Effects of enzymatic hydrolysis on the allergenicity of natural cow milk based on a BALB/c mouse model

Cow milk allergy is one of the most prevalent food allergies worldwide, particularly in infants and children. To the best of our knowledge, minimal research exists concerning the antigenicity of cow milk (CM). This study was performed to evaluate the allergenicity of enzymatically hydrolyzed cow milk (HM) in a BALB/c mouse model. The mice were randomly divided into 5 groups (n = 12/group), which were sensitized with phosphate-buffered saline, CM, and HM (Alcalase-, or Protamex-, or Flavorzyme-treated cow milk; Novo Nordisk; AT, PT, FT, respectively), respectively, using cholera toxin as adjuvant on d 0, 7, 14, 21. On d 28, the test mice were orally challenged with phosphate-buffered saline, CM, and HM (AT, PT, or FT) alone. Anaphylactic symptoms were monitored in the mice. Antibody, cytokine, histamine, and mouse mast cell protease-1 (mMCP-1) levels were measured using enzyme-linked immunosorbent assays. In addition, the numbers of T helper (Th)1 and Th2 cells, as well as the proportions of CD4⁺CD25⁺Foxp3⁺ Treg cells, in mouse spleens were detected using flow cytometry. Statistical significance was determined by one-way ANOVA. The results revealed significant differences between CM- and HM-challenged mice. Among these, the clinical scores of HM-challenged mice (AT, 1.50; PT, 2.00; FT, 1.92) were lower than those of CM-challenged mice (positive control, 2.83), but body weight and temperature of HM-challenged mice were higher than those of CM-challenged mice. In addition, significant reductions of allergen-specific IgE, IgG, histamine, and mMCP-1 were showed in HM-challenged mice, especially for histamine, ranging from 171.42 ng/mL to 214.94 ng/mL. Remarkable reductions of IL-4, IL-5, and IL-13 levels, as well as elevations of interferon-γ and IL-10 levels in the spleens of HM-challenged mice were also detected. Moreover, the number of Th2 cells decreased in the HM-challenged mice, to 2.36% (AT), 1.79% (PT), and 4.03% (FT), respectively, whereas the numbers of Th1 cells (AT, 6.30%; PT, 6.70%; FT, 6.56%) and the proportions of CD4⁺CD25⁺Foxp3⁺Tregs (AT, 8.86%; PT, 9.21%; FT, 9.16%) increased significantly. Our findings indicate that exposure to HM was sufficient to induce a shift toward a Th1 response, thereby reducing potential allergenicity. Importantly, these results will lay a theoretical foundation for the development of hypoallergenic CM products.     

Yogurt starter cultures of Streptococcus thermophilus and Lactobacillus bulgaricus ameliorate symptoms and modulate the immune response in a mouse model of dextran sulfate sodium-induced colitis

We investigated the yogurt starter cultures of Lactobacillus bulgaricus 151 and Streptococcus thermophilus MK-10 for their effect on the severity of experimental colitis, lymphocyte profile, and regulatory T-cell response. Colitis was induced in BALB/c mice via the administration of 3.5% dextran sulfate sodium salt (DSS) in drinking water for 6 d. Next, the mice were gavaged intragastrically with an active yogurt cultures (YC) mixture (～5 × 10⁹ cfu/mouse per day) or saline (vehicle) for 8 d. Mice receiving DSS or saline alone served as positive and negative controls, respectively. The length of the colon, disease activity index, histological scores, myeloperoxidase activity, epithelium-associated microbes, short-chain fatty acid profile, total IgA antibody-forming cells, CD3⁺CD8⁺, CD3⁺CD4⁺, CD3⁺CD4⁺CD25⁺, CD3⁺CD4⁺CD25⁺Foxp3⁺ T-cell subsets, and cytokine profiles (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and tumor necrosis factor) were examined after termination of the mice. Feeding mice with YC mixture reduced disease symptoms and modified intestinal microbiota and host inflammatory responsiveness to DSS. We observed limited weight loss and a decreased disease activity index score, lowered myeloperoxidase activity, and somewhat reduced damage of the intestine. The YC mixture upregulated the colon length, increased the amount and diversity of mucosa-associated microbes (enterobacteria, enterococci, and yeast), and decreased the concentration of putrefactive short-chain fatty acids in the cecal contents. It downregulated the input of cytotoxic CD3⁺CD8⁺ T cells and CD3⁺CD4⁺CD25⁺FoxP3⁺ regulatory T cells in Peyer's patches and enhanced CD3⁺CD4⁺CD25⁺ T cells in spleens and CD3⁺CD4⁺CD25⁺FoxP3⁺ cells in peripheral blood mononuclear cells. Simultaneously, IgA antibody-forming cells were downregulated in mesenteric lymph nodes (MLN) and enhanced in spleens (SPL). The cultures mostly enhanced the production of cytokines tested in MLN and SPL, except for IL-6, which was downregulated in MLN. Interleukin-2 and IL-4 were the most upregulated in MLN, whereas IL-10, IL-4, IL-2, IFN-γ, and tumor necrosis factor were most upregulated in SPL. In serum, the YC mixture downregulated IFN-γ and clearly increased IL-2. Based on these results, we recognize the high anti-inflammatory and immunomodulatory potential of the L. bulgaricus 151 and S. thermophilus MK-10 set. The strains possess the ability to modulate the intestinal mucosal and systemic immune system toward both IgA production and induction of regulatory T cells, shifting Th1/Th2 balance.     

