Analysis of iron superoxide dismutase-encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection.

The present work aimed to evaluate the immunogenicity of Leishmania amazonensis iron superoxide dismutase (SOD)-encoding DNA experimental vaccine and the protective properties of this DNA vaccine during infection. The SOD gene was subcloned into the pVAX1 plasmid, and it was used to immunize BALB/c mice. Twenty-one days after the last immunization, mice were sacrificed (immunogenicity studies) or subcutaneously challenged with L. amazonensis (studies of protection), and alterations in cellular and humoral immune responses were evaluated, as well as the course of infection. Mice only immunized with pVAX1-SOD presented increased frequencies of CD4(+) IFN-γ(+), CD8(+)IFN-γ(+) and CD8(+)IL-4(+) lymphocytes; moreover, high levels of IgG2a were detected. After challenge, mice that were immunized with pVAX1-SOD had increased frequencies of the CD4(+)IL-4(+), CD8(+)IFN-γ(+) and CD8(+)IL-4(+) T lymphocytes. In addition, the lymph node cells produced high amounts of IFN-γ and IL-4 cytokines. Increased IgG2a was also detected. The pattern of immunity induced by pVAX1-SOD partially protected the BALB/c mice from a challenge with L. amazonensis, as the animals presented reduced parasitism and lesion size when compared to controls. Taken together, these results indicate that leishmanial SOD modulates the lymphocyte response, and that the elevation in IFN-γ possibly accounted for the decreased skin parasitism observed in immunized animals.

HCV viremia drives an increment of CD86 expression by myeloid dendritic cells.

The host immune response, including innate and adaptive immunity, plays a critical role in determining the outcome of viral infection. Nevertheless, little is known about the exact reasons for the failure of the host immune system in controlling hepatitis C virus (HCV) infection. Impairment of dendritic cells (DCs) function is probably one of the mechanisms responsible for immune evasion of HCV. In this study, the frequency and phenotype of DCs subsets were analyzed in three groups: HCV-infected individuals who developed viral persistence (1), HCV-infected individuals who spontaneously cleared the virus (2) and HCV-seronegative uninfected subjects (3). The results showed that the frequency of DCs subsets was not statistically significant between groups. Plasmacytoid DCs circulating exhibited an immature phenotype characterized by low expression of CD86. On the other hand, CD86 expression in myeloid DCs was significantly higher in chronic infected individuals compared to healthy controls (P=0.037). A positive correlation was observed between CD86(+) myeloid DC (mDC) and HCV viral load (r=0.4121, P=0.0263). These results suggest that HCV did not have an inhibitory effect on mDC maturation and the HCV viremia drives the increase of CD86 expression in mDC. The regulation of DCs maturation and migration lies at the level of intracellular signaling. HCV can activate or block intracellular signaling pathways and alter DC function. In conclusion, the present study suggests that imbalance of DC maturation by the virus represents a mechanism of evasion of the immune system despite the fact that HCV viremia appears to exert a "stimulatory" effect on cell-surface immune phenotype.

Lower numbers of circulating Natural Killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV-1) associated neurological disease.

Human T lymphotropic virus type 1 (HTLV-1) infects 10-20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4(+) and fewer CD8(+) cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4(+) NK T subset are associated with HTLV-1 disease progression.

Genealogical evidence for positive selection in the nef gene of HIV-1.

The pattern and process of evolution in the nef gene of HIV-1 was analyzed within and among patients. Using a maximum likelihood method that allows for variable intensity of selection pressure among codons, strong positive selection was detected in a hemophiliac patient over 30 mo of infection. By reconstructing the process of allele substitution in this patient using parsimony, the synapomorphic amino acid changes separating each time point were found to have high probabilities of being under positive selection, with selective coefficients of at least 3.6%. Positive selection was also detected among 39 nef sequences from HIV-1 subtype B. In contrast, multiple pairwise comparisons of nonsynonymous and synonymous substitution rates provided no good evidence for positive selection and sliding window analyses failed to detect most positively selected sites. These findings demonstrate that positive selection is an important determinant of nef gene evolution and that genealogy-based methods outperform pairwise methods in the detection of adaptive evolution. Mapping the locations of positively selected sites may also be of use in identifying targets of the immune response and hence aid vaccine design.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6.

