Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens

The present report describes an alternative method for in vitro detection of HIV-1 -specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analized. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

Molecular and biological diversity of HIV-1 in Brazil

To determine the genomic polymorphism and biological properties present in HIV-1 Brazilian isolates, were analyzed five viral isolates obtained from patients residing in Rio de Janeiro (P1 and P5), São Paulo (P3) and Bahia (P2 and P4) states. For each viral isolate in vitro characteristics such as replication rate, syncytium-inducing capacity and cell death were observed in lymphoblastoid (H9, CEM and peripheral blood mononuclear cells) as well as monocytoid (U937) cells. In addition, the evaluation of the restriction fragment lenght polymorphism of these isolates was also performed using a panel of endonucleases such as Hind III, Bgl II, Sac I, Pst I, Kpn I and Eco RI. One of the isolates (P1), showed the highest phenotypic and genotypic divergence, when compared to others. The results found suggest a HIV heterogeneity in Brazil similar to that already described in other regions of the world.

Cytokines and dysregulation of the immune response in human malaria

The dysregulation of the immune response by malaria parasite has been considered as a possible constraint to the effectiveness of malaria vaccination. In spite of the important role interleukin-I (IL-1) in malaria are lacking. We found that only 2 out of 35 subjectswith acute malaria showed increased levels of serum IL-1 alpha by enzyme immunoassay. To assess whether IL-1 could interfere with T- lymphocyte responses, blood mononuclear cells from patients infected with Plasmodium falciparum, P. vivax, or healthy subjects were cultured with phytohemagglutinin, and lymphocyte proliferation measured 72h later by 3H-thymidine incorporation. Our data showed that T-lymphocyte responses are depressed both in P. falciparum (10,500 ñ 2,900) and P. vivax malaria (13,000 ñ 3,300), as compared to that of healthy individuals (27,000 ñ 3,000). Addition of IL-1 partially reserved depression of malaria lymphocytes, but had no effect on normal cells. On the other hand, T-lymphocytes from malaria infected-subjects presented a minimal decrease in proliferation, when cultured in the presence of exogenous PGE2. These data indicate the occurrence of two defects of immunoregulation in malaria: a deficiency of IL-1 production by monocytes/macrophages, and an increased resistance of lymphocytes to the antiproliferative effect of PGE2.

Vaccine strategies against schistosomiasis

In this review the authors analyze the effector and regulatory mechanisms in the immune response to schistosomiasis. To study these mechanisms two animal models were used, mouse and rat. The mouse totaly permissive host like human, show prominent-T cell control in the acquisition of resistance. But other mechanisms like antibody mediated cytotoxity (ADCC) involving eosinophils and IgG antibodies described in humans, are observed in rats. Also in this animal, it is observed specific IgE antibody high production and blood and tisssue eosinophilia. Using the rat model and schistosomula as target, some ADCC features have emerged: the cellular population involved are bone marrow derived inflammatory cell (mononuclear phagocytes, eosinophils and platelets), interacting with IgE through IgE Fc receptors. Immunization has been attempted using the recombinant protein Sm28/GST. Protection has been observed in rodents with significant decrease of parasite fecundity and egg viability affecting the number, size and volume of liver egg granulomas. The association of praziquantel and immunization with with Sm28/GST increases the resistance to infection and decreases egg viability. The authors suggest the possibility of the stablishment of a future vaccine against Schistosoma mansoni.

