Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.

Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice.

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.

Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.

An��lise fenot��pica de c��lulas dendr��ticas e de linf��citos T efetores, polifuncionais e reguladores no l��quen plano

INTRODU����O: L��quen plano (LP) �� uma doen��a mucocut��nea de natureza inflamat��ria cr��nica de etiologia ainda desconhecida. A estimula����o da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as c��lulas dendr��ticas e direcionar a resposta de c��lulas T CD4+ e CD8+ efetoras, assim como tamb��m favorecer o estado inflamat��rio do LP. OBJETIVOS: Avaliar o perfil fenot��pico de c��lulas dendr��ticas miel��ides (mDCs) e plasmocit��ides (pDCs) e de linf��citos T CD4+ e CD8+ ap��s est��mulo com agonistas de TLRs no sangue perif��rico de pacientes com LP. Al��m disto, avaliar a frequ��ncia, perfil de matura����o e os subtipos de c��lulas T CD4+ e TCD8+ reguladores. M��TODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 �� 4,73 anos de idade e um grupo controle com 22 indiv��duos sadios (18 mulheres, 4 homens), com 43,92 �� 7,83 anos de idade. As c��lulas mononucleares (CMNs) de sangue perif��rico foram avaliadas por citometria de fluxo quanto ��: 1) Produ����o de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) An��lise de c��lulas T CD4+ e CD8+ monofuncionais e polifuncionais ap��s est��mulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avalia����o de c��lulas Th17 e Th22/Tc22 em CMNs ap��s est��mulo com SEB; 4) Frequ��ncia, perfil de matura����o e subtipos de c��lulas T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequ��ncia de mDCs TNF-alfa+ ap��s est��mulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminui����o da express��o de CD83. J�� nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a express��o de CD80 e o CpG/TLR9 diminuiu a express��o de CD83 no LP. 2) As c��lulas T CD4+ secretoras de IL-10 mostraram aumento da frequ��ncia nos n��veis basais, que diminuiu ap��s est��mulo com LPS e SEB. Em contraste, a produ����o de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As c��lulas T CD4+ polifuncionais, secretoras de 5 citocinas simult��neas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminu��ram no LP ap��s est��mulo com CL097 e CpG. Entretanto, na aus��ncia de IL-10, houve aumento da frequ��ncia de c��lulas CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em c��lulas TCD4+ que expressam CD38, marcador de ativa����o cr��nica e na aus��ncia de IL-10. Similarmente, ��s TCD4+, uma diminui����o de c��lulas T CD8+ IFN-y+ e TNF+ foram detectadas ap��s est��mulo com CpG. 3) As c��lulas Th22/Tc22 nos n��veis basais e ap��s est��mulo com SEB se mostraram aumentadas. As c��lulas Th17 n��o mostraram diferen��as entre os grupos. 4) A frequ��ncia das c��lulas T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) est�� elevada no LP. Quanto aos perfis de matura����o, h�� aumento na frequ��ncia de c��lulas TCD4+ de mem��ria efetora enquanto que para as c��lulas T CD8+ h�� predom��nio das c��lulas de mem��ria central. Quanto aos subtipos, h�� aumento nas c��lulas T CD4+ regs perif��ricas (pT reg). CONCLUS��ES: O estado de ativa����o das mDCs ap��s ativa����o das vias de TLRs 4 e 7/8 pode influenciar na gera����o de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos est��mulos TLRs reflete a ativa����o destas c��lulas no sangue perif��rico. Al��m disso, o aumento de Th22/Tc22 e das c��lulas T regs indicam uma rela����o entre regula����o e c��lulas efetoras no sangue perif��rico evidenciando que existem altera����es extracut��neas no LP

Mapping the determinants in owl monkey CD4 and CCR5 that allow entry of early-stage HIV-1 Env variants

Thesis (Master's)--University of Washington, 2016-06

Immunohistological analysis of Tannerella forsythia-induced lesions in a murine model