Effects of age and nutrition on expression of CD25, CD44, and L-selectin (CD62L) on T-cells from neonatal calves

Effects of the plane of nutrition and age on the proliferation and activation of lymphocyte subsets from milk replacer-fed calves were investigated in vitro. Holstein calves were fed a standard (0.45 kg/d of a 20% crude protein, 20% fat milk replacer, n = 4) or intensified (1.14 kg/d of a 28% crude protein, 20% fat milk replacer, n = 4) diet from 1 to 8 wk of age. Average daily weight gain of intensified-diet (0.66 kg/d) calves was greater than that of standard-diet (0.27 kg/d) calves. Relative to the pokeweed mitogen-induced responses of CD4⁺ cells from steers (5 to 6 mo of age), CD4⁺ cells from 1-wk-old calves showed decreased proliferative activity, delayed increase in CD25 expression, and no demonstrable increase in CD44 expression or decrease in CD62L expression. Calf CD8⁺ and γδT-cell receptor⁺ cells, unlike T-cells from the older animals, did not demonstrate decreased expression of CD62L after stimulation with mitogen. The increased expression of CD44 by mitogen-stimulated γδT-cell receptor⁺ cells from older animals was not seen in γδT-cell receptor⁺ cells from 1-wk-old calves. At wk 8 of age, mitogen-induced proliferation and expression of activation antigens by T-cells from standard-fed calves were similar to responses of T-cells from steers indicating rapid maturation of T-cell function during the neonatal period. Feeding calves an intensified milk replacer was associated with decreased proliferation of mitogen-stimulated CD4⁺, CD8⁺, and γδT-cell receptor⁺ cells; decreased CD25 expression by mitogen-stimulated CD4⁺ and CD8⁺ cells; and decreased CD44 expression by mitogen-stimulated CD8⁺ cells. These results indicate that the functional capacity of the calf's T-cell population becomes more adult-like during the first weeks of life and suggest that nutrition modulates T-cell function during this period of immune maturation.     

Anti-allergic effect of strawberry extract

We examined the anti-allergic effect of strawberry extract on human peripheral blood mononuclear cells (PBMCs) and atopic dermatitis model mice NC/NgaTndCrlj. The addition of strawberry extract suppressed total IgE production in the cedar pollen antigen Cry j 1-stimulated PBMCs. Flow cytometric analysis showed that strawberry extract decreased the rate of CD3⁺CD4⁺ helper T cells by 17.3% and increased the rate of CD3⁺CD8⁺ cytotoxic T cells by 19.7% in PBMCs. Moreover, the extract inhibited the expression level of GATA3 that is the master regulator of type 2 helper T cells (Th2) in human primary pan T cells isolated from PBMCs. Oral administration of strawberry extract lowered dermatitis scores and serum IgE levels in mice. In addition, it also decreased the GATA3 expression level in mouse blood cells. These results revealed that strawberry extract suppressed the severity of atopic dermatitis through the down-regulation of serum IgE by inhibition of Th2 differentiation.     

