extracellular antigen gP43 showed no correlation with the in vitro physiopathogenicity of the analyzed strains . We demonstrate that the Vero model presented in this paper is a suitable system to study infection and virulence in vitro . We are currently assessing its usefulness as a tool for the analysis of the interaction between pathogen, host and antifungal agents. Oral Dis, 2000 Sep, 6(5), 327 - 9 Paracoccidioidomycosis in an HIV-positive patient: a case report with gingival aspects; Giovani EM et al.; Paracoccidioidomycosis is an important deep mycosis, endemic in some areas of the South American countryside, with great incidence in males bearing rural activities, being unusual in urban developed centres . Cell-mediated immunity is the main host defence against the P . brasiliensis, and HIV-positive patients have been increasingly affected by the disease, although only a few reports are available in the literature . We present a case of paracoccidiodomicosis in an HIV-positive female, with unusual clinical history and histopathological aspects. Biochemistry, 2000 Sep 12, 39(36), 10967 - 74 Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study; Sjogren T et al.; We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha) . Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed . This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0 . Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm . The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5) . After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release . The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A . The structure shows that CO binds to the iron of the d(1) heme in the active site . The ligation of the c heme is unchanged in the complex . A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme . Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site . Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1). Scand J Gastroenterol, 2000 Aug, 35(8), 824 - 31 Presence of bacterial 16S ribosomal RNA gene segments in human intestinal lymph follicles; Chiba M et al.; BACKGROUND: There is currently no information regarding microbial agents inside the intestinal lymph follicles . METHODS: Biopsy or resected specimens, mostly from macroscopically normal areas, were sectioned with a cryostat . DNA was extracted from microdissected samples, exclusively from the lymph follicle . Amplification of DNA was performed using universal primers designed from conserved regions of bacterial 16S ribosomal RNA (rRNA) . Several clones with inserts of around 400 base pairs were subjected to DNA sequence analysis followed by a database homology search . RESULTS: Bacterial 16S rRNA gene segments were detected in the lymph follicle in 2 of 14 (14%) non-inflammatory bowel disease (IBD) cases, 4 of 14 (28%) Crohn disease cases, and in 2 of 5 (40%) ulcerative colitis cases . Nineteen 16S rRNA gene segments were recognized in the eight positive cases . Five segments showed 100% identity to known bacterial 16S rRNAs, namely staphylococcus species, Streptococcus sanguis, and Paracoccus marcusii . However, the other 14 segments showed below 100% identity, indicating either the presence of unknown bacteria or of bacteria without known DNA data . No single identified or unidentified bacterium, characteristic of IBD, including Mycobacterium paratuberculosis and Listeria monocytogenes, was detected . CONCLUSIONS: The present study confirms the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles and paves the way for new investigations into the microbiology of the lymph follicle . Whether or not bacteria inside the lymph follicle is a primary stimulus in IBD has yet to be clarified. Med Mycol, 2000 Aug, 38(4), 317 - 22 Morphological aspects of Paracoccidioides brasiliensis in lymph nodes: implications for the prolonged latency of paracoccidioidomycosis? Restrepo A. In order to determine if fungal morphology in tissues would furnish indications on the viability of Paracoccidioides brasiliensis yeast cells, lymph node biopsies from five patients with paracoccidioidomycosis, including one with residual circumscribed lesions, were examined . A program that allows transferring of microscopic images to the computer for further processing was used . In the four active cases, the infected lymph nodes had over 49% of healthy-looking yeast cells while in the case of the residual lesion, this figure was smaller (21%) . The residual had a larger proportion of aberrant yeast cells, with predominance of shell-like, empty cells (33%) and crescent bodies (30%); balloon-like yeasts were also seen (16%) . The last two types of cells were also seen in the active lesions, but in smaller proportions, < or = 8% and < or = 9%, respectively . The number of multiple budding yeast cells, which clearly demonstrate fungal viability, ranged 24-33% in active cases but was only 5% in the residual lesion . Although the number of biopsies examined is small, the results tend to indicate that the morphology of P . brasiliensis yeast cells in walled-off tissues is abnormal and that the number of viable elements is small . There might be a connection between these findings and the long latency period illustrated by those patients with paracoccidioidomycosis that have been diagnosed in non-endemic areas . Additionally, if P . brasiliensis yeast cells were to be subjected to the microaerophilic environment present in walled-off lesions, they would probably require a long time to multiply . Under these circumstances, the mycosis would also need many years to manifest. Med Mycol, 2000 Aug, 38(4), 309 - 15 Experimental paracoccidioidomycosis in high and low antibody responder mice of Selection IV-A; Soares AM et al.; High (H) and low (L) responder mice were selected for their ability to produce antibodies against sheep and human erythrocytes (Selection IV-A) . In this selection, the difference in antibody responsiveness between H and L lines (HIV-A and LIV-A mice, respectively) was shown to depend mainly on macrophage function . The more rapid catabolism of antigens by macrophages in L mice has been suggested as the main cause of the low antibody production . Due to this high macrophage activity, L animals have been described as more resistant than H animals to intracellular pathogens . These animals were utilized as an experimental model of paracoccidioidomycosis . HIV-A and LIV-A mice were infected with Paracoccidioides brasiliensis by the intravenous route . As expected, H mice were more susceptible to P . brasiliensis with a shorter survival time and higher levels of