Dendritic cells (DCs) are potent antigen presenting cells (APCs) that link the innate and adaptive immune system by their unique ability to induce and direct immune responses towards various T helper (Th)-type of immune responses such as Th1-, Th2-, Th9-, Th17-, Th22- or T regulatory (TR). The type of Th response generated very much depends on the nature of the antigen encountered and allows for an effective and proficient immune response. For example, Th1 responses are used to clear intracellular pathogens while Th2 responses are needed to clear extracellular pathogens The ability to specifically modulate Th-responses is an area of intense research, as it allows for the development of more effective vaccines and immunotherapeutics. Immunomodulation of DCs is one strategy by which specific Th-type immune responses may be tailored. Current research is focused on identifying agents that have the capacity to immunomodulate DCs such as host defense peptides (HDPs). Apart from their anti-microbial activities, HDPs have a number of immune functions including recruitment and subsequent activation of DCs. The goal of this study was to examine the immunomodulatory effects of HDPs on porcine DC functions. This research was part of a larger multinational research project to develop a novel adjuvant platform for single-immunization vaccines against pertussis in neonates. The pig model was used for this research because of its physiological similarities to humans and the recently developed pertussis infection model in young piglets. A series of experiments was conducted to characterize and describe porcine DC functions. Two subsets of DCs were successfully characterized and tested for their response to stimulation with HDPs. Initial results demonstrated a minimal effect of HDPs on DC functions, therefore we expanded the number of HDPs used to include both synthetic derivatives of HDPs known as innate defense regulators (IDRs) and naturally- occurring HDPs. We examined these effects on peripheral blood mononuclear cells (PBMC) in vitro and found that HDPs induce expression of the chemokine interleukin (IL)-8, which resulted in PBMC recruitment in vitro. We then proceeded to evaluate the HDPs in vivo by intradermally administering them into the flank of pigs. Surprisingly, treatment with the HDPs did not result in recruitment of neutrophils in vivo. We also examined the effects of formulating IDR-1002 as an adjuvant with the academic antigen Keyhole Limpet Hemocyanin (KLH) on the development of KLH-specific immune responses in vaccinated pigs. While there was no difference in antibody titers between vaccinated and control animals, we found that co-formulation with IDR-1002 decreased both antigen-specific and mitogen-induced proliferation in KLH/IDR-1002 vaccinated animals as long as four weeks post-treatment. These results demonstrate that specific IDRs can suppress certain aspects of the pro-inflammatory immune response making them potentially highly versatile tools to modulate and tailor the immune response in disease states characterized by a pro-inflammatory component.

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.