Increase in Escherichia coli inoculum dose accelerates CD8+ T-cell trafficking in the primiparous bovine mammary gland

Although migration of leukocytes into the mammary gland is pivotal for a cow's response against intramammary invading pathogens, the contribution of lymphocyte subsets to this response remains unclear. To investigate the dynamics of lymphocyte populations during Escherichia coli mastitis, T-lymphocyte subsets, CD4⁺/CD8⁺ ratio, CD21⁺ cells, and lymphoproliferation were studied in blood and milk of primiparous cows exposed to different quantities of bacteria. The cows were intramammarily inoculated with 10⁴ cfu of E. coli (group A) and 10⁶ cfu (group B). Compared with group A, a much greater number of lymphocytes migrated into the infected quarters at postinfection hour (PIH) 6 to 24 in group B, and the CD8⁺ cells were the first-recruited T cells in the milk. There was a significant decline in the CD4⁺/CD8⁺ ratios at PIH 6 to 24 in group B. The decrease of CD4⁺/CD8⁺ ratios at PIH 6 to 24 resulted mainly from greater CD8⁺ cell concentrations in milk. In contrast, at PIH 72, CD4⁺/CD8⁺ ratios increased about 2-fold in both groups. This increase was mainly due to the increase in CD4⁺ cell concentration. The increased concentration of CD4⁺ cells coincided with an increase in the CD21⁺ cell population in the milk. In blood, the increase of CD8⁺ cells appeared much faster in group B (PIH 6 and 12) than in group A. The results from lymphoproliferation also indicated a greater increase in the proliferative response in both blood and milk lymphocytes of group B. Our study demonstrates for the first time that an increase of E. coli inoculum dose accelerates the trafficking of CD8+ cells during initiation of E. coli mastitis, and these cells are the predominant T cells in milk during the early hours of bovine E. coli mastitis.     

Immune cell populations residing in mesenteric adipose depots and mesenteric lymph nodes of lean dairy cows

Inconsistent evidence of inflammatory immune cell infiltrates in adipose tissues with extensive triglyceride mobilization raises the possibility that regulatory or anti-inflammatory immune cell populations reside within the mesenteric adipose tissue (MAT) and mesenteric lymph nodes (MLN). These resident immune cell populations may be involved in attenuating the inflammatory response. We explored the immune cell population of MAT and MLN collected from lean, lactating Holstein cows without apparent disease in an abattoir (n = 42). Lean cows had a body condition score of 2.6 ± 0.1 (mean ± SD) with a greater frequency of adipocyte area occurring in small rather than large adipocytes. Cells were labeled with monoclonal antibodies specific to bovine leukocyte antigens for enumeration by flow cytometry. Within both lymph node and adipose tissues, relatively large subpopulations of cells expressed the β2 integrins CD11b and CD11c, class II major histocompatibility antigens (MHCII), and the SIIRP-1α receptor (CD172a) typical of dendritic cells and macrophages. Macrophage/dendritic cell heterogeneity was marked by β2 integrin expression alone or in conjunction with CD172a or MHCII across subpopulations from both tissues; CD209, the DC-SIGN c-type lectin receptor of dendritic cells, was not detected by fluorescence-activated cell sorting in either tissue. Lymphocytes comprised 74.1 ± 3.7% and 13.7 ± 3.7% of the MLN and MAT cell populations, respectively, and CD3⁺CD4⁺ lymphocytes accounted for 49.8 ± 9.9% of the MLN and 6.13 ± 1.23% of the MAT cells. Fox P3⁺ regulatory lymphocytes comprised 15.3 ± 1.1% and 6.73 ± 0.52% of the MLN and MAT cells, whereas γδ⁺ lymphocytes accounted for 6.65 ± 0.74% and 3.91 ± 0.43% of the MLN and MAT cells, respectively. Subpopulations of CD3⁺CD8⁺ cytotoxic T cells and CD3⁺CD11c⁺ innate lymphocytes were present in MLN but not MAT. These results show that subpopulations of resident tissue macrophages, dendritic cells, T helper lymphocytes, regulatory T lymphocytes (Tregs), and γδ lymphocytes reside in mesenteric lymph nodes and adipose tissues. Balance in the innate and adaptive immune functions embedded in these tissues could support metabolic health.     