specific antibodies when compared to L mice . Contrasting with the survival time, the lungs, spleen and liver from H mice showed typical nodular granulomas containing epithelioid and giant cells and few fungi . On the other hand, in LIV-A mice, the lesions of these organs were characterized by looser granulomas with irregular borders and the presence of a large number of fungi . However, the adrenal gland showed different lesion patterns . In H mice these lesions were extensive and characterized by loose granulomas with numerous fungi, while in LIV-A mice the lesions were small and limited to the cortex . Moreover the HIV-A mice presented higher levels of serum corticosterone when compared to LIV-A ones . The higher susceptibility of H mice could be attributed to the extensive lesions of the adrenal glands . These results suggest the use of the H line from the IV-A Selection as an experimental model for further studies of adrenal involvement in paracoccidioidomycosis. Arq Neuropsiquiatr, 2000 Sep, 58(3A), 741 - 7 {Paracoccidioidomycosis of the central nervous system: case report}; da Silva CE et al.; The involvement of the central nervous system in paracoccidioidomycosis is more frequent than previously thought . The first reference to the possibility that Paracoccidioides brasiliensis could affect the central nervous system was by Pereira & Jacobs in 1919 . Since then, a great number of other studies has showed this form of clinical behavior and, in some of them, the frequency has ranged 27.27% . We report a clinical case of a 34-year-old white Brazilian woman admitted because of bacterial pneumonia . In the sixth day of admission, the patient developed cerebellar symptomatology with nausea, vomiting, dysmetria and gait disturbance . Central nervous system computer tomographic scanning disclosed a hypodense lesion in the right cerebellar hemisphere . The patient was submitted to surgery with total excision of the lesion . Histopathological examination confirmed the diagnosis of neuroparacoccidioidomycosis . Coadjuvant treatment with sulfamethoxazole-trimetoprim was introduced . The patient had a good outcome and was discharge 30 days after surgery. J Eur Acad Dermatol Venereol, 2000 Mar, 14(2), 113 - 7 Cutaneous primary paracoccidioidomycosis; Garcia Bustinduy M et al.; Paracoccidioidomycosis is a common fungal infection in Latin America . Few cases have been described in non-endemic countries and their diagnosis without the correct suspicion, bearing in mind that the latency period might be up to 60 years, may not be easy . We report the case of a 59-year-old man who worked as a taxi driver for 25 years in Venezuela . Cutaneous paracoccidioidomycosis was diagnosed one year after he returned to Spain . No internal signs or symptoms of the disease were found and cell-mediated immunity showed no depression . Our case suggested us to consider direct cutaneous infection, although respiratory tract is believed to be the most frequent portal of entry for this infection. J Clin Microbiol, 2000 Sep, 38(9), 3478 - 80 PCR for diagnosis of paracoccidioidomycosis; Gomes GM et al.; A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa . In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis. J Clin Microbiol, 2000 Sep, 38(9), 3190 - 3 Small subunit ribosomal DNA sequence shows Paracoccidioides brasiliensis closely related to Blastomyces dermatitidis; Bialek R et al.; The similarities of paracoccidioidomycosis and blastomycosis are highly suggestive of a close relation of the two etiological agents . Whereas the agent of the first disease is exclusively endemic in Latin America, the agent of the latter one is endemic in North America and Africa . In symptomatic travelers visiting both areas of endemicity, differentiation of the diseases might be impossible, even though therapy and prognosis for these two diseases differ significantly . In order to identify differences in the 18S rRNA gene (rDNA) for use as molecular diagnostic tools, we sequenced this gene from five isolates of Paracoccidioides brasiliensis and compared them to known sequences of other fungi . Neighbor-joining, maximum parsimony, and maximum likelihood analyses and, finally, the Kishino-Hasegawa test revealed that P . brasiliensis, Blastomyces dermatitidis, and Emmonsia parva are more closely related than Histoplasma capsulatum and B . dermatitidis, whose teleomorphic forms belong to one genus, Ajellomyces . In accordance with the work of other investigators who have used internal transcribed spacer and large subunit rDNA sequences, our small subunit rDNA data show that the dimorphic fungus P . brasiliensis must be grouped within the order Onygenales and is closely related to members of the family Onygenaceae . There are hints in the molecular phylogenetic analysis that the family Onygenaceae might be further divided into two families . The subgroup that includes P . brasiliensis comprises all zoopathogenic species . The differences in the 18S rDNAs appear to be too small to allow species identification of the members of the family Onygenaceae pathogenic for humans by use of target sequences within this gene. Rev Inst Med Trop Sao Paulo, 2000 Jul-Aug, 42(4), 231 - 4 Central nervous system paracoccidioidomycosis . Report of a case successfully treated with itraconazol; Villa LA et al.; Paracoccidioidomycosis (PCM) is a primary pulmonary infection that often disseminates to other organs and systems . Involvement of the central nervous system (CNS) is rare and due to the fact that both clinical alertness and establishment of the diagnosis are delayed, the disease progresses causing serious problems . We report here a case of neuroparacoccidioidomycosis (NPCM), observed in a 55 year-old male, who consulted due to neurological symptoms (left hemiparesis, paresthesias, right palpebral ptosis, headache, vomiting and tonic clonic seizures) of a month duration . Upon physical examination, an ulcerated granulomatous lesion was observed in the abdomen . To confirm the diagnosis a stereotactic biopsy was taken; additionally, mycological tests from the ulcerated lesion and a bronchoalveolar lavage were performed . In the latter specimens, P . brasiliensis yeast cells were visualized and later on, the brain biopsy revealed the presence of the fungus . Treatment with itraconazole (ITZ) was initiated but clinical improvement was unremarkable; due to the fact that the patient was taking sodium valproate for seizure control, drug interactions were suspected and confirmed by absence of ITZ plasma levels . The latter medication was changed