Control Glands Research Articles

Postnatal administration of 2-oxoglutaric acid improves the intestinal barrier affected by the prenatal action of dexamethasone in pigs

ObjectiveThe potential effects of the prenatal administration of dexamethasone and the postnatal treatment with 2-oxoglutaric acid on postnatal development of the small intestine of farm animals have not been examined experimentally. The aim of this study was to establish the changes in morphologic parameters of the small intestine damaged by the prenatal action of dexamethasone in piglets supplemented with 2-oxoglutaric acid. MethodsThree milligrams dexamethasone was administered intramuscularly every second day from day 70 of pregnancy to parturition and then piglets were supplemented with 2-oxoglutaric acid for 35 d of postnatal life (0.4 g/kg of body weight). The histomorphometry of the pig duodenum and jejunum was determined. Immunohistochemical staining with anti–Ki-67, CD3, null T cells, cadherin, claudin, and neurofilament antibodies was performed. ResultsMaternal treatment with dexamethasone decreased and limited the expression of claudin and cadherin in the epithelium. Dexamethasone led to thinning of the myenteron of the duodenum and the middle part of the jejunum in weaned piglets and influenced duodenal glands that became more elongated compared with control glands. Moreover, 2-oxoglutaric acid increased cell proliferation and the amount and maturity of peripheral blood lymphocytes in the duodenum and jejunum. It supported epithelial integrity and changed the circularity of the nerve plexuses. ConclusionThe 2-oxoglutaric acid administered to piglets while suckling induced a complete recovery from intestinal damage caused by the prenatal action of dexamethasone.

Relationship between implementation of the American Society of Clinical Oncology/College of American Pathologists guideline recommendations for breast core-needle biopsies and delay in therapy for patients with breast cancer.

26 Background: Recently the ASCO/CAP suggested new guideline recommendations for immunohistochemical (IHC) testing of biomarkers in breast cancer. An algorithm that relies on accurate, reproducible assay performance was proposed and preanalytical, analytical and postanalytical standards suggested. One such recommendation is to have appropriate internal controls when assessing hormone expression. In this retrospective study we evaluated 242 breast core biopsy specimens to assess the impact of the recently proposed ASCO/CAP guideline recommendations and its possible impact on routine daily practice. In addition, we explore the impact of blindly selecting a random core for potential future studies. Methods: A total 988 cores from 242 specimens corresponding to 224 patients diagnosed between 2008 and 2009 were examined. Each core was examined for the presence of normal glands, percentage of tumor involvement, biomarker status, and diagnostic adequacy. Results: Absence of normal glands was found on 61/242 specimens (25%). Of these, expression of ER, PR and HER2 was seen in 44 (72%), 28 (46%), and 21 specimens (35%) respectively. Negative staining for ER, PR and HER2 was observed in 7 specimens (12%). A total of 148 cores (15%) showed absence of tumor and 275 cores (28%) had <25% of tumor in them. 18 cores (2%) were insufficient for diagnosis due to either scant tumor present or crush artifact present. Conclusions: Although the presence of internal control glands is optimal, its presence may be unnecessary if proper IHC batch run controls are utilized and histologic/IHC concordance is performed. Tumor histology and IHC profile concordance is imperative specially in triple negative cases. The impact on patient health care of an “receptor uninterpretable” biopsy is yet to be determined, but likely would include assessment of the delay in care due to a repeat biopsy with procurement of additional tissue, additional heath care costs, and anxiety to the patient. Adequacy check using frozen section could be done to assess for the presence of tumor during the procedure.

Effect of parasympathectomy on the salivary secretion of submandibular gland in rats

To investigate the effect of parasympathectomy on secretion of submandibular glands and the feasibility of treatment for xerostomia in rats. Twenty Sprague-Dawley male rats weighing 200 - 300 g were randomly divided into the experimental group (n = 12), in which the right chorda-lingual nerve was cut, and the control group (n = 8). The secretion of submandibular gland was measured for 5 min by Schirmer test for both groups. The stimulated saliva flow rate decreased on 1st, 12th and 24th week after denervation in the right operated submandibular glands (P < 0.05). No difference in secretion was found between the left non-operated glands and the control group. Comparing with the left non-operated gland and the control gland, the saliva flow rate at rest in the right operated submandibular gland increased on the 1st, 12th and 24th week (P < 0.05). After parasympathectomy of rat submandibular glands, the saliva flow rate at rest increased in the denervated gland, which suggests that parasympathectomy of submandibular gland might be used as a therapy for xerostomia.

