Isolation of a specific antigen with alkaline phosphatase activity from soluble extracts of Paracoccidioides brasiliensis

A specific antigen of Paracoccidioides brasiliensis was isolated from a metabolic extract of the fungus. The extraction was made by specific adsorption to and subsequent elution from a column containing a cross linked polymer to which the antibodies of a monospecific rabbit serum had been covalently attached. The purity of the final product was demonstrated by immunodiffusion analysis of the eluate using immune serum produced in a sensitized rabbit. The purified antigen was shown to have cationic electrophoretic mobility and alkaline phosphatase activity.     

Isoenzyme profile of Paracoccidioides brasiliensis

Isoenzyme profiles of 10 strains of Paracoccidioides brasiliensis from different origins (nine strains from patients with different clinical forms of paracoccidioidomycosis and one from the faeces of a penguin) were determined by polyacrylamide gel electrophoresis using 37 different enzymes. Differences in carbonate dehydratase, phosphoglucomutase, phosphoglucoseisomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and beta-esterase were detected among the isolates studied allowing the characterization of nine zymodemes. Two isolates showed identical profiles. There was no correlation between the zymodeme patterns and virulence, clinical forms of the disease nor age of the cultures.     

Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with "apparent cure." The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.     

Cloning and nucleotide sequence of a specific DNA fragment from Paracoccidioides brasiliensis

We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates.     

Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis

The structured clinical interview for diagnosis (axis 1) according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-III-R) was used to assess psychiatric morbidity in 110 infertile patients. They were divided into two groups according to whether referral to the service of psychosomatic medicine was deemed advisable by the physician in charge. Psychiatric disorders were diagnosed in 39 of 56 (69.6%) patients in the referred group and in 13 of 54 (24.1%) in the non-referred group. Psychiatric morbidity was found in 61.1% of females and 21% of males. Adjustment disorders were found in 59.6% (31/52) of all patients, in 59% (24/39) of patients among the referred group and in 61.5% (8/13) of patients among the non-referred group. Fourteen (67%) of 21 women in the referred group with adjustment disorders suffered from anxiety. In addition, 33.3% of patients in the non-referred group showed important psychological dysfunction, although DSM-III-R criteria were not met. Psychiatric morbidity was significantly associated with the number of treatment cycles and female gender in the whole study population, as well as with the type and length of infertility in the non-referred group. Psychological services in an infertility clinic help to identify at an early stage those individuals who are more likely to be vulnerable. This would enable psychological interventions to be targeted towards those in greater need.     

In vitro amphotericin B effects on growth, viability and dimorphism of Paracoccidioides brasiliensis: reversal of the treatment

The reversed dermal graft has been employed in reconstructive plastic surgery for almost 80 years. It is recommended that the dermatologic surgeon use this graft technique whenever a case calls for the combined advantages of the split-thickness skin graft for poorly vascularized areas and the axial pedicle flap appropriate to areas subject to marked mechanical strain. We present cases in which defects on the scalp, sole, palm, and great toe have been covered by reversed dermal graft and split-thickness skin graft in a single operation with excellent results. Our modification of the technique has proved that simultaneous grafting with reversed dermis and split-thickness skin graft produces satisfactory results, reduces postoperative care by 2 weeks, and obviates repeated anesthesia.     

Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response

To assess human cellular immune response to paracoccidioidomycosis (PCM), lymphocyte proliferative responses to purified antigens from Paracoccidioides brasiliensis were determined in healthy persons previously infected by the fungus (positive donors), in healthy noninfected persons (controls), and in PCM patients. Affinity-purified gp70 and gp43, the two major antigens in humoral immune responses, were used. Both induced lymphocyte proliferation (gp43 species-specific) in positive donors but not in controls; healthy persons previously infected by Histoplasma capsulatum reacted to gp70 and not to gp43. A similar cross-reactivity in antibody response to gp70 was previously reported; however, antibody response to gp43 has been considered specific. Lymphocytes from PCM patients, who, unlike positive donors, have high levels of anti-gp43 and anti-gp70 antibodies, proliferated poorly with gp70 and gp43 but better with other stimuli. This dichotomy between humoral and cellular antigen-specific responses suggests a Th2 immune response in PCM, which may be related to failure to control the infection.     

