Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

Tumor infiltrating lymphocytes (TIL) reflect the host''s anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (P = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8⁺CD62L⁺ non-effector cells in TIL compared to PBL, implying age-related increase of CD8⁺CD62L⁻ effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages.     

Utilization of the Porcine System to Study Lymphotoxin-β Regulation in Intestinal Lymphoid Tissue

Lymphotoxin-β (LT-β) has been suggested to be a regulator of secondary lymphoid structure development. In the present study, we isolated porcine LT-β (poLT-β) from adult swine spleens. The open reading frame encoded a predicted 246-amino acid polypeptide exhibiting higher similarity to the human than the mouse LT-β protein. Expression of LT-β mRNA in various swine tissues was analyzed by real-time PCR, and it was found to be higher in the ileal Peyer's patches (Pps) of adults than in newborns. In addition, ligand stimulation of toll-like receptors 2, 4, and 9, which are activated by bacterial components, increased LT-β expression only in neonatal ileal Pps. These results suggest that colonization by commensal bacteria may affect the maturation of neonatal ileal Pps by the induction of LT-β via toll-like receptors. LT-β may therefore be useful for studying the development of the intestinal immune system at parturition in both swine and humans.     

Impact of Methimazole Treatment on Magnesium Concentration and Lymphocytes Activation in Adolescents with Graves’ Disease

The aim of this research was to assess plasma magnesium (Mg) concentration, the frequencies of activated T CD4+ and T CD8+ lymphocytes and B lymphocytes in adolescents with hyperthyroidism due to Graves’ disease (GD), and to assess changes in the above-mentioned parameters during methimazole (MMI) treatment. The frequencies of activated T and B cells were measured by flow cytometry method and plasma Mg concentration was determined by spectrophotometry method in 60 adolescents at the time of GD diagnosis and after receiving the normalisation of the thyroid hormones levels. The control group consisted of 20 healthy volunteers. We observed lower plasma Mg concentration, and higher frequencies of activated T and B lymphocytes in the study group before the treatment in comparison with healthy controls, and with study group in MMI-induced euthyreosis (p?<?0.01).Statistically significant negative correlations between the percentages of activated T CD3+, T CD4+, T CD8+ and B CD19+ lymphocytes, and plasma Mg concentration before the treatment were found (r?<??0.335, p?<?0.002). After the treatment no vital differences in plasma Mg concentration, and in percentages of activated cells between GD patients and controls were found, except CD8+CD25+ cells (p?=?0.03). The present study demonstrates that both activated T and B cells might play an important role in the pathogenesis of GD, and activation is related to Mg plasma level. The use of MMI in treatment of hyperthyroidism due to GD leads to decrease the frequencies of activated lymphocytes and normalisation of Mg levels.     

Squamous Tissue Lymphocytes in the Esophagus of Controls and Patients with Reflux Esophagitis and Barrett’s Esophagus Are Characterized by a Non-Inflammatory Phenotype

Background and Objective

Reflux esophagitis (RE) is characterized by inflammation of the squamous epithelium (SQ) of the esophagus and may progress to Barrett’s esophagus (BE) characterized by intestinal metaplasia. The role of inflammation in this transition has been postulated but lacks experimental evidence. Here, the inflammatory responses in the esophagus of these patients were investigated.

Patients and Methods

Fifty-one esophageal biopsies from with patients BE (n = 19), RE (n = 8) and controls (n = 23) were analyzed. T-cells were analyzed before and after ex vivo expansion (14 days) by multicolor flow cytometric analysis. The following markers were studied: CD3, CD4, CD8 (T-cell markers), Granzyme B (marker of cytotoxicity), CD103 (αE/epithelial integrin) and NKg2a (inhibitory receptor on T-cells and NK-cells).

