Immune reconstitution after autologous hematopoietic stem cell transplantation��

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B���T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin���s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-rela����es entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreens��o deste complexo sistema biol��gico. Tal �� particularmente verdade no caso das interac����es entre os linf��citos B e T, quer durante o desenvolvimento celular, quer ao n��vel das fun����es celulares efectoras. A compreens��o da interdepend��ncia entre linf��citos B e T e a possibilidade de manipular esta rela����o pode ser directamente aplic��vel a situa����es em que a imunidade est�� deficiente, como �� o caso das doen��as neopl��sicas ou da imunossupress��o ap��s radio ou quimioterapia. O trabalho apresentado nesta disserta����o iniciou-se com o desenvolvimento de um novo m��todo laboratorial para medir directamente a diversidade do report��rio celular (Cap��tulo III). Redu����es da diversidade do report��rio dos receptores de c��lulas T t��m sido relacionadas com um estado de imunodefici��ncia. O m��todo desenvolvido utiliza ���gene chips���, aos quais hibridizam os ��cidos nucleicos codificantes das cadeias proteicas dos receptores linfocit��rios. A diversidade �� calculada com base na frequ��ncia de hibridiza����o do ��cido nucleico da amostra aos oligonucle��tidos presentes no ���gene chip���. De seguida, e utilizando este novo m��todo e outras t��cnicas de quantifica����o celular examinei, num modelo animal, o papel que as c��lulas policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocit��rio T no timo, especificamente na aquisi����o de um report��rio diverso de receptores T (Cap��tulos IV e V). Testei, ent��o, a hip��tese de que a presen��a no timo de p��ptidos mais diversos, como a imunoglobulna policlonal, induzisse a g��nese de precursores T mais diversos. Demonstr��mos que a diversidade do compartimento T �� aumentado pela presen��a de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta mol��cula, representam as mol��culas aut��logas mais diversas presentes nos organismos vertebrados. Estes p��ptidos s��o apresentados por c��lulas apresentadoras de antig��nio ��s c��lulas precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a g��nese da diversidade dos receptores. Tamb��m demonstr��mos que a presen��a de um report��rio mais diverso de linf��citos T se associa a um incremento da fun����o imunol��gica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejei����o mais eficientes de um maior n��mero de agressores internos e externos. Demonstr��mos que ratinhos com receptores de c��lulas T (RCT) com maior diversidade rejeitam transplantes cut��neos discordantes para antig��nios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor report��rio T (Cap��tulo V). Por outro lado, uma redu����o da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobreviv��ncia de transplantes cut��neos incompat��veis para o antig��nio H-Y (antig��nio minor de histocompatibilidade), indicando uma diminui����o da fun����o linfocit��ria T. Al��m disso, a reconstitui����o da diversidade dos linf��citos T em ratinhos com uma diversidade de report��rio T diminu��da, induzida pela administra����o de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminui����o significativa da sobreviv��ncia dos enxertos cut��neos (Cap��tulo V). Estes resultados sugerem que o aumento do report��rio de c��lulas T contribui para uma melhoria das fun����es celulares T e poder��o ter