A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12.

Leishmania braziliensis causes cutaneous and mucosal leishmaniasis in humans. Most patients with cutaneous leishmaniasis heal spontaneously and may therefore have developed protective immunity. There appears to be a mixed cytokine profile associated with active cutaneous or mucosal disease, and a dominant T helper (Th)1-type response associated with healing. Leishmanial antigens that elicit these potent proliferative and cytokine responses from peripheral blood mononuclear cells (PBMC) are now being identified. Herein, we report on the cloning and expression of a L. braziliensis gene homologous to the eukaryotic ribosomal protein eIF4A (LeIF) and patient PBMC responses to rLeIF. Patients with mucosal and self-healing cutaneous disease had significantly higher proliferative responses than those with cutaneous lesions. Whereas the parasite lysate stimulated patient PBMC to produce a mixed Th1/Th2-type cytokine profile, LeIF stimulated the production of interferon gamma (IFN-gamma), interleukin 2 (IL-2), and tumor necrosis factor alpha but not IL-4 or IL-10. Recombinant LeIF (rLeIF) downregulated both IL-10 mRNA in the "resting" PBMC of leishmaniasis patients and LPS-induced IL-10 production by patient PBMC. rLeIF also stimulated the production of IL-12 in cultured PBMC from both patients and uninfected individuals. The production of IFN-gamma by patient PBMC stimulated with either rLeIF or parasite lysate was IL-12 dependent, whereas anti-IFN-gamma monoclonal antibody only partially blocked the LeIF-induced production of IL-12. In vitro production of both IFN-gamma and IL-12 was abrogated by exogenous human recombinant IL-10. Therefore, we have identified a recombinant leishmanial antigen that elicits IL-12 production and Th1-type responses in patients as well as IL-12 production in normal human PBMC.

Antigen-specific immunosuppression in visceral leishmaniasis is cell mediated.

Visceral leishmaniasis is associated with an antigen-specific immunosuppression during the acute disease. Patients become responsive to Leishmania antigen in both in vivo and in vitro assays after successful antimony therapy. The cell type involved in the suppression of lymphocyte reactivity to Leishmania antigen was studied by selective depletion of mononuclear cell (MNC) populations and in co-cultivation experiments. Adherent cells were depleted on plastic and by passage on nylon wool columns. High-avidity Fc+ cells were depleted by adherence to BSA-anti-BSA complexes and OKT4+ and OKT8+ cells were depleted by treatment with monoclonal antibody (anti-OKT4+ and OKT8+) and complement. Depletion of MNC preparations of adherent cells, high-avidity Fc+ cells, OKT4+ cells and OKT8+ cells failed to restore the lymphocyte reactivity to Leishmania antigen. Antimony therapy was associated with restoration of the proliferative responses of unseparated MNC (before treatment 460 +/- 76 cpm and after treatment 4,293 +/- 1,442 cpm). Co-culture of frozen cells obtained before chemotherapy with autologous MNC obtained after treatment reduced the response of posttreatment cells to Leishmania antigen by 80%. We conclude that the antigenic specific suppression of lymphocyte proliferation in visceral leishmaniasis is cell mediated.

Absence of gamma interferon and interleukin 2 production during active visceral leishmaniasis.

The lymphocytes from eight patients with active visceral leishmaniasis (VL), a disease associated with marked immunologic dysfunction, were examined for ability to produce interleukin 2 (IL-2) and gamma interferon during in vitro cultivation. It was found that both IL-2 and gamma interferon production, in response to leishmania antigen, was absent during the active disease, but was restored after successful chemotherapy. Untreated VL patients produced IL-2 and gamma interferon when stimulated with phytohemagglutinin (PHA). Six patients with either active cutaneous or mucosal leishmaniasis, a disease not associated with immunosuppression, showed high levels of gamma interferon in response to leishmania antigen and PHA. Since IL-2 and gamma interferon have been shown to have important roles in the immune response and in the killing of leishmania, their absence may represent a key defect in the immune response in VL.

Validation of criteria for nosocomial use of amikacin in Brazil with the Delphi technique.

