Hematopoietic systems in vertebrates have been the focus of intense study. However immunocyte development is well characterized in very few invertebrate groups. The sea urchin is an attractive model for the study of immune cell development. Larval immunocytes, pigment cells and derivatives of the blastocoelar cells, emerge from a small population of precursors specified at blastula stage. Analyses from the genome reveal a complex system of immune receptors and effectors and a near complete set of homologues of vertebrate transcriptional regulators. Characterization of the expression profile and function of sea urchin homologues of key vertebrate hematopoietic transcription factors imply a conserved role in immunocyte development. SpGatac, an orthologue of the vertebrate Gata-1/2/3 transcription factors and SpScl, an orthologue of Scl/Tal-2/Lyl-1 transcription factors are both required for immune cell specification in the embryo. An important cis-regulatory mechanism that restricts SpGatac expression to the blastocoelar cells involves repression by SpGcm in the pigment cells. Characterization of the expression of several additional transcription factors, including SpE2A, an orthologue of vertebrate E2A/HEB/ITF2, SpId, an orthologue of the Class V bHLH factors that modulate E-protein function, and SpLmo2, an orthologue of the cofactor part of the transcriptional complex that includes Scl and Gata family members, suggests the existence of a conserved regulatory complex for hematopoiesis. Two isoforms of the SpE2A gene were identified. The shorter isoform shares genomic organization and sequence conservation with the mouse paralogue of E2A, HEBAlt. Expression of SpE2A and SpE2AAlt is consistent with a function in immunocyte development in the sea urchin embryo. Findings of the counterpart to a key vertebrate regulatory system functioning in the development of immunocytes in the simple sea urchin embryo lay the foundation for comparative immunocyte developmental gene regulatory network analyses. These will in turn lead to a greater understanding of the evolution of immune systems across phyla and will provide simple invertebrate model systems for detailed comparative investigations of regulatory function with direct relevance to vertebrates.

Prenatal stressors have been reported to affect postnatal cognitive, metabolic, reproductive and immune functions. This study examined immune indices and function in Brahman calves prenatally stressed by transportation of their dams on d 60, 80, 100, 120 and 140 ± 5 d of gestation. Based on assessment of cow’s temperament and their reactions to repeated transportation it was evident that temperamental cows displayed greater pre-transport cortisol (P < 0.0001) and glucose (P < 0.03) concentrations, and habituated slower to the stressor compared to cows of calm and intermediate temperament. Serum concentration of cortisol at birth was greater (P < 0.03) in prenatally stressed versus control calves. Total and differential white blood cell counts and serum cortisol concentration in calves from birth through the age of weaning were determined. We identified a sexual dimorphism in neutrophil cell counts at birth (P = 0.0506) and cortisol concentration (P < 0.02) beginning at 14 d of age, with females having greater amounts of both. Whether weaning stress differentially affected cell counts, cortisol concentrations and neutrophil function of prenatally stressed and control male calves was examined. At 2 d post weaning, all calves had increased cortisol concentration (P < 0.0001) and neutrophil cell counts (P < 0.0001). However, in vitro production of reactive oxidative species by neutrophils was decreased (P = 0.0002) 2 d post weaning. Moreover, prenatally stressed calves demonstrated a larger (P = 0.0203) decrease in their immune function relative to control calves at 2 d post-weaning. Importantly, prenatally stressed calves took longer than controls to recover from the weaning stress. Additional studies are needed to clarify if prenatally stressed calves are more susceptible than control calves to pathogens during the post weaning period. Management practices to improve animal welfare and livestock production may need modification if follow-up studies demonstrate that prenatal stress also affects reproductive development, growth, performance and meat quality.

No description available.