Neuropilin-2 promotes branching morphogenesis in the mouse mammary gland

Although the neuropilins were characterized as semaphorin receptors that regulate axon guidance, they also function as vascular endothelial growth factor (VEGF) receptors and contribute to the development of other tissues. Here, we assessed the role of NRP2 in mouse mammary gland development based on our observation that NRP2 is expressed preferentially in the terminal end buds of developing glands. A floxed NRP2 mouse was bred with an MMTV-Cre strain to generate a mammary gland-specific knockout of NRP2. MMTV-Cre;NRP2(loxP/loxP) mice exhibited significant defects in branching morphogenesis and ductal outgrowth compared with either littermate MMTV-Cre;NRP2(+/loxP) or MMTV-Cre mice. Mechanistic insight into this morphological defect was obtained from a mouse mammary cell line in which we observed that VEGF(165), an NRP2 ligand, induces branching morphogenesis in 3D cultures and that branching is dependent upon NRP2 as shown using shRNAs and a function-blocking antibody. Epithelial cells in the mouse mammary gland express VEGF, supporting the hypothesis that this NRP2 ligand contributes to mammary gland morphogenesis. Importantly, we demonstrate that VEGF and NRP2 activate focal adhesion kinase (FAK) and promote FAK-dependent branching morphogenesis in vitro. The significance of this mechanism is substantiated by our finding that FAK activation is diminished significantly in developing MMTV-Cre;NRP2(loxP/loxP) mammary glands compared with control glands. Together, our data reveal a VEGF/NRP2/FAK signaling axis that is important for branching morphogenesis and mammary gland development. In a broader context, our data support an emerging hypothesis that directional outgrowth and branching morphogenesis in a variety of tissues are influenced by signals that were identified initially for their role in axon guidance.

Proteomic Analysis of Human Transplanted Submandibular Gland in Patients with Epiphora after Transplantation

Submandibular gland autotransplantation is effective for treating severe dry eye syndrome. However, more than 40% of patients show epiphora within 3-6 months after treatment. The mechanism underlying the hypersecretion in epiphora remains to be elucidated for developing novel interventions. Since salivary gland secretion is dependent on a variety of proteins, we analyzed the changes in protein expression in transplanted glands of epiphora patients with 2-D gel electrophoresis and electrospray ionization quadrupole/time-of-flight mass spectrometry and evaluated their possible roles in epiphora. There were 23 proteins that showed altered expression in the glands of epiphora patients, 15 being up-expressed and 8 being down-expressed. The expression of secretory proteins was decreased in these glands, including alpha-amylase, cystatin S, SA, and SN. In contrast, cytoskeletal proteins were all up-regulated, including actin and vimentin. Immunofluorescence revealed that the intensity ratio of F-actin in apical and lateral cytoplasm to total F-actin in acini was decreased in the glands of epiphora patients. Carbachol stimulation induced a similar redistribution of F-actin in the control glands. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was increased in both carbachol-stimulated and epiphora glands. Preincubation of submandibular glands with ERK1/2 inhibitors PD98059 or U0126 inhibited carbachol-induced F-actin redistribution. These results indicated that differentially expressed proteins participated in the hypersecretion of transplanted submandibular glands and the redistribution of F-actin might be involved in this hypersecretion in an ERK1/2-dependent manner.

Analysis of the cumulative changes in Graves’ disease thyroid glands points to IFN signature, plasmacytoid DCs and alternatively activated macrophages as chronicity determining factors

Graves’ disease (GD) is a chronic autoimmune process in the thyroid gland and involves IFN and IFN driven immune activation. Assuming the thyroid gland is the main site stimulating the autoimmune response, we investigated the role of IFNs and other factors in the chronic evolution of GD by comparing the transcriptomic profiles of thyroid glands from short clinical course (SC), long clinical course (LC) cases, and control glands (C). Over 200 differentially expressed genes of the immune system were identified. Results were extensively analyzed bioinformatically and validated by qPCR in 31 glands. The analysis indicated that GD involved a progressive accumulation of changes with clearly distinct profiles in the SC and LC glands. Humoral response, antigen presentation and chemokines & cytokines were overall the most represented gene ontology categories in LC cases. Ingenuity Pathway Analysis pointed to a few inflammatory pathways in SC cases whereas LC cases involved numerous complex pathways, such us “communication between innate and adaptive immune cells” and “autoimmune thyroid signaling”. A broad IFN signature consisted of the over-expression of 74 and 84 type I and type II IFN responsive genes respectively (overall 96 out of 211, 45%), but many of these genes can also be directly activated through cytoplasmic viral receptors. For the first time, plasmocytoid dendritic cells were identified in GD thyroid, but surprisingly, the main producers of IFN-alpha were cells with a myeloid cell phenotype. In addition, cells with the phenotype of alternatively activated macrophages were detected in abundance in GD thyroids, confirming data from the transcriptomic analysis. Collectively, these results confirmed the role of IFNs, suggested other natural immunity triggers, identified new cell types in the local disease process, and expanded our knowledge of the processes that may determine the chronicity of GD.