Immunological profiles of patients from endemic areas infected with Schistosoma mansoni

Crude extracts of eggs (SEA) adult worms (SWAP) or cercariae (Cerc) have been used to stimulate Peripheral Blood Mononuclear cells (PBMC) and have provided rather distinct profiles of responses in different types of patients. In genenral it is clear that patients with early infections respond strongly to SEA while response to SWAP are developed more slowly. As infection progresses into the more chronic phases, a general pattern is seen whic leads to lower anti-SEA proliferative responses in the face of higher responses to SWAP and variable anti-cerc responsiveness. Cured not re-exposed patients express very high levels of anti-SEA proliferation. It has recently been seen that those individuals who live in endemic areas and have continued water contact, but are reapeatedly stool-negative (who are presumed to have self-cured or be putatively resistant; endemic normals) are strongly responsive to antigenic extracts, particularly to SEA. Furthermore, our results show that endemic normal individuals have significantly higher IFN gamma production upon PBMC stimulation with schistosome antigens than infected individuals. With the emergence of more studies it is becoming apparent that both the intensity and the prevalence of a given area may influence or shape the general responsiveness of the population under study.

Evaluation of spleen cell population and effect of splenectomy on granuloma modulation in BALB/c mice infected with Schistosoma mansoni

A kinetic study of the cells present in the spleen of BALB/c mice infected with Schistosoma mansoni was carried out. The lymphocytes were evaluated phenotypically with monoclonal antibodies and the effect of splenectomy on the modulation of periovular granuloma was also investigated. The infected mice had proportional increases in the numbers of neutophils, plasma cells, macrophages and eosinophils in the spleen. The largest number of neutrophil, plasma cells and macrophage were found between the 8th and the 12th week of infection, while the amount of eosinophils were higher later on, around the 20th week. The lymphocytes phenotipically characterized as Thy 1.2, Lyt 1.2 (CD4) increased mildly in proportional numbers. However, the percentage of lymphocytes with the Lyt 2.2 (CD8) phenotype, which is characteristic of supressor and cytotoxic T cells, increased significantly with the progress of the disease. The numbers of B lymphocytes, which comprise 50% of the mononuclear cells present in the spleen, increased significantly till the 16th week they began to decrease. The mean diameters of periovular granulomas were comparatively similar in both experimental groups (splenectomized and non-splenectomized mice). However, the evolutive types of granuloma (exudative, intermediate and fibrous) in splenectomized mice were proprtionally different from those of non splenectomized mice in the 16th and 24th week of infection. It is inferred that lymphonodes or other secondary lymphoide organs, in the abscence of the spleen, assume a modulating action on periovular granulomas, although the evolution of the granulomas is somehow delayed in splenectomized mice.

CCR2(+) monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice.

Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.

Modulatory Role of the Ovarian Function in Neuroimmunoendocrine Axis Activity.

The aim of this study was to evaluate the effect of ovariectomy on the acute-phase response of inflammatory stress. Ex vivo adrenocortical, peripheral mononuclear cell (PMNC) and adipocyte activities were studied in intact and ovariectomized mice. Endotoxemia was mimicked by intraperitoneal administration of bacterial lipopolysaccharide (LPS; 25 mg per mouse) to sham-operated and 21-day ovariectomized mice. Circulating corticosterone, tumor necrosis factor-alpha (TNFalpha) and leptin concentrations were monitored before and 30-120 min after the administration of LPS. Additionally, in vitro experiments were performed with isolated corticoadrenal cells, PMNCs and omental adipocytes from sham-operated and ovariectomized mice incubated with specific secretagogues. The results indicate that while ovariectomy enhanced TNFalpha secretion after in vivo administration of LPS, it reduced corticoadrenal response and abrogated LPS-elicited leptin secretion into the circulation. While the corticoadrenal sensitivity to ACTH stimulation was reduced by ovariectomy, the LPS-induced PMNC response was not affected. Exogenous leptin enhanced baseline PMNC function regardless of surgery. Finally, ovariectomy drastically reduced in vitro adipocyte functionality. Our data support the notion that ovariectomy modified neuroendocrine-immune-adipocyte axis function and strongly suggest that ovarian activity could play a pivotal role in the development of an adequate immune defense mechanism after injury.