OBJECTIVE: T2D is a metabolic disease that is a significant health problem throughout many populations. Increased incidence of T2D across the age spectrum makes preventive measures for this disease a top healthcare priority. Physiological changes such as expression of pro-inflammatory T cell cytokines, insulin resistance, and pancreatic beta cell dysfunction play major roles in the onset of T2D. Current treatments include lifestyle changes with oral medications and/or synthetic insulin therapy. While treatments aim to normalize blood glucose and increase insulin sensitivity in patients diagnosed with T2D, efforts are growing to find preventative therapies for prediabetes, a condition where blood glucose levels are higher than normal but are under the threshold determined for a diabetes diagnosis. Metformin, a well-known first-line recommendation for treating T2D, in conjunction with lifestyle modification may be a viable preventative measure to delay the onset of T2D. Previous study results have created momentum to generate data promoting metformin use as an off-label preventative drug for T2D. To identify a therapeutic intervention that may help to shift T cell cytokine profiles from being pro-inflammatory and diabetogenic to anti-inflammatory, we investigated the effects of metformin on immune cell function in prediabetic patients. It is known that one effect of metformin is activating AMPK, which secondarily decreases inflammation. We therefore hypothesized metformin affects immune cell function by modulating genes in the AMPK pathway. METHODS: We recruited 49 subjects using EPIC database to screen patients with appointments at the Nutrition and Weight Management Center at Boston Medical Center. Forty-nine pre-metformin and 13 post- metformin blood samples were collected from subjects at baseline and after three months of taking metformin, respectively. Ficoll was utilized to separate and extract PBMCs. I activated PBMCs with LPS or CpG for 24 hours, and anti-CD3/CD28 for 24 or 40 hours. Then I isolated and reverse transcribed RNA, producing cDNA. We ran a human AMPK signaling qRT-PCR array on the 40-hour anti-CD3/CD28 activated PBMCs from 4 randomly chosen subjects and analyzed data to investigate candidate targets in the AMPK pathway possibly modulated by metformin. I designed primers for six chosen targets and ran qRT-PCR comparing the pre- and post-metformin dataset of 13 subjects, using the generated human gene-specific primers to see if these genes were affected across the dataset. RESULTS: Total sample population was n=13. The majority of subjects were African American females. The study participants were considered prediabetic when A1C measured between 5.7-6.4%. Median A1C and BMI averaged at 5.8% and 38.6 kg/m2  2.48 (mean  SEM), respectively. There was an expected decrease in BMI as metformin is associated with weight loss. To understand how metformin may affect genes in the AMPK pathway, qRT-PCR array analysis of the 40-hour anti-CD3/CD28 activated PBMCs in a subset of 4 subjects was used to create a volcano plot. The plot demonstrated that out of the possible gene candidates, SLC2A4, LIPE, INSR, CRY1, GAPDH, and STK11 had the greatest log2 fold change and –log (p-value). Further analysis on the 4 subjects compared delta Ct values and relative gene expression showing CRY1, a circadian function gene, had a significant decrease in expression (p=0.03, n=4, paired t-test). Primers were designed for the six candidate genes and used to run qRT-PCR on the entire dataset of 13 subjects. There was a significant decrease in expression of STK11 in 24-hour non-stimulated PBMCs (p=0.008, n=12, paired t-test) and CRY1 in 24-hour anti-CD3/CD28 activated PBMCs (p=0.04, n=12, paired t-test). There was a significant increase in expression of SLC2A4 in 24-hour CpG activated PBMCs (p=0.02, n=12, paired t-test). Furthermore, GLUT4 was detected in CpG activated immune cells and gene expression was increased in cells from subjects post-metformin treatment. CONCLUSIONS: Further investigation is required to examine how metformin decreases the expression of CRY1 and how this decrease associates with pro-inflammatory cytokine expression. STK11 expression was decreased in non-stimulated cells but did not show any trend in the activated conditions. Additional research is warranted to see if these results can be repeated, and if so, more work will be needed to define the link between CRY1/STK11 and metformin-driven AMPK activation in immune cells. Protein expression analysis will be required to support our gene expression data. Overall, these findings initiate our understanding of how AMPK activation and changes in cellular metabolism activate pathways leading to cytokine secretion by immune cells. Further study of the downstream effects of metformin and how it may change inflammatory cytokine profiles will strengthen the evidence identifying metformin as a viable preventative therapy for prediabetic patients.