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80 degrees C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Effects of interleukin-15 on in vitro human T cell proliferation and activation.

Interleukin-15 (IL-15) has been reported to have many activities on T cell populations, including a potential role in improving antigen-specific proliferation in HIV-1 disease. We tested this response in healthy adults by studying the response of T cell populations after stimulation with medium, tetanus, cytomegalovirus (CMV) antigens in cultures from 21 volunteers. IL-15 caused a dose-dependent increase in medium and antigen-induced proliferation. The expansion was due to CD8>natural killer (NK)>CD4 lymphocytes and memory > naive cells. The IL-15-stimulated CD8 cells had increased levels of the activation markers CD69 and DR. The published CMV-induced expression of CD57 on CD8+ cells was increased in CMV seronegative and seropositive subjects by IL-15. IL-15 appears to be a stimulator of T cell populations in healthy adults and may be useful in settings to enhance nonspecific NK activity or antigen-specific CD8 activity.

Production of influenza-stimulated tumor necrosis factor-alpha by monocytes following acute influenza infection in humans.

Tumor necrosis factor-alpha (TNF-alpha) is often measured in the serum or plasma of patients with severe infections, and marked elevation correlates with poor outcome. The relationship of TNF-alpha to protection from disease is frequently not observed because prospective studies of infectious agents are difficult to perform. We took advantage of a human antiviral influenza challenge study to correlate TNF-alpha production with seroconversion and symptom development. TNF-alpha production was measured by ELISA in the plasma compartment or was measured by intracellular production at the single cell level in the monocyte gated population. Monocyte TNF-alpha was associated with asymptomatic seroconversion, whereas there was no change in the plasma at the times measured. Measurement of TNF-alpha at the single cell level by flow cytometry may allow for better differentiation of the protective role of this cytokine in future studies.

Cytomegalovirus-specific IFNgamma and IL-4 are produced by antigen expanded human blood lymphocytes from seropositive volunteers.

The cytokine responses to cytomegalovirus (CMV) antigen in seropositive and seronegative individuals were measured using a combination of antigenic expansion and intracellular staining. Intracellular IFNgamma and IL-4 were produced in a dose-dependent manner by T cells in response to CMV only in the seropositive population. The potential for individual cells to produce both Th1 and Th2 cytokines simultaneously was clear, as IL-4 was most often produced in those cells with the highest IFNgamma production. The cytokine-specific nature of this response was demonstrated by fluorescence microscopy, which showed granular cytoplasmic staining, and at the mRNA level by ribonuclease protection assays. These methods expand our ability to evaluate the immune response to CMV, and can now be correlated to a number of clinical conditions.

Evaluation of canarypox-induced CD8(+) responses following immunization by measuring the effector population IFNgamma production.

CD8(+) cytolytic activity is traditionally measured by detecting the release of 51Cr after incubation of effector cells with HLA-matched, infected, radiolabeled targets. An alternative method to detect CD8+ activity is to measure the production of intracellular interferon gamma (IFNgamma) after antigen-specific stimulation, either by ELISPOT or by flow cytometry. Studies were performed in 19 volunteers enrolled in a phase 1 trial of candidate canarypox HIV-1 vaccines that encoded multiple HIV-1 genes. The vaccines including vCP205 (Env, Gag, and protease), vCP1433 (Env, Gag, protease, and CTL epitope-rich regions of pol and nef) and vCP1452 (equivalent to vCP1433 with additional immunomodulatory genes of vaccinia). PBMCs were stimulated in vitro with vaccinia constructs encoding env and gag or a lacZ control, and the effectors were cultured for 12-14 days. EBV-transformed B cell lines were infected overnight with the vaccinia vectors, and then incubated with the effector cells for 4 h in the presence of monensin. CD8(+) gene-specific activity was determined as a percentage of IFNgamma cells in the CD3(+)CD8(+)CD45RO(+) gate after subtracting both the isotype control and the lacZ control stimulation. CD4 memory IFNgamma production was simultaneously determined in the CD3(+)CD8(-)CD45RO(+) gate. Using these techniques in blinded studies, we found that CD8(+) IFNgamma activity could be measured in the majority of volunteers given four immunizations. Specifically, the responses to the gag gene were control -- 0/2; vCP205 -- 2/4; vCP1433 -- 5/6; vCP1452 -- 4/7. Most of the positive responses were detected after the fourth immunization. Flow cytometric techniques hold promise as a surrogate measure of CTL and for ease of phenotyping of the effector population.

