Positive Lymphoproliferative Responses Research Articles

LeishmaniaLiHyC protein is immunogenic and induces protection against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems by the toxicity of drugs, high cost and/or emergence of resistant strains. The diagnosis is hampered by variable sensitivity and/or specificity of tests. In this context, prophylactic vaccination could represent a control measure against disease. In this study, the protective efficacy of Leishmania LiHyC protein was evaluated in a murine model against Leishmania infantum infection. LiHyC was used as recombinant protein (rLiHyC) associated with saponin (rLiHyC/S) or Poloxamer 407-based polymeric micelles (rLiHyC/M) to immunize mice. Animals received also saline, saponin or empty micelles as controls. The immunogenicity was evaluated before and after the challenge, and results showed that vaccination with rLiHyC/S or rLiHyC/M induced the production of high levels of interferon-gamma (IFN-γ), interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor in cell culture supernatants, as well as higher IFN-γ expression evaluated by RT-qPCR and involvement from CD4+ and CD8+ T-cell subtypes producing IFN-γ, tumor necrosis factor-α and IL-2. A positive lymphoproliferative response was also found in cell cultures from vaccinated animals, besides high levels of rLiHyC- and parasite-specific nitrite and IgG2a antibodies. Immunological assays correlated with significant reductions in the parasite load in the spleens, livers, bone marrows and draining lymph nodes from vaccinated mice, when compared to values found in the controls. The micellar composition showed slightly better immunological and parasitological data, as compared to rLiHyC/S. Results suggest that rLiHyC associated with adjuvants could be considered for future studies as a vaccine candidate against VL.

A recombinant Leishmania amastigote-specific protein, rLiHyG, with adjuvants, protects against infection with Leishmania infantum

Vaccination against visceral leishmaniasis (VL) should be considered as a control measure to protect against disease, and amastigote-specific proteins could help to develop such vaccines, since this parasite form is in contact with the host immune system during the active disease. In this study, a Leishmania amastigote-specific protein, LiHyG, was evaluated as recombinant protein (rLiHyG) as vaccine candidate against Leishmania infantum infection in BALB/c mice. The protein was associated with saponin (rLiHyG/Sap) or Poloxamer 407-based polymeric micelles (rLiHyG/Mic) as adjuvants, and animals receiving saline, saponin or micelle as controls. Immunological and parasitological analyses were performed before (n = 8 per group; as primary endpoint) and after (n = 8 per group; as secondary endpoint) infection. Results showed that, in both endpoints, rLiHyG/Sap and rLiHyG/Mic induced higher levels of IFN-γ, IL-12 and GM-CSF in spleen cell cultures from vaccinated animals, besides elevated presence of IgG2a isotype antibodies. Decreased hepatotoxicity and ‘positive lymphoproliferative response were also found after challenge. Such findings reflected in significantly lower levels of parasite load found in their spleens, livers, bone marrows and draining lymph nodes. In conclusion, rLiHyG associated with Th1-type adjuvant could be considered for future studies as vaccine candidate to protect against VL.

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Asymptomatic immune responders to Leishmania among HIV positive patients.

Concomitant infection with human immunodeficiency virus (HIV) and the Leishmania parasite is a growing public health problem, the result of the former spreading to areas where the latter is endemic. Leishmania infection is usually asymptomatic in immunocompetent individuals, but the proportion of HIV+ individuals in contact with the parasite who remain asymptomatic is not known. The aim of the present work was to examine the use of cytokine release assays in the detection of asymptomatic immune responders to Leishmania among HIV+ patients with no previous leishmaniasis or current symptomatology. Eighty two HIV+ patients (all from Fuenlabrada, Madrid, Spain, where a leishmaniasis outbreak occurred in 2009) were examined for Leishmania infantum infection using molecular and humoral response-based methods. None returned a positive molecular or serological result for the parasite. Thirteen subjects showed a positive lymphoproliferative response to soluble Leishmania antigen (SLA), although the mean CD4+ T lymphocyte counts of these patients was below the normal range. Stimulation of peripheral blood mononuclear cells (PBMC) or whole blood with SLA (the lymphoproliferative assay and whole blood assay respectively), led to the production of specific cytokines and chemokines. Thus, despite being immunocompromised, HIV+ patients can maintain a Th1-type cellular response to Leishmania. In addition, cytokine release assays would appear to be useful tools for detecting these individuals via the identification of IFN-γ in the supernatants of SLA-stimulated PBMC, and of IFN-γ, MIG and IL-2 in SLA-stimulated whole blood. These biomarkers appear to be 100% reliable for detecting asymptomatic immune responders to Leishmania among HIV+ patients.