Structural characterization and optimization of antibody-selected phage library mimotopes of an antigen associated with autoimmune recurrent thrombosis

The presence of high titers of anti-cardiolipin antibodies (ACA's) of autoimmune origin, which are known to bind to plasma beta2-glycoprotein I (aka apolipoprotein H), correlates clinically with autoimmune recurrent thrombosis. Soluble beta2-glycoprotein I binds to solid-phase ACA (immobilized on a surface plasmon resonance chip) with a Kd of 1.4 microM, but if the reactants are reversed and beta2-glycoprotein I is on the solid-phase support, then the Kd is 52 nM. This 27-fold difference in affinity reflects the avidity/entropic advantage obtained for an antibody binding to an antigen that is made multivalent because it is attached to a solid phase. A mimotope of this antigen, selected from a phage display peptide library screen with an ACA, has been shown to bind to solid-phase ACA as a phage, using surface plasmon resonance. This peptide is representative of the motif from 37 peptides obtained in a previously reported phage library screen with this ACA (1). A synthetic version of this peptide, referred to as P4, has the sequence: A1G2P3C4I5L6L7A8R9D10R11C12P13G14, and binds to its selecting antibody with a Kd of 42 nM. NMR data indicate that proline-13 is present in both cis and trans configurations, and that these two geometries dramatically affect the overall tertiary structure of the molecule. The peptide lacking this proline binds severalfold better to the ACA, consistent with at least one of these structures having low affinity for binding ACA. Replacement of the arginine-9 position with a proline decreases binding affinity to ACA 10-fold. Another phage library-selected peptide has a proline in position 9, but also has a leucine in position 5, instead of isoleucine. Since its affinity for ACA is nearly as good as that for peptide P4, the phage library screening must have selected for a non-beta-branched amino acid in this position to compensate for the adverse effects of the arginine-9 to proline-9 substitution. The solution structure of a modified version of the antibody-selected phage peptide P4 with the central proline was determined. This peptide has one turn comprised of Ala-Pro-Asp-Arg, with the proline peptide bond in the cis configuration, and another turn that contains the disulfide and adjacent residues. If the disulfide is replaced by a thioether, and the central proline by an alpha-methyl proline, in an attempt to make the peptide more biologically stable, there is little adverse effect on affinity for ACA. The thioether bond/turn is fairly well defined with a Calpha to Calpha separation of 4.9 +/- 0.8 A. The alpha-methyl proline adopts the trans configuration, and this central Ala-(alpha-methyl-Pro)-Asp-Arg turn adopts a distorted type I turn conformation with a probable i to i+3 hydrogen bond. Modeling studies suggest that the proline peptide bond configuration switched from cis to trans in the presence of the alpha-methyl group on proline because of steric hindrance with the beta-carbon of the preceding residue. Overall, this peptidomimetic molecule is structurally very similar to the peptide with natural amino acids, with an rmsd difference of only 1.37 A, when comparing backbone atoms.     

Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans

The kinetics of the glutathione (GSH) conjugation of (+)- and (-)-enantiomers of anti- as well as syn-3,4-dihydroxy-1,2-oxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene (B[c]PDE) catalyzed by murine GSH S-transferase (GST) isoenzymes has been investigated. Murine GSTs exhibited significant differences in their enantioselectivity toward B[c]PDE stereoisomers. For example, while pi class isoenzyme mGSTP1-1 was virtually inactive toward stereoisomers with 1S configuration [(-)-syn-and (+)-anti-B[c]PDE], these stereoisomers were good substrates for alpha class isoenzyme mGSTA1-2. When GST activity was measured as a function of varying B[c]PDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isoenzyme examined obeyed Michaelis-Menten kinetics with all four B[c]PDE stereoisomers. Alpha class isoenzyme mGSTA4-4 exhibited negligible activity toward all four stereoisomers of B[c]PDE. The catalytic efficiency of mGSTA1-2 was approximately 1.5- to 15-fold higher than other murine GSTs in the GSH conjugation of (-)-anti-B[c]PDE, which among the four B[c]PDE stereoisomers is the most potent pulmonary carcinogen in the newborn mouse model and a potent skin tumor-initiator. While alpha class isoenzymes mGSTA3-3 and mGSTA1-2 were equally efficient in the GSH conjugation of (+)-anti-B[c]PDE, their catalytic efficiencies toward this stereoisomer were significantly higher than those of mGSTP1-1 and mGSTM1-1. Likewise, mGSTA1-2 was relatively more efficient than other GSTs in the GSH conjugation of both enantiomers of syn-B[c]PDE. In summary, our results indicate that (a) murine GSTs significantly differ in their enantioselectivity in the GSH conjugation of B[c]PDE stereoisomers, which may partially account for the observed differences in the carcinogenic potency of B[c]PDE stereoisomers, and (b) mGSTA1-2 and mGSTA3-3 play a major role in the detoxification of B[c]PDE.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

