Carryover effects of maternal late-gestation heat stress on granddaughters' growth and mammary gland development.

Maternal (F0) exposure to late-gestation heat stress reduces their daughter's (F1) mammary gland fat pad (FP) mass, parenchyma (PAR) mass, and epithelial cell proliferation when evaluated at birth and weaning, and the daughters go on to produce less milk in their first lactation. Herein, we investigated the effect of maternal late-gestation heat stress on whole-body growth and mammary development of their granddaughters (F2). Multiparous F0 cows had access to heat abatement (n = 41, shade, and active cooling via fans and water soakers) or not (n = 41, shade only) for the last 56 d of gestation during a subtropical summer. Consequently, the F1 daughters, born to F0 cows, were heat-stressed (HTF1, n = 36) or cooled (CLF1, n = 37) in utero during the last 2 mo of gestation. All F1 heifers were raised as an identically managed cohort until first calving. The F2 granddaughters, born to HTF1 (HTF2, n = 12) or CLF1 (CLF2, n = 17), were raised as an identically managed cohort until 70 d of age. Dry matter intake, BW, hip height, wither height, chest girth, head circumference, mammary gland teat length, and left-right and front-rear teat distances were measured. Average daily gain was calculated for the preweaning period (0-49 d). Mammary ultrasounds were performed on d 21, 49, and 70 (n = 9/group) on the rear left and right quarters to quantify PAR and FP areas. Mammary biopsies were collected for histological evaluation of epithelial structures (hematoxylin and eosin staining), and to quantify cells positive for estrogen receptor, α subunit (ERα), cell proliferation (Ki67), and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling, TUNEL). Heifer growth from birth to d 49 was similar between CLF2 and HTF2 for all parameters evaluated. Distances between teats and teat length were not different between groups. On d 70, CLF2 heifers tended to have a greater average PAR (right and left quarters) relative to HTF2 heifers. Although the left FP was smaller in HTF2 heifers relative to CLF2 heifers, the average FP was not different. The lumenal and nonlumenal epithelial structures in the PAR of HTF2 heifers were significantly smaller than those of CLF2 heifers. In addition, HTF2 heifers had a reduced percentage of proliferating cells in the epithelial and stromal compartments and a greater percentage of apoptotic cells, particularly in the stroma. The percentage of ERα positive cells was significantly reduced in HTF2 heifers. In summary, although HTF2 heifers' DMI was similar and they grew at the same rate as CLF2 heifers throughout the preweaning phase, their mammary glands had smaller PAR areas with fewer epithelial structures characterized by reduced cell turnover and lower ERα expression. These early changes in the microstructure and cellular turnover of the mammary gland may partly explain the reduction in lactation performance relative to CLF2 counterparts at maturity.

Feed restriction of lactating cows triggers acute downregulation of mammary mammalian target of rapamycin signaling and chronic reduction of mammary epithelial mass.

While there is generally no consensus about how nutrients determine milk synthesis in the mammary gland, it is likely that the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) plays a role as a key integrator of nutritional and mitogenic signals that can influence a multitude of catabolic and anabolic pathways. The objectives of this study were to evaluate acute changes (<24 h) in translational signaling, in addition to chronic changes (14 d) in mammary gland structure and composition, in response to a severe feed restriction. Fourteen lactating Holstein dairy cows were assigned to either ad libitum feeding (n = 7) or a restricted feeding program (n = 7). Feed-restricted cows had feed removed after the evening milking on d 0. Mammary biopsies and blood samples were collected 16 h after feed removal, after which cows in the restricted group were fed 60% of their previously observed ad libitum intake for the remainder of the study. On d 14, animals were slaughtered and their mammary glands dissected. In response to feed removal, an acute increase in plasma nonesterified fatty acid concentration was observed, concurrent to a decrease in milk yield. In mammary tissue, we observed downregulation of the mTORC1-S6K1 signaling cascade, in addition to reductions in mRNA expression of markers of protein synthesis, endoplasmic reticulum biogenesis, and cell turnover (i.e., transcripts associated with apoptosis or cell proliferation). During the 14 d of restricted feeding, animals underwent homeorhetic adaptation to 40% lower nutrient intake, achieving a new setpoint of 14% reduced milk yield with 18% and 29% smaller mammary secretory tissue DM and CP masses, respectively. On d 14, no treatment differences were observed in markers of protein synthesis or mammary cell turnover evaluated using gene transcripts and immunohistochemical staining. These findings implicate mTORC1-S6K1 in the early phase of the adaptation of the mammary gland's capacity for milk synthesis in response to changes in nutrient supply. Additionally, changes in rates of mammary cell turnover may be transient in nature, returning to basal levels following brief alterations that have sustained effects.

