RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gamma delta-positive (gamma delta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in super-natants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gamma delta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gamma delta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gamma delta(+) cell expansions were similar with the two vaccines.

The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity

Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3' end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Delta m131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Delta m131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Delta m131/129 was reduced compared with that of wt MCMV. Furthermore, the Delta m131/129 mutants failed to establish a high-titer infection in the salivary glands, These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Delta m131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4(+) CD8(+) phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Endostatin gene therapy enhances the efficacy of IL-2 in suppressing metastatic renal cell carcinoma in mice

We investigated whether the administration of IL-2 combined with endostatin gene therapy was able to produce additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma. Renca cells were injected into the tail vein of BALB/c mice. After 24 h, the animals were randomly divided into four groups (5 mice/group). One group of mice was the control, the second group received treatment with 100,000 UI of Recombinant IL-2 (Proleukin, Chiron) twice a day, 1 day per week during 2 weeks (IL-2), the third group received treatment with a subcutaneous inoculation of 3.6 x 10(6) endostatin-producing cells, and the fourth group received both therapies (IL-2 + ES). Mice were treated for 2 weeks. In the survival studies, 10 mice/group daily, mice were monitored daily until they died. The presence of metastases led to a twofold increase in endostatin levels. Subcutaneous inoculation of NIH/3T3-LendSN cells resulted in a 2.75 and 2.78-fold increase in endostatin levels in the ES and IL-2 + ES group, respectively. At the end of the study, there was a significant decrease in lung wet weight, lung nodules area, and microvascular area (MVA) in all treated groups compared with the control group (P < 0.001). The significant difference in lung wet weight and lung nodules area between groups IL-2 and IL-2 + ES revealed a synergistic antitumor effect of the combined treatment (P < 0.05). The IL-2 + ES therapy Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice treated with the combined therapy (log-rank test, P = 0.0028). Conjugated therapy caused an increase in the infiltration of CD4, CD8 and CD49b lymphocytes. An increase in the amount of CD8 cells (P < 0.01) was observed when animals received both ES and IL-2, suggesting an additive effect of ES over IL-2 treatment. A synergistic effect of ES on the infiltration of CD4 (P < 0.001) and CD49b cells (P < 0.01) was also observed over the effect of IL-2. Here, we show that ES led to an increase in CD4 T helper cells as well as cytotoxic lymphocytes, such as NK cells and CD8 cells, within tumors of IL-2 treated mice. This means that ES plays a role in supporting the actions of T cells.

Pulmonary periarterial inflammation in fatal asthma

P>Background To date, little information has been available about pulmonary artery pathology in asthma. The pulmonary artery supplies the distal parts of the lungs and likely represents a site of immunological reaction in allergic inflammation. The objective of this study was to describe the inflammatory cell phenotype of pulmonary artery adventitial inflammation in lung tissue from patients who died of asthma. Methods We quantified the different inflammatory cell types in the periarterial region of small pulmonary arteries in lung tissue from 22 patients who died of asthma [fatal asthma (FA)] and 10 control subjects. Using immunohistochemistry and image analysis, we quantified the cell density for T lymphocytes (CD3, CD4, CD8), B lymphocytes (CD20), eosinophils, mast cells (chymase and tryptase), and neutrophils in the adventitial layer of pulmonary arteries with a diameter smaller than 500 mu m. Results Our data (median/interquartile range) demonstrated increased cell density of mast cells [FA=271.8 (148.7) cells/mmCD4CD8ration of mast cells, eosinophils, and neutrophils, but not of T and B lymphocytes.

The effects of human herpesvirus 8 infection and interferon-gamma response in cutaneous lesions of Kaposi sarcoma differ among human immunodeficiency virus-infected and uninfected individuals

Background Kaposi sarcoma (KS) is associated with human herpesvirus 8 (HHV-8). The cutaneous immune response in this tumour is not well established and a better understanding is necessary. Objectives To evaluate the HHV-8 expression and immune response in cutaneous lesions of classic KS (CKS) and AIDS-associated KS (AIDS-KS). Methods We performed a quantitative immunohistochemical study of cells expressing HHV-8 latency-associated nuclear antigen (LANA), CD4, CD8 and interferon (IFN)-gamma in skin lesions from patients with CKS and AIDS-KS (with or without highly active antiretroviral therapy, HAART). Results CKS showed higher LANA expression compared with AIDS-KS, regardless of HAART. We also found higher LANA expression in nodules compared with patch/plaque lesions. The tissue CD4+ cell proportion was lower in AIDS-KS patients without HAART than in patients with CKS. In CKS lesions, CD4+ and CD8+ cells expressed IFN-gamma, as shown by double immunostaining. AIDS-KS presented low numbers of IFN-gamma-expressing cells. CD8+ cell numbers were similar in all groups, which appeared unrelated to the clinical or epidemiological type of KS. Conclusions. Our quantitative data on the pattern of KS lesions in selected groups of patients, as shown by in situ immune response, demonstrated a CD4+ T-cell involvement associated with IFN-gamma, an environment of immune response-modified human immunodeficiency virus (HIV) infection. In our sample, the promotion of KS in patients without HIV appears to be related to higher HHV-8 load or virulence than in those with AIDS. This higher resistance may be explained by a sustained immune response against this herpesvirus, that is only partially restored but effective after HAART.

Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS gene

We present a case of autoimmune lymphoproliferative syndrome (ALPS) caused by a previously undescribed minimal deletion in the death domain of the FAS gene. ALPS is an uncommon disease associated with an impaired Fas-mediated apoptosis. The patient presented with a history of splenomegaly since 4 months of age, associated with cervical lymphadenopathy, which improved with oral corticosteroid treatment. Relevant laboratory findings were the presence of anemia, thrombocytopenia, and positive direct and indirect Coombs tests. He was not an offspring of consanguineous parents. Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. In both lymph nodes, there was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation and a very high cell proliferating index. Some clear cells were also present, raising the suspicion of malignant lymphoma. In one of the lymph nodes, there was also a focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease. Immunostaining showed numerous CD3+ cells, many of which were double-negative (CD4- CD8-) and expressed CD57, especially around the follicles. Molecular studies of the lymph node biopsy showed a point deletion (4-base pair deletion) in exon 9 of the FAS gene (930del TGCT), which results in 3 missense amino acids. (c) 2008 Elsevier Inc. All rights reserved.

Immunoendocrinology of the Thymus in Chagas Disease

During immune response to infectious agents, the host develops an inflammatory response which could fail to eliminate the pathogen or may become dysregulated. In this case, the ongoing response acquires a new status and turns out to be detrimental. The same elements taking part in the establishment and regulation of the inflammatory response (cytokines, chemokines, regulatory T cells and counteracting compounds like glucocorticoids) may also mediate harmful effects. Thymic disturbances seen during Trypanosoma cruzi (T. cruzi) infection fit well with this conceptual framework. After infection, this organ suffers a severe atrophy due to apoptosis-induced thymocyte exhaustion, mainly affecting the immature double-positive (DP) CD4+CD8+ population. Thymus cellularity depletion, which occurs in the absence of main immunological mediators involved in anti-T. cruzi defense, seems to be linked to a systemic cytokine/hormonal imbalance, involving a dysregulated increase in Tumor Necrosis Factor alpha (TNF-alpha) and corticosterone hormone levels. Additionally, we have found an anomalous exit of potentially autoimmune DP cells to the periphery, in parallel to a shrinkage in the compartment of natural regulatory T cells. In this context, our data clearly point to the view that the thymus is a target organ of T. cruzi infection. Preserved thymus may be essential for the development of an effective immune response against T. cruzi, but this organ is severely affected by a dysregulated circuit of proinflammatory cytokines and glucocorticoids. Also, the alterations observed in the DP population might have potential implications for the autoimmune component of human Chagas disease. Copyright (C) 2011 S. Karger AG, Basel

Human mucosal leishmaniasis: Neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL-17-type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL-17 and IL-17-inducing cytokines (IL-1 beta, IL-23, IL-6 and TGF-beta) were detected by immunohistochemistry in ML patients. IL-17(+) cells exhibited CD4(+), CD8(+) or CD14(+) phenotypes, and numerous IL-17(+) cells co-expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17-mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP-9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL-17 is involved in ML pathogenesis.

Translocations t(X;14)(q28;q11) and t(Y;14)(q12;q11) in T-cell prolymphocytic leukemia

P>We report a case of T-cell prolymphocytic leukemia (T-PLL) in a 41-year-old male. Classical cytogenetic, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) studies of a blood sample obtained at diagnosis revealed the co-existence of t(X;14)(q28;q11), t(Y;14)(q12;q11) and a ring chromosome derivated from i(8)(q10). Immunophenotypic studies revealed involvement of T-cell lineage, with proliferation of CD4(-) CD8(+). The co-existence of two translocations involving both sex chromosomes in a case of T-PLL is rare. Chromosomal instability associated with the disease progression may have allowed the emergence of cell clones with translocations involving the sex chromosomes and the ring chromosome observed.

5-Lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection

This study provides evidence supporting the idea that although inflammatory cells migration to the cardiac tissue is necessary to control the growth of Trypanosoma cruzi, the excessive influx of such cells during acute myocarditis may be deleterious to the host. Production of lipid mediators of inflammation like leukotrienes (LTs) along with cytokines and chemokines largely influences the severity of inflammatory injury in response to tissue parasitism. T cruzi infection in mice deficient in 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of LTs and other lipid inflammatory mediators, resulted in transiently increased parasitemia, and improved survival rate compared with WT mice. Myocardia from 5-LO(-/-) mice exhibited reduced inflammation, collagen deposition, and migration of CD4(+), CD8(+), and IFN-gamma-producer cells compared with WT littermates. Moreover, decreased amounts of TNF-alpha, IFN-gamma, and nitric oxide synthase were found in the hearts of 5-LO(-/-) mice. Interestingly, despite of early higher parasitic load, 5-LO(-/-) mice survived, and controlled T cruzi infection. These results show that efficient parasite clearance is possible in a context of moderate inflammatory response, as occurred in 5-LO(-/-) mice, in which reduced myocarditis protects the animals during T cruzi infection. (c) 2010 Elsevier Masson SAS. All rights reserved.