implica����es importantes na terap��utica e reconstituti����o imunol��gica em contexto de SIDA, neoplasias, autoimunidade e ap��s tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hip��tese cl��nica de que doentes com neoplasias hematol��gicas sujeitos a transplanta����o de precursores hematopoi��ticos e com recupera����o imunol��gica precoce ap��s transplante teriam uma sobreviv��ncia mais longa do que doentes que n��o recuperassem t��o bem a sua imunidade. Analis��mos a sobreviv��ncia global e sobreviv��ncia sem doen��a de 42 doentes com linfoma n��o Hodgkin de c��lulas do manto sujeitos a transplante aut��logo de precursores hematopoi��ticos (Cap��tulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linf��citos imediatamente ap��s o transplante aut��logo, apresentaram uma sobreviv��ncia global e sem progress��o mais longa do que doentes que n��o recuperaram contagens linfocit��rias t��o precocemente. Estes resultados demonstram o efeito positivo de uma reconstituti����o imunol��gica robusta ap��s transplante de presursores hematopoi��ticos, sobre a sobreviv��ncia de doentes com neoplasias hematol��gicas. Do mesmo modo, estud��mos o efeito que a recupera����o de n��veis s��ricos normais de imunoglobulina policlonal tem na sobreviv��ncia de doentes com outras neoplasias hematol��gicas de linf��citos B, como o mieloma m��ltiplo,ap��s transplante aut��logo de precursos hematopoi��ticos (Cap��tulo VII). A sobreviv��ncia livre de doen��a dos 110 doentes com mieloma m��ltiplo analizados est�� associada com a sua capacidade de recuperar n��veis s��ricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a import��ncia da imunoglobulina policlonal para a g��nese de compet��ncia imunol��gica. Tamb��m estud��mos o impacto de um sistema imunit��rio eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma n��o Hodgkin (LNH) (Cap��tulo VIII). Os resultados mostram que doentes com valores mais elevados de linf��citos T CD4+ respondem melhor (em termos de maior sobrevida livre de doen��a) ao rituximab, do que doentes com valores mais baixos. Estas observa����es ilustram a necessidade de um sistema imunit��rio competente para o benef��cio cl��nico da terap��utica com rituximab em doentes com LNH. Em conclus��o, o trabalho apresentado nesta disserta����o demonstra que as c��lulas B e a imunoglobulina policlonal promovem a diversidade das c��lulas T no timo e melhoram a fun����o linfocit��ria T perif��rica. Concomitantemente, tamb��m demonstr��mos que, no contexto de reconstitui����o imune, por exemplo, ap��s transplante aut��logo de precursores hematopoi��ticos em doentes com linfomas de c��lulas do manto, o n��mero absoluto de linf��citos �� uma factor independente da sobreviv��ncia. Os resultados demonstram, tamb��m, a import��ncia dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princ��pio se prova pelo facto de que doentes com mieloma m��ltiplo sujeitos a transplante aut��logo de precursores hematopoi��ticos que recuperam valores normais s��ricos de imunoglobulinas policlonais, terem melhores taxas de resposta em compara����o com doentes que n��o recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplica����es na pr��tica cl��nica dado que a maioria dos tratamentos de doen��as neopl��sicas implica imunossupress��o e, subsequente, recupera����o imunol��gica. Estes estudos podem ser um instrumento fundamental para uma melhor compreens��o do sistema imune e guiar uma escolha mais eficiente de op����es terap��uticas bem como contribuir para a concep����o de futuros estudos cl��nicos.