UNLABELLED: The Delphi technique has been used since the 1950s to collect the opinions of experts; to gauge their indications, and in some instances, to develop a consensus. This systematic collection and aggregation of informed judgments from a group of experts on specific questions or issues is a highly efficient and cost-effective means to establish guidelines and policies, when compared to other strategies, such as committee meetings or personal interviews. OBJECTIVE: Examine the content validation process of the proposed criteria of the American Society of Health System Pharmacists (ASHP) for amikacin use in hospital settings. MATERIAL AND METHODS: The Delphi technique was applied using the proposed ASHP criteria questionnaire containing 102 specific questions related to the nosocomial use of amikacin by individual patients. The questionnaire contained six groups of questions: 1) Identification and basic demographic data, 2) Relevant data for the use of amikacin, 3) Justification of its usage, 4) Critical parameters of amikacin use, 5) Complications, 6) Measurement of results. Eight hospital specialist medical doctors were selected, including five in the area of infectious diseases, one surgeon, one nephrologist and one in critical care medicine. The questionnaire was e-mailed to the doctors and they were asked for their opinion about the appropriateness of the questions. They were to say whether the general concept seemed totally or partially adequate to the proposed process, what grade (0 to 10) they would give to each section, and if there were any perceived deficiencies, they could add, omit or modify individual questions. A second questionnaire containing the questions for which there had been no consensus based on the answers to the previous one was re-sent to the participants for consolidation. RESULTS: Feedback revealed an agreement of 75% concerning the utility and appropriateness of sections 1 and 2. The section about the justification of amikacin usage was agreed on by 50%. There was a total agreement of 62% for the critical parameters of amikacin use, and a partial agreement of 37%. The complication of usage of the questionnaire was agreed upon by 50% of the participants, and positive measurement of the results was totally agreed on by 62%, and partially by 37%. The overall score for the questionnaire was 8.77 +/- 0.25. CONCLUSION: The usage criteria for amikacin recommended by ASHP were validated by the Delphi technique for utilization in Brazilian hospital settings. The Delphi technique applied to validate a questionnaire instrument for monitoring the correct use of a specific strategic antibiotic indicated for the treatment and prophylaxis of serious antibiotic-resistant Gram-negative bacteria, proved to be a reliable and simple tool for designing guidelines and a consensus document for hospital use of antibiotics.

Identification of differentially expressed genes in human prostate cancer using subtraction and microarray.

We have identified human prostate cancer- and tissue-specific genes using cDNA library subtraction in conjunction with high throughput microarray screening. Subtracted cDNA libraries of prostate tumors and normal prostate tissue were generated. Characterization of subtracted libraries showed enrichment of both cancer- and tissue-specific genes. Highly redundant clones were eliminated by colony hybridization. The remaining clones were selected for microarray to determine gene expression levels in a variety of tumor and normal tissues. Clones showing overexpression in prostate tumors and/or normal prostate tissues were selected and sequenced. Here we report the identification of two genes, P503S and P504S, from subtracted libraries and a third gene, P510S, by subtraction followed by microarray screening. Their expression profiles were further confirmed by Northern blot, real-time PCR (TaqMan), and immunohistochemistry to be overexpressed in prostate tissues and/or prostate tumors. Full-length cDNA sequences were cloned, and their subcellular locations were predicted by a bioinformatic algorithm, PSORT, to be plasma membrane proteins. The genes identified through these approaches are potential candidates for cancer diagnosis and therapy.

Identification and characterization of prostein, a novel prostate-specific protein.

In this report, we describe the application of a systematic, genome-based approach to identify prostein, a novel prostate-specific protein expressed in normal and malignant prostate tissues. Characterization of the prostein gene shows that prostein cDNA encodes a 553-amino acid protein. The protein is predicted to be a type IIIa plasma membrane protein with a cleavable signal peptide and 11 transmembrane-spanning regions. The prostein gene is located on chromosome 1 at the WI-9641 locus between q32 and q42. Prostein mRNA is shown to be uniquely expressed in normal and cancerous prostate tissues using Northern blot, eDNA microarray, and real-time PCR analyses. Furthermore, prostein mRNA expression does not appear to be prostate tumor grade related and is restricted exclusively to prostate cell lines. Immunohistochemical staining using a mouse monoclonal antibody generated against prostein demonstrates that this protein is specifically detected in prostate tissues both at the plasma membrane and in the cytoplasm. Prostein expression is androgen responsive because treatment of LNCaP cells with androgen up-regulates prostein message and protein expression levels. These results validate prostein as a prostate-specific marker with potential utility in the diagnosis and treatment of prostate cancer.

