The immune system is composed of innate and adaptive mechanisms. Innate immune responses are significantly modulated by immunomodulatory factors that act through the induction of specific patterns of cytokine production in responding cells. Human leukocytes have been shown to respond to substance(s) present in acid extracts of commercial shark cartilage (SC). Shark cartilage is a food supplement taken by consumers as a prophylaxis and for the treatment of conditions ranging from arthritis to cancer. No reliable scientific evidence in the literature supports the alleged usefulness of shark cartilage supplements, but their use remains popular. Cartilage extracts exhibit immunomodulatory properties by inducing various inflammatory, Th1-type cytokines and potent chemokines in human peripheral blood leukocytes (HPBL) in vitro. The objectives of the study were to (1) to determine the nature of the active component(s), (2) to define the scope of cellular response to SC extract, and (3) to elucidate the molecular mechanisms underlying bioactivity. Results showed that there are at least two cytokine-inducing components which are acid stable. One anionic component has been identified as a small (14-21 kDa) glycoprotein with at least 40% carbohydrate content. Shark cartilage stimulated HPBL to produce cytokines resembling an inflammatory, Th1 polarized response. Leukocyte-specific responses consist of both initial cytokine responses to SC directly (i.e., TNF-a) and secondary responses such as the IFN-γ response by lymphocytes following initial SC stimulation. Response of RAW cells, a murine macrophage cell line, indicated that TNF-α could be induced in macrophages of another mammalian species in the absence of other cell types. The results suggest that the human monocyte/macrophage is most likely to be the initial responding cell to SC stimulation. Stimulation of cells appears to engage at least one ligand-receptor interaction with TLR 4, although the role of TLR 2 cannot be ruled out. Initial activation is likely followed by the activation of the JNK and p38 MAPK signal transduction pathways resulting in activation, release, and translocation of transcription factor nuclear factor κB (Nf-kB). This dissertation research study represents the first in-depth study into characterizing the bioactive component(s) of commercial shark cartilage responsible for its immunomodulating properties and defining cellular responses at the molecular level.

A desnutrição modifica a resistência à infecção, alterando grande variedade de processos fisiológicos, incluindo a hematopoiese e a resposta imune. Os mecanismos exatos que comprometem o sistema imunológico em condições de desnutrição ainda não estão totalmente compreendidos, sendo que a desnutrição modifica a funcionalidade das células efetoras na resposta inflamatória/infecciosa, causando redução da síntese de citocinas pró-inflamatórias, além de alterações na hematopoiese. Os linfócitos têm papel-chave tanto na resposta imune inata quanto adaptativa. Nesse contexto, propusemo-nos a avaliar alguns aspectos da resposta linfoide esplênica em um modelo de desnutrição proteica. Camundongos BALB/c, machos, submetidos à desnutrição proteica, após perda de aproximadamene 20% do peso corpóreo, foram eutanasiados. Hemograma, mielograma, esplenograma e análise das concentrações séricas de proteína totais, albumina, pré-albumina e dosagem de imunoglobulinas G e M foram realizados. As células do timo e do baço foram coletadas e analisadas por citometria de fluxo - e foi realizado o ciclo celular. Para avaliação da linfoproliferação, as células esplênicas foram cultivadas, in vitro, e estimuladas com LPS ou ConA durante 72 horas. Foram quantificadas a capacidade de produção de INF-γ, IL-2 e IL-10 e a expressão de STAT- 1 e STAT-3. Os animais desnutridos apresentaram anemia, leucopenia e severa redução na celulariedade da medula óssea, com comprometimento no setor mieloide e diminuição da celulariedade do baço, com redução da população de linfócitos. Os animais desnutridos apresentaram alterações no desenvolvimento celular linfoide, alteração na capacidade de proliferação, menor produção de citocinas pró-inflamatórias, como IL-2, e, ao mesmo tempo, aumento da produção de IL-10. Os animais também apresentaram maior porcentagem de células CD3+ e CD4+ no baço em relação aos animais controle. Quando as células esplênicas foram estimuladas, apresentaram maior expressão de STAT-3 e menor expressão de STAT-1. Baseando-se nestes resultados, discutimos se deficiências nutricionais alteram a imunocompetência e aumentam o risco de infecções. / Malnutrition modifies the resistance against infection by changing a variety of physiological processes, including the hematopoiesis and the immune response. The exact mechanisms that compromise the immune system in conditions of malnutrition is not yet thoroughly understood, since malnutrition modifies the functionality of the effector cells in inflammatory / infectious response, causing the reduction of the synthesis of proinflammatory cytokines, besides causing alterations in hematopoiesis. The lymphocytes have a key role in both innate and adaptive immune responses, in this context, we proposed to evaluate some aspects of lymphoid spleen cells response in a model of protein malnutrition. Male mice BALB / c submitted to protein malnutrition after approximately 20% loss of body weight were euthanized. The complete blood count, bone marrow examination, spleen cells count and analysis of serum total protein, albumin, pre-albumin and immunoglobulin G and M were performed. The thymus and spleen cells were collected and analyzed by flow cytometry as well as the cell cycle performed. For evaluation of the lymphocyte proliferation the spleen cells were cultivated in vitro and stimulated with LPS or ConA for 72 hours and the ability to produce IFN-γ, IL-2 and IL-10 as well as the expression of STAT-1 and STAT -3, were quantified. The malnourished animals presented anemia, leukopenia and severe reduction in celulariedade bone marrow with myeloid commitment in the sector and decreased celulariedade spleen, with reduced lymphocyte population. The malnourished animals showed changes in lymphoid cell development, abnormal proliferation capacity, reduced production of proinflammatory cytokines such as IL-2, and at the same time, increased production of IL-10. The animals also had higher percentages of CD3 + and CD4 + spleen compared to control animals. When spleen cells were stimulated, showed higher expression of STAT-3 and lower expression of STAT-1. Based on these results, we discussed whether nutritional deficiencies alter immunocompetence and increase the risk of infections.