Phosphorylation Regulates c-Myc's Oncogenic Activity in the Mammary Gland

Expression of the c-Myc oncoprotein is affected by conserved threonine 58 (T58) and serine 62 (S62) phosphorylation sites that help to regulate c-Myc protein stability, and altered ratios of T58 and S62 phosphorylation have been observed in human cancer. Here, we report the development of 3 unique c-myc knock-in mice that conditionally express either c-Myc(WT) or the c-Myc(T58A) or c-Myc(S62A) phosphorylation mutant from the constitutively active ROSA26 locus in response to Cre recombinase to study the role of these phosphorylation sites in vivo. Using a mammary-specific Cre model, we found that expression of c-Myc(WT) resulted in increased mammary gland density, but normal morphology and no tumors at the level expressed from the ROSA promoter. In contrast, c-Myc(T58A) expression yielded enhanced mammary gland density, hyperplastic foci, cellular dysplasia, and mammary carcinoma, associated with increased genomic instability and suppressed apoptosis relative to c-Myc(WT). Alternatively, c-Myc(S62A) expression reduced mammary gland density relative to control glands, and this was associated with increased genomic instability and normal apoptotic function. Our results indicate that specific activities of c-Myc are differentially affected by T58 and S62 phosphorylation. This model provides a robust platform to interrogate the role that these phosphorylation sites play in c-Myc function during development and tumorigenesis.

Adenoviral delivery of Tousled kinase for the protection of salivary glands against ionizing radiation damage

Oral complications of salivary hypofunction often afflict cancer patients undergoing radiotherapy for head and neck cancers. Dry mouth or xerostomia is an undesirable consequence of radiotherapy that compromises normal oral functions in addition to causing odynophagia and increasing the patient's risk of oral infections and dental caries. Radiation-induced xerostomia is irreversible, and palliative measures to provide symptomatic relief remain the mainstay of treatment. Previously, we identified a splice variant of a cellular kinase, Tousled-like kinase 1B (TLK1B), which when overexpressed protects normal epithelial cells against ionizing radiation (IR)-induced cell death. To address the need to protect salivary glands in patients undergoing regional radiotherapy, we investigated whether preemptive expression of TLK1B in salivary glands protects against IR. In stably-derived salivary cell lines in vitro, TLK1B expression increased cell survival after IR. Cells expressing exogenous TLK1B were less radiosensitive (A5-TLK1B, α/β=0.67 Gy; ParC5-TLK1B, α/β=4.3 Gy) compared to control cells (A5-BK, α/β=1.7 Gy; ParC5-BK, α/β=32.7 Gy). Using a recombinant adenovirus serotype 5 viral vector for TLK1B gene transfer into rat submandibular salivary glands in vivo, we demonstrated that TLK1B protects the saliva-secreting acinar cells and better preserves salivary gland function against IR relative to control glands. After a single fraction of 16 Gy, the decline in salivary function at 8 weeks was less pronounced in TLK1B-treated animals (40%) as compared to saline-treated controls (67%). Histopathological analysis demonstrated increase in acinar atrophy, decrease in acinar cell number, and increase in inflammatory infiltrate and fibrosis in irradiated control tissues relative to TLK1B-treated glands. These results show the radioprotective benefits of TLK1B and implicate its usefulness in the management of regional radiotherapy-induced xerostomia.

Reduced statherin reactivity of human submandibular gland in diabetes

Statherin is a salivary protein involved in the formation of enamel pellicle and in regulation of calcium homeostasis. Diabetes and other pathologies affect both salivary flow and protein secretion by salivary glands, causing increased susceptibility to mucosal infections, tooth demineralization, and caries. The purpose of this study was to compare the statherin expression in submandibular glands of healthy and diabetic subjects. Fragments of submandibular glands obtained from diabetic and non diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The results were statistically evaluated. Specific statherin labeling was demonstrated in secretory granules of acinar cells in both diabetic and normal samples. The staining was much more intense in the latter compared to those of diabetics. The labeling density was quantified by evaluating the number and spatial distribution of gold particles within the granules. The number of gold particles was significantly lower in glands from diabetics than in control glands. The results obtained suggest that a reduced statherin secretion by salivary glands might be partly responsible for a less effective protection of the oral tissues, resulting in an higher incidence of caries and oral infections associated with diabetes.