High growth rate fails to enhance adaptive immune responses of neonatal calves and is associated with reduced lymphocyte viability

The objective of the study was to evaluate the effects of 3 targeted growth rates on adaptive (i.e., antigen-specific) immune responses of preruminant, milk replacer-fed calves. Calves (9.1 ± 2.4 d of age) were assigned randomly to one of 3 dietary treatments to achieve 3 targeted daily rates of gain [no growth (maintenance) = 0.0 kg/d, low growth = 0.55 kg/d, or high growth = 1.2 kg/d] over an 8-wk period. The NRC Nutrient Requirements of Dairy Cattle calf model computer program was used to estimate the milk replacer intakes needed to achieve target growth rates. All calves were fed a 30% crude protein, 20% fat, all-milk protein milk replacer reconstituted to 14% dry matter. Diets were formulated to ensure that protein would not be limiting. All calves were vaccinated 3 wk after initiation of dietary treatments with Mycobacterium bovis, strain bacillus Calmette-Guerin and ovalbumin. Growth rates for no-growth (0.11 kg/d), low-growth (0.58 kg/d), and high-growth (1.16 kg/d) calves differed throughout the experimental period. Blood glucose concentrations in high-growth calves increased with time and were higher than in low- and no-growth calves. Mononuclear and polymorphonuclear leukocyte percentages in peripheral blood were unaffected by growth rate but did change with advancing age. Percentages of CD4⁺ T cells increased with age in no-growth and low-growth calves, a characteristic of maturation, but failed to increase in high-growth calves. Growth rate did not affect the percentages of CD45RO⁺ (memory) CD4⁺ and CD8⁺ T cells, antigen (i.e., ovalbumin)-specific serum IgG concentrations, or antigen (i.e., purified protein derivative)-induced IFN-γ and nitric oxide secretion by mononuclear cell cultures. Antigen-elicited cutaneous delayed-type hypersensitivity responses of no-growth calves exceeded responses of low-growth, but not high-growth, calves. In resting- and antigen-stimulated cell cultures, viabilities of CD4⁺, CD8⁺, and γδTCR⁺ T cells from high-growth calves were lower than those of the same T cell subsets from no-growth and low-growth calves. Alternatively, resting cultures of mononuclear leukocytes from high-growth calves produced more nitric oxide than those from no-growth and low-growth calves. In conclusion, adaptive immune responses were affected minimally by growth rate. The results suggest that protein-energy malnutrition in the absence of weight loss is not detrimental to antigen-specific responses of neonatal vaccinated calves and that a high growth rate does not enhance these responses. The negative effect of a high growth rate on the viability of circulating T cell populations may influence infectious disease resistance of the calf.     

Estimating genetic and phenotypic parameters of cellular immune-associated traits in dairy cows

Data collected from an experimental Holstein-Friesian research herd were used to determine genetic and phenotypic parameters of innate and adaptive cellular immune-associated traits. Relationships between immune-associated traits and production, health, and fertility traits were also investigated. Repeated blood leukocyte records were analyzed in 546 cows for 9 cellular immune-associated traits, including percent T cell subsets, B cells, NK cells, and granulocytes. Variance components were estimated by univariate analysis. Heritability estimates were obtained for all 9 traits, the highest of which were observed in the T cell subsets percent CD4⁺, percent CD8⁺, CD4⁺:CD8⁺ ratio, and percent NKp46⁺ cells (0.46, 0.41, 0.43 and 0.42, respectively), with between-individual variation accounting for 59 to 81% of total phenotypic variance. Associations between immune-associated traits and production, health, and fertility traits were investigated with bivariate analyses. Strong genetic correlations were observed between percent NKp46⁺ and stillbirth rate (0.61), and lameness episodes and percent CD8⁺ (?0.51). Regarding production traits, the strongest relationships were between CD4⁺:CD8⁺ ratio and weight phenotypes (?0.52 for live weight; ?0.51 for empty body weight). Associations between feed conversion traits and immune-associated traits were also observed. Our results provide evidence that cellular immune-associated traits are heritable and repeatable, and the noticeable variation between animals would permit selection for altered trait values, particularly in the case of the T cell subsets. The associations we observed between immune-associated, health, fertility, and production traits suggest that genetic selection for cellular immune-associated traits could provide a useful tool in improving animal health, fitness, and fertility.     