to clonazepam and after several weeks, clinical improvement began to be noticed and was accompanied by diminishing P . brasiliensis antigen and antibody titers . In the PCM endemic areas, CNS involvement should be considered more often and the efficacy of itraconazole therapy should also be taken into consideration. Rev Soc Bras Med Trop, 2000 May-Jun, 33(3), 309 - 12 Intestinal paracoccidioidomycosis simulating colon cancer; Chojniak R et al.; We report a case of intestinal involvement of Paracoccidioidomycosis, in a patient considered to have colonic cancer . The diagnosis of this mycosis should be considered when an abdominal mass associated with intra-lesional calcifications on X-ray is observed . CT scans increase the findings. Microbes Infect, 2000 Jul, 2(8), 877 - 84 Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells; Hanna SA et al.; Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans . The 43-kDa glycoprotein of P . brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin.Very little is known about early interactions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P.brasiliensis related to the adhesion and invasion process . We found that there is a permanent interaction after 30 min of contact between the fungus and the cells . The yeasts multiply in the cells for between 5 and 24 h . Different strains of P . brasiliensis were compared, and strain 18 (high virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M (mutant obtained by ultraviolet radiation, deficient in gp43) . P . brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process . An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P . brasiliensis to the Vero cells . Glycoprotein 43 (gp43) antiserum abolished 85% of the binding activity of P . brasiliensis . This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis. J Bacteriol, 2000 Sep, 182(17), 4677 - 87 Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17; Friedrich CG et al.; The gene region coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17 is located on a 13-kb insert of plasmid pEG12 . Upstream of the previously described six open reading frames (ORFs) soxABCDEF with a partial sequence of soxA and soxF (C . Wodara, F . Bardischewsky, and C . G . Friedrich, J . Bacteriol . 179:5014-5023, 1997), 4,350 bp were sequenced . The sequence completed soxA, and uncovered six new ORFs upstream of soxA, designated ORF1, ORF2, and ORF3, and soxXYZ . ORF1 could encode a 275-amino-acid polypeptide of 29,332 Da with a 61 to 63% similarity to LysR transcriptional regulators . ORF2 could encode a 245-amino-acid polypeptide of 26,022 Da with the potential to form six transmembrane helices and with a 48 to 51% similarity to proteins involved in redox transport in cytochrome c biogenesis . ORF3 could encode a periplasmic polypeptide of 186 amino acids of 20,638 Da with a similarity to thioredoxin-like proteins and with a putative signal peptide of 21 amino acids . Purified SoxXA, SoxYZ, and SoxB are essential for thiosulfate or sulfite-dependent cytochrome c reduction in vitro . N-terminal and internal amino acid sequences identified SoxX, SoxY, SoxZ, and SoxA to be coded by the respective genes . The molecular masses of the mature proteins determined by electrospray ionization spectroscopy (SoxX, 14,834 Da; SoxY, 11,094 Da; SoxZ, 11,717 Da; and SoxA, 30,452 Da) were identical or close to those deduced from the nucleotide sequence with differences for the covalent heme moieties . SoxXA represents a novel type of periplasmic c-type cytochromes, with SoxX as a monoheme and SoxA as a hybrid diheme cytochrome c . SoxYZ is an as-yet-unprecedented soluble protein . SoxY has a putative signal peptide with a twin arginine motif and possibly cotransports SoxZ to the periplasm . SoxYZ neither contains a metal nor a complex redox center, as proposed for proteins likely to be transported via the Tat system. Rev Latinoam Microbiol, 1999 Jul-Sep, 41(3), 139 - 43 Random amplified polymorphic DNA (RAPD) analysis of Paracoccidioides brasiliensis isolates; Totti DO et al.; Five Paracoccidioides brasiliensis isolates of humans origin were analyzed using three arbitrary primers (3301, 3304 and 3307 of 10, 9 and 10 oligonucleotides respectively) in random amplified polymorphic DNA (RAPD) analysis . The analysis of the complex RAPD profiles obtained were carried out using the Dice similarity coefficient that distinguished the isolated Pb 02 from the others (Pb 18, Pb 192, Pb 265 and Pb SN) . The results revealed limited intraspecific genomic variations in these P . brasiliensis isolates and indicate that RAPD can be useful for analysis of P . brasiliensis genome for characterization or differentiation within this genus. Biochemistry, 2000 Aug 8, 39(31), 9551 - 60 The Met99Gln mutant of amicyanin from Paracoccus versutus; Diederix RE et al.; The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus . Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement . M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin . In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin . For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M . These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin . A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand. J Biol Chem, 2000 Oct 27, 275(43), 33231 - 7 Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite; George SJ et al.; Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide . The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme . We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant . The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1) . This d(1) heme-NO complex is still readily observed after 15 min . EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed . Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+) . No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme . Implications for the mechanism of P . pantotrophus cytochrome cd(1) are discussed. J Clin Microbiol, 2000 Aug, 38(8), 3106 - 9 Molecular identification of Paracoccidioides brasiliensis by PCR amplification of ribosomal DNA; Motoyama AB et al.; We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01 . Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P . brasiliensis and other human pathogenic fungi by PCR . The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P . brasiliensis in Latin America. J Clin Microbiol, 2000 Aug, 38(8), 2940 - 2 Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay; Bialek R et al.; A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target . This gene encodes an outer membrane protein unique to this dimorphic fungus . DNA from six clinical isolates and the ATCC strain 60885 of P . brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions . PCR was done on DNA extracts of lung homogenates from 23 experimentally P . brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice . The results were compared to quantitative cultures . A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA . In all seven P . brasiliensis isolates, the expected 196-bp nested PCR product was found . Their sequences were 100% identical to the gp43 gene sequence in GenBank . DNA extracts of all other, related fungi were negative . The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 x 10(3) to 1.3 x 10(7) CFU of P . brasiliensis per g of lung . Uninfected BALB/c mice and H . capsulatum-infected mice samples gave negative results . The high sensitivity and specificity of this new nested PCR assay for the detection of P . brasiliensis in tissue samples were demonstrated . The assay may be useful for diagnosis in human tissue samples. J Clin Microbiol, 2000 Aug, 38(8), 2909 - 13 Comparison of an established antibody sandwich method with an inhibition method of Histoplasma capsulatum antigen detection; Garringer TO et al.; The Histoplasma antigen immunoassay utilizes an antibody sandwich method that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis . Inhibition assays have been developed for antigen detection and offer at least one potential advantage, namely, reduced antibody requirements . We have developed an inhibition assay using the polyclonal antibody employed in our standard sandwich assay . Urine and serum specimens from patients with culture-proven histoplasmosis and controls were tested using both methods . The two methods had similar sensitivities for detection of antigen in urine (antibody sandwich = 92.5% versus inhibition = 87.5%, P = 0.500) and serum (82.5% versus 80.0%, P = 1 . 000) . With serum, the specificities of both methods were similar (antibody sandwich assay = 95.0% versus inhibition assay = 92.5%, P = 1.000), and with urine, the specificity of the antibody sandwich method was superior (97.5% versus 80.0%, P = 0.039) . While the overall reproducibility of both methods was excellent (with urine, antibody sandwich assay intraclass correlation coefficient = 0.9975 and with serum = 0.9949; correlation coefficient of the inhibition assay with urine = 0.9736 and with serum = 0.9850), that of the inhibition method was only fair to poor for the controls: urine = -0 . 0152, serum = 0.5595 . Reproducibility was good for the controls using the sandwich method: urine = 0.7717, serum = 0.9470 . Cross-reactivity was observed in specimens from patients infected with Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Penicillium marneffei . In conclusion, the decreased specificity and inferior reproducibility with control specimens suggest that the inhibition assay has poorer precision toward the lower end of the detection range. Br J Dermatol, 2000 Jul, 143(1), 188 - 91 Paracoccidioidomycosis (South American blastomycosis) successfully treated with terbinafine: first case report; Ollague JM et al.; We describe a 63-year-old man who presented with painful malodorous lesions in the perianal, perineal and scrotal regions . Following definitive diagnosis of paracoccidioidomycosis, he was treated initially with trimethoprim/sulphamethoxazole, but there was no clinical improvement . He then received terbinafine (Lamisil) 250 mg twice daily for 6 months . There was rapid resolution of all lesions and complete relief of symptoms, without any associated side-effects . The patient remains clinically well and without any evidence of infection 2 years after discontinuation of terbinafine treatment. Med Mycol, 2000 Jun, 38(3), 225 - 9 Production of IFN-gamma is impaired in patients with paracoccidioidomycosis during active disease and is restored after clinical remission; Karhawi AS et al.; Cellular immunity is usually suppressed during paracoccidioidomycosis (PCM) and is restored after treatment . In this study we evaluated the induction of a type 1 (interferon gamma (IFN-gamma)), a type 2 (interleukin (IL)-10) and a primarily macrophage derived cytokine (tumor necrosis factor (TNF)-alpha) in peripheral blood mononuclear cells (PBMC) from patients with PCM . Eight male patients with active PCM, nine male patients with clinical remission of the disease and 10 healthy control subjects were enrolled in the study . Cytokines were induced with non-specific stimuli --phytohaemagglutin (PHA) (induces IL-10 and IFN-gamma), Lipopolysaccharide (induces TNF-alpha)--and Paracoccidioides brasiliensis antigen (PbAg) (induces IL-10, IFN-gamma and TNF-alpha) . Induction of IFN-gamma with PHA differed among the three groups (P < 0.01; Kruskal-Wallis test) and with PbAg was lower in patients with active disease compared to those in clinical remission (P = 0.05; Mann-Whitney) . Induction of IL-10 and of TNF-alpha was similar in the three groups . The suppressed production of IFN-gamma in patients with active disease may underscore the cellular immune deficiency seen in these patients. Med Mycol, 2000 Jun, 38(3), 193 - 9 Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus novemcinctus) in an area where the fungus was recently isolated from soil; Silva-Vergara ML et al.; Natural infection of armadillos (Dasypus novemcinctus) with Paracoccidioides brasiliensis in Northern Brazil was reported in 1986, raising great interest in the understanding of the role of this mammal in the epidemiological cycle of the fungus . Recently, P . brasiliensis was isolated from the soil of Ibia, State of Minas Gerais, southeastern Brazil . Armadillos captured in this area were evaluated for the presence of P . brasiliensis in the viscera and infection was detected in 4/16 animals (25%) . Fungal yeast phase cells were observed in three of the four infected armadillos by direct microscopic examination and by the indirect immunofluorescence test carried out on homogenized tissues . P . brasiliensis was isolated from three armadillos whose homogenized viscera had been injected into Swiss mice . The new strains (Ibia-T1, Ibia-T2 and Ibia-T3) were identified as P . brasiliensis on the basis of macro- and micromorphology, thermodimorphism, production and serologic activity of exoantigens, and by polymerase chain reaction (PCR)-detection of the gp43 gene . The lethality and lesions caused to the mice from which the strains were recovered confirmed the virulence of the isolates . We conclude that P . brasiliensis infects armadillos