Lanthanum activates calcium-sensing receptor and enhances sensitivity to calcium

The aim of this study was to investigate whether nanomolar concentrations of lanthanum could influence the calcium-sensing receptor (CaSR) response. Embryonic kidney (HEK-293) cells transiently transfected with the human CaSR were used to test the ability of lanthanum to activate the CaSR, either alone or in combination with calcium. CaSR activation was measured by flow cytometry. Parathyroid glands from 4-month-old male Wistar rats with normal renal function (n = 60) were also cultured ex vivo with different concentrations of lanthanum to measure parathyroid hormone (PTH) secreted to the medium and PTH mRNA. The maximal CaSR activation induced by 1 muM lanthanum chloride (LaCl(3)) was similar to that induced by 16 mM calcium chloride (CaCl(2) 16 mM: 294 +/- 14%; LaCl(3) 1 muM: 303 +/- 11%). Lanthanum half effective concentration (EC(50)) was 77.28 nM, lower than the 2.30 mM obtained for calcium, supporting the concept that this metal is a strong agonist of the CaSR. Moreover, lanthanum was also able to enhance CaSR sensitivity to calcium. The presence of 1 nM LaCl(3) significantly left-shifted the CaSR response curve, changing the EC(50) value for calcium from 2.30 mM (calcium alone) to 1.26 mM (calcium + 1 nM lanthanum). The parathyroid glands cultured with lanthanum showed a trend to secrete less PTH compared to the control glands: 1.51 +/- 0.23 (control), 0.91 +/- 0.17 (La 100 nM) and 1.04 +/- 0.18 (La 400 nM) [(pg/h)/(pg/h), mean +/- SEM] (ANOVA P = 0.0145). A similar trend was also observed in PTH synthesis measured by PTH mRNA levels. These in vitro findings demonstrate that lanthanum, at nanomolar concentrations, is an agonist of the CaSR able to activate it in the absence of calcium. In addition, it can also enhance CaSR sensitivity to calcium, modulating PTH synthesis and secretion.

Substance P stimulates human airway submucosal gland secretion mainly via a CFTR-dependent process

Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.

Analysis of SAFB1 and SAFB2 Knockout Mice Reveals Non-Redundant Functions of the Two Estrogen Receptor Co-Repressor Paralogs.

Abstract The Scaffold Attachment Factor B1 (SAFB1) and B2 (SAFB2) have been shown to be involved in chromatin organization, transcriptional regulation, and RNA processing. The paralogs SAFB1 and SAFB2 share 74% similarity at the amino acid level, with up to 98% similarity in some functional domains. These functional domains include DNA and RNA-binding domains, and a C-terminal repression domain. We have previously shown that SAFB1 and SAFB2 function as estrogen receptor (ER) corepressors – they directly bind to ER, and repress its transcriptional activity. There is also evidence that both proteins play a role in breast cancer; their shared chromosomal locus on chromosome 19p13 displays high rates of LOH, and protein loss was associated with worse survival of breast cancer patients.To understand the roles of SAFB1 and SAFB2 in development and function of hormone responsive tissues, we generated gene-specific knockout (KO) mouse models. Deletion of SAFB1 resulted in a high degree of embryonic and perinatal lethality. Surviving SAFB1 KO mice displayed severe growth retardation associated with low serum IGF-I levels, male infertility and female subfertility. In contrast, SAFB2 KO mice were born at the expected Mendelian ratio and did not show any obvious defects in growth or fertility. Initial gross pathology analysis has not yet revealed any significant defects.Since the SAFB proteins are known to play a role in estrogen response, we analyzed mammary gland development in the two mouse models. Young virgin SAFB1 KO mice showed delayed mammary gland development with a significantly reduced number of terminal end buds and decreased outgrowth compared to wild type (WT) controls. This was likely a result of delayed puberty, due to low IGF-I levels, since at the four month time point, mammary gland growth was restored in the virgin SAFB1 KO mice to that seen in WT controls. Interestingly, the KO glands exhibited increased alveolar development and side branching. To measure proliferation directly, we performed mammary gland transplantation experiments, thereby excluding secondary effects due to systemic defects. These studies showed a significant increase in proliferation in SAFB1 KO glands compared to WT control glands. In contrast to the SAFB1-null mice, we did not observe any obvious mammary gland defects in pubertal or adult SAFB2-null mice. However, mammary glands from aged virgin SAFB2 mice (1.5 years old) showed extensive side branching and precocious development of alveolar buds resembling glands of late pregnant mice. This was associated with increased proliferation of alveolar cells in the SAFB2 KO glands.In summary, genetic ablation of the ER co-repressors SAFB1 and SAFB2 results in defects in mammary gland development. In general, the two mouse models have very different phenotypes, revealing diverse and non-redundant functions of SAFB1 and SAFB2, findings that were unexpected based on their high sequence similarity. We are currently performing additional studies to finalize characterization of the in vivo phenotypes, focusing on hormone responsive tissues, and also to understand the mechanism underlying the observed phenotypes. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2162.