A 3-YEAR FOLLOW-UP OF A BRAZILIAN AIDS PATIENT WITH PROTRACTED DIARRHEA CAUSED BY Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most prevalent microsporidian parasite that causes gastrointestinal infection in persons with AIDS. Microsporidia are increasingly recognized as important opportunistic pathogens all over the world but in Brazil only few cases have been reported due either to the non awareness of the clinical presentation of the disease or to difficulties in the laboratory diagnosis. We report a 3-year follow-up of a Brazilian HIV-positive patient in whom microsporidial spores were detected in stools and were identified as E. bieneusi using electron microscopy and PCR. The patient presented with chronic diarrhea, CD4 T-lymphocytes count below 100/mm3 and microsporidial spores were consistently detected in stools. Albendazole was given to the patient in several occasions with transient relief of the diarrhea, which reappeared as soon as the drug was discontinued. Nevertheless, a diarrhea-free period with weight gain up to 18 Kg occurred when a combination of nucleoside and protease inhibitors was initiated as part of the antiviral treatment.

PARACOCCIDIOIDOMYCOSIS AND AIDS: REPORT OF THE FIRST TWO COLOMBIAN CASES

The records of the first two Colombian patients with AIDS and paracoccidioidomycosis are presented. Both patients were males and had no known risk factors for HIV although in the past they had worked in the field where they could have been infected with the fungus. They exhibited the juvenile type of disease with multiple organ system involvement and symptoms of short duration. They were deeply immunodepressed as indicated by less than 100 CD4 T lymphocytes per mL; however, serologic tests revealed circulating anti-Paracoccidioides brasiliensis antibodies and in one patient the first diagnostic clue came from such tests. In one case, the mycosis preceded the AIDS diagnosis while in the other, both pathologies were discovered simultaneously. Antimycotic therapy with itraconazole was administered for over 10 months, with an initial dose of 200 mg/day followed by 100 mg/day; marked improvement of the mycotic signs and symptoms was soon noticed an there have been no signs of relapse. The patients�� improvement was also due to the combined retroviral treatment that was instituted. In spite of the rarity of the AIDS-paracoccidioidomycosis association, physicians practicing in endemic areas should consider the presence of the mycosis in immunosuppressed patients, since a prompt diagnosis and institution of combined antimycotic-anti-retroviral treatments would result in patient improvement and survival. It appears possible that the longer survival time of today's AIDS patients would give the quiescent fungus the opportunity to revive, multiply and cause overt disease.

Cytokine serum levels in patients infected by human immunodeficiency virus with and without Trypanosoma cruzi coinfection

This study assessed the number of CD4 T lymphocytes, the parasitemia and serum levels of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-4 and IL-10 of patients infected by human immunodeficiency virus (HIV) and human immunodeficiency virus/Chagas' disease coinfection. CD4 T lymphocytes were low in the two groups of patients, although significantly lower in patients without Chagas' disease. Serum levels of IFN-gamma, IL-4 and TNF-alpha were significantly higher in patients with HIV/Chagas' disease. IL-4/IFN-gamma ratios were higher in patients with HIV/Chagas' disease, which showed a clear balance in favor of Th2-like cytokines in this group of patients. This Th2 balance was higher in patients with detectable parasitemia. We conclude that, although immunosuppression was observed, with CD4 T lymphocytes bellow 200/��m��, these patients did not display reactivation of T. cruzi infection and that a balance favorable to Th2 was associated with the presence of parasitemia.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Bel��m, State of Par��, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p &gt; 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p &gt; 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Coinfection with Mycoplasma Pneumoniae and Chlamydia Pneumoniae in ruptured plaques associated with acute myocardial infarction

OBJECTIVE: To study atheromas, Mycoplasma pneumoniae (M. pneumoniae), and Chlamydia pneumoniae (C. pneumoniae). METHODS: C. pneumoniae was studied with immunohistochemistry and M. pneumoniae with in situ hybridization (ISH), in segments of coronary arteries (SCA) as follows: group A - thrombosed ruptured plaques (TRP) of 23 patients who died due to acute myocardial infarction (AMI); group B - 23 nonruptured plaques (NRP) of group A patients; group C - NRP of 11 coronary patients who did not die due to AMI; and group D - 11 SCA from patients with dilated cardiomyopathy or Chagas' disease without atherosclerosis. RESULTS: The mean number of C. pneumoniae+ cells/400x in groups A, B, C, and D was, respectively, 3.3��3.6; 1.0��1.3; 1.2��2.4; and 0.4��0.3; and the percentage of M. pneumoniae area was, respectively, 3.9��3.5; 1.5�� 1.6; 0.9��0.9; and 0.4��0.2. More M. pneumoniae and C. pneumoniae were found in of group A than in group B (P<0.01). Good correlation was seen between the area of the vessel and the M. pneumoniae area in the plaque (r = 0.46; P=0.001) and between C. pneumoniae+ cells and CD4+ T lymphocytes (r = 0.42; P<0.01). The number of C. pneumoniae+ cells correlated with CD20+ B cells (r=0.48; P<0.01). CONCLUSION: M. pneumoniae and C. pneumoniae are more frequently found in TRP correlate with the intensity of the inflammation and diameter of the vessel (positive remodeling).

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.