Identifying t-cell epitopes of the VP1 capsid protein of human rhinovirus

Background The newly identified HRV-C has been linked to more severe symptoms of asthma and an increased risk of prior and subsequent hospital admission. Our research team has recently reported that despite their sequence dissimilarity, the HRV-A and HRV-C species have a high degree of antibody cross-reactivity and the HRV-C specific antibody response is low (Iwasaki et al., 2013). This study aims to identify species-specific and cross-reactive T-cell epitopes for the HRV-A and HRV-C species. Methods In total, 110 overlapping synthetic peptides of HRV-A (genotype HRV-34) and HRV-C (genotype QPM) were purchased from Mimotopes (Vic, Australia) covering the entire VP1 protein of both genotypes. T-cell proliferation was measured by 3 H-thymidine incorporation. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 20 adults from the general population of Perth (WA), with no evidence of current respiratory illness. The PBMCs were cultured in AIM-V media with the individual peptides of HRV-A and HRV-C in triplicate. PHA and PPD were included as positive controls and negative controls contained only media. 3 H-thymidine incorporation was measured after a 6-day culture and a stimulation index of greater than 2 (calculated by the mean of wells containing antigen divided by the mean of the negative control wells) was considered a positive lymphoproliferative response. Variation on replicate cultures was lower than 20%. Results We have identified four species-specific peptides for HRV-QPM and three for HRV-34 that triggered T-cell proliferation in more than 50% of the tested subjects. Four pairs of cross-reactive peptides were identified for HRV-A and HRV-C that stimulated the cells of more than 40% of the subjects tested. Conclusions To our knowledge, this is the first study to map T-cell epitopes in a genotype of HRV-C and also the first to map the entire HRV VP1 protein. Future HLA typing and in silico studies for other HRV-A and HRV-C genotypes based on the reactive peptides will be conducted. These findings will help to identify differences in the immune recognition of the two most prevalent HRV species and are valuable tools for future vaccine development.

Antigen-specific T lymphocyte proliferation decreases over time in advanced chronic hepatitis C.

To evaluate T cell immunity in advanced liver disease, antigen-specific lymphoproliferative (LP) responses were prospectively studied in the context of the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial. Peripheral blood responses to hepatitis C virus (HCV), tetanus and Candida protein antigens were measured at baseline, month 12 (M12), M24, M36 and M48 in 186 patients randomized to either low-dose peginterferon-alfa-2a (PEG-IFN) only or observation. Liver histology was evaluated at baseline, M24 and M48. Patients with cirrhosis (Ishak 5-6) were less likely to have positive LP responses to HCV at baseline than patients with fibrosis (15%vs 29%, P = 0.03) and had lower levels of HCV c100 responses at baseline, M24 and M48 (P = 0.11, P = 0.05, P = 0.02, respectively). For 97 patients with complete longitudinal data, the frequency of positive LP responses to HCV, tetanus and Candida antigens declined over time (P < 0.003), and the slope of this decline was greater in the PEG-IFN treatment group than the observation group (P < 0.02). Lower levels of tetanus LP responses were associated with fibrosis progression and clinical outcomes (P = 0.009). Poorer CD4+ T cell proliferative function was associated with more advanced liver disease in chronic hepatitis C and may be further affected by long-term PEG-IFN treatment.