Cell Proliferation and Apoptosis-Key Players in the Lung Aging Process.

Currently, the global lifespan has increased, resulting in a higher proportion of the population over 65 years. Changes that occur in the lung during aging increase the risk of developing acute and chronic lung diseases, such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung cancer. During normal tissue homeostasis, cell proliferation and apoptosis create a dynamic balance that constitutes the physiological cell turnover. In basal conditions, the lungs have a low rate of cell turnover compared to other organs. During aging, changes in the rate of cell turnover in the lung are observed. In this work, we review the literature that evaluates the role of molecules involved in cell proliferation and apoptosis in lung aging and in the development of age-related lung diseases. The list of molecules that regulate cell proliferation, apoptosis, or both processes in lung aging includes TNC, FOXM1, DNA-PKcs, MicroRNAs, BCL-W, BCL-XL, TCF21, p16, NOX4, NRF2, MDM4, RPIA, DHEA, and MMP28. However, despite the studies carried out to date, the complete signaling pathways that regulate cell turnover in lung aging are still unknown. More research is needed to understand the changes that lead to the development of age-related lung diseases.

The bHLH transcription factor ASCL1 promotes differentiation of endocrine cells in the stomach and is regulated by Notch signaling.

Notch signaling regulates gastrointestinal stem cell proliferation and differentiation yet Notch-regulated transcriptional effectors of gastric epithelial cell differentiation are poorly understood. Here we tested the role of the bHLH transcription factor Achaete-Scute homolog 1 (ASCL1) in gastric epithelial cell differentiation, and its regulation by Notch. Newborn Ascl1 null mice showed a loss of expression of markers of neurogenin-3-dependent enteroendocrine cells, with normal expression of enterochromaffin-like cells, mucous cells, chief cells, and parietal cells. In adult mice, Ascl1 gene expression was observed in the stomach, but not the intestine, with higher expression in antral than corpus epithelium. Lineage tracing in Ascl1-CreERT2; Rosa26-LSL-tdTomato mice revealed single, scattered ASCL1+ cells in the gastric epithelium, demonstrating expression in antral gastrin- and serotonin-producing endocrine cells. ASCL1-expressing endocrine cells persisted for several weeks posttamoxifen labeling with a half-life of approximately 2 months. Lineage tracing in Gastrin-CreERT2 mice demonstrated a similar lifespan for gastrin-producing cells, confirming that gastric endocrine cells are long-lived. Finally, treatment of Ascl1-CreERT2; Rosa26-LSL-tdTomato mice with the pan-Notch inhibitor dibenzazepine increased the number of lineage-labeled cells in the gastric antrum, suggesting that Notch signaling normally inhibits Ascl1 expression. Notch regulation of Ascl1 was also demonstrated in a genetic mouse model of Notch activation, as well as Notch-manipulated antral organoid cultures, thus suggesting that ASCL1 is a key downstream Notch pathway effector promoting endocrine cell differentiation in the gastric epithelium.NEW & NOTEWORTHY Although Notch signaling is known to regulate cellular differentiation in the stomach, downstream effectors are poorly described. Here we demonstrate that the bHLH transcription factor ASCL1 is expressed in endocrine cells in the stomach and is required for formation of neurogenin-3-dependent enteroendocrine cells but not enterochromaffin-like cells. We also demonstrate that Ascl1 expression is inhibited by Notch signaling, suggesting that ASCL1 is a Notch-regulated transcriptional effector directing enteroendocrine cell fate in the mouse stomach.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows.

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Placental expression of estrogen-related receptor gamma is reduced in fetal growth restriction pregnancies and is mediated by hypoxia†.

