Streptococcus pneumoniae[italicized everytime] (S. pneumoniae) causes a spectrum of disease severity, and human host factors likely play a role in this variation. One candidate factor is macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine and upstream regulator of innate immunity. The MIF[italicized when not in parenthesis] promoter contains two functional polymorphisms, a tetranucleotide (CATT) repeat such that MIF expression increases with repeat number from 5-8 and a single nucleotide polymorphism (SNP) leading to a G-to-C transition, which results in increased MIF expression in cell line reporter assays. Emerging data suggest an association between high-expression MIF alleles and inflammatory disease. This study comprised two parts. For the in vitro portion, we hypothesized that peripheral blood monocytic cells (pBMCs) cultured from healthy individuals with low-expressing MIF genotypes (5-CATT alleles or SNP-GG) would have lower MIF content and release than those from individuals with high-expressing MIF genotypes (7-CATT or SNP-C alleles). For the in vivo study, we hypothesized that individuals with low-expressing MIF genotypes would have less severe systemic inflammatory responses than individuals with high-expressing MIF genotypes in response to S. pneumoniae infection. Blood samples and chart findings were collected prospectively at three Connecticut hospitals from 30 inpatients with documented invasive S. pneumoniae infections. Genomic DNA was isolated from host blood, amplified, and genotyped using fragment analysis (CATT repeat) and allelic discrimination (SNP) methods. Fishers exact tests were used to compare genotypes and disease severity. For the in vitro experiments, there were no differences observed in serum MIF levels or MIF content or release from pBMCs based on MIF genotype. In the cohort of patients infected with S. pneumoniae, serum MIF levels among enrolled subjects were significantly higher than the reported normal values, but levels did not vary with genotype or disease severity. The SNP genotype was not correlated with disease severity or occurrence of meningitis. The CATT genotype did not correlate significantly with disease severity or occurrence of meningitis, although there was a trend suggesting an association between the 7-CATT allele and meningitis (p = 0.1188, 8% without meningitis had a 7-CATT allele vs. 40% with meningitis). More patient samples will need to be analyzed in order to definitively elucidate the role of MIF genetics in infection with S. pneumoniae

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Acute graft versus host disease (aGVHD) affects more than 40% of patients undergoing haematopoietic stem cell transplantation. aGVHD occurs after transplantation of donor haematopoietic cells into hosts incapable of rejecting the donor cells, when donor T cells attack host tissue. Despite extensive efforts, aGVHD remains problematic to prevent and difficult to control. Current therapies to prevent aGVHD induce profound immunosuppression, leaving patients at increased risk of infection and leukaemic relapse. Dendritic cells (DC) are professional antigen presenting cells of haematopoietic origin and are the primary stimulators of the immune system, uniquely being able to activate naïve T cells. A growing body of evidence suggests that DC are responsible for the stimulation of the donor T cells which cause aGVHD. I have used a model of aGVHD which utilizes conditioned severe combined immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMC). In this model human CD4+ T cells appear to be responsible for an aGVHD-like syndrome which results in death 15-30 days post transplant. I have shown, using in vitro depletion of individual populations, that other subpopulations of human PBMC did not affect the survival of the mice. I have also demonstrated that human DC are required for the induction of aGVHD in the majority of mice. This novel finding validated the use of this model to test the primary hypothesis; that antibody mediated depletion of DC would prevent aGVHD. The murine IgM monoclonal antibody (Mab), CMRF-44 Mab, is specific for an unknown molecule expressed on the surface of activated human DC. Previous work had shown that when mixed lymphocyte reaction stimulator cells were depleted of CMRF-44+ cells, there was a significant reduction in the proliferation of responder cells. Here I tested the efficacy of CMRF-44 as a therapy for the prevention of aGVHD in the model. CMRF-44 Mab did not improve survival of mice treated with human PBMC, despite recent data showing that CMRF-44 expression on DC was predictive of aGVHD in patients. In vitro depletion of CMRF-44+ cells from human PBMC prior to transplantation also did not reduce incidence of aGVHD. An alternate target for the depletion of human DC was CD83 which is also expressed on the surface of activated human DC. I generated a rabbit polyclonal antibody using a human CD83 fusion protein, which was then affinity purified in a multi-step process which yielded only antibody specific for human CD83. Treatment with this antibody greatly improved survival of transplanted mice. Further experiments showed that anti-CD83 treatment did not abrogate human leucocytes including CD8+ memory T cells suggesting that a therapy using an anti-CD83 antibody has the potential to prevent aGVHD without the immunosuppression associated with current anti-aGVHD therapies. The work described here has validated the use of a human mouse chimeric model as an in vivo assay of human DC function and shown that targeting CD83 has the potential to reduce the incidence of clinical aGVHD whilst preserving donor memory T cells.