Propietats protectores de la lipoproteïna de alta densitat (HDL) sobre l'acció inflamatòria de la lipoproteïna de baixa densitat electronegativa (LDL(-)) en monòcits humans

Electronegative low-density lipoprotein (LDL(-)) is a modified fraction of LDL present in peripheral blood whose proportion is elevated in subjects with increased cardiovascular risk. LDL(-) has been shown to have an inflammatory effect on human endothelial cells and mononuclear blood cells. On the other hand, high-density lipoprotein (HDL) is known to have a protective effect against cardiovascular disease, partly mediated by its anti-inflammatory properties. The objective of the current work is to study the putative protective properties of HDL towards the inflammatory effect of LDL(-) in human monocytes, in order to elucidate the mechanisms behind their interaction. Total LDL and HDL were isolated by ultracentrifugation and LDL(-) was obtained from total LDL by anion exchange chromatography. HDL and LDL(-) were incubated together and then re-isolated, and their characteristics were compared to those of untreated lipoproteins. The inflammatory activity of the lipoproteins was determined by incubating monocytes with lipoproteins and measuring cytokine release from the cultured monocytes. The biochemical composition and electrophoretic mobility of the lipoproteins were also determined before and after their interaction. Incubation of HDL with LDL(-) reduced the inflammatory effect of LDL(-) and, in turn, HDL gained inflammatory properties. This indicates a transfer of inflammatory potential taking place during the interaction of LDL(-) and HDL. Additionally, LDL(-) lost non-esterified fatty acids (NEFAs) while HDL gained the same. We conclude that a transfer of NEFAs takes place between LDL(-) and HDL. These observations suggest that NEFAs play a role in the inflammatory effect mediated by LDL(-).

Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology

Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC) and cloned. These T cell clones (TCC) were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%). On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%), bradycardia with megacolon (75 %) and bradycardia (75%). Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

Sustained improvement in left ventricular function after bone marrow derived cell therapy in patients with acute ST elevation myocardial infarction. A 5-year follow-up from the Stem Cell Transplantation in Ischaemic Myocardium Study.

BACKGROUND: Intracoronary injection of autologous bone marrow-derived mononucleated cells (BM-MNC) may improve LV function shortly after acute ST elevation myocardial infarction (STEMI), but little is known about the long-term durability of the treatment effect. METHODS: In a single-centre trial a total of 60 patients with acute anterior STEMI, successful reperfusion therapy and a left ventricular ejection fraction (LVEF) of <50% were screened for the study. 23 patients were actively treated with intracoronary infusion of BM-MNC within a median of 3 days. The open-label control group consisted of 19 patients who did not consent to undergo BM-MNC treatment but agreed to undergo regular clinical and echocardiographic follow-up for up to 5 years after AMI. RESULTS: Whereas at 4 months there was no significant difference between the increase in LVEF in the BM-MNC group and the control group (+7.0%, 95%CI 3.6; 10.4) vs. +3.9%, 95%CI -2.1; 10), the absolute increase at 5 years remained stable in the BM-MNC but not in the control group (+7.95%, 95%CI 3.5; 12.4 vs. -0.5%, 95%CI -5.4; 4.4; p for interaction between groups = 0.035). DISCUSSION: In this single-centre, open-labelled study, intracoronary administration of BM-MNC is feasible and safe in the short term. It is also associated with sustained improvement of left ventricular function in patients with acute myocardial infarction, encouraging phase III studies to examine the potential BM-MNC effect on clinical outcome.

Chronic experimental infection by Trypanosoma cruzi in Cebus apella monkeys

Twenty young male Cebus apella monkeys were infected with CAl Trypanosoma cruzi strain and reinfected with CA l or Tulahuen T.cruzi strains, with different doses and parasite source. Subpatent parasitemia was usually demonstrated in acute and chronic phases. Patent parasitemia was evident in one monkey in the acute phase and in four of them in the chronic phase after re-inoculations with high doses of CAl strain. Serological conversion was observed in all monkeys; titers were low, regardless of the methods used to investigate anti-T. cruzi specific antibodies. Higher titers were induced only when re-inoculations were perfomed with the virulent Tulahuén strain or high doses of CAl strain. Clinical electrocardiographic and ajmaline test evaluations did not reveal changes between infected and control monkeys. Histopathologically, cardiac lesions were always characterized by focal or multifocal mononuclear infiltrates and/or isolated fibrosis, as seen during the acute and chronic phases; neither amastigote nests nor active inflammation and fibrogenic processes characteristic of human acute and chronic myocarditis respectively, were observed. These morphological aspects more closely resemble those found in the "indeterminate phase" and contrast with the more diffuse and progressive pattern of the human chagasic myocarditis. All monkeys survived and no mortality was observed.