Primary isolate neutralization by HIV type 1-infected patient sera in the era of highly active antiretroviral therapy.

Sera from highly selected HIV-1-positive patients are known to have the ability to neutralize a diverse array of primary isolates of HIV-1. The human osteosarcoma cell line that expresses CD4 and chemokine receptors (GHOST cells) was adapted to study HIV-1 neutralization in 37 HIV-1-infected individuals who were selected because of slow disease progression or nonprogression. Many of these individuals were receiving combination drug therapy. Molecularly cloned HIV-1 JR-FL and NL4-3 viruses were used as prototypes to define assay conditions. Sera were then tested at a 1:40 dilution against six additional primary isolates, three of which utilized CCR5 and three of which used both CCR5 and CXCR4. The assay was highly reproducible and independent of viral input titer, with a readout at 48 hr equivalent to that at later time points. As previously reported, neutralization sensitivity was entirely independent of coreceptor usage. Only a few sera from slow progressors were able to neutralize a broad array of primary isolates at a 1:40 dilution, and the best clinical predictor of broadly neutralizing antibody for primary isolates was the present use of antiretroviral agents. In further studies it was found that purified antibody accounted for the majority of the measured neutralization. However, experiments with exogenous addition of antiviral agents showed that the use of nucleosides also greatly contributed to the measured neutralization in some patients. Measurement of neutralization of HIV-1 primary isolates by sera from patients receiving antiretroviral therapy must be carried out with some caution.

Pathogenetic aspects of sepsis and possible targets for adjunctive therapy.

The outcome of patients with sepsis arises from multiple factors affecting both the host and the invading microorganisms. Age, presence of underlying disease, source of infection and some specific etiological agents have been related to prognosis. Appropriateness of antimicrobial therapy, considering the in vitro susceptibility tests for the infecting bacteria, has been strongly associated with the outcome. Therefore even after the cascade of sepsis has been triggered, the control of bacteria growth is still fundamental for the outcome of the infection. This is a major distinction point from experimental studies in which whole killed bacteria and their products are used as model of sepsis. However, even within the setting of adequate antimicrobial use, patients still die of sepsis. Thus, strategies focusing on further therapy targets are an important area of interest for basic and clinical research. Although such adjunctive sepsis therapy has failed to achieve consistent better survival rates so far, nevertheless, it improved our understanding of the pathophysiological events seen in sepsis that the possibility that a new and effective treatment may arise has been warmly considered. In this paper we aim to review some aspects of the pathogenesis of sepsis, focusing on recent advances and on possible targets for adjunctive therapy. Published clinical trials and experimental data supporting such trials are commented on.

Azole resistance among oral Candida species isolates from AIDS patients under ketoconazole exposure.

This is a prospective study designed to investigate species distribution and azole susceptibility profile among Candida spp. isolated from the oral cavities of AIDS patients. One hundred thirty-two AIDS patients sequentially admitted at a teaching tertiary care hospital were enrolled in this study. Samples were obtained by swabbing the oral cavities of the patients. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was further determined according to the NCCLS microbroth assay. Among all patients with prescriptions of systemic antifungal drugs, ketoconazole had been elected to treat 56% of patients. We found 82% of oral yeast carriage, 22% of them harboring non-albicans species. Overall rates of susceptibility dose dependent/resistance to azoles was 16% for itraconazole, 13% for ketoconazole, and 10% for fluconazole with a high agreement rate among the susceptibility profiles of all isolates tested against the triazoles.