Tannerella forsythia has been implicated as a defined periodontal pathogen. In the present study a mouse model was used to determine the phenotype of leukocytes in the lesions induced by subcutaneous injections of either live (group A) or nonviable (group B) T. forsythia. Control mice (group C) received the vehicle only. Lesions were excised at days 1, 2, 4, and 7. An avidin-biotin immunoperoxidase method was used to stain infiltrating CD4(+) and CD8(+) T cells, CD14(+) macrophages, CD19(+) B cells, and neutrophils. Hematoxylin and eosin sections demonstrated lesions with central necrotic cores surrounded by neutrophils, macrophages and lymphocytes in both group A and group B mice. Lesions from control mice exhibited no or only occasional solitary leukocytes. In both groups A and B, neutrophils were the dominant leukocyte in the lesion 1 day after injection, the numbers decreasing over the 7-day experimental period. There was a relatively low mean percent of CD4(+) and CD8(+) T cells in the lesions and, whereas the percent of CD8(+) T cells remained constant, there was a significant increase in the percent of CD4(+) T cells at day 7. This increase was more evident in group A mice. The mean percent of CD14(+) macrophages and CD19(+) B cells remained low over the experimental period, although there was a significantly higher mean percent of CD19(+) B cells at day 1. In conclusion, the results showed that immunization of mice with live T. forsythia induced a stronger immune response than nonviable organisms. The inflammatory response presented as a nonspecific immune response with evidence of an adaptive (T-cell) response by day 7. Unlike Porphyromonas gingivalis, there was no inhibition of neutrophil migration.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity

Modification of human islets prior to transplantation may improve long-term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo-rejection. Intragraft incorporation of stem cells secreting beta (��)-cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three-dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture-assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo-response in discrete T-cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo-response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T-cell subsets to AEC-bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre-transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo-reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long-term graft survival in islet transplantation.

The relationship of thymulin activity, immune parameters of HIV disease progression and substance use in a cohort of HIV seropositive alcohol and drug users

Zinc is essential for the activity of thymulin, a thymic hormone involved in T-lymphocyte differentiation and activation. Zinc deficiency is widespread in populations with HIV infection, and HIV+ drug users are particularly susceptible to zinc deficiency and immune suppression. This dissertation explored the relationship of zinc-bound active thymulin to plasma zinc, CD4+ and CD8+ cell count, the CD4+/CD8+ ratio, and drug use in HIV-infected drug users. Zinc-bound active thymulin was assessed in plasma of HIV+ drug users who were participating in a 30 month zinc supplementation trial. Plasma from 80 participants at the 12 month visit, and 40 of these same participants, randomly selected, at the baseline visit were assessed for zinc-bound active thymulin levels using a modification of the rosette inhibition assay. Thymulin activity was directly associated with CD4+ cell count (�� = 0.127, p = 0.002) and inversely associated with cocaine use (�� = ���0.908, p = 0.026; R2 = 0.188, p = 0.019) independent of HIV viral load, age, gender and antiretroviral use. An increase in thymulin activity was 1.4 times more likely when CD4+ cell count increased (OR = 1.402, 95%CI: 1.006���1.956), independent of change in viral load, antiretroviral use, and age. Participants who used cocaine consistently, were 7.6 times less likely to have an increase in thymulin activity (OR = 0.133, 95%CI: 0.017���1.061). There was a direct correlation between change in plasma zinc and change in zinc-bound active thymulin (r = 0.243, p = 0.13). Analysis of CD4+ cell count decline in 222 participants in the zinc supplementation trial across the 30 months showed that both crack cocaine use and heavy alcohol use accelerated CD4+ cell count decline. Thymulin activity is directly associated with HIV disease progression, measured by CD4+ cell count, and is depressed with cocaine use independent of antiretroviral use and HIV viral load. Cocaine and heavy alcohol accelerate CD4+ cell count decline. The effect of cocaine on thymic output requires further evaluation as a mechanism for the association of cocaine use with faster HIV disease progression.