Results

Analysis of ex vivo cultures from normal looking SQ from controls, RE patients, and BE patients revealed no significant differences in the number and phenotypes of T-cells. In contrast, tissue from RE was different to normal SQ in four aspects: 1) higher percentages of CD3⁺CD4⁺-cells (72±7% vs 48±6%, p = 0.01) and 2) CD8⁺GranzymeB⁺ -cells (53±11% vs 26±4%, p<0.05), while 3) lower percentages of CD4⁺CD103⁺-cells (45±19% vs 80±3%, p = 0.02) and 4) CD8⁺NKg2a⁺- cells (31±12% vs 44±5%).

Conclusion

Despite the fact that both tissues are exposed to the same reflux associated inflammatory triggers, the immune response observed in RE is clearly distinct from that in SQ of BE. The differences in immune responses in BE tissue might contribute to its susceptibility for transformation into intestinal metaplasia.     

Infections in the tissue material and their impact on the loss of transplants in the Laboratory of in vitro Cell and Tissue Culture with Tissue Bank in the years 2011–2015

Radiation sterilization eliminates microbiological infections but causes the degradation of the cell factor. The negative result of microbiological examination for tissue transplants is one of the conditions for approval for distribution in patients. The study attempts to verify impact of the presence of microbes onto material for transplant loss. In the 2011–2015 period, we analyzed 293 donors of skin and amnion. Microbiological sampling was performed. The total of 21 strains of bacteria, molds and fungi was identified in collected tissue. The widest spectrum of strains was found in skin (17), followed by amnia (8). The total number of positive findings was 147 and was again highest in skin (129), while the number of positive findings in amnia was 18 only. The general percentage of fungal infections was very low. The presence of fungal strains was only observed in allogeneic skin (2%). Large number of microorganisms isolated from the skin before sterilization was observed, so it seems impossible to use allogeneic intravital skin. However, the intravital application of allogeneic amnion obtained from cesarean section remains to be considered.     

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

Increase in the occurrence of abortions associated with exposure to the Bluetongue virus serotype 8 in naïve dairy herds

The transplacental transmission capacity demonstrated for Bluetongue virus serotype 8 (BTV-8) in cattle probably is associated with an increased occurrence of abortions. The objectives of this study were to quantify the effect of BTV-8 exposure on the occurrence of abortions in previously naive dairy cow herds under natural infection conditions, and to determine a possible risk period during pregnancy associated with this increase. Two criteria were considered in order to estimate the occurrence of abortion: late return-to-service after a first artificial insemination (AI), and short gestations. A late return-to-service was defined as a return taking place 90 to 200 days after a first AI. These criteria were compared between cows in herds exposed during the 2007 epizootic in France and cows in herds that were not exposed. To determine the risk period during a pregnancy, variations in the occurrence of abortions were quantified according to the stage of the pregnancy during which the exposure took place. Survival analyses were used to estimate the risk of increased occurrence of abortion associated with BTV-8 exposure, adjusted by the principal factors known to influence the risk of abortion. Exposure to the BTV-8 virus under natural conditions in previously naive dairy herds notified after clinical suspicion during the 2007 epizootic was associated with an increase in the occurrence of abortions, regardless of the stage of pregnancy. The at-risk gestation period depended on the criteria used to detect abortions. The mean effect of BTV-8 exposure in the ensemble of detected outbreaks corresponded to an increase of 6.7% in late return-to-service. BTV-8 exposure during the first 3 mo of gestation was associated with a 15% increase in late return-to-service for cows with no return-to-service at 90 days, while this increase was 6% for exposure starting from the third month of gestation (in outbreaks detected in September). BTV-8 exposure from the third month of gestation was associated with a 1.9% increase of short gestations. The effect of exposure was more pronounced for outbreaks detected early in the epizootic compared with those detected later.     

Increase in the production of β-carotene in recombinant Escherichia coli cultured in a chemically defined medium supplemented with amino acids

Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of β-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of β-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest β-carotene production that has been reported among the recombinant cells to date.     