Co-infection with HTLV-1 is associated with a shorter survival time for HIV-1-infected patients in Bahia, Brazil.

Co-infection with HTLV-1 reaches 20% among patients infected by HIV-1 in Bahia, Brazil. To evaluate its impact on survival, we conducted a retrospective, case-control study involving 198 patients (63 cases). Co-infection was associated with parenteral exposure (P = 0.0001) and female sex (P = 0.02). Co-infected patients had a shorter mean survival (1849 days) than controls (2430 days, P = 0.001), regardless of sex or baseline CD4 cell count. In Bahia, Brazil, co-infection with HIV-1 and HTLV-1 is associated with a shorter survival time.

Genetic heterogeneity of the V3 region of the HIV-1 envelope glycoprotein in Brazil. Brazilian Collaborative AIDS Research Group.

OBJECTIVE: To examine the genetic heterogeneity of the V3 region of HIV-1 gp120 from 22 Brazilian HIV-1 specimens. DESIGN: Genetic heterogeneity was examined by DNA sequencing of the C2 V3 region of the HIV-1 envelope (env) gene from polymerase chain reaction (PCR)-amplified HIV-1 DNA. Deduced amino-acid sequences were compared to determine the extent of amino-acid conservation among the Brazilian specimens. Genetic similarity among and between the Brazilian specimens and other previously published HIV-1 isolates was analyzed by principal co-ordinate and DNA parsimony methods. METHODS: A 282 base pair (bp) region of a 1.5 kilo (k) bp PCR-amplified HIV-1 env fragment was sequenced by a Taq dye-labeled primer cycle sequencing reaction. Nucleotide sequences were used to analyze inter-specimen relationships based on overall nucleotide sequence similarity and DNA parsimony principles. RESULTS: Amino-acid comparison showed that 15 of the 35 (43%) residues of the V3 loop were conserved among the Brazilian specimens. Nine of the 22 (40%) Brazilian specimens contained the North American-European GPGR tetrapeptide motif, while eight (36%) contained the GWGR motif, previously reported in Japanese isolates. Principal co-ordinate analysis demonstrated that 19 of the 20 examined Brazilian HIV-1 specimens were more similar to North American and Haitian isolates than to African isolates. Similar results were also obtained by DNA parsimony analysis. CONCLUSION: The majority of the Brazilian specimens examined are more genetically related to North American and Haitian HIV-1 isolates than to African isolates. This finding and the presence of a GWGR V3 loop motif in some Brazilian isolates may be important for vaccine development.

Serologic validation of HIV infection in a tropical area.

We have defined human immunodeficiency virus type 1 (HIV-1) serologic reactivity in Brazilians living in an area endemic for tropical diseases. Enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analyses were performed on 342 patients with diseases including Chagas' disease, schistosomiasis, typhoid fever, helminthiasis, and cutaneous and visceral leishmaniasis. Nine percent of the visceral leishmaniasis patients' sera reacted in the HIV-1 ELISA but all were WB negative. All other sera from these patients were HIV negative. A total of 224 HIV-1 ELISA repeatedly positive sera also were HIV-1 WB tested. They were drawn from a total population of 19,230 individuals, including AIDS patients, blood donors, homosexual men, intravenous drug users, pregnant women, individuals with hemophiliac, and tuberculosis and sexually transmitted disease patients. The WB results were analyzed using five different interpretive criteria for WB positivity. The Centers for Disease Control (CDC) and the World Health Organization (WHO) criteria were the most sensitive and specific for identifying HIV-1-infected individuals. The WB pattern was similar to that seen in the United States. Envelope (ENV) protein antibodies were highly predictive of HIV-1 infection; none of the AIDS patients lacked ENV protein reactivity. We conclude that among the tropical diseases studied, only visceral leishmaniasis is associated with false-positive HIV-1 ELISA tests. Current CDC and WHO criteria for interpretation of HIV-1 WB tests are appropriate for Brazil.

