Autoantibodies and High-Risk HLA Susceptibility Markers in First-Degree Relatives of Brazilian Patients with Type 1 Diabetes Mellitus: A Progression to Disease Based Study

The objective of this study was to determine the frequencies of autoantibodies to heterogeneous islet-cell cytoplasmic antigens (ICA), glutamic acid decarboxylase(65) (GAD(65)A), insulinoma-associated antigen-2 (IA-2A) and insulin (IAA)-and human leukocyte antigen (HLA) class II markers (HLA-DR and -DQ) in first degree relatives of heterogeneous Brazilian patients with type I diabetes(T1DM). A major focus of this study was to determine the influence of age, gender, proband characteristics and ancestry on the prevalence of autoantibodies and HLA-DR and -DQ alleles on disease progression and genetic predisposition to T1DM among the first-degree relatives. IAA, ICA, GAD(65)A, IA-2A and HLA- class II alleles were determined in 546 first-degree-relatives, 244 siblings, 55 offspring and 233 parents of 178 Brazilian patients with T1DM. Overall, 8.9% of the relatives were positive for one or more autoantibodies. IAA was the only antibody detected in parents. GAD(65) was the most prevalent antibody in offspring and siblings as compared to parents and it was the sole antibody detected in offspring. Five siblings were positive for the IA-2 antibody. A significant number (62.1%) of siblings had 1 or 2 high risk HLA haplotypes. During a 4-year follow-up study, 5 siblings (expressing HLA-DR3 or -DR4 alleles) and 1 offspring positive for GAD(65)A progressed to diabetes. The data indicated that the GAD(65) and IA-2 antibodies were the strongest predictors of T1DM in our study population. The high risk HLA haplotypes alone were not predictive of progression to overt diabetes.

Conjunctival inflammation in patients under topical glaucoma treatment with indication to surgery

PURPOSE: To compare the frequency of conjunctival HLA-DR expression (a surrogate marker for inflammation) in eyes treated with topical prostaglandin analogues versus eyes treated with other topical antiglaucomatous drugs. METHODS: Patients diagnosed with primary open-angle glaucoma presenting indication for trabeculectomy were divided in groups according to the use or not of prostaglandin analogues. All subjects were treated with the maximum tolerated dose of antiglaucomatous drugs until the date of the surgery. At the beginning of the surgical procedure, a 5 x 5 mm biopsy of the bulbar conjunctiva was collected, incubated with monoclonal anti-HLA-DR antibody and processed for histological analysis. RESULTS: Of the 31 eyes included (31 patients), 25 were under topical prostaglandin analogues (Group 1) and six under other topical pharmacological agents (Group 2). Fourteen eyes of Group 1 (56%) and three of Group 2 (50 %) were positive for the inflammatory marker HLA-DR (P=1.0). The percentage of stained cells ranged from 15.49 to 48.09% (median: 27.61) in Group 1, and from 18.35 to 28 (median: 20.71) in Group 2, with no differences statistically significant (p=0.33). CONCLUSION: The use of prostaglandin analogues did not increase conjunctival expression of HLA-DR compared to other topical antiglaucomatous agents.

Depletion of Langerhans cells in the tongue from patients with advanced-stage acquired immune deficiency syndrome: relation to opportunistic infections

Aims: To quantify and compare the expression of Langerhans cells (LCs) in the tongue mucosa of AIDS patients with different opportunistic infections, and from acquired immune deficiency syndrome (AIDS) and non-AIDS patients with normal tongues, using autopsy material. Methods and results: Human leucocyte antigen D-related (HLA-DR), CD1a and CD83 antibodies were used to identify and quantify LCs by immunohistochemistry in tongue tissue of 40 AIDS patients (10 with lingual candidiasis, 10 with lingual herpes, 10 with oral hairy leukoplakia and 10 with no lesions) and 23 tongues from human immunodeficiency virus (HIV)negative control patients. Quantification was performed by means of conventional morphometry in four different regions (anterior, middle, posterior and lateral) of the tongue. The results were expressed as positive cells per area of epithelium. The AIDS patients presented a lower density of CD1a(+) cells (P < 0.001), HLA-DR (P < 0.003) and CD83 (P < 0.001) in all regions of the tongue compared to the non-AIDS control group. However, no differences in any of the markers were found when AIDS patients with different opportunistic infections were compared with AIDS patients without tongue infection. Conclusions: Advanced stage AIDS patients showed a depletion of LCs in the tongue mucosa. HIV infection induces cytopathic changes in LCs, contributing to their depletion regardless of the presence of oral infections.

