Probiotics, immunity and exercise : a review

Nutritional practices that promote good health and optimal athletic performance are of interest to athletes, coaches, exercise scientists and dietitians. Probiotic supplements modulate the intestinal microbial flora and offer promise as a practical means of enhancing gut and immune function. The intestinal microbial flora consists of diverse bacterial species that inhabit the gastrointestinal tract. These bacteria are integral to the ontogeny and regulation of the immune system, protection of the body from injection, and maintenance of intestinal homeostasis. The interaction of the gut microbial flora with intestinal epithelial cells and immune cells exerts beneficial effects on the upper respiratory tract, skin and uro-genital tract. The capacity for probiotics to modulate perturbations in immune function after exercise highlight their potential for use in individuals exposed to high degrees of physical and environment stress. Future studies are required to address issues of dose-response in various exercise settings, the magnitude of species-specific effects, mechanisms of action and clinical outcomes in terms of health and performance.

Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

Vaccination to protect against infection of the female reproductive tract

Infection of the female genital tract can result in serious morbidities and mortalities from reproductive disability, pelvic inflammatory disease and cancer, to impacts on the fetus, such as infant blindness. While therapeutic agents are available, frequent testing and treatment is required to prevent the occurrence of the severe disease sequelae. Hence, sexually transmitted infections remain a major public health burden with ongoing social and economic barriers to prevention and treatment. Unfortunately, while there are two success stories in the development of vaccines to protect against HPV infection of the female reproductive tract, many serious infectious agents impacting on the female reproductive tract still have no vaccines available. Vaccination to prevent infection of the female reproductive tract is an inherently difficult target, with many impacting factors, such as appropriate vaccination strategies/mechanisms to induce a suitable protective response locally in the genital tract, variation in the local immune responses due to the hormonal cycle, selection of vaccine antigen(s) that confers effective protection against multiple variants of a single pathogen (e.g., the different serovars of Chlamydia trachomatis) and timing of the vaccine administration prior to infection exposure. Despite these difficulties, there are numerous ongoing efforts to develop effective vaccines against these infectious agents and it is likely that this important human health field will see further major developments in the next 5 years.

Human Kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

Setting up a platform for plant-based influenza virus vaccine production in South Africa

Background During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. Results For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. Conclusions We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4 + and CD8 + T cells from the cervix responded to the HPV-16 antigens and that interferon-γ (IFN-γ) production was HPV type-specific. Comparing HPV-specific T-cell IFN-γ responses at the cervix with those in the blood, we found that while CD4 + and CD8 + T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-γ responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4 + and CD8 + T-cell IFN-γ responses to E7 were of similar magnitude in both compartments and CD8 + responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.

Report on the AIDS Vaccine 2008 Conference

The "AIDS Vaccine 2008" Conference was held in Cape Town, South Africa (October 13 to 16, 2008) and organized, under the aegis of the Global HIV Vaccine Enterprise, by Dr. Lynn Morris (Chair of the Conference) National Institute of Communicable Diseases; Dr. Koleka Mlisana from CAPRISA, University KwaZulu-Natal, Durban, Dr. Glenda Gray from Perinatal HIV Research Unit, University Witwatersrand, Johannesburg and Dr. Carolyn Williamson from Institute of Infectious Diseses. and Molecular Medicine, UCT, Cape Town (Co-Chairs of the Conference). Since the first AIDS Vaccine conference, organized in Paris in 2000, this was the first time it was held outside of the U.S. and Europe, and involved nearly 1,000 participants. Besides three Plenary Sessions with ten state-of-the-art plenary lectures and one Keynote Lecture given by Dr. A.S. Fauci (Director of NIAID, NIH, USA), the Conference was organized in nine oral sessions, four poster discussion groups covering a wide spectrum of scientific information relating to HIV vaccine research and development. Moreover three Symposia, two Special Sessions, one Roundtable as well as two Debates were held, the latter focusing on current controversial topics. The conference opening was memorable for a number of reasons: among these was the presence of South Africa's new Minister of Health, Barbara Hogan who, in her first speech in a major forum as a senior member of the SA Government, affirmed that HIV causes AIDS, and that the search for a vaccine is of paramount importance to SA and the rest of the world. A scientific summary of the Conference is reported in the present article, divided into four major topics: (1) vaccine concepts and design; (2) T-cell immunology and innate immunity; (3) B-cell immunology, neutralizing antibodies and mucosal immunology; and (4) clinical trials. © 2009 Landes Bioscience.