No description available.

Magister Scientiae - MSc (Chemistry) / Tuberculosis (TB) is one of the major infectious diseases that affect the health of people all over the world. South Africa is one of the countries that account for most of the TB cases; it is the leading cause of death in South Africa and is known to be lethal when combined with HIV in patients. Various tests have been used to diagnose tuberculosis infected patients, but some of these tests give false positive results. Studies have shown that tuberculosis-related cytokines can serve as biological markers for the diagnosis of TB. Cytokines are signalling proteins secreted by immune cells and one such cytokine is interferon-gamma (IFN-?). Interferon-gamma is secreted by immune cells in response to various pathogens and has many physiological roles in the immune system and inflammatory stimuli. IFN-? was first detected using antibody-based immunosensing techniques but this approach is expensive, time consuming and has low stability, it is therefore vital that an alternative detection method for IFN-? be developed.

No description available.

Human immunodeficiency virus infection is a chronic condition causing significant changes in the immune system, which are reflected in the altered gene expression patterns of the immune cells. By studying these patterns through gene expression profiling it is possible to describe not only the current states the cells are in, but also to extrapolate the proximal signals that resulted in the observed patterns. In the studies described herein, we have applied this approach to better understand the alterations in the immune function that occur in HIV infection. First, we have obtained transcriptional profiles of CD4+ and CD8+ T cells from patients in early infection, in chronic infection, and in non-progressive infection, and we compared these profiles to each other and to the profiles from uninfected donors. The analyses of the profiles revealed no discernable changes in the T cells of the non-progressive patients when compared to the uninfected individuals. On the other hand, T cells from patients with progressive infection, both early and late, showed patterns characteristic of type I interferon (IFN) exposure. We next examined experimentally the possible proximal causes of the observed transcriptional profiles. We analyzed the gene expression patterns induced by TGFβ, 13 type I interferons, as well as recombinant HIV Tat protein, in T cells and peripheral blood mononuclear cells. The TGFβ responses were inconsistent with the transcriptional profiles seen in HIV-infected patients, whereas both type I IFNs and HIV Tat induced genes in patterns consistent with those seen in patients. In fact, the thirteen IFN-induced patterns were indistinguishable from each other. Tat treatments induced interferon-stimulated genes (ISGs) as well as other genes and the response was not dependent on the presence of plasmacytoid dendritic cells (pDCs), suggesting monocytes as the possible source of the interferon response. In the last study, we examined the responses of plasmacytoid dendritic cells (pDCs) to HIV and other stimuli in healthy and HIV-infected subjects. We observed induction of IFN genes in pDCs of all subjects in response to influenza virus and TLR7 agonist imiquimod, but not to HIV virus. In summary, HIV infection results in chronic induction of type I IFN response in cells of the immune system. The source of this response is likely to be type I IFNs produced by monocytes/macrophages rather than plasmacytoid cells. The monocytic production of type I IFN may be a Tat-dependent response.