This paper was published in Nature 2009 Aug 13;460(7257):904-8. doi: 10.1038/nature08240. http://www.nature.com/nature/journal/v460/n7257/full/nature08240.html / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12855号 / 論医博第2085号 / 新制||医||1006(附属図書館) / 31535 / (主査)教授 髙折 晃史, 教授 羽賀 博典, 教授 岩井 一宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23440号 / 生博第461号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Autism spectrum disorder (ASD) is a neurodevelopmental disorder marked by social deficits and repetitive actions. A communication pathway exists between the brain and gut called the gut-brain axis. It is thought that gut bacteria can secrete signaling molecules, triggering inflammation across the body. These studies attempt to determine if markers are expressed in two mouse models of ASD behaviors, BTBR and a valproic acid model. Immunohistochemistry of ionized calcium binding adaptor molecule 1 from male mouse brain tissue showed no microglial activation in any group. Cytokine analysis did exhibit an increase in interleukin 1 (IL-1) in male adult mice only. Sequencing of bacterial profiles demonstrated differences between groups. Altogether, it appears that microbiome differences do not trigger robust differences in inflammatory pathways in these animals in this study. It is imperative that a reliable animal model of behaviors be identified for novel studies that can impact the development of the disorder.

Adipose tissue is a key regulator of energy metabolism and glucose homeostasis by promoting triglyceride storage and breakdown in various physiological states. Obesity, however, alters adipose tissue metabolism, inducing chronic inflammation, followed by excessive lipolysis. This results in higher systemic free fatty acid (FFA) levels, leading to desensitization of insulin signaling and ultimately to insulin resistance. Although the link between obesity and progression of insulin resistance and type 2 diabetes mellitus (T2DM) remains unclear, tumor necrosis factor-alpha (TNF-alpha) has been proposed to be a key player in promoting obesity-related development of T2DM through chronic inflammation of adipose tissue. TNF-alpha has direct and indirect mechanisms by which it elicits insulin resistance in adipocytes. TNF-alpha attenuates insulin signaling by directly inhibiting insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1). Indirectly, TNF-alpha activates signaling pathways to increase lipolysis and FFA release into circulation, leading to insulin resistance. Lipid droplet-associated fat specific protein 27 (FSP27) protects adipocytes from lipolysis by regulating the lipolytic capacity as well as transcription of adipose triglyceride lipase (ATGL). It has been observed that TNF-alpha promotes lipolysis by reducing the expression of FSP27 in murine adipocytes. The effect of TNF-alpha on lipolysis human adipocytes has also been studied; yet its effect on promoting insulin resistance in human adipocytes still remains elusive. In the present study, we examined the effect of FSP27 on TNF-alpha induced lipolysis and insulin resistance in human adipocytes. TNF-alpha enhanced lipolysis in cultured human adipocytes. In addition, TNF-alpha reduced the expression of endogenous FSP7 and the phosphorylation of AKT, inhibiting the activation of insulin signaling pathway in cultured human adipocytes. FSP27 overexpression, however, attenuated TNF-alpha induced lipolysis and restored activation of insulin signaling through phosphorylation of AKT in cultured human adipocytes. Taken together, these data suggest that FSP27 has a protective effect against TNF-alpha induced lipolysis and insulin resistance through regulating lipolysis and insulin signaling in human adipocytes.

Dopamine (DA) transmission is a key player in the rewarding aspects of ethanol as well as ethanol dependence. The current dogma is that DA transmission is increased during ethanol via the inhibition of ventral tegmental area (VTA) GABA neurons and that excitation of VTA GABA neurons during withdrawal results in decreased DA transmission. Microglia, the major neuroimmune effector in the brain, may be a key mediator in this process by releasing cytokines following activation. We evaluated the effect of ethanol on cytokine concentrations in the VTA and NAc using a cytometric bead array, and found that low dose ethanol (1.0 g/kg) decreased interleukin (IL)-10 levels, but high dose ethanol increased IL-10 levels (4.0 g/kg). We also used standard cell-attached mode electrophysiological techniques to evaluate the effects of select cytokines on VTA neuron firing rate in vitro. We found no change in firing rate in response to IL-6, but an increase in firing rate in VTA DA neurons response to IL-10. Consistent with the changes in firing rate, optically-evoked IPSCs were also found to be decreased in response to IL-10. Ex vivo voltammetry and in vivo microdialysis were done to determine whether IL-10 can directly result in an increase in DA release. Although ex vivo voltammetry showed no change in DA release, IL-10 increased DA release in vivo. These findings suggest that the rewarding and/or addictive effects of ethanol are mediated by cytokines, specifically the anti-inflammatory cytokine IL-10.