A inflamação associada à obesidade é caracterizada por uma ativação crônica e de baixa intensidade do sistema imune. Diversos autores demonstraram mudanças em parâmetros inflamatórios após perda de peso. A cirurgia bariátrica é um método para tratar obesidade com alta eficiência e menor risco de recidiva. Os mononucleares de sangue periférico (MNSP) constituem um material biológico interessante para pesquisa, visto a capacidade de refletir alterações de expressão gênica de diferentes tecidos e o fácil acesso para análise. O presente estudo teve por objetivo avaliar os efeitos da obesidade e da perda de peso induzida por cirurgia bariátrica sobre a atividade imunológica, por meio de cultura primária de MNSP de pacientes com obesidade grau III (IMC >= 40 kg/m2). Foram coletadas amostras de sangue de veia periférica de 10 voluntários com peso normal (grupo controle) e antes e após a cirurgia de 20 voluntários com obesidade grau III. Após a separação dos mononucleares pelo gradiente de Ficoll-HyPaque, as células foram estimuladas por lipopolissacarídeo (LPS) ou concanavalina A (Con-A) e os sobrenadantes das culturas coletados para dosagem de IL-1?, IL-6, TNF-?, IFN-?, IL-10 e IL-17 por teste ELISA. As amostras de sangue também foram utilizadas para exames bioquímicos, dosagens de adiponectina, leptina e citocinas séricas. Os resultados evidenciaram maiores concentrações de IL-6, TNF-?, IL-1? e IL-10 nos sobrenadantes das culturas de MNSP do grupo com obesidade em relação ao grupo controle. Na comparação entre dosagens de citocinas do grupo com obesidade, observamos redução de TNF-?, IL-1? e IL-10 após 6 meses da cirurgia, a qual não foi observada após 1 ano, e aumento de IL-17 após 1 ano de tratamento. Não houve diferença significativa nas concentrações de citocinas séricas na comparação entre os grupos com obesidade e controle ou pré e pós-operatório. Observamos correlações das citocinas de sobrenadante das culturas de MNSP e séricas com resultados laboratoriais relacionados à homeostase glicêmica em pacientes com obesidade antes e após a cirurgia bariátrica, além da correlação entre citocinas do sobrenadante e o estado de adiposidade no pós-operatório. Concluímos que a obesidade grau III está associada a modificações da produção de citocinas por MNSP e a perda de peso induzida por cirurgia bariátrica influencia esta produção no primeiro ano de tratamento. / The obesity-associated inflammation is characterized by a chronic and low intensity activation of the immune system. Several authors have shown changes in inflammatory parameters after weight loss. Bariatric surgery is a method for treating obesity with high efficiency and less risk of recurrence. Peripheral blood mononuclear cells (PBMC) are an interesting biological material for research, due to the ability to reflect changes in gene expression in different tissues and easy access to analysis. This study aimed to evaluate the effects of obesity and weight loss induced by bariatric surgery on immune activity through PBMC culture of morbidly obese patients (BMI >= 40 kg/m2). Peripheral vein blood samples were collected from 10 volunteers with normal weight (control group) and before and after the surgery in 20 volunteers with morbid obesity. After separation of the mononuclear cells by Ficoll-Hypaque gradient centrifugation, cells were stimulated with lipopolysaccharide (LPS) and concanavalin A (Con-A) and culture supernatants collected for IL-1?, IL-6, TNF-?, IFN-?, IL-10 and IL-17 dosage by ELISA. Blood samples were also used for biochemical examinations and adiponectin, leptin and serum cytokine dosage. The results showed higher concentrations of IL-6, TNF-?, IL-1? and IL-10 in the supernatants of the MNSP cultures of the obesity group in relation to the control group. In the comparison between cytokine dosages of the obesity group, we observed reduction of TNF-?, IL-1? and IL-10 after 6 months of surgery, which was not observed after 1 year, and IL-17 increased after 1 year of treatment. There was no significant difference in serum cytokine concentrations in the comparison between obesity and control groups or operated group. We observed correlations of cytokines obtained in PBMC culture supernatant and serum with laboratory results related to glucose homeostasis in patients with obesity before and after bariatric surgery, as well as correlation between cytokines of the supernatant and the state of adiposity postoperatively. We conclude that morbid obesity is associated with changes in cytokine production by PMNC and weight loss induced by bariatric surgery influences cytokine production in the first year of treatment.