Milk basic protein supplementation exerts an anti-inflammatory effect in a food-allergic enteropathy model mouse

To examine novel functions of milk basic protein (MBP) in T-cell-related inflammatory diseases, such as autoimmune diseases and allergies, we evaluated the effects of MBP on the causative responses of ovalbumin (OVA)-specific T cells in a food-allergic enteropathy model, OVA23–3 mice, which express an OVA-specific T-cell receptor gene. The OVA-specific CD4⁺ T cells of the mesenteric lymph nodes (MLN) from OVA23–3 mice were cultured with CD11c⁺ dendritic cells of MLN from BALB/cA mice in the absence or presence of MBP following stimulation with OVA; then the levels of CD69 expression and the levels of cytokine production by CD4⁺ T cells were measured to evaluate activation. The effects of MBP supplementation of OVA 23–3 mice were assessed by feeding a diet containing OVA (OVA diet) with or without MBP for 28 d. Intestinal inflammation, together with activation and cytokine production of CD4⁺ T cells by MLN, as well as femoral bone mineral density, were measured. In in vitro culture, MBP inhibited excess activation and IL-4 production by CD4⁺ T cells. The supplementation of MBP to the OVA diet attenuated OVA-specific IgE production in OVA-diet-fed OVA23–3 mice and slightly resolved developing enteropathy caused by excess IL-4 production by CD4⁺ T cells. Feeding OVA diet to OVA23–3 mice exhibited bone loss accompanied with enteropathy, whereas MBP supplementation prevented bone loss and increased osteoprotegerin, an osteoclastogenesis inhibitory factor, in the mice. The inhibition of T-cell-activation in both MLN and bone marrow by MBP supplementation may help prevent increased IgE levels caused by excessive IL-4 production and bone loss accompanied by enteropathy. Our findings show that MBP may help attenuate both T-cell-related inflammation and bone loss.     

Low molecular weight β-glucan stimulates doxorubicin-induced suppression of immune functions in mice

The aim of this study was to evaluate the protective effect of low molecular weight β-glucan (LMG) against doxorubicin (DOX)-induced immune suppression of tumor-bearing mice. The tumor size and spleen cell functions such as spleen cell proliferation, cytokine production (interferon-γ and interleukin-2), and the population of CD4⁺ and CD8⁺ T cells were estimated. In the tumorbearing mice, the tumor size was significantly (p<0.05) decreased by DOX treatment. However, there was no significant difference between mice treated with high molecular weight β-glucan (HMG) and mice treated with LMG. Spleen cell proliferation and cytokine production were significantly (p<0.05) decreased in only DOX treated group, but increased in all β-glucan treated groups with DOX. Moreover, the populations of CD4⁺ and CD8⁺ T cells were also increased in the LMG-treated group. It appears that LMG effectively reduces the DOX-induced immune toxicity through activation of immune cells such as splenocytes.     

Active hexose correlated compound exerts therapeutic effects in lymphocyte driven colitis

Active hexose correlated compound (AHCC) is a commercial extract of Basidiomycetes fungi enriched in oligosaccharides that is used as a human nutritional supplement for various purposes in humans. Our aim was to study the anti‐inflammatory effect of AHCC in the CD4+ CD62L⁺ T cell transfer model of colitis, considered one of the closest to the human disease. Colitis was induced by transfer of CD4⁺ CD62L⁺ T cells to recombination activating gene 1^?/? mice. AHCC (75 mg/d) was administered by gavage as a post‐treatment. Three groups were established: noncolitic, colitic (CD4⁺ CD62L⁺ transferred mice treated with vehicle), and AHCC (colitic treated with AHCC). AHCC improved colitis, as evidenced by a 24% lower colonic myeloperoxidase and a 21% lower alkaline phosphatase activity. In addition, a decreased secretion of proinflammatory genes assessed by RT‐qPCR was observed, particularly TNF‐α and IL‐1β. Ex vivo mesenteric lymph node cells obtained from AHCC treated mice exhibited a fully normalized production of IL‐6, IL‐17, and IL‐10 (p < 0.05). Also, AHCC treated mice exhibited decreased STAT4 and IκB‐α phosphorylation in splenic CD4⁺ cells. Our data provide validation of AHCC colonic anti‐inflammatory activity in a chronic, T cell driven model of inflammatory bowel disease.     