Cytomegalovirus antigenemia in acquired immunodeficiency syndrome patients with untreated cytomegalovirus retinitis.

PURPOSE: To determine the frequency of cytomegalovirus (CMV) viremia in patients with acquired immunodeficiency syndrome (AIDS) and untreated CMV retinitis using conventional cell culture isolation and the sensitive CMV antigenemia assay. METHODS: We examined 24 AIDS patients with ophthalmologic diagnosis of untreated CMV retinitis and 24 AIDS patients without present or past retinitis (control patients) from three medical centers between September 1992 and March 1994. Cytomegalovirus antigenemia was detected by an indirect peroxidase staining in 300,000 cytocentrifuged neutrophils, using a mixture of murine monoclonal antibodies directed against the pp65 lower matrix protein of CMV. RESULTS: Positive antigenemia was demonstrated in eight (33.3%) of the 24 retinitis patients and in none of the 24 control patients (P < .001). Only two of the eight antigenemia-positive patients had a concurrent positive CMV isolation from blood leukocytes by conventional cell culture assay. CONCLUSIONS: These results emphasize the risk of extraocular disease in AIDS patients with CMV retinitis because the virus is often present in peripheral blood leukocytes. The CMV antigenemia assay may be a simple and rapid means of identifying those patients with unilateral retinitis at highest risk of developing CMV retinitis of the fellow eye or of visceral CMV disease if intravitreal injections or implants are used as sole treatment for CMV retinitis.

High prevalence of hepatitis C infection in a Brazilian prison: identification of risk factors for infection.

Hepatitis C virus (HCV) causes infectious hepatitis worldwide. It is transmitted mainly by blood products and sharing of intravenous paraphernalia during illicit drug use. High prevalence rates have been described among specific groups considered to be at higher risk for HCV infection, including prison inmates. The objectives of this study were: to determine the HCV seroprevalence among inmates of Casa de Detenção de São Paulo; to identify risk factors for HCV infection; and to compare the seroprevalence of HCV to other blood borne or sexually transmitted diseases. From December, 1993, to January, 1994, a total of 779 inmates were interviewed to collect information on sociodemographic status, sexual behavior, and past experience with illicit drugs. Blood samples were obtained from 756 inmates for serological tests. 310 (41%) blood samples were positive for anti-HCV, 425 (56.2%) were negative, and 21 (2.8%) showed indeterminate results. In this population, we found a seroprevalence of 13.7% for HIV, 3.3% for syphilis (VDRL), and 68.1% for hepatitis B virus previous infection. Four variables were each identified as associated with a positive anti-HCV serologic test: a positive VDRL (OR = 2.63 IC 95% 1.08 to 6.36); a time of current imprisonment longer than 130 months (OR = 2.44 IC 95% 1.04 to 5.71); previous incarceration at Casa de Detenção de São Paulo (OR = 1.73 IC 95% 1.19 to 2.52) and; illicit drug use before admission to the Casa de Detenção de São Paulo (OR = 1.64 IC 95% 1.15 to 2.33). The seroprevalence of HCV antibodies among the study population was high (41%), indeed, one of the highest clusters of HCV infection recorded until now. Four variables were each shown to be associated with HCV infection. The simultaneous presence of these 4 variables is associated with an 82% probability of being anti-HCV positive. Although risk factor analysis indicates most HCV infections occur prior to inprisonment, initiation of control measures to prevent continued transmission after incarceration should be done.

Etiological drug treatment of human infection by Trypanosoma cruzi.