Malignant lymphomas are a group of cancerous diseases that develop from lymphocytes and primarily affect lymph nodes. Being the sixth most common cancer type in Sweden, lymphoma is a societal problem that needs to be tackled by improving care and treatment of patients. This study was designed to examine the blood cell composition in lymphoma patients and well as determine whether the use of cryopreserved cells affected the analysis outcome. An evaluation of the methods used was also performed. Frozen peripheral blood from lymphoma patients as well as fresh and frozen blood from healthy controls was used. The cells of interest were monocytes, granulocytes, Treg, NKT, iNKT, B and T cells plus the dendritic cell activation protein CCR7. Three immunophenotyping methods were used. Method one was used in staining surface cell markers while the other two were for both surface and intracellular staining using two distinctive kits. The results showed no significant difference in immune cell composition between patients and blood donors. Limited patient samples and the lack of female blood donors could explain the unexpected result. A substantial difference in Treg cells was observed in fresh and frozen tested samples as well as T cell outcomes in method one compared to the other two methods. There were fewer Treg cells in frozen samples, which probably was due to cryopreservation while the lack of fixation in method one led to the loss of CD4+ T cells. Overall, the methods used were adequate but definitely require some improvements.

Dans l’optique de produire de maîtriser les conditions d’expansion ex vivo de greffons hématopoïétiques produits à partir de sang périphérique en homéostasie, l’objectif de ce projet a été de caractériser fonctionnellement, métaboliquement et transcriptomiquement les cellules souches hématopoïétiques (CSH). Compte tenu de l’impossibilité technique de sélectionner spécifiquement les CSH humaines, nous avons utilisé un modèle cellulaire enrichi en CSH, le modèle Side Population(SP). Dans un premier temps, nos travaux ont confirmé que les CSH du sang périphérique étaient majoritairement dans la population SP et qu’elles possédaient des caractéristiques fonctionnelles proches des CSH des autres compartiments hématopoïétiques. Nous avons également démontré l’implication des basses concentrations d’O2 sur le maintien des CSH du sang périphérique. Dans un second temps, nos résultats ont prouvé que les CSH du sang périphérique utilisaient à la fois la glycolyse et la phosphorylation oxydative pour produire l’énergie nécessaire à leur maintien. Enfin, ce projet a permis d’apporter des résultats préliminaires concernant les régulations transcriptomiques des CSH du sang périphérique. Ces données montrent donc que le sang périphérique en homéostasie pourrait constituer une source potentielle de cellules pour la production de greffons hématopoïétiques tout en apportant les premiers éléments de compréhension de la physiologie de ces cellules, afin, dans un plus long terme de maîtriser leur maintien ou leur différenciation ex vivo. / To evaluate the possibility to control ex vivo expansion conditions, a key point to produce hematopoietic graft from steady state peripheral blood (SSPB), the objective of this project to characterize the functional properties, the metabolism and the transcriptomic regulations of hematopoietic stem cell (HSC) from SSPB. Due to the lack of strong HSC’s marker in human, we choose to use the Side Population (SP) model, previously described as enriched in HSC in other hematopoietic compartments. In a first part of our work, we showed that HSC from SSPB are mainly inside the SP population. Indeed, SP cells from SSPB exhibit functional properties very closed from HSC. In addition, we found they strongly affected by low O2 concentrations, as HSC from bone marrow. In a second part, our results showed that HSC from SSPB use as much glycolysis as oxidative phosphorylation to produce energy they need to maintain their properties. All together, these data give some interesting information about HSC regulation and needs. They also suggest that HSC from SSPB could be considering as a potential source of hematopoietic graft for therapy.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Several papers have reported the enhancing effects of opiate peptides, like Met-enkephalin, on natural killer (NK) cell activity. We examined the actions of Met-enkephalin on NK activity in blood obtained from 18 different donors, of different ages, many of them on several occasions, at several ratios of effector to target cells, several concentrations of peptide, in several types of flasks, with the purity and identity of the pentapeptide verified by chromatography, in a system responsive to interferon, and with results calculated in different ways. No significant increase was found for the peptide for any ratio of cells, any concentration of peptide, or any single subject, even when the subjects with the lowest baseline NK cell activity were used or when the subjects were more than 60 years old. Instead of an increase, the combined results for all subjects at all ratios at all concentrations of Met-enkephalin showed an overall decrease of 31.3 % specific cytotoxicity. These results fail to support the reports of an enhancing effect of Met-enkephalin on NK cell activity.

Our previous report showed that the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) blocks the inhibitory effect of morphine sulfate on E-rosette formation by human peripheral blood lymphocytes (PBL). In this study, additional in vitro effects of Tyr-MIF-1 on human PBL were studied. The percentages of positive cells for CD 2, a sheep erythrocyte receptor, CD 4 and CD 8 were unchanged after incubation of PBL with morphine or morphine plus Tyr-MIF-1. Tyr-MIF-1 was not mitogenic by itself. The addition of Tyr-MIF-1 did not increase the proliferative response of PBL to Con A, although morphine did. Tyr-MIF-1 did not activate PBL to produce IL 2 nor did it affect the production of IL 2 by Con A-stimulated PBL. The results suggest that Tyr-MIF-1 does not directly modulate CD 2, CD 4 and CD 8 expression, does not alter the proliferative response of PBL, and does not affect the production of IL 2.

No description available.