Esta pesquisa avaliou o efeito da transferência passiva dos leucócitos do colostro na imunidade específica de bezerras recém-nascidas. Foram acompanhadas 20 bezerras Holandesas durante o período neonatal, distribuídas em dois grupos experimentais: grupo COL+ recebeu colostro fresco (4L) proveniente de suas respectivas mães; e grupo COL- recebeu colostro congelado e acelular (4L), oriundo de vacas doadoras de colostro. As avaliações foram realizadas antes da mamada do colostro (M0), 1-2 (M1), 7 (M2), 14 (M3), 21 (M4) e 28 dias pós-nascimento (M5). As bezerras foram submetidas ao exame clínico, seguido da colheita das amostras sanguíneas para realização de hemograma, imunofenotipagem e cultivo celular. Os dois grupos foram colostrados com colostro de igual qualidade com relação à concentração de imunoglobulinas (70-120 g/L). A concentração de células do colostro fresco fornecido ao grupo COL+ foi de 1.895.849 células/mL. Não foi possível encontrar diferenças para as funções vitais em relação aos grupos experimentais. O exame específico dos sistemas revelou um caso de broncopneumonia, três de inflamação umbilical e maior frequência de escore de fezes 3 no COL-. As alterações clínicas foram refletidas no eritrograma das bezerras, sendo encontrado menor valor médio para a taxa de hemoglobina (HGB) no COL- em M3. Em relação à idade, observou-se redução gradativa dos valores médios para He (hemácias), HGB, HCT (hematócrito) e índices hematimétricos no primeiro mês de vida. A frequência de bezerras anêmicas foi maior no grupo COL- nos momentos M4 e M5. Em relação ao leucograma, observou-se diferença entre os grupos para linfócitos no M0 e M2 com valores superiores no COL-. Em relação aos momentos foi possível detectar leucocitose por neutrofilia no M0 e M1, observando-se inversão da relação neutrófilo:linfócito a partir desses momentos. Os valores de CD45+CD45RO- foram maiores em M0 no COL-, além disso, observou-se aumento da expressão do marcador de memória celular CD45RO+ do M0 ao M1 nos dois grupos experimentais. O CD3+gamma-delta- aumentou no decorrer do estudo, em contrapartida as células CD3+gamma-delta+ foram menores em M5 com relação ao M0-M3. Foi detectado também aumento dos valores de CD14+MHCII+ no primeiro mês de vida indicando maturação das células apresentadoras de antígeno. Em relação à produção de citocinas pelas células mononucleares sanguíneas, foi possível identificar maior concentração de IFN-gamma em M4, quando as células do COL- foram estimuladas com S. aureus (1 mononuclear:10 bactérias inativadas). A concentração de IL-17 detectada a partir das células do COL+ foi maior em M3, quando as células foram estimuladas com ConA. Com base nos resultados obtidos, pode-se concluir que: a) bezerras COL- apresentaram maior frequência e intensidade de doenças que evoluíram para anemia da inflamação; b) bezerras COL- apresentaram maior número absoluto de linfócitos, representadas especialmente pela subpopulação CD3+gamma-delta+ nos episódios de maior frequência de diarreias; c) Linfócitos de memória CD45RO+ aumentaram após a colostragem em ambos os grupos, sugerindo que outros componentes acelulares do colostro podem apresentar papel fundamental no desenvolvimento da resposta imunológica de bezerras recém-nascidas; d) a subpopulação CD3+gamma-delta- e as células CD14+MHCII- e CD14+MHCII+ aumentaram durante o primeiro mês de vida, indicando maturação imunológica; e) as células mononucleares das bezerras não responderam ao Herpesvírus Bovino tipo 1, porém responderam aos estímulos bacterianos, especialmente para a Escherichia coli; a interpretação do leucograma em conjunto com a análise das variações apresentadas para as citocinas inflamatórias IFN-gamma e IL-17 permitem afirmar que as bezerras apresentaram resposta inflamatória retardada e de menor magnitude no COL-. / This study evaluated the effect of leukocytes passive transference from bovine colostrum in specific immunity of newborn calves. During neonatal period, 20 Holstein calves were followed. Animals were distributed in two experimental groups: COL+ which received fresh colostrum (4L) from their mothers, and COL- which received frozen and acellular colostrum (4L) that came from donor cows. The evaluations were performed in the following moments: before colostrum intake (M0), 1-2 (M1), 7 (M2), 14 (M3), 21 (M4) and 28 days after birth (M5). Heifers were submitted to clinical examination. Then, blood samples were harvested for hemogram, immunophenotyping and cell culture. Both groups were fed with the same quality of colostrum (immunoglobulin concentration 70-120 g/L). The cell concentration of fresh colostrum that was provided to COL+ group was 1.895.849 cells/mL. It was not possible to detect differences in vital functions concerning the experimental groups. The system specific examination reveled one case of bronchopneumonia, three cases of umbilical inflammation and major rates of diarrhea score 3 in group COL-. Clinical alterations were reflected in calves erythrogram. It was found lower mean value for hemoglobin (HGB) in M3 for COL-. Regarding age, a gradual reduction in mean values for erythrocytes, HGB, HCT (hematocrit) and hematimetric rates were observed in the first month of life. The frequency of anemic heifers was higher in COL- group at moments M4 and M5. Regarding leukogram, it was observed difference between groups for lymphocytes in M0 and M2 with higher values in COL-. Concerning moments, it was possible to detect leukocytosis by neutrophilia from M0 up to M1 and inversion of neutrophil:lymphocyte relation from this moment. Values of CD45+CD45RO- was higher in M0 for COL-, furthermore, increase of cellular memory marker expression CD45RO+ was observed from M0 to M1 in both groups. The CD3+gamma-delta- increased during the study. On the other hand, CD3+gamma-delta+ were lower in M5 in relation to M0-M3. Increase of CD14+MHCII+ values were also detected in the first month of life, indicating maturation of antigen presenting cells. Regarding cytokine production by mononuclear cells of heifers blood, it was possible to identify higher concentration of IFN-gamma in M4 when cells of COL- were stimulated with S. aureus (1 mononuclear: 10 inactivated bacteria). The concentration of IL-17 detected from COL+ cells was higher in M3, when cells were stimulated with ConA. Based on these results, it can be concluded that: a) COL- heifers presented higher frequency and intensity of diseases that evolved to anemia of inflammation; b) COL- heifers presented higher lymphocyte absolute number, represented specially by CD3+gamma-delta+ subsets in episodes of higher frequency of diarrhea; c) memory lymphocytes CD45RO+ increased after colostrum intake in both groups, suggesting that other acellular colostrum components can present fundamental role in development of immunological response in newborn heifers; d) the subset of CD3+gamma-delta- and the cells CD14+MHCII- and CD14+MHCII+ increased during the first month of life, indicating immunological maturation; e) heifers mononuclear cells did not respond for herpes virus bovine type 1, however, responded for bacterial stimulus, specially Escherichia coli. The interpretation of leukogram with the variation of presented analyses for inflammatory cytokines IFN-gamma and IL-17, allow to state that heifers presented delayed inflammatory response and of lesser magnitude in COL-.