One of the major healthcare issues found almost worldwide, especially in the United States, is the obesity epidemic. Obesity is known to have deleterious effects on many body/organ systems. C1q TNF-related protein-3 (CTRP3) is effective at preventing high-fat diet-induced fatty liver. With these two factors taken into consideration, this study explores the possible effects of a high caloric diet on the muscle wall of the uterus, i.e., the myometrium, and how over-expression of CTRP3 may modify those effects. We hypothesize that consumption of excessive amounts of fat and sugar will have detrimental effects on the dual layers of the mouse myometrium. For this study, 17 mice were divided into 4 treatment groups: wild type/low fat diet, wild type/high fat diet, CTRP3-overexpressing/low fat diet, and CTRP3-overexpressing/high fat diet. The mice were placed on their respective diets at 7 weeks of age with a feeding duration of 12 weeks. At the conclusion of the feeding protocol, the female reproductive tissues were harvested and fixed in 4% paraformaldehyde and subsequently paraffin embedded. The uterine horns of each mouse were painstakingly paraffin embedded in a vertical position so that cross sections of the uterus could be obtained and measured. These 4µ sections were stained using standard H&E staining techniques and visualized under light microscopy. A randomization grid was utilized to determine measurement locations on the tissue. For each animal, 15 measurements were made of the outer longitudinal layer of the uterine horn, as well as 15 measurements of the inner circular layer, and 15 measurements of the thickness of the two layers combined. Two-way ANOVA was used to determine if any changes seen were statistically significant. At the time of the writing of this abstract, no appreciable differences have been found between the treatment groups, although there will be more data and final statistics completed before the presentation of our findings.

Background: IL-4 and IL-13 are related cytokines with similar functional properties. Differential regulation of IL-4 and IL-13 has not been described. Objective: We have examined the effects of IFN-α on antigen-driven proliferation, IL-4 generation, and IL-13 generation from human PBMCs and T-cell clones. Methods: Proliferation was assessed by 3H-thymidine incorporation. Cytokine generation was assessed by reverse transcription PCR and ELISA. Messenger RNA stability was assessed in the presence of actinomycin D. Results: IFN-α induced a concentration-dependent inhibition of antigen-driven proliferation of TH1 and TH2 clones (median effective concentration, 150 to 200 U/mL); the sensitivity of TH1 and TH2 clones to IFN-α was not significantly different (P = .6). IFN-α induced an analogous concentration-dependent inhibition of antigen-driven IL-13 generation from TH1 and TH2 clones (median effective concentration, 100 U/mL); this effect was evident by 12 hours of culture and persisted beyond 48 hours. However, IL-4 generation from TH2 clones was insensitive to IFN-α at all concentrations and times tested (1 to 10,000 U/mL). A similar inhibitory effect of IFN-α on mitogen-driven proliferation and IL-13 generation from PBMCs was demonstrated; once again, IL-4 generation from PBMCs was insensitive to IFN-α. IL-13 mRNA stability was unaffected by IFN-α, suggesting transcriptional regulation. Conclusion: IFN-α differentially regulates antigen-stimulated IL-4 and IL-13 generation.

IFN-γ and/or LPS induced nitrite production and inhibition of Chlamynia trachomatis (CT) replication in the murine macrophage cell line, RAW264.7. Linear regression analysis demonstrated a strong correlation between nitrite production and inhibition of CT replication (correlation coefficients: -0.93, P < 0.001). L-NMMA specifically inhibited nitrite production and restored CT replication (55-71%). Inducible nitric oxide synthase (iNOS) mRNA was analyzed by Northern and dot blot hybridization with an iNOS cDNA probe. A strong correlation between iNOS mRNA expression and inhibition of CT replication also was observed (correlation coefficient: -0.97, P < 0.05). Furthermore, anti-TNF-α antibody, which completely neutralized biological activity of the secreted TNF-α neither inhibited nitrite production nor restored CT replication in the LPS- and/or IFN-γ-treated RAW264.7 cells. In mouse peritoneal macrophages treated with IFN-γ, both L-NMMA and anti-TNF-α antibody inhibited nitrite production and restored CT replication. However, L-NMMA and the antibody had no effect upon nitrite production and CT inhibition in LPS-treated peritoneal macrophages. These data indicate that NO production is one mechanism for inhibition of CT replication in IFN-γ-activated murine macrophages.