Simultaneous Occurrence of Biphenotypic T Cell/Myeloid Lesions Involving t(12;13)(p13;q14) in a Pediatric Patient

This paper chronicles a 2-year-old girl who presented with acute leukemia/lymphoma syndrome of the T cell immuno-phenotype. At this time, the cytogenetic analysis of her bone marrow cells showed a reciprocal translocation between the short arm of chromosome 12 and the long arm of chromosome 13, t(12;13)(p13;q14). The immunophenotyping of bone marrow blast cells by flow cytometry revealed a population of cells positive for CD56, CD117, CD45, partial CD33, partial HLA-DR, CD13, CD7, CD2 and CD5. Therefore, a diagnosis of acute leukemia with a mixed T cell/myeloid phenotype was made. The patient had a poor response to classic T cell acute lymphocytic leukemia/lymphoma therapy; thus, her treatment was changed to a myeloid leukemia protocol, which produced a good response. She underwent a successful cord blood transplantation from an unrelated HLA partially matched donor. The coexistence of these two phenotypes prompts questions about the existence of clonal instability, which might influence the choice of therapy. The rarity of the t(12;13)(p13;q14) and the coexistence of T cell/myeloid markers suggest a nonrandom association. To the best of our knowledge, this is the first reported case in which a cell clone bearing a t(12;13)(p13;q14) translocation in a mixed T cell/myeloid lesion was detected. Copyright (C) 2012 S. Karger AG, Basel

Dendritic cells from X-linked hyper-IgM patients present impaired responses to Candida albicans and Paracoccidioides brasiliensis

Background: Patients with X-linked hyper-IgM syndrome (X-HIGM) due to CD40 ligand (CD40L) mutations are susceptible to fungal pathogens; however, the underlying susceptibility mechanisms remain poorly understood. Objective: To determine whether monocyte-derived dendritic cells (DCs) from patients with X-HIGM exhibit normal responses to fungal pathogens. Methods: DCs from patients and controls were evaluated for the expression of costimulatory (CD80 and CD86) and MHC class II molecules and for their ability to produce IL-12 and IL-10 in response to Candida albicans and Paracoccidioides brasiliensis. We also evaluated the ability of C albicans- and P brasiliensis-pulsed mature DCs to induce autologous T-cell proliferation, generation of T helper (T-H) 17 cells, and production of IFN-gamma, TGF-beta, IL-4, IL-5, and IL-17. Results: Immature DCs from patients with X-HIGM showed reduced expression of CD80, CD86, and HLA-DR, which could be reversed by exogenous trimeric soluble CD40L. Most important, mature DCs from patients with X-HIGM differentiated by coculturing DCs with fungi secreted minimal amounts of IL-12 but substantial amounts of IL-10 compared with mature DCs from normal individuals. Coculture of mature DCs from X-HIGM patients with autologous T cells led to low IFN-g production, whereas IL-4 and IL-5 production was increased. T-cell proliferation and IL-17 secretion were normal. Finally, in vitro incubation with soluble CD40L reversed the decreased IL-12 production and the skewed T-H(2) pattern response. Conclusion: Absence of CD40L during monocyte/DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with X-HIGM. (J Allergy Clin Immunol 2012; 129: 778-86.)

Immunological parameters in elderly women: Correlations with aerobic power, muscle strength and mood state