Mol��culas coestimulat��rias na leishmaniose visceral

Visceral leishmaniasis (VL) is endemic in many countries, including Brazil. The protozoan Leishmania infantum, is the etiological agent of VL, and is transmitted by the bite of female sandflies during the blood meal. The majority of subjects when exposed to the parasite do not develop the disease, because of development of Th1 cellular responses. Those who have develop signs of VL such as fever, weight loss, hepatosplenomegaly, have impairment of the cellular immune response, specific to the Leishmania antigens. We evaluated whether the specififc anergy during symptomatic VL, may be associated with changes in T cells costimulatory molecules or their ligands in CD14+ monocytes. There is an increase in CTLA-4 porcentage on CD4+ T lymphocytes (p=0.001) and ICOS on CD4+ and CD8+ T cells (p=0.002 to CD4+ and p=0.003 to CD8+), after stimulation by soluble Leishmania antigen (SLA) during active visceral leishmaniasis, and that there is a higher percentage of these molecules ex vivo, when comparing symptomatic to recovered individuals (p=0.04 to CTLA-4 in CD4+, and p=0.001 to ICOS in CD4+ and p=0.026 to CD8+). Moreover, we found a high gene expression of CTLA-4, OX-40 and ICOS during active VL. CD40, CD80, CD86, HLA-DR and ICOSL molecules do not suffer changes during disease. There is IFN-�� production by the peripheral blood cells, after SLA stimulation, by peripheral blood cells in symptomatic subjects; however, there is a decrease of the ratio IFN-��/IL-10, which is reversed after clinical recovery. The impairment of some costimulatory molecules pathways during symptomatic VL could inhibit the ability of phagocytes to kill Leishmania and could facilitate their survival and the proliferation inside macrophages.

��volution des r��ponses CD4 et CD8 dirig��s contre le VIH au cours de l'infection

M��moire num��ris�� par la Direction des biblioth��ques de l'Universit�� de Montr��al.

L'interleukine-21 : un nouveau joueur dans l'hom��ostasie des lymphocytes T m��moires CD8 ��� et dans l'h��matopo����se

M��moire num��ris�� par la Direction des biblioth��ques de l'Universit�� de Montr��al.

��volution des r��ponses CD4 et CD8 dirig��s contre le VIH au cours de l'infection

M��moire num��ris�� par la Direction des biblioth��ques de l'Universit�� de Montr��al.

L'interleukine-21 : un nouveau joueur dans l'hom��ostasie des lymphocytes T m��moires CD8 ��� et dans l'h��matopo����se

M��moire num��ris�� par la Direction des biblioth��ques de l'Universit�� de Montr��al.

Impact of weight reduction program on serum alanine aminotransferase activity and immunologic response in obese hepatitis B patients.

Background: Globally, chronic B viral hepatitis (HBV) is a major health problem. Obesity is a common problem among patients with HBV. Several studies have reported that obesity is an important risk factor that alters immune system response in individuals with no underlying cause of liver disease. However, there is a strong association between BMI and the human immune system among HBV patients. Objective: This study was to examine the correlation between body mass index, serum alanine aminotransferase activity (ALT) and immunologic response in obese hepatitis B patients. Material and methods: One hundred fifty male patients with chronic hepatitis B virus, their age ranged from 30 to 45 (38.64 �� 7.12) years and their BMI ranged from 30-35 kg/m2. All Subjects were included in two groups: The first group received weight reduction program in the form of treadmill aerobic exercises in addition to diet control whereas the second group received no therapeutic intervention. Parameters of serum alanine aminotransferase (ALT), CD3, CD4 and CD8 were quantified; Leukocyte, differential counts and body mass index (BMI) were measured before and after 3 months at the end of the study. Results: There was a 24.7%, 36.8%, 30.8%, 40.7%, 28.6%, 25.9%, 33.3% and 14.3 % reduction in mean values of alanine aminotransferase (ALT), white blood cells, total neutrophil count, monocytes, CD3, CD4 ,CD8 and BMI respectively in group (A) at the end of the study. In addition, there were significant differences between mean levels of the investigated parameters in groups. Conclusion: Based on our findings, weight loss modulates serum alanine aminotransferase and immune system parameters of patients with hepatitis B virus infection.

Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 ��g/100 ��L) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNF��, IFN �� and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p &lt; 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNF��, IFN �� and IL2 and CD69 expression in the group of animals immunized at a dose of 10 ��g when compared to control group (p &lt; 0.05). Conclusion: A 10 ��g dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.