Lymphoproliferative Response to Herpes Simplex Virus, Cytomegalovirus, Epstein-Barr Virus, Varicella Zoster Virus, Human Herpes Virus 6, 7 and 8 Antigen Stimulation in Pediatric Allogeneic Stem Cell Transplant Recipients.

Abstract 4663 IntroductionDelayed immune reconstitution is a major cause of transplant-related mortality in allogeneic stem cell transplant setting. Reactivation of herpes viruses is a common post-transplant complication. In this study, we evaluate the recovery of cell-mediated immunity (CMI) to various herpes viruses by measuring lymphoproliferative response (LPR) to specific recall antigens. Materials and MethodsCell-mediated immunity was evaluated by the in-vitro lymphoproliferative response of peripheral blood mononuclear cells (PBMC) to specific purified HSV, VZV, CMV, EBV, HHV-6, 7, 8 antigens. Results were expressed as stimulation index (SI). SI≥a3 was regarded as positive lymphoproliferative response (LPR). Serial measurements were made at before transplant, and monthly during the initial 6 months post-transplant then quarterly till 12 months post-transplant. ResultsFrom 2001-2004, 36 patients (M=19; F=17) with median age 10.5 years old were recruited. Hematological malignancies accounted for 58.3% of cases. Most transplants were from matched siblings (MSD) with peripheral blood stem cells (PBSC) as the source of stem cells. Altogether 50% of subjects showed positive LPR to HSV, CMV and VZV antigens at 2-4 months post-transplant; A significant upsurge of LPR were observed at 4-6 months post-transplant. However, no positive LPR to HSV, HHV-6,7 and 8 antigens were observed. The antibody status of donor and recipient for HSV, CMV and VZV were associated with the timing of recovery of CMI. Donor choice and stem cell source were important determinants of eventual LPR at day 100 post-transplant. ConclusionMore than half of transplant recipients developed satisfactory LPR to HSV, CMV and VZV at 2-4 months after transplant. Pre-transplant serostatus of donor and recipient, donor choice and stem cell are important determinants of LPR to herpes viruses. Disclosures:No relevant conflicts of interest to declare.

Immune responses against recombinant tick antigen, Bm95, for the control of Rhipicephalus ( Boophilus) microplus ticks in cattle

Immune responses against Bm95 recombinant cattle tick antigen and its protective efficacy for control of Rhipicephalus ( Boophilus) microplus ticks were determined in experimental crossbred cow calves. Anti-Bm95 antibody titers, as assessed by indirect ELISA, in immunized calves ranged from 196.1 ± 13.7 on day 0 to 7979.9 ± 312.5 on day 110 post-primary immunization. The rise in antibody titer was statistically significant ( p < 0.01) throughout the study period. Besides this, constantly higher lymphoproliferative response (LPR), as assessed by lymphocyte stimulation test, was observed from 10 days post-immunization, but a positive LPR of antigen stimulated cells in immunized animals was recorded only on day 50 and day 70 post-immunization. Following challenge of immunized calves with larvae of R. microplus, significant increase ( p < 0.01) in rejection percentage, mean number of damaged ticks, mean percentage of dead ticks, and decrease in engorgement weight were recorded in immunized animals. Also, there were significant differences ( p < 0.01) in preoviposition period, oviposition period, egg mass weight and percent hatchability between the immunized and control calves. The percent reduction in number of adult females in vaccinated calves, reduction in mean weight of egg masses, percent reduction in mean weight and reduction in fertility of engorged females collected from vaccinated calves were determined and the efficacy of Bm95 recombinant cattle tick antigen was 81.27%.