Antigen-specific B-cell responses by neonatal calves after early vaccination

The objective of this research was to evaluate the effects of early vaccination on the phenotype (i.e., activation marker expression) and functional capacity of B cell populations in neonatal calves. In the first of 2 experiments, 6 calves were vaccinated with ovalbumin at 3 and 5 wk of age. Three of the 6 calves also were vaccinated with Mycobacterium bovis, strain bacillus Calmette-Guerin (BCG) at 3 wk of age. Mycobacterium bovis lipoarabinomannan-reactive IgG₁ and IgG₂ were detected in calf sera prior to vaccination, indicative of colostral transfer of maternal Ig cross-specific to BCG. Ovalbumin-specific IgG₁ and IgG₂ were not detected before vaccination. Vaccination of 3-wk-old calves with ovalbumin elicited antigen-specific IgG₁ and IgG₂ anti-body responses that were amplified by secondary vaccination. Vaccination with BCG did not elicit a measurable antibody response. In the second experiment, 6 calves were vaccinated with ovalbumin at 3 and 5 wk of age in addition to BCG at 3 wk of age. Lymph node cell populations stimulated with ovalbumin had decreased CD5, CD21, and CD40 expression and increased B-B2, CD25, and CD80 expression on IgM⁺ cells. Stimulation of the same population with purified-protein derivative increased CD25 and CD80 expression on IgM⁺ cells. Expression of activation molecules on ovalbumin- and purified protein derivative-stimulated CD5⁺IgM⁺ cells was similar to expression on the larger IgM⁺ cell population. An increased expression of major histocompatibility class II on CD5⁺IgM⁺ cells after stimulation was the only exception. Interestingly, IgM⁺ cells isolated from the superficial cervical lymph node draining the vaccination site, but not from the opposing cervical lymph node, responded to antigen stimulation in vitro. In conclusion, calves generated B cell responses to ovalbumin and BCG after vaccination. Additional studies are necessary to determine whether maternal immunologic experience transferred via colostral immunoglobulin inhibits production of mycobacteria-specific immunoglobulin production in the calf.     

Glycation of the Major Milk Allergen β‐Lactoglobulin Changes Its Allergenicity by Alterations in Cellular Uptake and Degradation

1 Scope

During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β‐lactoglobulin (BLG), in their interactions with cells crucially involved in allergy.

2 Methods and results

BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T‐cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco‐2 monolayer. Uptake of glycated BLG by bone marrow–derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor‐mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4⁺ T‐cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG‐specific IgE sensitized basophil cells.

3 Conclusions

This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy.     

Decreasing pro-inflammatory cytokine and reversing the immunosenescence with extracts of Pu-erh tea in senescence accelerated mouse (SAM)

Immunosenescence, the progressive decline of adaptive immunity and chronic inflammation with ageing has been demonstrated to be the main factor responsible for infections, cancer and autoimmune conditions in the elderly. Senescence-accelerated mouse (SAM) was used to study the protective effects of Pu-erh tea in the elderly. The senile-prone sub-strain, SAM-P8 mice were administered individually with ripened or crude Pu-erh tea at 125, 250 or 500 mg/kg. The results showed that Pu-erh tea significantly increased the fractions of naïve T lymphocytes, CD8⁺CD28⁺ T lymphocytes and NK cells in the peripheral blood, but decreased the levels of IL-6 in aged mice. These data suggested that the Pu-erh tea reversed the immunosenescence by restoring the immune deficiency and decreasing pro-inflammatory cytokine. Thus, long term drinking of Pu-erh tea may be beneficial for the aged population in terms of increasing the body’s resistance to infection and cancer.     

Effects of Wnt-10b on proliferation and differentiation of adult murine skin-derived CD34 and CD49f double-positive cells

Although mouse Wnt-10b has been shown to play various roles in a wide range of biological actions, the effects on epithelial stem/progenitor cells in the skin have not been reported. In the present study, we investigated the effects of Wnt-10b on proliferation and differentiation of murine skin-derived CD34 and CD49f double-positive (CD34⁺CD49f⁺) cells, a supposed fraction as enriched epithelial stem/progenitor cells. The cells were prepared from dorsal skin samples obtained from young adult mice as α6 integrin (CD49f) and CD34 double-positive cells by fluorescent activated cell sorting (FACS), and they were cultured with or without Wnt-10b to investigate its effects on proliferation and differentiation. Involvement of canonical Wnt signaling pathway was confirmed by TOPFLASH assay, and differentiation of the CD34⁺CD49f⁺ cells was assessed by RT-PCR analysis and immunocytochemical examinations. The skin-derived CD34⁺CD49f⁺ cells were immunopositive for Lhx2 and expressed mRNA of classical markers for bulge stem cells, including Lhx2, keratin15, Sox9, S100a6, and NFATc1. Their proliferation was suppressed by Wnt-10b, and the markers for differentiated epithelial cells became to be expressed in the culture with Wnt-10b. These results suggest that Wnt-10b promotes differentiation of epithelial stem/progenitor cells in the skin.     