Increase in Unemployment over the 2000’s: Comparison between People Living with HIV and the French General Population

BackgroundDespite improved health, unemployment has increased among people living with HIV (PlwHIV) over the last decade. However, since the economic recession of 2008, unemployment also increased in the French general population. This paper aimed to determine if the increase in the unemployment rate in the HIV population was higher than that in the French general population.MethodsWe used data from the ANRS-Vespa study, a repeated cross-sectional survey among two national representative samples of PlwHIV followed at hospitals in France in 2003 and 2011. We compared employment and unemployment rates between HIV-infected people (overall and according to period of HIV diagnosis) and the French general population in 2003 and 2011, using multivariate Poisson regressions adjusted for individual sociodemographic characteristics.ResultsThe employment rate among PlwHIV was consistently lower than that in the general population in 2003 and 2011. In contrast, there was a trend of an increasing unemployment rate difference between PlwHIV and the general population: PlwHIV’s unemployment rate was 1.48 (95% confidence interval [CI]: 1.16–1.90) times higher than that of the general population in 2003, versus 1.62 (95% CI: 1.34–1.96) times higher in 2011. This unemployment rate difference was the highest for PlwHIV diagnosed in or after 2008 (adjusted prevalence rate ratio: 2.06; 95% CI: 1.59–2.67).ConclusionsThese results suggest that in time of economic recession, an increasing proportion of PlwHIV may be excluded from the labor market although they are willing to re-enter it. This constitutes a major issue relative to social consequences of chronic disease.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Changes in the Mutagenic and Estrogenic Activities of 17β-Estradiol after Treatment with Nitrite

We determined the changes in the mutagenic and estrogenic activities of 17β-estradiol after a nitrite treatment. Nitrite-treated 17β-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17β-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17β-estradiol to have been formed in the reaction mixture. 2-Nitro-17β-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17β-estradiol and 2-nitro-17β-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17β-estradiol was found to be lower than that of 17β-estradiol. These data suggest that a daily oral intake of 17β-estradiol and nitrite might induce the formation of mutagenic compounds in our body.     

Treatment of growth hormone excess in dogs with the progesterone receptor antagonist aglépristone

Acromegaly or hypersomatotropism in dogs is almost always due to progestin-induced hypersecretion of GH originating from the mammary gland. The aim of this study was to investigate whether aglépristone, a progesterone receptor antagonist, can be used to treat this form of canine acromegaly. In five Beagle bitches hypersomatotropism was induced by administration of MPA for over 1 year. Subsequently, aglépristone was administered. Blood samples were collected before MPA administration, immediately before, during, and 3.5 and 5.5 weeks after the last administration of aglépristone for determination of the plasma concentrations of GH and IGF-I. In addition, blood samples for the determination of the 6-h plasma profile of GH were collected before MPA administration, before aglépristone administration, and 1 week after the last aglépristone treatment. MPA administration resulted in a significant increase of the mean plasma IGF-I concentration, whereas analysis of the pulsatile plasma profile demonstrated a trend (P=0.06) for a higher mean basal plasma GH concentration and a higher mean AUC(0) for GH. Treatment with aglépristone resulted in a significant decrease of the mean plasma GH and IGF-I concentrations. Analysis of the pulsatile plasma profile showed a trend (P=0.06) for a lower mean basal plasma GH concentration and a lower mean AUC(0) for GH 1 week after the last aglépristone treatment compared with these values before aglépristone administration. Three and a half and 5.5 weeks after the last aglépristone administration the mean plasma IGF-I concentration increased again. In conclusion, aglépristone can be used successfully to treat dogs with progestin-induced hypersomatotropism.     

Is Remodelling of Corticospinal Tract Terminations Originating in the Intact Hemisphere Associated with Recovery following Transient Ischaemic Stroke in the Rat?

Following large strokes that encompass the cerebral cortex, it has been suggested that the corticospinal tract originating from the non-ischaemic hemisphere reorganises its pattern of terminal arborisation within the spinal cord to compensate for loss of function. However many strokes in humans predominantly affect subcortical structures with minimal involvement of the cerebral cortex. The aim of the present study was to determine whether remodelling of corticospinal terminals arising from the non-ischaemic hemisphere was associated with spontaneous recovery in rats with subcortical infarcts. Rats were subjected to transient middle cerebral artery occlusion or sham surgery and 28 days later, when animals exhibited functional recovery, cholera toxin b subunit was injected into the contralesional, intact forelimb motor cortex in order to anterogradely label terminals within cervical spinal cord segments. Infarcts were limited to subcortical structures and resulted in partial loss of corticospinal tract axons from the ischaemic hemisphere. Quantitative analysis revealed there was no significant difference in the numbers of terminals on the contralesional side of the spinal grey matter between ischaemic and sham rats. The results indicate that significant remodelling of the corticospinal tract from the non-ischaemic hemisphere is not associated with functional recovery in animals with subcortical infarcts.     