Seroepidemiology of human T-cell lymphotropic virus type I/II in northeastern Brazil.

We investigated the prevalence of human T-lymphotropic virus I/II (HTLV-I/II) infection in Bahia, a state in Northeastern Brazil. Healthy individuals (n = 327) and patients (n = 337) with a variety of diseases were screened for antibodies to HTLV-I/II using an enzyme immunoassay and Western blot. The overall prevalence among healthy subjects was 1.8% (six of 327); among patients it was 18.4% (62 of 337). Patients with AIDS had the highest prevalence of HTLV-I/II infection, 22.7% (20/88), followed by randomly selected patients from an infectious disease hospital, 19.4% (25 of 129), and tuberculosis patients, 11.1% (10 of 90). Four of 14 patients with myelopathy and three of 16 patients with lymphoid leukemia or lymphoma were seropositive for HTLV-I/II. Sixty-three of 68 HTLV-I/II-positive specimens were then typed: 53 patients were HTLV-I positive, three patients were HTLV-II positive, and in seven patients the assay could not distinguish infection by HTLV-I or II. The finding among HIV-seropositive intravenous drug users in Bahia of coinfection with HTLV-I is contrasted with reports from other areas in which dual infection occurs with HTLV-II. Although high prevalence of HTLV-I infection was found in Bahia, the extent and clinical manifestations of HTLV-I/II infection in Brazil remains imprecisely defined, and further studies are needed.

Cloning, characterization and serological evaluation of K9 and K26: two related hydrophilic antigens of Leishmania chagasi.

We report here the molecular cloning and characterization of two related hydrophilic antigens of Leishmania chagasi. These two antigens have predicted molecular weights of approximately 9 and 26 kDa and detect antibodies in sera of patients with kala-azar (k). Thus, to maintain consistency with nomenclature of the previously described 39 kDa diagnostic antigen of L. chagasi (k39 [1]), these antigens are being referred to as k9 and k26. A significant difference between k9 and k26 is the presence of 11 copies of a 14 amino acid repeat in the open reading frame of k26. The region flanking the repeats of k26 shares a 69% identity with the open reading frame of k9. The recombinant proteins encoded by both antigens are very hydrophilic and show aberrant migration on SDS PAGE. Results of Southern blot analysis reveal that k9 and k26 are conserved to varying degrees among various Leishmania species. Interestingly, the repeat region of k26 is specific to L. chagasi and L. donovani while the flanking region is conserved among several other species. Transcript levels of k26 are significantly upregulated in the amastigote stage of the parasite. Our results show that recombinant K26 is specific in detecting antibodies in infection sera from visceral leishmaniasis (VL) patients. Thus rK26 may complement rK39 in a more accurate diagnosis of VL in the old and the new world.

Evaluation of DPPD, a single recombinant Mycobacterium tuberculosis protein as an alternative antigen for the Mantoux test.

Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult particularly because sensitization with non-tuberculous mycobacteria leads to false positive tests. Using the guinea pig model of tuberculosis, we have recently described a recombinant antigen (DPPD) that could circumvent this problem. The DPPD gene is unique to the M. tuberculosis complex organisms and is absent in the organisms representative of all other members of the Mycobacterium genus. Moreover, DPPD induced strong DTH in 100% of the guinea pigs infected with M. tuberculosis and in none of the guinea pigs immunized with nine different species of Mycobacterium. Here we present results of a clinical investigation using DPPD. Mantoux test using both PPD and DPPD was initially performed in 26 patients with confirmed pulmonary tuberculosis and in 25 healthy PPD negative individuals. The results indicated that both PPD and DPPD elicited DTH in 24 out of the 26 patients. No DTH was observed in any of the PPD negative individuals. In addition, a small clinical trial was performed in a population of 270 clinically healthy and randomly selected individuals. DPPD produced a bimodal histogram of skin reaction size and PPD produced a skewed histogram. Because the DPPD gene is not present in non-tuberculous bacilli, these results suggest that this molecule can be an additional tool for a more specific diagnosis of tuberculosis.

Immunofluorescent antibody test in American visceral leishmaniasis: sensitivity and specificity of different morphological forms of two Leishmania species.