Oleic acid modulation of the immune response in wound healing: A new approach for skin repair

Injury triggers inflammatory responses and tissue repair. Several treatments are currently in use to accelerate healing: however, more efficient formulations are still needed for specific injuries. Since unsaturated fatty acids modulate immune responses, we aimed to evaluate their therapeutic effects on wound healing. Skin wounds were induced in BALB/c mice and treated for 5 days with n-3, n-9 fatty acids or vehicle (control). n-9 treated mice presented smaller wounds than control and n-3 at 120 h post-surgery (p.s.). Collagen III mRNA,TIMP1 and MMP9 were significantly elevated in n-9 group compared to n-3 or vehicle at 120 h p.s. Among the inflammatory mediators studied we found that IL-10, TNF-alpha and IL-17 were also higher in n-9 treated group compared to n-3 or vehicle at 120 h p.s. Interestingly, COX2 had decreased expression on wound tissue treated with n-9. Inflammatory infiltrate analysis revealed diminished frequency of CD4(+), CD8(+) and CD11b(+) cells in n-9 wounds at 24 and 120 h p.s., which was not related to cell death, since in vitro apoptosis experiments did not show any cell damage after fatty acids administration. These results suggested that unsaturated fatty acids, specifically n-9, modulate the inflammation in the wound and enhance reparative response in vivo. n-9 may be a useful tool in the treatment of cutaneous wounds. (C) 2010 Elsevier GmbH. All rights reserved.

Chemokines in human periodontal disease tissues

An immunoperoxidase technique was used to examine IP-10 (interferon-gamma inducible protein 10), RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemoattractant protein-1), and MIP-1alpha (macrophage inflammatory protein-1alpha) in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups according to the size of infiltrate. MIP-1alpha+ cells were more abundant than the other chemokines with few MCP-1+ cells. The mean percent MIP-1alpha+ cells was higher than the percent MCP-1+ cells (P = 0.02) in group 2 (intermediate size infiltrates) lesions from periodontitis subjects, other differences not being significant due to the large variations between tissue samples. Analysis of positive cells in relation to CD4/CD8 ratios showed that with an increased proportion of CD8+ cells, the mean percent MIP-1alpha+ cells was significantly higher in comparison with the mean percent RANTES+ and MCP-1+ cells (P < 0.015). Endothelial cells were MCP-1+ although positive capillaries were found on the periphery of infiltrates only. Keratinocyte expression of chemokines was weak and while the numbers of healthy/gingivitis and periodontitis tissue sections positive for IP-10, RANTES and MCP-1 reduced with increasing inflammation, those positive for MIP-1alpha remained constant for all groups. In conclusion, fewer leucocytes expressed MCP-1 in gingival tissue sections, however, the percent MIP-1alpha+ cells was increased particularly in tissues with increased proportions of CD8 cells and B cells with increasing inflammation and also in tissues with higher numbers of macrophages with little inflammation. Further studies are required to determine the significance of MIP-1alpha in periodontal disease.

Aids e estomatite aftóide recidivante

O estado de imunodeficiência em pacientes HIV positivos tem sido causa de episódios severos de Estomatite Aftóide Recidivante (EAR). OBJETIVO: Este estudo objetiva estabelecer evidências da relação entre o surgimento (ou agravamento) de EAR com o estado de imunossupressão causado pelo vírus HIV, por meio da contagem de células CD4+, CD8+ e da carga viral infectante. FORMA DE ESTUDO: estudo de série. MATERIAL E MÉTODO: Noventa e quatro pacientes HIV (1)-positivos (25 mulheres e 69 homens) com EAR foram acompanhados no ambulatório de Aids da Divisão de Clínica ORL do Hospital das Clínicas da FMUSP no período de janeiro de 1998 a dezembro de 2003. A idade dos pacientes variou de 19 a 63 anos (média = 35,3 anos). RESULTADO: Os pacientes com Aids e soropositivos apresentaram, respectivamente, oito aftas e duas aftas por surto. Da mesma maneira, os pacientes portadores de úlceras do tipo major apresentaram menor contagem de células CD8+, CD4+ e relação CD4+/CD8+ e maior valor médio da carga viral do que os pacientes portadores de aftas herpetiformes e minor. Entre os portadores de aftas minor e herpetiforme não houve diferença estatística. CONCLUSÕES O aparecimento das lesões, principalmente as do tipo major, está diretamente relacionado ao estado imunitário do paciente soropositivo, acarretando déficits nutricionais e piora na qualidade de vida. Desta forma, o diagnóstico e tratamento da EAR é um desafio que não deve ser desprezado.