in locations with different geoclimatic characteristics and vegetation cover . The direct observation of yeast cells in tissues and the multiple visceral involvement, including the lungs, suggests the occurrence of paracoccidioidomycosis disease in these mammals and supports their role as wild hosts in the epidemiological cycle of the fungus. Med Mycol, 2000 Jun, 38(3), 185 - 91 A critical analysis of isolation of Paracoccidioides brasiliensis from soil; Franco M et al.; The present review is a critical analysis of positive and negative reports of the isolation of Paracoccidioides brasiliensis from soil . The strains isolated from soil or soil-contaminated material (dogfood, penguin feces) by Batista et al . in Recife, Brazil, Negroni in the Argentinian Chaco, Albornoz in rural Venezuela, Silva-Vergara et al . in Ibia, Brazil, Ferreira et al . in Uberlandia, Brazil, and Gezuele et al . at the Uruguayan base in the Antarctic region, presented mycological characteristics consistent with P . brasiliensis . In most of these studies, morphological characterization was complemented with an evaluation of virulence and antigenicity, and biochemical or molecular analysis . These isolations, therefore, can be considered true, supporting the concept of soil as an important element in the ecology of the pathogen . The large number of negative reports in attempts involving soil samples and the low repeatability of isolation of the fungus from the same area indicate that the specific conditions supporting growth of the pathogen in soil have not been fully clarified. Rev Inst Med Trop Sao Paulo, 2000 May-Jun, 42(3), 167 - 70 Gallium-67 imaging in a patient with paracoccidioidomycosis: a case report; Teixeira AB et al.; A 26 year-old female was admitted with abdominal pain, fever and weight loss . The clinical and laboratory investigations led to the diagnosis of paracoccidioidomycosis . Gallium-67 whole body images correlated well with the clinical course of the disease and with the patient's prognosis. Rev Soc Bras Med Trop, 1999 Sep-Oct, 32(5), 571 - 5 {Paracoccidioidomycosis in Brazilian Indians of the Suruí tribe: clinical-laboratory study of 2 cases}; Forjaz MH et al.; Paracoccidioidomycosis has been considered the most frequent endemic systemic mycosis in Latin America . Although most cases of paracoccidioidomycosis involve rural workers, this systemic fungal disease has been scarcely reported among Amerindian populations from Brazil . We report two cases of paracoccidioidomycosis in Tupi-Monde Amerindians from Cacoal, state of Rondonia, Brazil . Both cases exhibited positive serological results by a specific immunodiffusion test only when the assay was performed with antigens obtained from the mycelial form of P . brasiliensis . The authors present a literature review of paracoccidioidomycosis in Brazilian Amerindians and discuss the need for further investigations about the impact of the antigenic diversity of P . brasiliensis from different geographic areas on the serological diagnosis of PCM. Mycoses, 1999, 42 Suppl 2, 11 - 8 Yeast tissue phase of Emmonsia pasteuriana inoculated in golden hamster by intratesticular way; Drouhet E et al.; The scope of our study was to present an experimental model reproducing the dimorphic yeast-like population (as for Histoplasma capsulatum, Blastomyces dermatitidis) similar to that observed in the cutaneous biopsy of an Italian woman who had never traveled abroad, being intravenous drug user and HIV positive for 10 years, finally infected with the new dimorphic fungus Emmonsia pasteuriana . Experimental inoculation was unsuccessful by intraperitoneal (i.p.) and intravenous (i.v.) ways in a mouse and in a guinea-pig model inoculated by cutaneous or subcutaneous routes, reason for that we chose the golden hamster, highly sensitive to dimorphic fungi as agents of systemic mycoses as histoplasmosis, blastomycosis, sporotrichosis, penicilliosis marneffei, paracoccidioidomycosis when the inoculation was done by intraperitoneal route . We inoculated young golden hamsters by i.p . and intratesticular ways . Only by this last route we reproduced an orchiepididymitis with necrosis, haemorrhages and a polymorphic yeast-like population similar to the polymorphism observed in the cutaneous biopsy of the patient . The intratesticular affinity of E . pasteuriana provided an interesting model for this infection. Antimicrob Agents Chemother, 2000 Jul, 44(7), 1997 - 2000 Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis; Hahn RC et al.; Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively . The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC. Protein Sci, 2000 May, 9(5), 846 - 58 The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus; Thompson GS et al.; The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods . The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35+/-0.06 A, using 1,498 distance and 55 torsion angle constraints . The protein has a double-wound Greek-key fold with two alpha-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography . Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements . Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale. J Biol Chem, 2000 Aug 18, 275(33), 25089 - 94 X-ray crystallographic study of cyanide binding provides insights into the structure-function relationship for cytochrome cd1 nitrite reductase from Paracoccus pantotrophus; Jafferji A et al.; We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme . Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer . The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B . The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme . Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme . No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme. Vaccine, 2000 Jul 1, 18(26), 3050 - 8 DNA-based vaccination against murine paracoccidioidomycosis using the gp43 gene from paracoccidioides brasiliensis; Pinto AR et al.; Gp43, the major 43-kDa antigenic glycoprotein of Paracoccidioides brasiliensis, or its 15-amino acid inner peptide (P10), induces a T-CD4(+), Th1 cellular immune response which protects BALB/c mice from intratracheal infection by virulent yeast forms . We investigated whether DNA vaccination using the gp43 gene could elicit protective immunity against P . brasiliensis . Animals immunised intramuscularly (i.m.) or intradermally (i.d.) with plasmid DNA containing the gp43 gene induced a specific, long lasting humoral and cellular immune response . A mixed Th1/Th2 cellular immune response in DNA-immunized mice was modulated in vivo by IFN-gamma and was protective in BALB/c mice . A significant decrease in the lung colony forming units (CFUs) and reduced, or no dissemination to the spleen and liver of immunised mice were observed. Mycopathologia, 1999, 146(3), 131 - 4 Comparative efficacy of fluconazole and amphotericin B in the parenteral treatment of experimental paracoccidioidomycosis in the rat; Martinez R et al.; Patients with severe and complicated paracoccidioidomycosis are treated with amphotericin B by the intravenous route . Fluconazole is active in vitro against Paracoccidioides brasiliensis and can also be administered intravenously, but few clinical or experimental data are available about its action against the infection caused by this fungus . In the present study, the efficacy of fluconazole and amphotericin B was assessed comparatively in rats inoculated parenterally with P . brasiliensis . The treatment was performed 3 times a week for 4 weeks starting one week after infection . Fluconazole administered intraperitoneally (14 mg/kg body weight/dose) was more effective (P < 0.001) than amphotericin B (2 mg/kg body weight/dose) in reducing the number of colony forming units in the lungs and spleen . When administered intravenously at the dose of 3 mg/kg body weight, fluconazole was as effective as amphotericin B (0.8 mg/kg body weight) in reducing the pulmonary fungal burden . Under these conditions, the rats treated with fluconazole had a smaller number of colony forming units than untreated animals (P < 0.001), but amphotericin B was more effective than fluconazole in reducing spleen infection (P < 0.005) . Except for this result obtained with a low dose, fluconazole showed an antifungal action equal to or higher than that of amphotericin B . The activity of fluconazole at doses equivalent to those used for human treatment suggests that this antifungal agent may be an alternative to amphotericin B for the early intravenous treatment of patients with paracoccidioidomycosis. Med Mycol, 2000 Apr, 38(2), 177 - 82 Effect of cytokines on antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis; Kurita N et al.; In our previous study, it was observed that human peripheral blood polymorphonuclear leucocytes (PMNs) exhibited a fungistatic effect on yeast cells of Paracoccidioides brasiliensis, and that interferon-gamma (IFN-gamma), but not tumor necrosis factor-alpha or interleukin-8 (IL-8), enhanced the antifungal activity of PMNs . In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) also enhanced the PMN activity . GM-CSF-activated PMNs exhibited a killing effect on P . brasiliensis isolate Bt-4 and an enhanced fungistatic effect on isolate Aoki . IL-1beta activated PMNs to kill isolate Bt-4 . Granulocyte colony-stimulating factor had no effect . Combinations of IFN-gamma with GM-CSF or IL-1beta, but not a combination of GM-CSF and IL-1beta, exhibited a synergistic effect in enhancing the antifungal activity of PMNs . These results strongly suggest that PMNs activated with IFN-gamma, GM-CSF and/or IL-1beta might play an important role in host defense in early infection with P . brasiliensis due to their enhanced antifungal activity. Rev Inst Med Trop Sao Paulo, 2000 Mar-Apr, 42(2), 59 - 66 Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable paracoccidioides brasiliensis propagules; Cock AM et al.; Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations . Based on an already established animal model, the contribution of viable and non-viable P . brasiliensis propagules to the development of fibrosis was investigated . BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6) viable conidia (Group I), or 6 . 5x10(6) fragmented yeast cells (Group II) . Control animals received PBS . Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late) . Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocot&tacute;s . During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed . Later on, mononuclear cells predominated . In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose . Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively . Collagen III, thick fibers became apparent in some animals at 4 weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements . In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas . Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week . Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis. Am J Pathol, 2000 May, 156(5), 1811 - 20 Interferon-gamma and tumor necrosis factor-alpha determine resistance to Paracoccidioides brasiliensis infection in mice; Souto JT et al.; To investigate the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-gamma (GKO) or TNF-alpha receptor p55 (p55KO) were infected with the parasite . GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively . Typical inflammatory granulomas were found only in WT mice . In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice . Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs . Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-alpha levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-alpha but higher levels of IFN-gamma . Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO) resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice . On the contrary, the addition of IFN-gamma to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation . Taken together, these findings suggests that endogenous TNF-alpha, acting through the p55 receptor, and IFN-gamma mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness. Infection, 2000 Mar-Apr, 28(2), 119 - 20 A case of paracoccidioidomycosis: experience with long-term therapy; Borgia G et al.; We describe long-term therapy for paracoccidioidomycosis occurring in a 61-year-old house-painter from Venezuela . The diagnostic examinations made in South America had shown pulmonary granulomatous lesions and an osteolytic pattern of the left knee that had been considered suspect of malignant disease with an indication for limb amputation . With the aid of fine needle aspiration biopsy (FNAB) and culture examination we diagnosed an osteomyelitis by Paracoccidioides brasiliensis and initiated therapy with itraconazole, 400 mg per day, reduced to 200 mg per day after 2 months . At the end of 2 years of drug therapy, we observed complete regression of the pulmonary lesions and of the osteolytic area of the left knee . Moreover, we have periodically observed our patient to verify his clinical development and he is still in good health . We suggest that this pathology be considered in differential diagnosis of leprosy, tuberculosis, leishmaniasis, and