Leishmania (Viannia) guyanensis Induces Low Immunologic Responsiveness in Leishmaniasis Patients from an Endemic Area of the Brazilian Amazon Highland

Cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis (CL-Lguy) is endemic in the Brazilian Amazon, differing from L. braziliensis infection in clinical, diagnostic, and therapeutic aspects. T-cell reactivity to leishmanial antigens possibly involved in the pathogenesis of CL-Lguy was studied herein. Variable lymphoproliferative responses (LPRs) to Leishmania antigens were found among the 23 studied patients, and 50% of them showed low or no response to these antigens. Active disease was associated with an enrichment of leishmanial-reactive T lymphocytes, mainly TCD4(+). High and low interferon (IFN)-gamma producers were observed. TNF-alpha, interleukin (IL)-10, and IL-5 were consistently detected. CL-Lguy displayed low antibody response in comparison to L. braziliensis patients. CL caused by L. braziliensis presented positive LPRs and higher IFN-gamma production but undetectable IL-5. L. guyanensis seems to induce a down-regulation of the immune system compared with L. braziliensis. This finding could explain some aspects of clinical presentation of CL-Lguy, such as high tissue parasite burden and frequent resistance to therapy.

Lymphoproliferative and Cytokine Responses to Cryptosporidium parvum in Patients Coinfected with C. parvum and Human Immunodeficiency Virus

We compared the lymphoproliferative and cytokine responses to Cryptosporidium parvum in human immunodeficiency virus (HIV)-seropositive and -seronegative patients. The lymphoproliferative and cytokine responses (interleukin-2 [IL-2], IL-4, IL-5, IL-10, gamma interferon, and tumor necrosis factor alpha) were assessed for 11 HIV-seropositive, Cryptosporidium-positive (group I) patients; 20 HIV-seropositive, Cryptosporidium-negative (group II) patients; 10 HIV-seronegative, Cryptosporidium-positive (group III) patients, including four post-renal transplant (group IIIa) and 6 presumably immunocompetent (group IIIb) patients; and 20 HIV-seronegative, Cryptosporidium-negative healthy individuals (group IV). No significant difference was observed in the number of patients showing positive lymphoproliferative responses in group I compared to group III (post-renal transplant [group IIIa] or immunocompetent [group IIIb]) patients, while a comparison of the median stimulation indices shows that responses were significantly lower in Cryptosporidium-infected, immunosuppressed (group I and IIIa) patients than in immunocompetent (group IIIb) patients. The number of patients showing positive responses and median stimulation indices was significantly higher for Cryptosporidium-infected (HIV-seropositive and -seronegative) individuals than for uninfected individuals, suggesting that Cryptosporidium induces significant in vitro lymphoproliferative responses in infected individuals. Cytokine levels, except for that of IL-5, were significantly higher in Cryptosporidium-infected (groups I and III) individuals than in uninfected (groups II and IV) individuals. There was no significant difference between the group I and III patients and between Cryptosporidium-infected immunosuppressed (group I or IIIa) and immunocompetent (group IIIb) patients.

Analysis of the cells involved in the lymphoproliferative response toCoxiella burnetiiantigens

Vaccination with an inactivated, whole cell, Q fever vaccine (Q-vax) induces lasting antibody conversion and a positive delayed-type hypersensitivity (DTH) skin reaction in about 60% of recipients but a long-lasting positive lymphoproliferative or mitogenic response to C. burnetii antigens with peripheral blood mononuclear cells (PBMC) in 85-95% of subjects. Analysis of the lymphoproliferative response to C. burnetii antigens has now been made by fractionation-reconstitution experiments with PBMC from vaccines, from past infections, and from healthy controls. The major contributor to the response in immune subjects proved to be the T lymphocyte. T cells were stimulated by both the phase I and phase II antigens of two prototype strains of C. burnetii and responses were greatly amplified by addition of IL-2. Similar T lymphocyte stimulation profiles were obtained with the 'Priscilla' strain of C. burnetii which represents a different biotype of Coxiella isolated from Q fever endocarditis; Q-vax is therefore likely to protect against endocarditis strains. Fractionation-reconstitution experiments with T and B cells from vaccines and subjects infected in the past, using various antigenic or haptenic fractions from C. burnetii indicate that protein, non-lipopolysaccharide components of the organism are responsible for the mitogenic response of immune T cells. However, the role of the lipopolysaccharide in the protective immunogen has still to be defined.