Dados mostram que o aparecimento do diabetes mellitus (DM), em pacientes previamente asmáticos, diminui os sintomas da asma, enquanto a insulina agrava a asma. Devido a dados na literatura e por dados prévios do nosso grupo, o presente estudo teve como objetivo avaliar o efeito modulatório da insulina na inflamação alérgica pulmonar em camundongos diabéticos e saudáveis. Camundongos machos dibéticos BALB/c (aloxana, 50mg/kg, iv, 10 dias) foram sensibilizados com ovalbumina (OVA, 20 &#181;g e Al (OH)3, 2 mg) 10 dias após a injeção de aloxana e uma dose reforço foi dada, após 12 dias da primeira de sensibilização, após 6 dias da dose reforço, os animais foram expostos a nebulização durante 7 dias com solução de OVA (1mg/mL) ou solução salina (SAL). Animais diabéticos foram tratados com doses múltiplas de Protamine Hagedorn Neutro (NPH) 2UI e 1UI, respectivamente, por via subcutânea 12 horas antes do desafio com OVA (às 19h) e 1UI (às 7h) 2h antes de cada desafio com OVA. Os animais não diabéticos receberam 1UI de insulina, pela mesma via 2h antes de cada desafio (às 7h), 24h após o último desafio, realizaram-se as seguintes análises: a) expressão de proteína quinase p38, proteína quinase regulada por sinais extracelulares 1 e 2 (ERK 1/2), proteína quinase ativada por estresse ou c-jun NH2- terminal (JNK) , transdutor de sinal e ativador de transcrição 3 (STAT 3) e transdutor de sinal e ativador de transcrição 6 (pSTAT 6) no homogenato de pulmão; b) perfil de imunoglobulinas presentes no soro; c) concentrações de interleucina (IL) IL-4, IL-5, IL-10, IL-13, fator de necrose tumoral alfa (TNF-&#945;), fator de crescimento endotelial vascular (VEGF), fator de crescimento transformador beta (TGF-&#946;) e interferon-gamma IFN-&#947; em homogenato de pulmão; d) migração celular em fluído do lavado broncoalveolar (LBA); e) perfil de células imunes na medula óssea, pulmão, timo e baço; f) mecânica pulmonar por BUXCO e FlexiVent. Em comparação com camundongos não diabéticos desafiados com OVA, os animais diabéticos desafiados com OVA mostraram diminuição em: ERK 1, ERK 2, JNK (fosfo54), JNK / SAPK, STAT3, pSTAT6 estava ausente; concentração da imunoglobulinas IgE, IgG1; perfil de citocinas Th2 como IL-4, IL-5, IL-13, TNF-&#945;, VEGF, TGF-&#946;; infiltrado inflamatório e) ausência de eosinofilia no LBA; células T, células B e eosinófilos na medula óssea, pulmão, timo e baço, e hiper-reatividade das vias aéreas. O tratamento com insulina restaubeleceu todos os parâmetros estudados. Portanto, sugerem que a insulina modula a inflamação alérgica pulmonar tardia em camundongos diabéticos. / Data show that the onset of diabetes mellitus (DM) in previously asthmatic patients decreases asthma symptoms while insulin worsens asthma. Due to data in the literature and previous data from our group, the present study aimed to evaluate the modulatory effect of insulin on pulmonary allergic inflammation in diabetic and healthy mice. Ovalbumin (OVA, 20 &#181;g and Al (OH)3, 2 mg) were sensitized at 10 days after alloxan injection and a booster dose was given , after 12 days of the first sensitization, after 6 days of booster dose, the animals were exposed to nebulization for 7 days with OVA solution (1mg / mL) or saline solution (SAL). Diabetic animals were treated with multiple doses of Protamine Hagedorn Neutral (NPH) 2UI and 1UI, respectively, subcutaneously 12 hours prior to challenge with OVA (at 7pm) and 1UI (at 7h) 2h before each challenge with OVA. Non-diabetic animals received 1UI of insulin, via the same route 2h before each challenge (at 7h), 24h after the last challenge, the following analyzes were performed: a) expression of protein kinase p38, protein kinase regulated by extracellular signals 1 and 2 (ERK 1/2), stress-activated or c-jun NH2-terminal protein kinase (JNK), signal transducer and transcriptional activator 3 (STAT 3) and signal transducer and transcriptional activator 6 (pSTAT 6) in the lung homogenate; b) profile of immunoglobulins present in serum; c) concentrations of interleukin (IL) IL-4, IL-5, IL-10, IL-13, tumor necrosis factor alpha (TNF-&#945;), vascular endothelial growth factor (VEGF), transforming TGF-&#946;) and interferon-gamma IFN-&#947; in lung homogenate; d) cell migration in bronchoalveolar lavage fluid (BAL); e) profile of immune cells in the bone marrow, lung, thymus and spleen; f) Pulmonary mechanics by BUXCO and FlexiVent. In contrast to non-diabetic mice challenged with OVA, diabetic animals challenged with OVA showed decrease in: ERK 1, ERK 2, JNK (phospho54), JNK / SAPK, STAT3, pSTAT6 was absent; IgE immunoglobulin levels, IgG1; profile of Th2 cytokines such as IL-4, IL-5, IL-13, TNF-&#945;, VEGF, TGF-&#946;; inflammatory infiltrate e) absence of eosinophilia in BAL; T cells, B cells and eosinophils in the bone marrow, lung, thymus and spleen, and airway hyperreactivity. The insulin treatment restored all parameters studied. Therefore, they suggest that insulin modulates late pulmonary allergic inflammation in diabetic mice.