This study was designed to determine which morphologic form and species of Leishmania is most suitable for detection of antibody in sera from American visceral leishmaniasis (AVL) patients by the indirect fluorescent antibody test (IFAT). Promastigotes and amastigotes of Leishmania mexicana or Leishmania donovani chagasi were used as sources of antigen. A total of 70 sera, including 30 from AVL patients, 30 from healthy subjects and 10 from Chagas' disease patients, were used in the study. Titers of antibody up to a dilution of 1:64 were found with all four antigens. At a titer of 1:128, the sensitivity of the IFAT using L. d. chagasi promastigotes as a source of antigen was 100% and the specificity at a titer of 1:32 was 98%. Although the sensitivity of the amastigote forms was close to 100% at a similar titer, the specificity at a titer of 32 using the L. d. chagasi amastigotes was 91% and using L. mexicana amastigotes was only 80%. The L. d. chagasi promastigote antigen was also the one that showed less cross reaction with sera from Chagas' disease patients. Since cross reactivity between Trypanosoma cruzi and Leishmania species is well known in serological tests, and minimizing of such cross reactivity is of critical importance for diagnosis, we suggest that L. d. chagasi promastigotes should be the antigen of choice for diagnosis of visceral leishmaniasis by IFAT in areas also endemic for trypanosomiasis.

Suppression of lymphocyte proliferative responses by sera from patients with American visceral leishmaniasis.

We examined the effect of sera from 11 patients with American visceral leishmaniasis on mitogen-driven lymphocyte proliferative capacity. All sera inhibited lymphocyte proliferation of patients' peripheral blood mononuclear cells (PBMC) when stimulated by either phytohemagglutinin, Concanavalin A or pokeweed mitogen. Serum was also strongly inhibitory for Concanavalin A-pulsed normal volunteers' PMBC. The effect of the serum was not due to cytotoxicity, inadequate nutritional support or altered kinetics of DNA synthesis. High levels of IgM or IgG (both total and antiparasite) and high levels of triglycerides were found in patients' sera.

Isolation of Leishmania mexicana amazonensis from the bone marrow in a case of American visceral leishmaniasis.

The first documented human case of visceral leishmaniasis caused by L. mexicana amazonensis is reported. Leishmania were isolated from bone marrow aspirate material from a typical visceral leishmaniasis patient. Further characterization by isoenzyme electrophoresis and by a panel of species- and subspecies-specific monoclonal antibodies established its classification as L. m. amazonensis.

Evaluation of the micro enzyme-linked immunosorbent assay (ELISA) for antibodies in American visceral leishmaniasis: antigen selection for detection of infection-specific responses.

This study was designed to evaluate the ELISA for diagnosis of American visceral leishmaniasis (AVL) using antigen prepared from different Leishmania isolates and from a strain of Trypanosoma cruzi. Two Leishmania donovani chagasi isolates from Bahia and Maranhão (both states of northern Brazil), one L. donovani from Sudan, one L. mexicana amazonensis isolate, and one T. cruzi isolate were used. A total of 375 sera were tested, including 119 from AVL patients, 96 from nonleishmaniasis hospitalized patients, 20 from healthy persons, 30 from patients with mucocutaneous leishmaniasis, 28 from patients with Chagas' disease, 20 from patients with tuberculosis, 21 from leprosy patients, 27 from schistosomiasis patients and 14 from patients with systemic mycoses. The antigens prepared from L. d. chagasi (Bahia) and L. m. amazonensis showed the highest sensitivity (98% and 99%, respectively) for detecting antibodies in sera from AVL patients. However, the specificity of L. d. chagasi (Bahia) antigen was better than that of L. m. amazonensis (96% vs. 86%). Comparison among the three L. donovani isolates demonstrated that the antigen prepared with the isolate from the same area where the sera originated yielded higher mean absorbance than the others. By using spectrophotometric absorbance values it was possible to use a single dilution of serum (between 1/100-1/400) since a clear separation was seen between AVL patients and controls. No patients with the other diseases who were tested gave positive results. We suggest that ELISA can be a very convenient, sensitive, and specific test for diagnosis of AVL when soluble antigen, preferably from an isolate from the test area, is used.