A necessidade de tratamentos mais eficazes e menos invasivos para os pacientes, em adição à capacidade de diferenciação celular da medula óssea sugere que o transplante de células mononucleares totais poderia ser uma das melhores formas de tratamento para as diversas patologias existentes. Entretanto, vários fatores implicam sobre a viabilidade das células da medula óssea dos quais destacamos a ausência de padronização de protocolo de criopreservação que permita a manutenção da viabilidade celular, sendo altamente necessário o desenvolvimento de estudos nesta área. Deste modo, neste estudo, após a anestesia de um grupo de animais foi realizada punção da medula óssea, separação das células mononucleares e avaliação da viabilidade. Foram testados oito meios diferentes para criopreservação das células. O meio de congelamento A é composto por 20% dimetilsulfóxido (DMSO), 40% Dulbecco´s Modified Eagle´s Médium (DMEM) e 40% de Plasma Autólogo, o meio de congelamento B contém 20% dimetilsulfóxido (DMSO), 40% Roswell Park Memorial Institute (RPMI) e 40% de Plasma Autólogo, o meio de congelamento C tem em sua composição 20% dimetilsulfóxido (DMSO), 40% soro fetal bovino (SFB) e 40% plasma autólogo, o meio de congelamento D é composto de 20% dimetilsulfóxido (DMSO) e 80% de plasma autólogo, o meio E constitui-se de 5% dimetilsulfóxido (DMSO), 47,5% de Dulbecco\'s Modified Eagle\'s Médium (DMEM) e 47,5% de plasma autólogo, o meio F contém 5% dimetilsulfóxido (DMSO), 47,5% de Roswell Park Memorial Institute (RPMI) e 47,5% de plasma autólogo, o meio G contém 5% dimetilsulfóxido (DMSO), 47,5% de soro fetal bovino (SFB) e 47,5% de plasma autólogo e o meio H constitui-se de 5% dimetilsulfóxido (DMSO) e 95% de plasma autólogo. Após as análises realizadas pela técnica de citometria de fluxo, o meio mais eficiente na criopreservação das células mononucleares de suínos foi o protocolo D por possuir maior concentração de plasma autólogo e crioprotetor. / The necessity of the most efficient and less invasive treatments for the patients, in addition to the capacity of cellular differentiation of the bone marrow suggests that the transplant of mononuclear total cells might be one of the best treatment for several pathologies. Meantime, several factors influence on the viability of the cells of the bone marrow as the absence of standardization of criopreservação protocol which could allow the maintenance of the cellular viability, being an important issue of investigation. In this study, after the anaesthesia of a group of animals, samples of bone marrow were collected, following the separation of the mononuclear cells and evaluation of the viability. Eight different protocols were tested for cells criopreservation. The protocol A by 20% dimetilsulfóxido (DMSO), 40% Dulbecco\'s Modified Eagle\'s Médium (DMEM) and 40% autologus plasma, B protocol conatained 20% dimetilsulfóxido (DMSO), 40% Roswell Park Memorial Institute (RPMI) and 40% autologus plasma, the C protocol was composed 20 % dimetilsulfóxido (DMSO), 40% bovine fetal serum (SFB) e 40% autologus plasma, D protocol was made using 20% dimetilsulfóxido (DMSO) and 80% autologus plasma, E protocol was constituted by 5% dimetilsulfóxido (DMSO), 47,5% de Dulbecco\'s Modified Eagle\'s Médium (DMEM) and 47,5% autólogo plasma, the F contains 5% dimetilsulfóxido (DMSO), 47,5% de Roswell Park Memorial Institute (RPMI) and 47,5% autologo plasma, the protocol G was compoed by 5% dimetilsulfóxido (DMSO), 47,5% de bovine feta serum (SFB) and 47,5% autologus plasma and the H protocol was 5% dimetilsulfóxido (DMSO) and 95% autologus plasma. After flux citometer analyses, the most efficient protocol in criopreservation of the mononuclear cells of pigs was the protocol D which was composed the by the most amount of autologus plasma and crioprotectant.

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.

Immunological and ultrastructural changes in mononuclear cells exposed to Mycobacterium avium serovar-specific glycopeptidolipid (GPL) and the chemically derived R-lipid (lipopeptide fragment) were examined.