Sjögren’s Syndrome-Like Ocular Surface Disease in Thrombospondin-1 Deficient Mice

Thrombospondin-1 (TSP-1) is a major activator of latent transforming growth factor-beta in vitro as well as in vivo. Mice deficient in TSP-1, despite appearing normal at birth, develop a chronic form of ocular surface disease that is marked by increased apoptosis and deterioration in the lacrimal gland, associated dysfunction, and development of inflammatory infiltrates that result in abnormal tears. The increase in CD4(+) T cells in the inflammatory infiltrates of the lacrimal gland, and the presence of anti-Sjögren's syndrome antigen A and anti-Sjögren's syndrome antigen B antibodies in the serum resemble autoimmune Sjögren's syndrome. These mice develop an ocular surface disorder dry eye that includes disruption of the corneal epithelial layer, corneal edema, and a significant decline in conjuctival goblet cells. Externally, several mice develop dry crusty eyes that eventually close. The inflammatory CD4(+) T cells detected in the lacrimal gland, as well as those in the periphery of older TSP-1 null mice, secrete interleukin-17A, a cytokine associated with chronic inflammatory diseases. Antigen-presenting cells, derived from TSP-1 null, but not from wild-type mice, activate T cells to promote the Th17 response. Together, these results indicate that TSP-1 deficiency results in a spontaneous form of chronic dry eye and aberrant histopathology associated with Sjögren's syndrome.

A sentinel function for teat tissues in dairy cows: dominant innate immune response elements define early response to E. coli mastitis

Escherichia coli intramammary infection elicits localized and systemic responses, some of which have been characterized in mammary secretory tissue. Our objective was to characterize gene expression patterns that become activated in different regions of the mammary gland during the acute phase of experimentally induced E. coli mastitis. Tissues evaluated were from Fürstenburg's rosette, teat cistern (TC), gland cistern (GC), and lobulo-alveolar (LA) regions of control and infected mammary glands, 12 and 24 h after bacterial (or control) infusions. The main networks activated by E. coli infection pertained to immune and inflammatory response, with marked induction of genes encoding proteins that function in chemotaxis and leukocyte activation and signaling. Genomic response at 12 h post-infection was greatest in tissues of the TC and GC. Only at 24 h post-infection did tissue from the LA region respond, at which time the response was the greatest of all regions. Similar genetic networks were impacted in all regions during early phases of intramammary infection, although regional differences throughout the gland were noted. Data support an important sentinel function for the teat, as these tissues responded rapidly and intensely, with production of cytokines and antimicrobial peptides.

A Functional Connection between pRB and Transforming Growth Factor β in Growth Inhibition and Mammary Gland Development

Transforming growth factor beta (TGF-beta) is a crucial mediator of breast development, and loss of TGF-beta-induced growth arrest is a hallmark of breast cancer. TGF-beta has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-beta cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1(DeltaL) and Rb1(NF)), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-beta growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-beta signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-beta cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-beta in growth control and mammary gland development.

Substance P stimulates human airway submucosal gland secretion mainly via a CFTR-dependent process

Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.