Role of eosinophilic airway inflammation in models of asthma

Eosinophils play a central role in the establishment and outcome of bronchial inflammation in asthma. Animal models of allergy are useful to answer questions related to mechanisms of allergic inflammation. We have used models of sensitized and boosted guinea pigs to investigate the nature of bronchial inflammation in allergic conditions. These animals develop marked bronchial infiltration composed mainly of CD4+ T-lymphocytes and eosinophils. Further provocation with antigen leads to degranulation of eosinophils and ulceration of the bronchial mucosa. Eosinophils are the first cells to increase in numbers in the mucosa after antigen challenge and depend on the expression of alpha 4 integrin to adhere to the vascular endothelium and transmigrate to the mucosa. Blockage of alpha4 integrin expression with specific antibody prevents not only the transmigration of eosinophils but also the development of bronchial hyperresponsiveness (BHR) to agonists in sensitized and challenged animals, clearly suggesting a role for this cell type in this altered functional state. Moreover, introduction of antibody against Major Basic Protein into the airways also prevents the development of BHR in similar model. BHR can also be suppressed by the use of FK506, an immunosuppressor that reduces in almost 100% the infiltration of eosinophils into the bronchi of allergic animals. These data support the concept that eosinophil is the most important pro-inflammatory factor in bronchial inflammation associated with allergy.

The tumor suppressor p16Ink4a regulates T lymphocyte survival.

In contrast to other cell cycle inhibitors, the tumor suppressor p16Ink4a is not detectable or expressed at very low levels in embryonic and adult mouse tissues, and therefore it has often been considered as a specialized checkpoint protein that does not participate in the control of normal cell cycle progression. However, Ink4a-/- mice possess increased thymus size and cellularity, thus suggesting the involvement of p16(Ink4a) in the control of thymocyte proliferation. In this study, we found increased numbers of CD8 and CD4 T lymphocytes in thymus and spleen from Ink4a-/- mice. Unexpectedly, this was not related to an increase in T-cell division rates, which were similar in lymphoid organs of Ink4a-/- and wild-type mice. In contrast, T-cell apoptosis rates were significantly decreased in thymus and spleen from Ink4a-/- mice. Moreover, whereas p16Ink4a-deficient and wild-type T cells were equally sensitive to Fas or TCR-mediated apoptosis, the former were clearly more resistant to apoptosis induced by oxidative stress or gamma irradiation. Our results indicate that p16Ink4a function is associated with T-cell apoptosis, and subsequently contributes to the control of T-cell population size in lymphoid organs.

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 ��l of an emulsion containing 50 ��g of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-&#945;, TGF-&#946; and CD4+ and acinar IFN-&#947;, TNF-&#945;, IL-1&#946; and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-&#946;. Significant positive correlations were found between portal CD4+ and TNF-&#945;, portal CD8+ and TGF-&#946;, portal CD45+RO and TNF-&#945;, acinar CD45+RO and IFN-&#947; and acinar CD57+ and TGF-&#946;. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4+ T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Levels of immune cells in transcendental meditation practitioners.

CONTEXT Relationships between mind and body have gradually become accepted. Yogic practices cause modulation of the immune system. Transcendental meditation (TM) is a specific form of mantra meditation. We reported previously different plasma levels of catecholamines and pituitary hormones in TM practitioners comparing with a control group, and patterns of the daytime secretion of these hormones different from those normally described. AIMS The aim of the following study is to evaluate the immune system in these meditation practitioners, by determining leukocytes and lymphocytes subsets. METHODS TM group consisted of 19 subjects who regularly practice either TM or the more advanced Sidhi-TM technique. A control group consisted of 16 healthy subjects who had not previously used any relaxation technique. Total leukocytes, granulocytes, lymphocytes and monocytes were counted by an automated quantitative hematology analyzer, whereas lymphocytes subsets were determined by flow cytometry. Samples were taken from each subject at 0900 h after an overnight fast. RESULTS The results indicated that the TM group had higher values than the control group in CD3+CD4-CD8+ lymphocytes (P < 0.05), B lymphocytes (P < 0.01) and natural killer cells (P < 0.01), whereas CD3+CD4+CD8- lymphocytes showed low levels in meditation practitioners (P < 0.001). No significant differences were observed in total leukocytes, granulocytes, monocytes, total lymphocytes or CD3+ lymphocytes comparing both groups. CONCLUSIONS The technique of meditation studied seems to have a significant effect on immune cells, manifesting in the different circulating levels of lymphocyte subsets analyzed. The significant effect of TM on the neuroendocrine axis and its relationship with the immune system may partly explain our results.

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8+ T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-��, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1��, MIP-1��, RANTES, IP-10, MIG, and IFN-��). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-��, IL-2, TNF-��, MIP-1��, MIP-1��, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.