Enteric viruses replicate in salivary glands and infect through saliva.

Enteric viruses like norovirus, rotavirus and astrovirus have long been accepted as spreading in the population through fecal-oral transmission: viruses are shed into feces from one host and enter the oral cavity of another, bypassing salivary glands (SGs) and reaching the intestines to replicate, be shed in feces and repeat the transmission cycle1. Yet there are viruses (for example, rabies) that infect the SGs2,3, making the oral cavity one site of replication and saliva one conduit of transmission. Here we report that enteric viruses productively and persistently infect SGs, reaching titres comparable to those in the intestines. We demonstrate that enteric viruses get released into the saliva, identifying a second route of viral transmission. This is particularly significant for infected infants, whose saliva directly transmits enteric viruses to their mothers' mammary glands through backflow during suckling. This sidesteps the conventional gut-mammary axis route4 and leads to a rapid surge in maternal milk secretory IgA antibodies5,6. Lastly, we show that SG-derived spheroids7 and cell lines8 can replicate and propagate enteric viruses, generating a scalable and manageable system of production. Collectively, our research uncovers a new transmission route for enteric viruses with implications for therapeutics, diagnostics and importantly sanitation measures to prevent spread through saliva.

Salivary gland progenitor cell biology provides a rationale for therapeutic salivary gland regeneration.

An irreversible loss of salivary gland function often occurs in humans after removal of salivary tumors, after therapeutic radiation of head and neck tumors, as a result of Sjögren's syndrome and in genetic syndromes affecting gland development. The permanent loss of gland function impairs the oral health of these patients and broadly affects their quality of life. The regeneration of functional salivary gland tissue is thus an important therapeutic goal for the field of regenerative medicine and will likely involve stem/progenitor cell biology and/or tissue engineering approaches. Recent reports demonstrate how both innervation of the salivary gland epithelium and certain growth factors influence progenitor cell growth during mouse salivary gland development. These advances in our understanding suggest that developmental mechanisms of mouse salivary gland development may provide a paradigm for postnatal regeneration of both mice and human salivary glands. Herein, we will discuss the developmental mechanisms that influence progenitor cell biology and the implications for salivary gland regeneration.

Epithelial Cell Lineage and Signaling in Murine Salivary Glands.

Maintaining salivary gland function is critical for oral health. Loss of saliva is a common side effect of therapeutic irradiation for head and neck cancer or autoimmune diseases such as Sjögren's syndrome. There is no curative treatment, and current strategies proposed for functional regeneration include gene therapy to reengineer surviving salivary gland tissue, cell-based transplant therapy, use of bioengineered glands, and development of drugs/biologics to stimulate in vivo regeneration or increase secretion. Understanding the genetic and cellular mechanisms required for development and homeostasis of adult glands is essential to the success of these proposed treatments. Recent advances in genetic lineage tracing provide insight into epithelial lineage relationships during murine salivary gland development. During early fetal gland development, epithelial cells expressing keratin 14 (K14) Sox2, Sox9, Sox10, and Trp63 give rise to all adult epithelium, but as development proceeds, lineage restriction occurs, resulting in separate lineages of myoepithelial, ductal, and acinar cells in postnatal glands. Several niche signals have been identified that regulate epithelial development and lineage restriction. Fibroblast growth factor signaling is essential for gland development, and other important factors that influence epithelial patterning and maturation include the Wnt, Hedgehog, retinoic acid, and Hippo signaling pathways. In addition, other cell types in the local microenvironment, such as endothelial and neuronal cells, can influence epithelial development. Emerging evidence also suggests that specific epithelial cells will respond to different types of salivary gland damage, depending on the cause and severity of damage and the resulting damaged microenvironment. Understanding how regeneration occurs and which cell types are affected, as well as which signaling factors drive cell lineage decisions, provides specific targets to manipulate cell fate and improve regeneration. Taken together, these recent advances in understanding cell lineages and the signaling factors that drive cell fate changes provide a guide to develop novel regenerative treatments.

Neurturin-containing laminin matrices support innervated branching epithelium from adult epithelial salispheres.