Fetal growth restriction (FGR) describes a fetus which has not achieved its genetic growth potential; it is closely linked to placental dysfunction and uteroplacental hypoxia. Estrogen-related receptor gamma (ESRRG) is regulated by hypoxia and is highly expressed in the placenta. We hypothesized ESRRG is a regulator of hypoxia-mediated placental dysfunction in FGR pregnancies. Placentas were collected from women delivering appropriate for gestational age (AGA; n = 14) or FGR (n = 14) infants. Placental explants (n = 15) from uncomplicated pregnancies were cultured for up to 4 days in 21% or 1% O2, or with 200 µM cobalt chloride (CoCl2), or treated with the ESRRG agonists DY131 under different oxygen concentrations. RT-PCR, Western blotting, and immunochemistry were used to assess mRNA and protein levels of ESRRG and its localization in placental tissue from FGR or AGA pregnancies, and in cultured placental explants. ESRRG mRNA and protein expression were significantly reduced in FGR placentas, as was mRNA expression of the downstream targets of ESRRG, hydroxysteroid 11-beta dehydrogenase 2 (HSD11B2), and cytochrome P-450 (CYP19A1.1). Hypoxia-inducible factor 1-alpha protein localized to the nuclei of the cytotrophoblasts and stromal cells in the explants exposed to CoCl2 or 1% O2. Both hypoxia and CoCl2 treatment decreased ESRRG and its downstream genes' mRNA expression, but not ESRRG protein expression. DY131 increased the expression of ESRRG signaling pathways and prevented abnormal cell turnover induced by hypoxia. These data show that placental ESRRG is hypoxia-sensitive and altered ESRRG-mediated signaling may contribute to hypoxia-induced placental dysfunction in FGR. Furthermore, DY131 could be used as a novel therapeutic approach for the treatment of placental dysfunction.

Tissue homeostasis in sponges: Quantitative analysis of cell proliferation and apoptosis.

Tissues of multicellular animals are maintained due to a tight balance between cell proliferation and programmed cell death. Sponges are early branching metazoans essential to understanding the key mechanisms of tissue homeostasis. This article is dedicated to the comparative analysis of proliferation and apoptosis in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia variabilis (class Calcarea). Labeled nucleotides EdU and anti-phosphorylated histone 3 antibodies reveal a considerable number of cycling cells in intact tissues of both species. Quantitative DNA staining reveals the classic cell cycle distribution curve. The main type of cycling cells are choanocytes - flagellated cells of the aquiferous system. The rate of proliferation remains constant throughout various areas of sponge bodies that contain choanocytes. The EdU tracking experiments conducted in H. dujardinii indicate that choanocytes may give rise to mesohyl cells through migration. The number of apoptotic cells in tissues of both species is insignificant, although being comparable to the renewing tissues of other animals. In vivo studies with tetramethylrhodamine ethyl ester and CellEvent Caspase-3/7 indicate that apoptosis might be independent of mitochondrial outer membrane permeabilization. Altogether, a combination of confocal laser scanning microscopy and flow cytometry provides a quantitative description of cell proliferation and apoptosis in sponges displaying either rapid growth or cell turnover.

Symposium review: Determinants of milk production: Understanding population dynamics in the bovine mammary epithelium.

The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary epithelial cells largely accounts for the decline in milk production with advancing lactation. The net decline in cell number (approx. 50%) is due to cell death but is simultaneously accompanied by cell renewal. Although the rate of cell proliferation is slow, by the end of lactation most cells in the gland were formed after calving. Typically milking is terminated when cows are in the final 2 mo of pregnancy. This causes regenerative involution, wherein extensive cell replacement and mammary growth occurs. We hypothesized that replacement of senescent secretory cells and progenitor cells during the dry period increases milk yield in the next lactation. Analysis of global gene expression revealed networks and canonical pathways during regenerative involution that support cell turnover and mammary growth, and reflect oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Immune responses consistent with influx of neutrophils, macrophages, and lymphocytes, and processes that support mammary differentiation and lactogenesis were also evident. Data also suggest that replication of stem and progenitor cells occurs during the dry period. Relying on long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine mammary stem cells. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%), predominantly estrogen receptor-negative, and localized in a basal or suprabasal layer of the epithelium. Analyses of gene expression in laser-microdissected LREC are consistent with the concept that LREC represent stem cells and progenitor cells, which differ in properties and location within the epithelial layer. We identified potential markers for these cells and have increased their number by infusing xanthosine through the teat canal of prepubertal heifers. Altering population dynamics of mammary stem and progenitor cells during the mammary cycle may be a means to increase efficiency of milk production.