Contribution of influenza immunity and virosomal-formulated synthetic peptide to cellular immune responses in a phase I subunit malaria vaccine trial

We have demonstrated recently in a phase Ia clinical trial that synthetic malaria peptides delivered by immuno-potentiating reconstituted influenza virosomes (IRIV) induced long-lived peptide-specific antibody responses in all volunteers. In the current ancillary study to this clinical trial we have investigated the cellular immune responses specific for IRIV and the surface bound synthetic malaria peptides tested. After vaccination, in 50% (8/16) of the volunteers at least one positive lymphoproliferative response specific for the 49mer peptide derived from the Plasmodium falciparum apical membrane antigen-1 (AMA-1) was observed with stimulation indices ranging from 2 to 4.5. All volunteers showed pre-existing IRIV specific cellular immunity assessed by ex vivo IFN-γ ELISpot analysis and lymphoproliferation. The pre-existing influenza specific T cell responses did not interfere negatively with the induction of malaria peptide-specific humoral and cellular immune responses. Our results support the view that IRIV constitute a safe antigen delivery system for induction of peptide-specific immune responses in human populations.

Hepatitis C virus (HCV) core protein enhances the immunogenicity of a co‐delivered DNA vaccine encoding HCV structural antigens in mice

In the present study, recombinant HCV (hepatitis C virus) core proteins enhanced the immune response elicited by a co-delivered DNA vaccine encoding HCV core and envelope proteins. A mixture of the plasmid pIDKE2 and Co.173, a protein comprising the first 173 amino acids of HCV core, in particular induces a strong humoral response, including antibodies that recognized peptides representing hypervariable region I from different viral isolates. Moreover, positive lymphoproliferative responses against the HCV structural proteins, encoded by the plasmid, were detected after two doses with this mixture. When the HCV core protein used in the mixture with pIDKE2 was Co.120, a protein comprising the first 120 amino acids of the viral antigen, a strong humoral response and a positive lymphoproliferative response were also detected. The effectiveness of this formulation was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV core protein. A 2 log reduction in vaccinia-virus titre was observed in mice immunized with the mixture of pIDKE2 and Co.120. Humoral and cellular immune responses elicited for the mixture of pIDKE2 with either Co.173 or Co.120 was stronger and more diverse than those generated by individual components. In conclusion, our results indicate that formulations comprising both DNA constructs and protein subunit vaccine candidates are able to elicit strong humoral and cellular immunity against several antigens. Particularly, HCV core protein might be used as a feasible vehicle/adjuvant for DNA vaccines.

Correlates of lymphoproliferative responses to measles, mumps, and rubella (MMR) virus vaccines following MMR-II vaccination in healthy children

Cell-mediated immunity (CMI) to measles, mumps, and rubella viral antigens plays a critical role in providing long-term protection against these infectious diseases. We examined CMI by measuring lymphoproliferative response induced in response to stimulation with the above three antigens following two doses of measles, mumps, and rubella-II (MMR-II) vaccine in a randomly selected, population-based cohort of healthy children. We determined if a correlative and predictive intraclass relationship exists between CMI to the three components of MMR-II. We detected positive lymphoproliferative responses to measles, mumps, and rubella vaccines. Mumps vaccine used as an antigen had the highest median stimulation index followed by measles and rubella vaccines. The overall intraclass correlation value for lymphoproliferative response to measles, mumps, and rubella using Pearson's correlation was 0.61 (95% confidence interval = 0.56, 0.66). We observed a significant pairwise association to individual vaccine components between subjects in the upper and lower 10th percentile of immune response. This study demonstrates recall CMI post-MMR-II vaccination with significant intraclass correlation among the CMI responses to the three vaccine components.

Assessment of human cytomegalovirus specific T cell immunity in human immunodeficiency virus infected patients in different disease stages following HAART and in long-term non-progressors.