The presence of > or = 25% blasts in a marrow aspirate obtained on day 7 of induction followed by a remission at day 28 has been associated with a poor prognosis in children with acute lymphoblastic leukemia (ALL). We evaluated whether a day 7 marrow biopsy may be used to more accurately assess therapeutic reduction of leukemia tumor burden. Studied were 76 children with ALL enrolled on CCG protocols at B.C's Children's Hospital who received both a day 7 aspirate and biopsy and were in remission by day 28. Evaluation for the correlation of the percentage aspirate blasts on day 7 with the biopsy demonstrated a moderate correlation with the percentage biopsy blasts (R = 61), but not correlation with the biopsy cellularity. We saw a similar prediction of outcome by the percentage blasts on day 7 marrow aspirate in the study as reported previously although it was not significant. Outcome analysis was done using leukemia burden as measured by the day 7 absolute blast index-aspirate (ABI-aspirate) calculated as the product of the biopsy cellularity with the percentage blasts on the aspirate. The ABI-aspirate significantly predicted patient outcome with 83% survival in those with an ABI-aspirate of < .06 compared to 51% in those > or = .06 (P = .01) and was highly significant when analyzed as a continuous predictor (P = .004). This is the first study to demonstrate that information gained from the day 7 marrow biopsy can improve prediction of outcome in children with ALL. Based on this preliminary study, we recommend that large population ALL therapy trials evaluate the role of the day 7 marrow biopsy for outcome prediction in children with ALL.     

Effect of rifampin on CD1b expression and double-negative T cell responses against mycobacteria-derived glycolipid antigen

Non-classical antigen-presentation by CD1 molecules expressed on cytokine-activated monocytes (CAM), and cell-mediated responses supported by double-negative (DN) and by CD8+ responder alphabeta T cells, are involved in host resistance against mycobacterial infections. The CD1b protein is responsible for presentation of non-peptide, lipid antigens to T cells. In this context, a pivotal role is played by induction of CD1b protein on the membrane of human monocytes activated by GM-CSF alone, and more efficiently by GM-CSF combined with IL-4. Rifampin (RFP), a drug which is extensively utilized for chemoprophylaxis or treatment of Mycobacterium tuberculosis, is known to reduce a number of B, or T cell-dependent responses. Therefore we undertook immunopharmacological studies on RFP, to determine the effects of this agent on human macrophage function, relative to antigen presentation by CD1b molecules and on DN T cell cytolytic function. The results showed that: (a) graded concentration of RFP (2 or 10 microg/ml) induced a significant increase of CD1b expression, in CAM as evaluated by FACS analysis; (b) RFP increased significantly the specific mAb binding to CD1b on CAM surface; (c) treatment of effector cells with RFP did not reduce DN T cell-mediated cytolysis against lymphoblastoid cells transfected with CD1b cDNA (C1R.b6 cells), pulsed with M. tuberculosis. These results suggest that RFP could be of potential value in improving mycobacterial antigen presentation without impairing responder T cell function.     

Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen

The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide-major histocompatibility complex (pMHC) antigen H-2Kb-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR beta chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC alpha helices suggests a generalized orientation that is mediated by the Valpha domain of the TCR and rationalizes how TCRs can effectively "scan" different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.     

Immunochemical identification of a new embryonal antigen in ovarian tumor tissue

The peripheral blood smears of 214 thermally injured patients studied during a thirty month period revealed a 20% incidence of normoblastemia (51 patients). Nucleated red blood cells were most common in patients with the largest burns. The absolute number of normoblasts was not related to the hematocrit and was not correlated with other determinants of erythropoietic function.     

Structural characterization of red wine rhamnogalacturonan II

A series of experiments is described that elucidates the sources of Ca2+ that contribute to activity-dependent neuronal facilitation in Hermissenda B photoreceptors during associative conditioning. In an in vitro preparation, pairings of a 4-s light with a 3-s mechanical stimulation of presynaptic hair cells increased the input resistance and elicited spike rate (i.e., excitability) of the B photoreceptors in the Hermissenda eye, indicative of a Ca(2+)-dependent process that is analogous to associative conditioning in the intact animal. This increase in excitability was reduced but not eliminated when hyperpolarizing current was applied to the B cell during the pairings, suggesting that voltage-dependent influx of Ca2+ contributed only a portion of the total calcium signal necessary for facilitation. Moreover, no increase in excitability was observed when a comparable current-induced depolarization of the photoreceptor was substituted for light-induced depolarization. In other experiments, Ca(2+)-dependent inactivation of a light-induced Na+ current was used as an index of intracellular Ca2+ concentration. It was determined that light caused a large increase in intracellular Ca2+ concentration regardless of whether the photoreceptor was allowed to freely depolarize in response to light or was voltage clamped at its resting membrane potential. Current-induced depolarization produced a smaller increase, while presynaptic stimulation had no measurable effect. Intracellular injections of either heparin, an antagonist of intracellular Ca2+ release, or EGTA, a general Ca2+ chelator, induced comparable reductions of light-induced Ca2+ accumulation. Finally, intracellular injections of heparin blocked the pairing-induced increases in B cell excitability as effectively as injections of EGTA. Taken as a whole, these data suggest that Ca2+ release from intracellular stores may be sufficient for the induction of facilitation in this preparation, while Ca2+ influx through voltage-dependent channels may have an additive effect and provide further evidence for the ubiquitous role of Ca2+ in learning-related forms of neuronal plasticity.     