Bee pollens originating from different species have unique effects on ovarian cell functions.

CONTEXT: The species-specific differences and mechanisms of action of bee pollen on reproduction have not been well studied. OBJECTIVE: We compared the effects of bee pollen extracts from different plants on ovarian cell functions. MATERIALS AND METHODS: We compared the effects of pollens from black alder, dandelion, maize, rapeseed, and willow at 0, 0.01, 0.1, 1, 10, or 100 µg/mL on cultured porcine ovarian granulosa cells. Cell viability was assessed with a Trypan blue test, the cell proliferation marker (PCNA), and an apoptosis marker (BAX) were assessed by immunocytochemistry. Insulin-like growth factor (IGF-I) release was measured by an enzyme-linked immunosorbent assay. RESULTS: Addition of any bee pollen reduced cell viability, promoted accumulation of both proliferation and apoptosis markers, and promoted IGF-I release. The ability of various pollens to suppress cell viability ranked as follows: rapeseed > dandelion > alder > maize > willow. The biological activity of bee pollens regarding their stimulatory action on ovarian cell proliferation ranked as follows: dandelion > willow > maize > alder > rapeseed. Cell apoptosis was promoted by pollens as follows: range > dandelion > alder > rapeseed > willow > maize. The ability of the pollens to stimulate IGF-I output are as follows: willow > dandelion > rapeseed > maize > alder. DISCUSSION: Bee pollen can promote ovarian cell proliferation by promoting IGF-I release, but it induces the dominance of apoptosis over proliferation and the reduction in ovarian cell viability in a species-specific manner. CONCLUSIONS: This is the first demonstration of adverse effects of bee pollen on ovarian cell viability and of its direct stimulatory influence on proliferation, apoptosis, and IGF-I release. The biological potency of bee pollen is dependent on the plant species.

DjERas plays an important role in planarian regeneration and homeostasis.

The mechanisms of cell turnover including cell proliferation and cell differentiation were complex. Planarians possess amazing regeneration ability and undergo cell turnover throughout life. We identified a homologous gene of ERas by RNAi in Dugesia japonica. Knocking-down DjERas resulted in regeneration and homeostasis defects. Furthermore, we found that the expression of neoblasts and late progeny marker gene decreased in DjERas RNAi planarians. Our studies indicated that down-regulation of DjERas inhibited the proliferation and differentiation of stem cells through the conserved signaling pathway, resulted in the inability of the planarian to regenerate and maintain homeostasis. Our results suggest that DjERas plays a crucial role in the process of cell turnover.

Ocular surface and chronic pesticide exposure: Evaluating the alterations in corneal cellular turnover concerning cell cycle and apoptosis.

The consequences of chronic pesticide exposure on the ocular surface are not yet fully known and lacunae exist regarding the repercussions of this xenobiotic insult on cellular turnover. The present work aims to establish the mechanistic relationship between ocular morbidity and chronic pesticide exposure by analyzing the impact on key regulators responsible for cell cycle and death. Vital components of cell cycle and death were primarily explored in this study by mimicking the on-field scenario regarding chronic pesticide exposure in a murine model. Various cellular aspects were taken into consideration through culture analyses, flowcytometric evaluation, fluorescence microscopic studies etc. We observed downregulation of key players of the cell-cycle at different stages (viz. Cyclin-D1, CDK4, pRb, PCNA, PP1, PP2A, p-cdc 25c and Aurora kinase A) with a corresponding increase in the expression of cell-cycle inhibitors like p18 and p21, which lead to hypoproliferation of corneal epithelial cells post pesticide exposure. The expression of GSK 3ß, a master-molecule involved in both cell cycle and apoptotic pathways corresponded well with the scientific theme and indicated towards cellular hypoproliferation and increase of apoptosis. Key players of both the intrinsic (viz. Bax/Bcl2, JNK) and extrinsic (viz. CD 95) apoptotic pathways were found to be activated leading to enhanced cleaved Caspase 3 expression and corresponding cell death. We tried to highlight the mechanistic correlation between the alterations in cellular turnover as the reason behind the heightened ocular morbidity due to 'chronic pesticide exposure'- the xenobiotic stress exerted by these 'farmers' friends'.

Glutaminolysis and lipoproteins are key factors in late immune recovery in successfully treated HIV-infected patients.