Effects on adhesion molecule expression and lymphocytes in the bovine mammary gland following intra-mammary immunisation

Changes to adhesion molecule expression and lymphocyte populations were evaluated in alveolar mammary tissue collected from cows following an immunisation protocol that involved intra-mammary inoculation to induce an IgA response in mammary secretions. The right quarters of the udder were immunised; the left side acted as a control. Antibody titres in secretions showed that at least two animals responded with antigen-specific IgA. Numbers of T-lymphocytes were 4-fold higher in immunised glands compared with controls ( P < 0.05). IgA-, IgM- and IgG-positive cell numbers were significantly higher ( P < 0.01) in immunised glands compared with controls in three of the four cows. No mucosal addressin molecule-1 (MAdCAM-1), vascular cell-adhesion molecule-1 (VCAM-1) or peripheral node addressin (PNAd) protein expression was detected on smaller venules that stained positively for von Willebrand factor in alveolar mammary tissues, from either immunised or control glands. Both VCAM-1 and PNAd were detected on smaller venules in supramammary lymph nodes, however, there was no significant difference between immunised and control glands. Quantification of MAdCAM-1 mRNA showed very low expression in both immunised and control alveolar tissue compared with Peyer's patch positive-control tissue. These findings suggest that the bovine mammary gland is capable of a mucosal antibody response; however, MAdCAM-1 is not involved with lymphocyte homing to the mammary gland in this species.

In Vivo Expansion of the Mammary Stem/ Progenitor Cell Population by Xanthosine Infusion

Mammary stem cells provide for growth and maintenance of the mammary gland and are therefore of considerable interest as determinants of productivity and efficiency of dairy animals and as targets of carcinogenesis in humans. Xanthosine treatment was previously shown to promote expansion of hepatic stem cells in vitro. The objective of this study was to determine if in vivo treatment with xanthosine can increase the mammary stem cell population. Xanthosine was infused into the right mammary glands of four female Holstein calves for 5 consecutive days. Immediately after each xanthosine treatment, calves were injected intravenously with 5-bromo-2-deoxyuridine (BrdU). Forty days after the final treatment, calves were euthanized and mammary tissue harvested. BrdU-label retaining epithelial cells (LREC) were detected immunohistochemically and quantified. Retention of BrdU was used as a marker for putative bovine mammary stem cells. Infusion of xanthosine into the bovine mammary gland significantly increased the number of LREC in treated glands compared to contralateral control glands (P < 0.05). LREC averaged 0.4% of epithelial cells in control glands and 0.8% in xanthosine-treated glands. The increase in LREC in xanthosine-treated glands was supported by a concomitant increase in telomerase activity (P < 0.01) and a correlation between LREC and telomerase (P < 0.05; r (2) = 0.7). Data indicate that in vivo treatment with xanthosine can be used to increase the number of mammary stem cells. This is the first demonstration of an in vivo treatment to increase the endogenous population of mammary stem cells, with utility for biomedical research and dairy management.

Spt6 enhances the elongation rate of RNA polymerase II in vivo

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.

Healthy human salivary glands contain a DHEA‐sulphate processing intracrine machinery, which is deranged in primary Sjögren's syndrome

Sjögren’s syndrome (SS) patients have low salivary dehydroepiandrosterone (DHEA) and androgen biomarker levels, but high salivary oestrogen levels. The hypothesis was that the healthy glands contain DHEA-sulphate processing intracrine machinery; the local androgen/oestrogen imbalance suggests that this is disarranged in SS. Indirect immunofluorescence and quantitative real-time PCR (qRT-PCR) of steroid sulphatase, sulfotransferase, 3β- and 17β-hydroxysteroid dehydrogenases (3β- and 17β-HSD), 5α-reductase and aromatase were performed for labial salivary glands of healthy controls and persons with SS. In control acini steroid sulphatase and sulfotransferase immunoreactivities were located in the basolateral cell parts. 3β- and 17β-HSD formed strong, interrupted bands along the basal cell parts. 5α-reductase was mainly located in acinar cell nuclei and aromatase in the apical cell membrane. All enzymes were more widespread in ducts. In SS, steroid sulphatase was weak and deranged, 3β- and 17β-HSD had lost their strict basal acinar cell localization and 5α-reductase was mainly found in the cytoplasm of the acinar cells, whereas aromatase showed similar staining in SS and controls. qRT-PCR of labial salivary glands disclosed all corresponding enzyme mRNAs with the levels of 3β-HSD in SS being the lowest. Healthy tubuloacinar epithelial cells contain complete intracrine machineries for DHEA(-sulphate) pro-hormone processing. These enzymes have in healthy acini an organized architecture, which corresponds with DHEA uptake from the circulation, nuclear site of production of the active dihydrotestosterone (DHT) end product and production of oestrogens into saliva for export to ductal and oral epithelial cells. SS is characterized by low 3β-HSD levels, which together with impaired subcellular compartmentalization of HSDs and 5α-reductase may explain the low local DHT and androgen biomarker levels in SS.