Increase in the Level of Skeletal Muscle α-Actin mRNA in Rats Fed a Non-protein Diet Supplemented with Cystine and Threonine

Pinguisone accumulated in cultured gametophytes of Aneurapinguis to a significantly high level. A biosynthetic study on the formation of pinguisone was carried out by feeding [2-¹³C]-acetate to the cultured gametophytes. Pinguisone was labeled at an adequate level to determine the labeling positions by a ¹³C-NMR analysis. The labeling pattern indicated two-methyl migration and C-C bond cleavage of the main chain in farnesyl diphosphate in the formation of pinguisone.     

Distribution of Laminin Variants and Their Integrin Receptors in Human Secondary Lymphoid Tissue: Colocalization suggests that the α6β4-integrin is a receptor for laminin-5 in lymphoid follicles

Laminins are a family of multifunctional basement membrane glycoproteins. Over the last years, many laminin isoforms have been characterized, which were shown to be composed of distinct combinations of variant α β and γ chains. Some of these isoforms show remark-able tissue specificity, which suggests functional involvement in local processes. In this study the previously described mAb 4C7. which recognize epithelial basement membranes as well as endothelial basement membranes in lymphoid follicles, was identified as an anti-laminin-5 antibody. Using a set of mAbs against various variant laminin chains we established that specifically the γ₂ chain of laminin-5 was confined to the endothelial basement membranes of vessels in lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid tissue. Additionally. the expression of the known integrin receptors of laminin-5 was also examined. The α6β4 integrin-receptor for laminin was found to be colocalized with the laminin-5 γ₂ chain on the abluminal surface of endothelial cells, whereas the α3 integrin chain could not be detected in lymphoid follicles. This finding suggests that the α6β4 integrin (and not the α3β1 integrin) serves as a laminin-5 receptor on endothelial cells in the follicular compartment of lymphoid tissue. Furthermore, α6β4 was also found in the same punctuated pattern on FDCs as laminin-5. The function of the laminin-α6β4 complex in this particular localisation is still obscure, but a role in the maintainance of the follicular compartment via hemidesmosome-like attachment sites is postulated.     

Comparing the Recurrence of Vulvovaginal Candidiasis in Patients Undergoing Prophylactic Treatment with Probiotic and Placebo During the 6 Months

This is a randomized, double-blind, clinical/comparative trial study, involving the recurrence of vaginal candidiasis (VVC) after initial treatment with oral fluconazole in patients undergoing prophylactic management with a probiotic and placebo for 6 months. Fifty-nine VVC patients who were diagnosed based on their history, physical examination, and culture of vaginal discharge were initially treated by a single dose of 150 mg fluconazole. According to the table of random numbers, the sample was divided into two groups. The patients from one group took probiotics, while those from the other group took a placebo, with all of them being continuously monitored for 6 months. The patients complaining of vaginal candidiasis symptoms, such as burning, pruritus, and a vaginal (curd-like) discharge, were examined and the discharge was cultured for candida. The positive cultures were considered to be recurring for the patients in each group. Thirty-one cases from the placebo group and 28 cases from the probiotic group were carefully observed. In total, the 6-month recurrence in the control group was eleven (35.5 %) and in the research group was two (7.2 %). The results from Fisher’s exact test for the value p = 0.01 and OR 0.14 95 % CI (0.028–0.7) showed significant recurrence in the placebo group. The findings demonstrated that taking probiotics withazole antifungal drugs could be highly effective in treating VVC, resulting in a lower recurrence rate as well.