The immunological, biochemical and molecular mechanisms associated with poor immune recovery are far from known, and metabolomic profiling offers additional value to traditional soluble markers. Here, we present novel and relevant data that could contribute to better understanding of the molecular mechanisms preceding a discordant response and HIV progression under suppressive combined antiretroviral therapy (cART). Integrated data from nuclear magnetic resonance (NMR)-based lipoprotein profiles, mass spectrometry (MS)-based metabolomics and soluble plasma biomarkers help to build prognostic and immunological progression tools that enable the differentiation of HIV-infected subjects based on their immune recovery status after 96 weeks of suppressive cART. The metabolomic signature of ART-naïve HIV subjects with a subsequent late immune recovery is the expression of pro-inflammatory molecules and glutaminolysis, which is likely related to elevate T-cell turnover in these patients. The knowledge about how these metabolic pathways are interconnected and regulated provides new targets for future therapeutic interventions not only in HIV infection but also in other metabolic disorders such as human cancers where glutaminolysis is the alternative pathway for energy production in tumor cells to meet their requirement of rapid proliferation.

BCL-2 levels do not predict azathioprine treatment response in inflammatory bowel disease, but inhibition induces lymphocyte apoptosis and ameliorates colitis in mice.

In inflammatory bowel disease (IBD), inflammation is sustained by an exaggerated response of lymphocytes. This results from enhanced expression of anti-apoptotic B cell lymphoma (BCL-2) and BCL-XL associated with a diminished turnover. Azathioprine (AZA) directly targets BCL-2 family-mediated apoptosis. We investigated whether the BCL-2 family expression pattern could be used to predict treatment response to AZA and determined whether BCL-2 inhibitor A-1211212 effectively diminishes lymphocytes and ameliorates inflammation in a model of colitis. BCL-2 family expression pattern was determined by next-generation sequencing (NGS). BCL-2 inhibitor was administered orally to Il10-/- mice. Haematological analyses were performed with an ADVIA 2120 and changes in immune cells were investigated using quantitative polymerase chain reaction (qPCR) and fluorescence activated cell sorter (FACS). We determined similar expression levels of BCL-2 family members in patients with remission and patients refractory to treatment, showing that BCL-2 family expression can not predict AZA treatment response. Expression was not correlated with the modified Truelove and Witts activity index (MTWAI). BCL-2 inhibitor initiated cell death in T cells from patients refractory to AZA and reduced lymphocyte count in Il10-/- mice. FACS revealed diminished CD8+ T cells upon BCL-2 inhibitor in Il10-/- mice without influencing platelets. Tnf, Il1ß, IfnƔ and Mcp-1 were decreased upon BCL-2 inhibitor. A-1211212 positively altered the colonic mucosa and ameliorated inflammation in mice. Pro-apoptotic BCL-2 inhibitor A-1211212 diminishes lymphocytes and ameliorates colitis in Il10-/- mice without inducing thrombocytopenia. BCL-2 inhibition could be a new therapy option for patients refractory to AZA.

The role of intermediate filaments in maintaining integrity and function of intestinal epithelial cells after massive bowel resection in a rat.

PURPOSE: Intermediate filaments (IFs) are a part of the cytoskeleton that extend throughout the cytoplasm of all cells and function in the maintenance of cell-shape by bearing tension and serving as structural components of the nuclear lamina. In normal intestine, IFs provide a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. The purpose of this study was to evaluate the role of IFs during intestinal adaptation in a rat model of short bowel syndrome (SBS). MATERIALS AND METHODS: Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75% bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's Digital Gene Expression (DGE) analysis was used to determine the cytoskeleton-related gene expression profiling. IF-related genes and protein expression were determined using real-time PCR, Western blotting and immunohistochemistry. RESULTS: Massive small bowel resection resulted in a significant increase in enterocyte proliferation and concomitant increase in cell apoptosis. From the total number of 20,000 probes, 16 cytoskeleton-related genes were investigated. Between these genes, only myosin and tubulin levels were upregulated in SBS compared to sham animals. Between IF-related genes, desmin, vimentin and lamin levels were down-regulated and keratin and neurofilament remain unchanged. The levels of TGF-ß, vimentin and desmin gene and protein were down-regulated in resected rats (vs sham animals). CONCLUSIONS: Two weeks following massive bowel resection in rats, the accelerated cell turnover was accompanied by a stimulated microfilaments and microtubules, and by inhibited intermediate filaments. Resistance to cell compression rather that maintenance of cell-shape by bearing tension are responsible for contraction, motility and postmitotic cell separation in a late stage of intestinal adaptation.