Induction of circulating tumor-reactive CD8+ T cells after vaccination of melanoma patients with the gp100 209-2M peptide.

Patients with stage I-III melanoma were vaccinated with the modified HLA-A2-binding gp100(209-2M)-peptide after complete surgical resection of their primary lesion and sentinel node biopsy. Cytoplasmic interferon-gamma production by freshly thawed peripheral blood mononuclear cells (direct ex vivo analysis) or by peripheral blood mononuclear cells subjected to 1 cycle of in vitro sensitization with peptide, interleukin-2, and interleukin-15 was measured following restimulation with the modified and native gp100 peptides, and also A2gp100 melanoma cell lines. Peptide-reactive and tumor-reactive T cells were detected in 79% and 66% of selected patients, respectively. Patients could be classified into 3 groups according to their vaccine-elicited T-cell responses. One group of patients responded only to the modified peptide used for immunization, whereas another group of patients reacted to both the modified and native gp100 peptides, but not to naturally processed gp100 antigen on melanoma cells. In the third group of patients, circulating CD8 T cells recognized A2gp100 melanoma cell lines and also both the modified and native peptides. T cells with a low functional avidity, which were capable of lysing tumor cells only if tumor cells were first pulsed by the exogenous administration of native gp100(209-217) peptide were identified in most patients. These results indicate that vaccination with a modified gp100 peptide induced a heterogeneous group of gp100-specific T cells with a spectrum of functional avidities; however, high avidity, tumor-reactive T cells were detected in the majority of patients.

Individual contributions of mutant protease and reverse transcriptase to viral infectivity, replication, and protein maturation of antiretroviral drug-resistant human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity. The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as "fitness") in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background. Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs. In peripheral blood mononuclear cells, replication rates (means +/- standard deviations) of RT recombinants were 72.5% +/- 27.3% and replication rates of PR recombinants were 60.5% +/- 33.6% of the rates of NL4-3. PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% +/- 23.5% of NL4-3 replication rates. Cloning of the cognate RT improved fitness of some PR mutant clones. For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% +/- 10.7%, and integrase content diminished by 23.3% +/- 18.4%), a novel mechanism for a decreased-fitness phenotype. Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT. Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure. These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Evaluation of the rabbit as a model for Chagas disease - II: histopathologic studies of the heart, digestive tract and skeletal muscle

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, seventy one animals were inoculated with different Trypanosoma cruzi strains and routes. The rabbits were submitted to necropsy in acute (earlier than three months of infection), recent chronic (three to six months) and late chronic (later than six months) phases. Myocarditis, generally focal and endomysial, occurred in 94.1%, 66.7% and 70.8% of the infected rabbits respectively in the acute, recent chronic and late chronic phases. The myocardial inflammatory exudate was composed by mononuclear cells, and also polymorphonuclear cells in the acute phase. In most cases of the late chronic phase, the myocarditis was similar to that described in the indeterminate form of human chagasic patients. Initial fibrosis occurred in the three phases but was more severe and frequent in the early chronic. Advanced fibrosis occurred only in the late chronic phase. Tissue parasites occurred only in the acute phase. The digestive tract and skeletal muscles showed mild and occasional lesions. Our data indicate that experimentally infected chagasic rabbits repeat some lesions similar to that of humans chagasic patients, specially that of the indeterminate form. So, it may be a useful, however not an ideal, model.