Vitronectin – master controller or micromanager?

The concept of the cellular glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knock-out mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that vitronectin occupies a role in the earliest events of thrombogenesis and tissue repair. That role is as a foundation upon which the thrombus grows in an organised structure. In addition to closing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators and provides a provisional scaffold for the repairing tissue. In the absence of vitronectin (e.g. VN-KO animal) this cascade is disrupted before it begins. Our data demonstrates that a wide variety of biologically active species associate with VN. While initial studies were focused on mitogens, other classes of bioactives (e.g. glycosaminoglycans, metalloproteinases) are now also known to specifically interact with VN. Many of these interactions are long-lived, often resulting in multi-protein complexes, while others are stable for prolonged periods. Multiprotein complexes provide several advantages: prolonging molecular interaction; sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular / biological responses. We contend that these, or equivalent, multi-protein complexes mediate vitronectin functionality in vivo. It is also likely that many of the species demonstrated to associate with vitronectin in vitro, also associate with vitronectin in vivo in similar multi-protein complexes. Thus the predominant biological function of vitronectin is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave, and ‘how’ to behave (i.e. appropriately for the current circumstance).

Antenatal ureaplasma infection impairs development of the fetal ovine gut in an IL-1-dependent manner

Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outcomes. We tested whether interleukin-1 (IL-1) signaling underlies intestinal pathology following ureaplasma exposure in fetal sheep. Pregnant ewes received intra-amniotic injections of ureaplasma or culture media for controls at 3, 7, and 14 d before preterm delivery at 124 d gestation (term 150 d). Intra-amniotic injections of recombinant human interleukin IL-1 receptor antagonist (rhIL-1ra) or saline for controls  were given 3 h before and every 2 d after Ureaplasma injection. Ureaplasma exposure caused fetal gut inflammation within 7 d with damaged villus epithelium and gut barrier loss. Proliferation, differentiation, and maturation of enterocytes were significantly reduced after 7 d of ureaplasma exposure, leading to severe villus atrophy at 14 d. Inflammation, impaired development and villus atrophy of the fetal gut was largely prevented by intra-uterine rhIL-1ra treatment. These data form the basis for a clinical understanding of the role of ureaplasma in postnatal intestinal pathologies.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

Irritable bowel syndrome : the role of gut neuroendocrine peptides

Irritable bowel syndrome (IBS) is a common chronic disorder with a prevalence ranging from 5 to 10 % of the world's population. This condition is characterised by abdominal discomfort or pain, altered bowel habits, and often bloating and abdominal distension. IBS reduces quality of life in the same degree of impairment as major chronic diseases such as congestive heart failure and diabetes and the economic burden on the health care system and society is high. Abnormalities have been reported in the neuroendocrine peptides/amines of the stomach, small- and large intestine in patients with IBS. These abnormalities would cause disturbances in digestion, gastrointestinal motility and visceral hypersensitivity, which have been reported in patients with IBS. These abnormalities seem to contribute to the symptom development and appear to play a central role in the pathogenesis of IBS. Neuroendocrine peptides/amines are potential tools in the treatment and diagnosis of IBS. In particular, the cell density of duodenal chromogranin A expressing cells appears to be a good histopathological marker for the diagnosis of IBS with high sensitivity and specificity.

Bovine colostrum modulates cytokine production in human peripheral blood mononuclear cells stimulated with lipopolysaccharide and phytohemagglutinin