A Lower Baseline CD4/CD8 T-Cell Ratio Is Independently Associated with Immunodiscordant Response to Antiretroviral Therapy in HIV-Infected Subjects.

We explored if baseline CD4/CD8 T-cell ratio is associated with immunodiscordant response to antiretroviral therapy in HIV-infected subjects. Comparing immunodiscordant and immunoconcordant subjects matched by pretreatment CD4 counts, we observed a lower pretreatment CD4/CD8 T-cell ratio in immunodiscordant subjects. Furthermore, pretreatment CD4/CD8 T-cell ratio, but not CD4 counts, correlated with the main immunological alterations observed in immunodiscordants, including increased regulatory T-cell (Treg) frequency and T-cell turnover-related markers. Then, in a larger cohort, only baseline CD4/CD8 T-cell ratio was independently associated with immunodiscordance, after adjusting by the viral CXCR4-tropic HIV variants. Our results suggest that the CD4/CD8 T-cell ratio could be an accurate biomarker of the subjacent immunological damage triggering immunodiscordance.

Expression of stem cell factors in the adult sea cucumber digestive tube.

Homeostatic cell turnover has been extensively characterized in mammals. In their adult tissues, lost or aging differentiated cells are replenished by a self-renewing cohort of stem cells. The stem cells have been particularly well studied in the intestine and are clearly identified by the expression of marker genes including Lgr5 and Bmi1. It is, however, unknown if the established principles of tissue renewal learned from mammals would be operating in non-mammalian systems. Here, we study homeostatic cell turnover in the sea cucumber digestive tube, the organ with high tissue plasticity even in adult animals. Both the luminal epithelium and mesothelium express orthologs of mammalian Lgr5 and Bmi1. However, unlike in mammals, there is no segregation of these positively labeled cells to specific regions in the luminal epithelium, where most of the cell proliferation would take place. In the mesothelium, the cells expressing the stem cell markers are tentatively identified as peritoneocytes. There are significant differences among the five anatomical gut regions in cell renewal dynamics and stem factor expression. The cloaca differs from the rest of the digestive tube as the region with the highest expression of the Lgr5 ortholog, lowest level of Bmi1 and the longest retention of BrdU-labeled cells.

Analysis of Cell Turnover in the Bronchiolar Epithelium Through the Normal Aging Process.

PURPOSE: Aging is associated with changes in the lung that leads to a decrease in its function. Alterations in structure and function in the small airways are well recognized in chronic lung diseases. The aim of this study was the assessment of cell turnover in the bronchiolar epithelium of mouse through the normal aging process. METHODS: Lungs from CD1 mice at the age of 2, 6, 12, 18, or 24 months were fixed in neutral-buffered formalin and paraffin-embedded. Proliferating cell nuclear antigen was examined by immunohistochemistry. Apoptosis was analyzed by in situ end-labeling of fragmented DNA. Epithelial dimensions were analyzed by morphometry. RESULTS: The 2-month-old mice showed significantly higher number of proliferating cells when compared with mice at all other age groups. The number of apoptotic cells in mice at 24 months of age was significantly greater than in mice at all other age groups. Thus, the number of epithelial cells decreased as the age of the subject increased. We also found reductions in both area and height of the bronchiolar epithelium in mice at 18 and 24 months of age. CONCLUSIONS: We found a decrease in the total number of epithelial cells in the aged mice, which was accompanied by a thinning of the epithelium. These changes reflect a dysregulated tissue regeneration process in the bronchiolar epithelium that might predispose to respiratory diseases in elderly subjects.

Increased lymphocyte apoptosis in mouse models of colitis upon ABT-737 treatment is dependent upon BIM expression.