CD2/CD21 index: A new marker to evaluate udder health in dairy cows

Lymphocytes play a significant role in the immunological processes of the bovine mammary gland and were found to be the dominant cell population in the milk of healthy udder quarters. The objective of this study was to investigate the quantitative relationship between CD2⁺ T and CD21⁺ B lymphocytes using flow cytometry. In a first study, quarter foremilk samples from apparently healthy udder quarters [somatic cell counts (SCC) ≤100,000 cells/mL; n = 65] were analyzed and compared with diseased quarters (SCC >100,000 cells/mL; n = 15). Percentages of CD2⁺ T cells were significantly higher in milk samples with SCC ≤100,000 cells/mL than in those with SCC >100,000 cells/mL, whereas percentages of CD21⁺ B cells developed in the opposite direction. As a result of this opposing trend, a new variable, the CD2/CD21 index—representing the percentages of CD2⁺ cells per CD21⁺ cells—was defined. Although diseased quarters with SCC >100,000 cells/mL and the detection of major pathogens revealed generally CD2/CD21 indices <10, values >10 were observed in apparently healthy quarters. Hence, a CD2/CD21 index cutoff value of 10 may be suitable to aid differentiation between unsuspicious and microbiologically suspicious or diseased udder quarters. To test whether CD2/CD21 indices <10 were primarily related to pathogens, quarters with SCC ≤100,000 cells/mL and >100,000 cells/mL with different bacteriological status (culture negative, or minor or major pathogens) were selectively examined in a second biphasic study. In the first trial, 63 udder quarters were analyzed and 55 of these quarters were able to be sampled again in the second trial carried out 14 d later. In both trials, results of the first study were confirmed. Indeed, CD2/CD21 indices <10 were also found in quarters showing SCC ≤100,000 cells/mL and containing minor or major pathogens at the time of the current or previous bacteriological analysis. The results of our examinations indicated a clear relationship between the CD2/CD21 index and the bacteriological status of the mammary gland. In combination with SCC, it offers a new marker for quick differentiation of unsuspicious and microbiologically suspicious or diseased udder quarters.     

3,10-Dihydroxy-decanoic acid,isolated from royal jelly,stimulates Th1 polarising capability of human monocyte-derived dendritic cells

Different pharmacologically active components have been isolated from royal jelly. Some of them possess imunomodulatory activity, but the mechanisms of their effect on the immune system have not been elucidated yet. In this study we tested the effect of 3,10-dihydroxy-decanoic acid (3,10-DDA), a fatty acid isolated from royal jelly, on maturation and functions of human monocyte-derived dendritic cells (MoDCs). We showed that 3,10-DDA stimulated maturation of MoDCs by up-regulating the expression of CD40, CD54, CD86 and CD1a, and increased their allostimulatory potential in co-culture with allogeneic CD4⁺T cells. 3,10-DDA-treated MoDCs enhanced the production of IL-12 and IL-18, and stimulated the production of interferon-γ in co-culture with allogeneic CD4⁺T cells, compared to control MoDCs. In contrast, the production of IL-10 was down-regulated. In conclusion, our results suggest that 3,10-DDA stimulates maturation and Th1 polarising capability of human MoDCs in vitro, which could be beneficial for anti-tumour and anti-viral immune responses.     

Anthraquinones of edible wild vegetable Cassia tora stimulate proliferation of human CD4 T lymphocytes and secretion of interferon-gamma or interleukin 10

Cassia tora L. is an edible wild plant. This study evaluated the immunostimulatory activities of four anthraquinones of C. tora (aloe-emodin, emodin, chrysophanol, and rhein) on human peripheral blood mononuclear cells (PBMC). Studies were conducted on lymphocyte proliferation by BrdU immunoassay, secretion of interferon-gamma (IFN-γ) and interleukin 10 (IL-10) by an ELISA assay and elucidation of responding immune cells by flow cytometry. The results showed that at non-cytotoxic concentrations, the tested anthraquinones were effective in stimulating the proliferation of resting human PBMC and/or secretion of IFN-γ. However, at the concentration of 10 μg/ml (35 μM), rhein significantly stimulated proliferation of resting human PBMC (stimulation index (SI) = 1.53), but inhibited IFN-γ secretion (74.5% of control). The augmentation of lymphocyte proliferation was correlated to the increase in number of CD4⁺ T cells, while the elevated secretion of IFN-γ and IL-10 might have been due to the activated CD4⁺ T cells.