Characteristics of the acquired immunodeficiency syndrome in Brazil.

The clinical and epidemiologic characteristics of the acquired immunodeficiency syndrome (AIDS) were studied in a tropical area of Brazil. During an 18-month period (July 1989-January 1991), 111 consecutive AIDS patients (102 men and nine women) were evaluated. Patients reported homosexual/bisexual activities (60%), intravenous drug use (19%), or both (6%), heterosexual activities (11%), blood transfusions (2%), and 2% belonged to an undetermined category. Weight loss, fever, oral thrush, and diarrhea were present in > or = 70% of the patients at presentation. An unexpected high frequency of hepatomegaly (49%) was detected, and found to be significantly associated with tuberculosis (P < 0.0001). Although the epidemiologic features of human immunodeficiency virus transmission were comparable to the United States/European pattern, the clinical spectrum of opportunistic infections more closely resembled that reported in Africa and Haiti, with a greater frequency of fungal and mycobacterial infections than Pneumocystis carinii pneumonia and viral infections.

A rapid and simple diagnostic test for active visceral leishmaniasis.

We have developed an immunodot assay for the serodiagnosis of active visceral leishmaniasis (AVL) which utilizes protein A colloidal gold as the visualizing agent. The test is simple, requires few reagents, and can be completed in two hours. It is sensitive and specific for active visceral leishmaniasis, and generally correlates with the ELISA. Either whole blood or sera in minute quantities may be used as test antibody. In addition, the use of the protein A gold immunodot is shown to detect anti-leishmania antibodies in infected dogs.

Selection of a skin test antigen for American visceral leishmaniasis.

Studies were designed to examine skin test responses to leishmanial antigens in American visceral leishmaniasis (AVL) in Brazil. We found that after recovery from AVL, patients had positive delayed hypersensitivity reactions to Leishmania. Different amounts of a soluble extract obtained from Leishmania donovani chagasi promastigotes were compared with whole L. d. chagasi promastigotes in persons with past AVL. The most effective soluble preparations tested contained 25 and 50 micrograms leishmanial protein. These produced positive responses in 95%-100% of the individuals with past AVL. The 25 micrograms protein dose was used in further studies. This preparation produced no positive responses in either normal controls, tuberculosis patients, or schistosomiasis patients, and less than 5% positive responses in persons with Chagas' disease. The same amount of soluble extract prepared from L. mexicana amazonensis produced 82% positive skin test responses in persons with past AVL. When persons living in an area endemic for AVL were skin tested with the 25 micrograms preparation of L. d. chagasi extract, 34.1% yielded positive tests with a low number of positive responses in young children and 48% positive in adults. Only 3.1% of the population studied had a history of AVL. We have found that positive delayed hypersensitivity response to a soluble Leishmania extract is a sensitive and specific indicator of previous infection with AVL.

Studies on the control of visceral leishmaniasis: validation of the Falcon assay screening test--enzyme-linked immunosorbent assay (FAST-ELISA) for field diagnosis of canine visceral leishmaniasis.

The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA), the latest version of the enzyme-linked immunosorbent assay, uses antigen-coated beads. A 96-well plate can be run in 20 min without electricity or expensive equipment. We compared the FAST-ELISA, a standard ELISA, and an indirect immunofluorescent assay (IFA) for evaluation of canine leishmaniasis under field conditions using samples from 161 dogs from our longitudinal study in the endemic area of Jacobina, Bahia, Brazil. Organisms were isolated by culture (NN medium) or by inoculation of hamsters with samples from 59 of the dogs. When plasma were tested, we found a sensitivity of 88% and a specificity of 90% using the FAST-ELISA with a spectrophotometer. Using the same plasma samples, the IFA had a sensitivity of 75% and a specificity of 93%. The standard ELISA had a sensitivity of 90% and a specificity of 85%. When whole blood was tested with the FAST-ELISA, we found a sensitivity of 85%. There was no significant difference between visual and spectrophotometric results with plasma or whole blood. The FAST-ELISA system provides a sensitive, specific, and field-adaptable test for canine visceral leishmaniasis, which can be evaluated quickly without the use of a microscope or spectrophotometer.