Bovine colostrum has been shown to influence the cytokine production of bovine leukocytes. However, it remains unknown whether processed bovine colostrum, a supplement popular among athletes to enhance immune function, is able to modulate cytokine secretion of human lymphocytes and monocytes. The aim of this investigation was to determine the influence of a commercially available bovine colostrum protein concentrate (CPC) to stimulate cytokine production by human peripheral blood mononuclear cells (PBMCs). Blood was sampled from four healthy male endurance athletes who had abstained from exercise for 48 h. PBMCs were separated and cultured with bovine CPC concentrations of 0 (control), 1.25, 2.5, and 5% with and without lipopolysaccharide (LPS) (3 microg/mL) and phytohemagglutinin (PHA) (2.5 microg/mL). Cell supernatants were collected at 6 and 24 h of culture for the determination of tumor necrosis factor (TNF), interferon (IFN)-gamma, interleukin (IL)-10, IL-6, IL-4, and IL-2 concentrations. Bovine CPC significantly stimulated the release of IFN-gamma, IL-10, and IL-2 (p < 0.03). The addition of LPS to PBMCs cocultured with bovine CPC significantly stimulated the release of IL-2 and inhibited the early release of TNF, IL-6, and IL-4 (p < 0.02). Phytohemagglutinin stimulation in combination with bovine CPC significantly increased the secretion of IL-10 and IL-2 at 6 h of culture and inhibited IFN-gamma and TNF (p < 0.05). This data show that a commercial bovine CPC is able to modulate in vitro cytokine production of human PBMCs. Alterations in cytokine secretion may be a potential mechanism for reported benefits associated with supplementation.

The effects of acute exercise-induced cortisol on CCR2 expression on human monocytes

CC-chemokine receptor 2 (CCR2) and its ligand, monocyte chemotactic protein-1 (MCP-1, also known as CCL2), are crucial for the recruitment of monocytes/macrophages to sites of inflammation. We conducted a series of experiments to investigate the relationship between stress, monocyte CCR2 expression and migration activity. First, we collected peripheral blood mononuclear cells (PBMC) from untrained subjects (n=8) and measured CCR2 expression on CD14(+) monocytes cultured with cortisol, epinephrine and norepinephrine. Second, we collected PBMC from the subjects before and after they cycled for 60 min at 70% peak O(2) uptake (VO2(peak)), and measured alterations in CCR2 expression on monocytes following exercise. Third, we cultured PBMC with serum obtained before and after exercise and the glucocorticoid antagonist RU-486 to determine the effect of cortisol on CCR2 expression in vitro. Last, we measured the ability of PBMC treated with serum or cortisol to migrate through membrane filters in response to CCL2. Cortisol (but not epinephrine or norepinephrine) increased CCR2 expression on monocytes in a dose- and time-dependent manner. Exercise did not influence CCR2 expression on PBMC, whereas incubation of PBMC with post-exercise serum significantly increased CCR2 expression. Both cortisol and post-exercise serum increased the migration of PBMC toward CCL2. The increase in CCR2 expression on PBMC following stimulation with cortisol and serum was blocked by the glucocorticoid receptor antagonist RU-486. In conclusion, cortisol released during exercise increased monocyte CCR2 expression and migration activity in vitro. These alterations may influence inflammation and regeneration of damaged tissue after acute stress.

Carbohydrate gel ingestion and immunoendocrine responses to cycling in temperate and hot conditions

PURPOSE: Heat stress might attenuate the effects of carbohydrate on immunoendocrine responses to exercise by increasing endogenous glucose production and reducing the rate of exogenous carbohydrate oxidation. The authors compared the efficacy of carbohydrate consumption on immune responses to exercise in temperate vs. hot conditions. METHODS: Ten male cyclists exercised on 2 separate occasions in temperate (18.1 +/- 0.4 degrees C, 58% +/- 8% relative humidity) and on another 2 occasions in hot conditions (32.2 +/- 0.7 degrees C, 55% +/- 2% relative humidity). On each occasion, the cyclists exercised in a fed state for 90 min at approximately 60% VO2max and then completed a 16.1-km time trial. Every 15 min during the first 90 min of exercise, they consumed 0.24 g/kg body mass of a carbohydrate or placebo gel. RESULTS: Neutrophil counts increased during exercise in all trials (p < .05) and were significantly lower (40%, p = .006) after the carbohydrate than after the placebo trial in 32 degrees C. The concentrations of serum interleukin (IL)-6, IL-8, and IL-10 and plasma granulocyte-colony-stimulating factor, myeloperoxidase, and calprotectin also increased during exercise in all trials but did not differ significantly between the carbohydrate and placebo trials. Plasma norepinephrine concentration increased during exercise in all trials and was significantly higher (50%, p = .01) after the carbohydrate vs. the placebo trial in 32 degrees C. CONCLUSION: Carbohydrate ingestion attenuated neutrophil counts during exercise in hot conditions, whereas it had no effect on any other immune variables in either temperate or hot conditions.