Forty-nine American Trypanosomiasis (Chagas' disease) patients, with xenodiagnosis proven parasitemia were treated by the authors. Forty-one of these patients were given benznidazole, at dosages ranging from 5mg/kg/day to 8mg/kg/day, during a pre-established period of 60 days. In this group, 17 patients had an undetermined form of the disease, whereas 22 had cardiologic disease and 4 had digestive disease (two patients had a mixed form of the disease). Side effects were frequent, and led to the discontinuation of treatment in 17 patients. The follow-up period ranged from 1 to 20 years (mean follow-up period of 6 yrs. 7 mo). 26 (63.4%) of the patients became parasitemia-negative. The other eight patients were treated with nifurtimox, during 120 days, following a variable dose regime of 5mg/kg/day (initial dose) to 17 mg/kg/day (final dose). Six of them had severe side effects, and only one patient remained parasitemia-negative throughout the observation period (ranging from 1 to 18 years). Benznidazole proved to be better tolerated and more effective in the management of parasitemia when compared to nifurtimox, although more effective and less toxic drugs are still desirable.

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus.

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/high CD127 Ø/low FoxP3+, and effector T cells were defined as CD25+CD127+FoxP3 Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Peripheral blood telomere content is greater in patients with endometriosis than in controls.

UNLABELLED: The etiology of endometriosis remains poorly understood but circulating stem cells may contribute. Telomeres shorten with cell divisions and age. Stem cells attempt to compensate for telomere attrition through the action of telomerase. Since circulating stem cells may contribute to endometriosis, we compared telomere content in lymphocytes of patients with and without endometriosis. METHODS: Observational study comparing peripheral lymphocytes telomere content, measured by quantitative polymerase chain reaction, in patients with (n = 86) and without endometriosis (n = 21). FINDINGS: Patients with endometriosis had longer telomeres than that of matched, endometriosis-free controls (telomere to single copy gene ratio [T/S ratio] of 1.62 vs 1.34, respectively, P = .00002). Patients with endometriosis were 8.1-fold more likely to have long telomeres. (odds ratio = 8.1, 95% confidence interval: 1.28-51.57, P = .0264). INTERPRETATION: Longer telomeres could be consistent with a stem cell origin of endometriosis.

Investigation of human parvovirus B19 occurrence and genetic variability in different leukaemia entities.

Human parvovirus B19V (B19V) has been associated with various haematological disorders, but data on its prevalence in leukaemia are scarce. In this cross-sectional study, we investigated patients in Sao Paulo, Brazil with leukaemia to determine the molecular frequency of B19 variants and characterize the viral genetic variability by partial and complete sequencing of the coding of non-structural protein 1 (NS1)/viral capsid proteins 1 and 2 (VP1/VP2). The presence of B19V infections was investigated by PCR amplification of the viral NS1 gene fragment and confirmed by sequencing analysis. The NS1/VP1/VP2 and partially larger gene fragments of the NS1-positive samples were determined by overlapping nested PCR and direct sequencing results. The B19V NS1 was detected in 40 (16%) of 249 bone marrow samples including 12/78 (15.4%) acute lymphoblastic leukaemia, 25/155 (16.1%) acute myeloid leukaemia and 3/16 (18.7%) chronic myeloid leukaemia samples. Of the 40 participants, 25 (62.5%) were infected with genotype 1a and 15 (37.5%) with genotype 3b. The phylogenetic analysis of other regions revealed that 12/40 (30%) of the patients with leukaemia were co-infected with genotypes 1a and 3b. In addition, a new B19V intergenotypic recombinant (1a/3b) and an NS1 non-recombinant genotype 1a were detected in one patient. Our findings demonstrated a relatively high prevalence of B19V monoinfections and dual infections and provide, for the first time, evidence of inter-genotypic recombination in adults with leukaemia that may contribute to the genetic diversity of B19V and may also be a source of new emerging viral strains with future implications for diagnosis, therapy and efficient vaccine development.

Chemokines in the pathogenesis of endometriosis and infertility.

Endometriosis is a chronic benign disease that affects women of reproductive age causing abdominal pain and infertility. Its pathogenesis remains obscure despite all the research conducted over the past 100 years. However, there is a consensus among the specialists that the basis of its pathophysiology would be multifactorial. Many publications have demonstrated that chemokines are somehow associated with the development of endometriosis and infertility. In this study, we reviewed all PubMed literature using MeSH terms "chemokines" and "endometriosis" as well as "chemokines" and "female infertility" to establish what we know and what we do not yet know about this relationship.