T cell immunity to human cytomegalovirus (HCMV) was assessed in HAART-treated HIV-1 infected patients (9 asymptomatic, CDC group A; and 22 symptomatic, CDC group B), and in eight HIV-1 long term non-progressors. Patients were either prospectively or cross-sectionally examined for CD4(+) T cell counts, HIV RNA load, HCMV leukoDNAemia, HCMV DNA in urine, lymphoproliferative response (LPR) to HCMV and phytohemagglutinin (PHA), and cytokine secretion (IFN-gamma and IL-4) by HCMV-stimulated peripheral blood mononuclear cell (PBMC) cultures. No patient either progressed to clinical AIDS or developed HCMV active infection during the study period. Twenty-nine patients responded to HAART, though 12 patients failed to recover the LPR to HCMV over the study period (three from CDC group A and nine from CDC group B). In contrast to healthy control individuals, most patients displaying positive LPRs LPRs to HCMV had unstable responses. Sustained LPRs to HCMV were significantly associated with high pre-HAART nadir CD4(+) T cell counts. Long-term suppression of HIV viremia correlated with recovery of LPR to HCMV. Sequential PBMC cultures from most patients secreted IFN-gamma (but not IL-4) at normal levels upon HCMV stimulation, irrespective of the pre-HAART nadir CD4(+) T cell counts and CDC group to which patients belonged. Failure to reconstitute IFN-gamma response was associated with very low pre-HAART nadir CD4(+) T cell counts. Control of HCMV infection in the cohort was associated with either recovery or maintenance of IFN-gamma response rather than with reconstitution of LPR to HCMV. A LPR to HCMV was absent in three out of eight long term non-progressors; contrarily, all patients showed preserved IFN-gamma responses.

Nuclear factor kappa B activation in human cord blood mononuclear cells.

The immunologic signals participating in immune responses early in life have not been completely elucidated. Regarding the characterization of neonatal cells, little is known concerning the activity of transcription factor nuclear factor kappa B (NF-kappaB), which regulates inflammatory genes and cytokine production. The aim of this study was to characterize NF-kappaB activation in cord blood mononuclear cells (CBMC). We analyzed the potential association of NF-kappaB activity with lymphocyte proliferation and influences on cytokine secretion in the early immune system. To determine the contribution of a disease whereby inheritance may impact neonatal immunity, we assessed the influence of maternal allergic disease on NF-kappaB regulation and cytokine secretion. CBMC from healthy newborns were isolated and stimulated with mitogen (n = 28). Nuclear extracts were analyzed by electrophoretic mobility shift assay, cytokine secretion by ELISA. FISH analysis excluded relevant maternal contamination of CBMC. All samples showed a positive lymphoproliferative response, and NF-kappaB activity was both increased and decreased after mitogen stimulation. Increased NF-kappaB activation was significantly associated with decreased TNF-alpha secretion (median 6.1 versus 50.3 pg/mL) in unstimulated CBMC. Mitogen stimulation resulted in increased NF-kappaB activity with a trend to increased IL-13 production. Maternal allergic disease was associated with higher TNF-alpha (median 982 versus 173 pg/mL) and IL-13 secretion (median 1328 versus 1120 pg/mL) after mitogen stimulation. Together, NF-kappaB activity is differentially activated in cord blood and associated with a distinct cytokine pattern. Whether differential NF-kappaB activity in cord blood is related to the subsequent development of immune diseases requires further investigation.

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin.

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon gamma (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.

Prospective Analyses of HIV‐1–Specific Proliferative Responses, Recall Antigen Proliferative Responses, and Clinical Outcomes in an HIV‐1–Seropositive Cohort