Exaggerated activation of lymphocytes contributes to the pathogenesis of inflammatory bowel disease (IBD). Medical therapies are linked to the BCL-2 family-mediated apoptosis. Imbalance in BCL-2 family proteins may cause failure in therapeutic responses. We investigated the role of BCL-2 inhibitor ABT-737 for lymphocyte apoptosis in mice under inflammatory conditions. B.6129P2-interleukin (IL)-10(tm1Cgn) /J (IL-10(-/-) ) weighing 25-30 g with ongoing colitis were used. Fifty mg/kg/day ABT-737 was injected intraperitoneally (i.p.). Haematological analyses were performed with an ADVIA 2120 flow cytometer and mass cytometry with a CyTOF 2. Following i.p. administration, ABT-737 was detected in both spontaneous and acute colitis in peripheral blood (PBL) and colon tissue. Treatment led to lymphopenia. CD4(+) CD44(+) CD62L(+) central memory and CD8(+) , CD44(+) CD62L(-) central memory T cells were decreased in PBL upon ABT-737 compared to vehicle-receiving controls. Increased apoptosis upon ABT-737 was determined in blood lymphocytes, splenocytes and Peyer's patches and was accompanied by a decrease in TNF and IL-1B. ABT-737 positively altered the colonic mucosa and ameliorated inflammation, as shown by colonoscopy, histology and colon length. A decreased BIM/BCL-2 ratio or absence of BIM in both Bim(-) (/) (-) and Il10(-) (/) (-) × Bim(-) (/) (-) impeded the protective effect of ABT-737. The BIM/BCL-2 ratio decreased with age and during the course of treatment. Thus, long-term treatment resulted in adapted TNF levels and macroscopic mucosal damage. ABT-737 was efficacious in diminishing lymphocytes and ameliorating colitis in a BIM-dependent manner. Regulation of inappropriate survival of lymphocytes by ABT-737 may provide a therapeutic strategy in IBD.

Increased T-cell turnover is associated with spondyloarthritis in virally suppressed patients with HIV-1 infection.

OBJECTIVES: Spondyloarthritis (SpA) is one of the most frequently observed inflammatory joint diseases in HIV-1-seropositive patients. T-cells were described frequently as one of the major driving forces in SpA, therefore we tried to look for T-cell aberrancies in our HIV-positive patients with SpA. METHODS: A total of 1098 files for HIV-positive patients who attended the HIV out-patient clinic of the Department of Clinical Immunology and Rheumatology at the Medical University Hanover for at least one visit between January 2004 and December 2010 were screened for the presence of a diagnosis of SpA. A cross-sectional study was conducted to investigate aberrancies in T-cell homeostasis induced by HIV-1 in these subjects. RESULTS: The prevalence of SpA in the HIV-positive patients was 1.6% (18 of 1098). Interestingly, the percentage of patients with SpA who were human leucocyte antigen (HLA)-B27 negative in our HIV-positive cohort was 80%. Despite combination antiretroviral therapy (cART) and viral suppression, an incomplete immune recovery of T-cell naïve/memory distribution and turnover, as identified by intracellular Ki-67 expression, was observed in HIV-positive patients with SpA. CONCLUSIONS: Independent of HLA-B27 status and despite cART, HIV-positive patients can develop SpA and exhibit an increased T-cell turnover rate.

Increased intestinal epithelial cell turnover and intestinal motility in Gymnophalloides seoi-infected C57BL/6 mice.

The changing patterns of goblet cell hyperplasia, intestinal epithelial cell turnover, and intestinal motility were studied in ICR and C57BL/6 mice infected with Gymnophalloides seoi (Digenea: Gymnophallidae). Whereas ICR mice retained G. seoi worms until day 7 post-infection (PI), C57BL/6 mice showed a rapid worm expulsion within day 3 PI. Immunosuppression with Depo-Medrol significantly delayed the worm expulsion in C57BL/6 mice. Goblet cell counts were increased in both strains of mice, peaking at day 1 PI in C57BL/6 mice and slowly increasing until day 7 PI in ICR mice. In C57BL/6 mice infected with G. seoi, newly proliferating intestinal epithelial cells were remarkably increased in the crypt, and the increase was the highest at day 1 PI. However, in ICR mice, newly proliferating intestinal epithelial cells increased slowly from day 1 to day 7 PI. Intestinal motility was increased in G. seoi-infected mice, and its chronological pattern was highly correlated with the worm load in both strains of mice. Meanwhile, immunosuppression of C57BL/6 mice abrogated the goblet cell proliferation, reduced the epithelial cell proliferation, and suppressed the intestinal motility. Goblet cell hyperplasia, increased intestinal epithelial cell turnover, and increased intestinal motility should be important mucosal defense mechanisms in G. seoi-infected C57BL/6 mice.