Vaccines are a highly effective means of preventing infectious disease. However, for many diseases of livestock the available vaccines are ineffective or sub-optimal. This is partly due to challenges surrounding the specific targeting of antigen presenting cells (APCs). In order to improve the delivery of protective antigens to host APCs, a novel lentiviral vector derived from visna / maedi virus (VMV) has been developed. Initial characterisation using an enhanced green fluorescent protein (eGFP) reporter transgene found that the novel VMV vector efficiently transduced a wide range of cell lines including cells of ovine, human, murine, bovine and caprine origin. In addition, the VMV vector was found to elicit sustained transgene expression for at least 4 weeks in rapidly dividing cell lines. One of the most important factors for acceptable vaccines is their safety. Therefore, in order to increase the bio-safety of the VMV vector, integration-defective and self-inactivating forms were produced. Integration-defective VMV lentiviral vectors (IDLVs) were found to produce 1-LTR circular episomes favourably over integrated provirus following the transduction of target feline and ovine cell lines. This led to a decrease in transgene expression over time in dividing cells. In contrast, in non-dividing cells transgene expression was maintained at a similar level to integration-competent VMV vectors. Self-inactivating (SIN) VMV vectors were constructed and found to have a significant decrease in LTR activity. Transgene expression was maintained by the insertion of an internal promoter derived from human cytomegalovirus (CMV) acting directly on the transgene. When self-inactivating and integration-defective modifications were incorporated into the same vector particle, a 4-fold decrease in transduction relative to the parent vector was observed. Ovine monocyte-derived dendritic cells (MDDCs) and macrophages (MDMs) were found to be efficiently transduced by the VMV vector, whereas lentiviral vectors derived from HIV-1 poorly transduced both of these primary cell populations. Following this work, the ability to deliver pathogen genes into APCs was studied using the Chlamydia abortus (C. abortus) major outer membrane protein (MOMP) as the transgene. C. abortus is the most common infectious cause of ovine abortion worldwide and MOMP has previously been shown to stimulate strong antibody responses after vaccination. Unexpectedly, the VMV vector encoding either eGFP or MOMP was found to induce apoptosis in MDDCs and MDMs using Annexin V staining. Apoptotic cells were detectable as early as 6 hours post-transduction of cells. Furthermore, release of the pro-inflammatory cytokine IL-1β was associated with the formation of late apoptotic cells. Apoptotic bodies produced post-transduction were able to be phagocytosed by immature MDDCs and the transgene efficiently cross-presented to T-cells. The ability of the novel VMV vector to induce a suitable recall immune responses was investigated using an in vitro model. Here, an autologous population of MDDCs were cultured with the apoptotic bodies produced post-transduction before the addition of autologous PBMC. Proteins from the apoptotic bodies were presented by the MDDCs to PBMC leading to a strong, antigen specific recall immune response against C. abortus MOMP. This was proven by the detection of cytokines IFNγ and IL-10 in the co-culture supernatant from PBMC activated by the MOMP transgene cross-presented by MDDCs. No release of IL-4 or IL-17A could be detected. These data presented in this thesis show the potential for improving delivery of antigens in livestock vaccines by the use of lentiviral vectors. In addition, this vector system provides a strong base for the study of other potential protective antigens in vitro.