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep® and RobosepTM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.

No / Recent studies have gained positive results using nanoparticles (NPs) in treating atherosclerosis on animals. But their toxicity and application in treating other heart diseases such as heart failure and endocarditis still need proper investigation. Gold nanoparticles (Au-NPs) were chosen as model substances as they have been successfully used in treating cancer. In this study, we use both H9C2 myoblasts and rat peripheral blood mononuclear cells to determine the influence of Au-NP size on their cytotoxicity and cell apoptosis. H9C2 cells were treated with Au-NPs of a diameter of 5, 20, 40 and 100nmfor 24 hrs before their cell viabilities tested by MTT assay, cell apoptosis measured by flow cytometry, and the generation of reactive oxygen species (ROS) detected by Fluorometric Intracellular ROS Kit. Distribution of the Au-NPs and their effects on the structure of mitochondria and lysosome were detected by electron microscopy. In addition, we obtained rat peripheral blood mononuclear cells and treated them with Au-NPs same with H9C2 cell line. Our results showed NPs of 5, 40, and 100 nm reduced cell viabilities on H9C2 cells while20nm showed no change on cell viability (Ctrl: 100±8.2 vs 20nm: 95.39±9.13, P>0.05, n=6) and some protect effect on ISO induced H9C2 cells apoptosis (ISO: 100±13.5 vs 20nm: 80.19±17.36, P>0.05, n=6). All size of Au-NPs reduced cell viabilities on rat peripheral blood mononuclear cells while 40nm showed the least reduction on cell viability (Ctrl: 100.0±3.0 vs 40nm: 76.31±3.68, P<0.001, n=6) and significant protect effect on ISO-induced rat peripheral monocytes apoptosis (ISO: 100±1.86 vs 40nm: 45.34±10.32, P<0.05, n=6). In addition, 20nm Au-NP showed some protect effect on ROS generation on ISO-induced H9C2 cells (ISO: 100±3.79 vs 20nm: 94.84±4.98, P>0.05, n=6), while 40nm produced more ROS (ISO: 100±3.79 vs 40nm: 141.63±42.81, P>0.05, n=6). Electron microscopy detection showed correlated results in structure. These results on H9C2 cell line are basically in agreeable to our animal study. The protective effect of 20nm may due to its ability to protect ISO-induced ROS generation. The results on rat peripheral monocytes are slightly different to those on H9C2 cells. Further investigation need to focus on the role of NPs size on cell apoptosis by detecting autophagy specific protein through western blotting. / Abstract of conference paper.

BACKGROUND: The course of disease for patients with idiopathic pulmonary fibrosis (IPF) is highly heterogeneous. Prognostic models rely on demographic and clinical characteristics and are not reproducible. Integrating data from genomic analyses may identify novel prognostic models and provide mechanistic insights into IPF. METHODS: Total RNA of peripheral blood mononuclear cells was subjected to microarray profiling in a training (45 IPF individuals) and two independent validation cohorts (21 IPF/10 controls, and 75 IPF individuals, respectively). To identify a gene set predictive of IPF prognosis, we incorporated genomic, clinical, and outcome data from the training cohort. Predictor genes were selected if all the following criteria were met: 1) Present in a gene co-expression module from Weighted Gene Co-expression Network Analysis (WGCNA) that correlated with pulmonary function (p < 0.05); 2) Differentially expressed between observed "good" vs. "poor" prognosis with fold change (FC) >1.5 and false discovery rate (FDR) < 2 %; and 3) Predictive of mortality (p < 0.05) in univariate Cox regression analysis. "Survival risk group prediction" was adopted to construct a functional genomic model that used the IPF prognostic predictor gene set to derive a prognostic index (PI) for each patient into either high or low risk for survival outcomes. Prediction accuracy was assessed with a repeated 10-fold cross-validation algorithm and independently assessed in two validation cohorts through multivariate Cox regression survival analysis. RESULTS: A set of 118 IPF prognostic predictor genes was used to derive the functional genomic model and PI. In the training cohort, high-risk IPF patients predicted by PI had significantly shorter survival compared to those labeled as low-risk patients (log rank p < 0.001). The prediction accuracy was further validated in two independent cohorts (log rank p < 0.001 and 0.002). Functional pathway analysis revealed that the canonical pathways enriched with the IPF prognostic predictor gene set were involved in T-cell biology, including iCOS, T-cell receptor, and CD28 signaling. CONCLUSIONS: Using supervised and unsupervised analyses, we identified a set of IPF prognostic predictor genes and derived a functional genomic model that predicted high and low-risk IPF patients with high accuracy. This genomic model may complement current prognostic tools to deliver more personalized care for IPF patients.