systemic mycoses, even in non-endemic areas. Infect Immun, 2000 May, 68(5), 2546 - 52 Nitric oxide participation in the fungicidal mechanism of gamma interferon-activated murine macrophages against Paracoccidioides brasiliensis conidia; Gonzalez A et al.; Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungus Paracoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form . The macrophage (Mphi) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-gamma), murine Mphis kill the fungus by an oxygen-independent mechanism . Our goal was to determine the role of nitric oxide in the fungicidal effect of Mphis on P . brasiliensis conidia . The results revealed that IFN-gamma-activated murine Mphis inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mphis activated with 50 U/ml and incubated for 96 h at 37 degrees C . When Mphis were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect . The inhibitory effect was reversed by the addition of anti-IFN-gamma monoclonal antibodies . Activation by IFN-gamma also enhanced Mphi nitric oxide production, as revealed by increasing NO(2) values (8 +/- 3 microM in nonactivated Mphis versus 43 +/- 13 microM in activated Mphis) . The neutralization of IFN-gamma also reversed nitric oxide production at basal levels (8 +/- 5 microM) . Additionally, we found that there was a significant inverse correlation (r = -0.8975) between NO(2)(-) concentration and transformation of P . brasiliensis conidia . Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase, N(G)-monomethyl-L-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production . These results show that IFN-gamma-activated murine Mphis kill P . brasiliensis conidia through the L-arginine-nitric oxide pathway. Biochemistry, 2000 Apr 18, 39(15), 4243 - 9 Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior; Koppenhofer A et al.; Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center . Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1 . Reproducible hysteresis is demonstrated in the redox titrations . In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV . However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2 . A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated . In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1) . The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes. Biochemistry, 2000 Apr 11, 39(14), 4028 - 36 Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus; Koppenhofer A et al.; Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction) . Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration . Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron . Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen . On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed . Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically . These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme . At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme . Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen . The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species . This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical . Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle. Med Mycol, 2000 Feb, 38(1), 51 - 60 Experimental paracoccidioidomycosis of the Syrian hamster: fungicidal activity and production of inflammatory cytokines by macrophages; Parise-Fortes MR et al.; Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action . In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection . The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection . TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection . A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals . These results suggest that during the early stages of infection with P . brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination . In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death . These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis. Med Mycol, 2000 Feb, 38(1), 31 - 9 Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis; Nino-Vega GA et al.; Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains . These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V . Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis . One gene (PbrCHS3) was not expressed at detectable levels . The others were regulated by morphology and/or by the growth phase of the organism . Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4, and PbrCHS5 were higher in hyphal cells than in yeast cells . This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis. Rev Inst Med Trop Sao Paulo, 2000 Jan-Feb, 42(1), 38 - 40 Paracoccidioidomycosis of the male genital tract . Report of eleven cases and a review of Brazilian literature; Severo LC et al.; Eleven cases of involvement of the genital tract in paracoccidioidomycosis were collected in a retrospective study of the clinical records of 683 patients seen in Porto Alegre, Rio Grande do Sul, Brazil . These cases are herein summarily reported . Eighteen similar cases were gathered in review of the Brazilian literature . Obtained data are discussed. Mycopathologia, 1999, 146(1), 13 - 7 Production of proteinase and phospholipase by Paracoccidioides brasiliensis; de Assis CM et al.; We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis . Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 degrees C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 degrees C . After 30 days net enzyme activity was visualized and quantitatively evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it . Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P . brasiliensis. Clin Diagn Lab Immunol, 2000 Mar, 7(2), 175 - 81 Anti-idiotypic antibodies in patients with different clinical forms of paracoccidioidomycosis; Souza AR et al.; Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America . Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors . Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form . The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43) . All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43 . Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system . The possible correlation of Ab2 titers with different clinical forms of disease was also verified . Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a) . Spontaneous anti-Id antibodies were found in all clinical forms of disease . The majority of patients (88%, n = 8) with the acute form of PCM had high titers of Ab2 . However, among patients with the multifocal chronic form of the disease, only 29% (n = 14) had high titers of Ab2; 70% (n = 10) of patients with the unifocal chronic form had low titers of Ab2 . A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment . Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans. Med Microbiol Immunol (Berl), 1999 Aug, 188(1), 41 - 9 Influence of the genetic background on the pattern of lesions developed by resistant and susceptible mice infected with Paracoccidioides brasiliensis; Xidieh CF et al.; To compare the sequential evolution of lesions developed by resistant (A/Sn) and susceptible (B10.A) mice to Paracoccidioides brasiliensis infection we inoculated a virulent isolate of the fungus and collected the pancreas/peripancreatic omentum monthly (from 1 to 6 months) post infection . After fixation, tissue sections were stained by conventional methods for light microscopy to investigate the cellular composition, the extracellular matrix (ECM) patterns and the morphology of the yeasts in the lesions . In both strains, the fungal lesions were localized mostly in the omentum; a few lesions in the pancreatic parenchyma were observed, mostly in B10.A mice . In both strains, macrophages and plasmocytes were the predominant cells in all lesions, followed by neutrophils (PMN) and macrophages transformed into giant and epithelioid cells . Remarkable differences were observed between resistant and susceptible mice, specially related to the ECM structure of the granulomatous lesions . In A/Sn mice, from the 1st month on, the coexistence of two types of lesions was observed: one type showed a well-defined encapsulated nodule, constituted mainly of type I collagen . Neutrophils were abundant in areas of massive fungal destruction and few viable yeasts were observed . The other type showed residual characteristics, with sparse collagen deposits and presence of xantomatous-like macrophages, containing degenerated fungi . Such residual lesions predominated after the 2nd month and were the only type observed from the 4th month on, indicating the control of the infection . In B10.A mice, on the contrary, only one type of lesion was observed, showing less tendency to encapsulation and the formation of multiple small granulomatous foci, individualized by reticular type III collagen fibers . There were many plasmocytes in the periphery and large numbers of budding yeasts, with no evidence of fungal destruction . In the course of the infection the lesions progressively increased in number and size . Altogether, the comparative histopathological analysis demonstrates the influence of the genetic pattern of the host on the lesions developed by resistant and susceptible mice to P . brasiliensis infection. Rev Inst Med Trop Sao Paulo, 1999 Nov-Dec, 41(6), 359 - 64 Jorge Lobo's disease: experimental inoculation in Swiss mice; Opromolla DV et al.; Sixty-four isogenic Swiss mice were intradermically inoculated in both hind foot pads . The inocula, consisting of fungal suspensions from biopsies obtained from Jorge Lobo's Disease patients, had the total number of fungi and the viability index determined using a Neubauer chamber and the fluorescein diacetate-ethidium bromide technique (FD-EB), respectively . The animals were sacrificed at times ranging from ten days to eighteen months after inoculation . The cellular infiltrate, mainly consisting of macrophages containing fungi, increased progressively up to end of the study; however, no macroscopic alterations were observed in the inoculated feet . After nine months, small numbers of Langhans' giant cells started to appear in the infiltrate . A considerable number of fungi was observed at the end of the experimental period, but only a few were viable when stained by the FD-EB technique . This fact suggests that there is a multiplication of fungal cells, which are destroyed by the macrophages but remain in the tissue for a long time due perhaps to the difficulties in their elimination . These findings led us to conclude that in spite of the maintenance of the infection in these animals, Swiss mice cannot be considered an ideal model to study Jorge Lobo's Disease . However, the authors call attention to the possibility of other mouse strains being more susceptible to Paracoccidioides loboi. Rev Inst Med Trop Sao Paulo, 1999 Nov-Dec, 41(6), 351 - 8 PCR with Paracoccidioides brasiliensis specific primers: potential use in ecological studies; Diez S et al.; The precise microenvironment of Paracoccidioides brasiliensis has not yet been discovered perhaps because the methods used are not sensitive enough . We applied to this purpose the polymerase chain reaction (PCR) using three sets of specific primers corresponding to two P . brasiliensis genes . This fungus as well as several other fungi, were grown and their DNA obtained by mechanical disruption and a phenol chloroform isoamylalcohol-based purification method . The DNA served for a PCR reaction that employed specific primers from two P . brasiliensis genes that codify for antigenic proteins, namely, the 27 kDa and the 43 kDa . The lowest detection range for the 27 kDa gene was 3 pg . The amplification for both genes was positive only with DNA from P . brasiliensis; additionally, the mRNA for the 27 kDa gene was present only in P . brasiliensis, as indicated by the Northern analysis . The standardization of PCR technology permitted the amplification of P . brasiliensis DNA in artificially contaminated soils and in tissues of armadillos naturally infected with the fungus . These results indicate that PCR technology could play an important role in the search for P . brasiliensis' habitat and could also be used in other ecological studies. J Interferon Cytokine Res, 2000 Jan, 20(1), 89 - 97 Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-gamma production; Kashino SS et al.; The secretion of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, and IL-10 by antigen-stimulated lymph node cells, eosinophil maturation, and the antibody isotypes produced were examined during intraperitoneal infection of susceptible (B10.A) and resistant (A/Sn) mice with Paracoccidioides brasiliensis . Lymph node cells from resistant mice produced early and sustained levels of IFN-gamma and IL-2, whereas susceptible animals secreted low to undetectable amounts of these type 1 cytokines . Both mouse strains presented late and transient production of IL-4, whereas IL-10 was produced constantly throughout the course of disease . Resistant animals produced increasing levels of IL-5 in the chronic phase of the infection (from the eighth week on), whereas susceptible mice showed two peaks of IL-5 producti