Clinical profile of a new monoclonal antibody-based immunoassay for tissue polypeptide antigen

Our preliminary evaluation of a new monoclonal antibody-based assay for tissue polypeptide antigen (TPA) has shown it to be clinically equivalent to the polyclonal antibody-based assay for TPA. The new assay (TPA-M) employs three monoclonal antibodies to epitopes on cytokeratins 8, 18 and 19. This multicenter, multinational study included 266 patients with newly diagnosed carcinomas of the lung, breast, large bowel and urinary bladder. TPA values from the two assays were compared with three other cytokeratin markers (TPS, CYFRA 21-1 and TPACyk) and with the established reference markers for these malignancies (CEA and NSE for lung, CA 15-3 for breast, CEA and CA 19-9 for colorectal tumors). Analysis of receiver operating characteristic (ROC) curves in lung, colorectal and bladder cancer showed similar sensitivities for the two assays, ranging from 50% to 80% with a specificity of 95%. In breast cancer all the markers studied showed poor sensitivity. However, TPA determination by either method could discriminate advanced stage (stages III and IV) from early stage disease (stages 0 to II). TPA showed similar discriminating ability in bladder cancer. On the basis of the results obtained in our patient series, it seems that of the cytokeratin markers studied, TPA and TPA-M are the most sensitive and offer a wide range of clinical applications.     

Serodiagnosis of tuberculosis by detection of antituberculous glycolipid antigen (TBGL antigen) antibodies in serum using enzyme-linked immunosorbent assay: clinical evaluation of anti-TBGL antibodies assay kit

Kyowa Medex Co., Ltd. developed the kit for the sero-diagnosis of tuberculosis, which detects IgG antibodies against tuberculous glycolipids antigen containing cord factor (TBGL antigen) prepared from M. tuberculosis using the enzyme-linked immunosorbent assay technique. We evaluated the kit using clinical specimens and the results are as follows: 1) In total, 34 out of 39 cases (87.2%) with active pulmonary tuberculosis showed positive anti-TBGL antibody. 2) Patients with cavity, patients with extensive lesions and patients excreting large amount of acid fast bacilli tended to show high positivity rates. 3) The antibody titers increased in 7 out of 11 cases after starting the antituberculous chemotherapy. 4) The use of the antibody is unsuitable for the determination of the activity of tuberculosis since the antibody titers only slightly decreased even after chemotherapy for two years. 5) Two out of four nontuberculous mycobacteriosis cases showed high antibody titers 6) All three AIDS patients with tuberculosis showed low antibody titers. 7) The antibody was negative in almost all healthy controls showing a positive PPD skin test after vaccination with BCG, and it was therefore assumed that the antibody titer is not increased by BCG vaccination. 8) The antibody titers of the staff members working in the tuberculosis wards were not high compared with those of staff members working in the other wards.     

新型含银配合物的合成与表征

合成了一种由银直接连接Anderson型钼氧酸盐阴离子形成的新型二维配合物,并用单晶X射线衍射仪测定了其晶体结构,用元素分析仪及红外光谱等进一步表征了化合物的结构。含银配合物为三斜晶系,P-1空间群,晶胞参数为a=1.1676(3)nm,b=1.2205(4)nm,c=1.2566(6)nm;α=95.313(5)°,β=113.974(5)°,γ=117.079(3)°;V=1.3695(9)nm3,Z=1。配合物中,相邻的4个{Fe(OH)6Mo6O18}3-之间存在两种连接方式,一种是被孤立的{AgO6}八面体通过端氧沿b轴连接起来;另一种则是被2个共边相连的{AgO6}八面体通过端氧沿b轴桥连起来。这两种连接方式沿c轴方向交替排列形成了二维网状结构。