Purpose: Our objective was to relate immunological data for healthy but sedentary elderly women to aerobic power, strength, and mood state. Methods: We measured peak aerobic power and one-repetition maximum strength along with mood (depression and fatigue), quality of life and carbohydrate intake on 42 women aged 60-77 years. Standard immunological techniques determined natural killer cell count and cytotoxic activity (NKCA), proliferative responses to phytohemaglutinin and OKT3, various lymphocyte subpopulations (CD3(+), CD3(-)CD19(+), CD56(+), CD4(+), CD8(+), CD56(dim) and CD56(bright)), and markers of activation, maturation, down-regulation and susceptibility to apoptosis (CD25(+), CD28(+), CD45RA(+), CD45RO(+), CD69(+), CD95(+), HLA-DR+). Results: Correlations of immune parameters with aerobic power and strength were very similar for absolute and relative immunological data. In the group as a whole, the only correlation with aerobic power was -0.35 (relative CD4(+)CD69(+) count), but in subjects with values <22.6 mL kg(-1) min(-1) correlations ranged from -0.57 (relative CD4(+)CD45RO(+)) to 0.92 (absolute CD56(dim)HLA-DR+). In terms of muscle strength, univariate correlation coefficients ranged from -0.34 (relative and absolute CD3(+)CD4(+)CD8(+)) to +0.48 (absolute CD3(+)HLA-DR+.) and +0.50 (absolute CD8(+)CD45RA(+)CD45RO(+)). Neither NKCA nor lymphocyte proliferation were correlated with aerobic power or muscle strength. Although mood state and quality of life can sometimes be influenced by an individual's fitness level, our multivariate analyses suggested that depression, fatigue and quality of life were more important determinants of immune profile than our fitness measures. Conclusions: Psychological changes associated with aging may have a substantial adverse effect upon the immune system, and immunological function may be enhanced more by addressing these issues than by focusing upon aerobic or resistance training. (C) 2012 Elsevier Inc. All rights reserved.

Broad and Cross-Clade CD4(+) T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-gamma secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-gamma and TNF-alpha, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

Cellular Infiltrates and NF kappa B Subunit c-Rel Signaling in Kidney Allografts of Patients With Clinical Operational Tolerance

Background. Nuclear factor kappa B (NF kappa B) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NF kappa B1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). Methods. Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NF kappa B pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean +/- SEM. Results. Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191 +/- 81; IR, 291 +/- 62; BC, 178 +/- 45; and NR, 210 +/- 42 cells/mm(2)) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%+/- 1.7%; IR, 3.5%+/- 0.70%; BC, 3.4%+/- 0.57%; and NR, 3.7%+/- 0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm(2) (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). Conclusions. Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NF kappa B pathway and a higher proportion of forkhead box P3-positive cells.

Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Immunomodulation of human monocytes following exposure to Lutzomyia intermedia saliva

Abstract Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS) of Lutzomyia intermedia, the natural vector of Leishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC) of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected with L. braziliensis. Results Pre-treatment of non-infected human monocytes with L. intermedia SGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation and L. braziliensis infection led to a significant increase in TNF-α, IL-6, and IL-8 production without significant alterations in co-stimulatory molecule expression. However, pre-treatment with L. intermedia SGS did not result in significant changes in the infection rate of human monocytes. Conclusion Our data indicate that L. intermedia saliva is able to modulate monocyte response, and, although this modulation is dissociated from enhanced infection with L. braziliensis, it may be associated with successful parasitism.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

Altered phenotype and function of dendritic cells in individuals with chronic periodontitis

OBJECTIVE: To investigate the effects of periodontal bacterial lysates on maturation and function of mature monocyte-derived dendritic cells (m-MDDCs) derived from individuals with chronic periodontitis (CP) or healthy periodontal tissue (HP). DESIGN: m-MDDCs derived from peripheral blood monocytes, cultured for 7 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF), were stimulated with lysates of Streptococcus sanguinis, Prevotella intermedia, Porphyromonas gingivalis, or Treponema denticola on day 4, and were then phenotyped. IL-10, IL-12 and IFN-gamma concentration in the supernatant of cultures were measured. RESULTS: Expression of HLA-DR was lower in bacterial-unstimulated mature m-MDDC from CP compared to HP (p=0.04), while expression of CD1a and CD123 were higher in CP. The expression pattern of HLA-DR, CD11c, CD123, and CD1a did not change on bacterial stimulation, regardless of the bacteria. Stimulation with P. intermedia upregulated CD80 and CD86 in CP cells (p≤0.05). Production of IL-12p70 by bacterial-unstimulated m-MDDCs was 5.8-fold greater in CP compared to HP. Bacterial stimulation further increased IL-12p70 production while decreasing IL-10. Significantly more IFN-gamma was produced in co-cultures of CP m-MDDCs than with HP m-MDDCs when cells were stimulated with P. intermedia (p=0.009). CONCLUSIONS: Bacterial-unstimulated m-MDDC from CP exhibited a more immature phenotype but a cytokine profile biased towards proinflammatory response; this pattern was maintained/exacerbated after bacterial stimulation. P. intermedia upregulated co-stimulatory molecules and IFN-gamma expression in CP m-MDDC. These events might contribute to periodontitis pathogenesis