In cross-sectional studies of chronically infected individuals, lymphoproliferative responses to human immunodeficiency virus (HIV) type 1 p24 Gag antigen have previously been associated with lower virus load. It was not known whether this association would be predictive of better clinical outcome in longitudinal studies. In blood samples from 608 HIV-seropositive individuals enrolled in a trial of glycoprotein 160 vaccine therapy over the course of 3-5 years, lymphoproliferative responses to HIV-1 antigens, tetanus toxoid (TT), and mitogens were measured and correlated with clinical outcome and other parameters of progression. Baseline lymphoproliferative responses to antigens and mitogens were used to categorize the cohort into responders or nonresponders. Although response to recall antigens did not correlate with clinical indices of disease progression, positive baseline lymphoproliferative responses to p24 and TT were associated with lower plasma levels of HIV-1 RNA. Persistently positive lymphoproliferative responses to the antigens also inversely correlated with repeated measurements of virus load, although the significance was lost once the measurements were adjusted for virus load and CD4(+) cell count at baseline, by use of generalized estimating equation analysis. These observations suggest that the effect of the association between lymphoproliferative response and virus load is established early during HIV-1 infection and does not increase over time and suggest that antigen-specific lymphoproliferative responses reflect the dynamic state of HIV-1 infection and are inversely associated with virus load.

Leprosy reactions: humoral and cellular immune responses to M. leprae, 65kDa, 28kDa, and 18 kDa antigens.

This study examines the immune responses against some stress proteins of Mycobacterium leprae in leprosy patients with and without leprosy reactions. Leprosy patients showed a higher level of antibodies to all antigens compared to healthy controls. The antibody response to 18kDa antigen was significantly higher in patients with Type 1 reaction compared to those of TT or borderline patients without Type 1 reaction, or those with Type 2 reaction. Borderline (BT/BL), lepromatous (LL) and patients with reactions (Type 1 and Type 2) had higher levels of antibodies to M. leprae soluble extract (MLSE) and 65kDa than those of the tuberculoid (TT) group. LL, borderline patients, and patients with Type 1 reaction had a higher level of antibody to 28kDa than those of healthy controls. However, no significant differences could be observed in antibody response to these antigens (MLSE, 65kDa, and 28kDa) between patients with reaction and without reaction. A significant proportion of TT/BT patients showed positive lymphoproliferative response to MLSE compared to BL/LL patients. In addition, the lymphoproliferative response to MLSE was significantly greater in patients with Type 1 reaction compared to patients without reaction. No difference in proliferative response to 65kDa could be observed in any of these groups. The finding of high levels of antibodies against stress proteins in patients with Type 1 reactions, especially to 18 kDa antigen, along with a heightened lymphoproliferative response to MLSE is suggestive of a coexistence of cell mediated and humoral immunity in leprosy patients during Type 1 reactions. On the other hand, in Type 2 reactions no significant role of stress proteins could be demonstrated except a heightened lymphoproliferative response to the 28 kDa antigen.

Clinical and immunological effects of a 6 week immunotherapy cycle with murabutide in HIV-1 patients with unsuccessful long-term antiretroviral treatment

In an effort to evaluate the potential of non-specific immunotherapy in restoring global immunity, we have examined the clinical tolerance and biological effects of a 6 week administration of the immunomodulator, murabutide, in chronically infected HIV-1 patients. Forty-two subjects, presenting weak immune reconstitution and ineffective virus suppression following long-term highly active antiretroviral therapy (HAART), were randomized to receive, or not, murabutide 7 mg/day on five consecutive days/week. Clinical and immunological parameters were monitored before and after the immunotherapy period. Administration of murabutide was generally well tolerated, although some grade III adverse events, reversible on treatment cessation, were observed. Interestingly, in comparison with pre-inclusion levels, at 1 week after the immunotherapy cycle, only murabutide recipients presented a significant increase in CD4 cells, platelet counts, and in the percentage of patients with undetectable viral loads (<50 copies/mL). Statistical significance between the two groups was only evident with the latter parameter. Some of these clinical changes were maintained even up to 12 weeks after murabutide administration, and were accompanied by an increased ability to mount cellular responses to active immunization with a recall antigen, and by a significant increase in the percentage of patients presenting positive lymphoproliferative responses to the viral antigen gp160. These results warrant further evaluation of extended periods or cycles of murabutide immunotherapy as adjunct to HAART.