Cell transplantation of autologous adult biopsies, grown ex vivo as epithelial organoids or expanded as spheroids, are proposed treatments to regenerate damaged branching organs. However, it is not clear whether transplantation of adult organoids or spheroids alone is sufficient to initiate a fetal-like program of branching morphogenesis in which coordinated branching of multiple cell types including nerves, mesenchyme and blood vessels occurs. Yet this is an essential concept for the regeneration of branching organs such as lung, pancreas, and lacrimal and salivary glands. Here, we used factors identified from fetal organogenesis to maintain and expand adult murine and human epithelial salivary gland progenitors in non-adherent spheroid cultures, called salispheres. These factors stimulated critical developmental pathways, and increased expression of epithelial progenitor markers such as Keratin5, Keratin14, FGFR2b and KIT. Moreover, physical recombination of adult salispheres in a laminin-111 extracellular matrix with fetal salivary mesenchyme, containing endothelial and neuronal cells, only induced branching morphogenesis when neurturin, a neurotrophic factor, was added to the matrix. Neurturin was essential to improve neuronal survival, axon outgrowth, innervation of the salispheres, and resulted in the formation of branching structures with a proximal-distal axis that mimicked fetal branching morphogenesis, thus recapitulating organogenesis. Epithelial progenitors were also maintained, and developmental differentiation programs were initiated, showing that the fetal microenvironment provides a template for adult epithelial progenitors to initiate branching and differentiation. Further delineation of secreted and physical cues from the fetal niche will be useful to develop novel regenerative therapies that instruct adult salispheres to resume a developmental-like program in vitro and to regenerate branching organs in vivo.

NOVH increases MMP3 expression and cell migration in glioblastoma cells via a PDGFR-alpha-dependent mechanism.

Nephroblastoma overexpressed gene (NOV) is highly expressed in the nervous system. We investigated its biological activity by expressing the human NOV gene (NOVH) in a human glioblastoma cell line that is negative for NOVH and by analyzing four clones with different levels of NOVH expression. There was no difference in cell proliferation between the NOVH-expressing cell lines, but there was increased cell adhesion and migration that correlated with increasing NOVH expression. Gene expression profiling was used to investigate the mechanisms by which NOVH expression regulated cell activity. We identified two induced genes in NOVH-expressing cells that are involved in cell migration: matrix metalloprotease (MMP)3 and platelet-derived growth factor receptor (PDGFR)-alpha. Our studies show that PDGFR-alpha induced MMP3 gene expression and increased cell proliferation and cell migration upon stimulation by platelet-derived growth factor (PDGF)-AA. We also show that the induction of MMP3 in cells expressing NOVH is potentiated by either cell density, serum, or PDGF-BB. Thus, expression of NOVH in glioblastoma cells triggers a cascade of gene expression resulting in increased cell adhesion and migration.

A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization.

A plaque growth chamber was developed for long-term growth of five separate plaques from the same plaque or saliva sample under identical conditions of temperature and gas phase. Reagent addition and growth conditions for each plaque could be independently controlled, and each was accessible for sequential sampling and electrode insertion. Plaques were cultured for over six weeks on pellicle-coated Lux (TM) 25-mm diameter cover-slips at 35 degrees C under 5% CO2 in N2, and supplied with a medium containing 0.25% mucin (BMM) at 3.6 mL/h, and with periodic 5% sucrose. Electron microscopy and flora analysis of microcosm plaques showed that they had close similarities to reported characteristics of natural dental plaques. Diverse motile bacteria were present. Sucrose-induced Stephan pH curves and urea-induced pH rises were also similar to those reported for natural plaques. Changes in plaque urease, calcium, phosphate concentrations, and the flora were followed over five weeks in a plaque supplied with BMM containing additional 2.5 mmol/L calcium and 7.5 mmol/L phosphate. Despite this high environmental calcium phosphate concentration, there was no continuing increase in calcium levels, although plaque phosphate doubled. Urease levels fluctuated. Changes in the cultivable flora were minor. A urea-containing calcium phosphate/mono-fluorophosphate pH 5 solution, applied for six min every two h for seven days, increased plaque calcium, phosphate, and fluoride to high levels. Thus, plaques grown over several weeks in the multi-station artificial mouth exhibited metabolic and pH behavior typical of natural plaques, could be analyzed during development, and the system allowed manipulation of environmental variables important in plaque pH control and calcification.

Laminin-1 and SIKVAV a laminin-1-derived peptide, regulate the morphology and protease activity of a human salivary gland adenoid cystic carcinoma cell line.

In a previous paper, we demonstrated that laminin-1 and its derived peptide SIKVAV modulates the morphology of an adenoid cystic carcinoma cell line (CAC2 cells). Light microscopy of CAC2 cells grown in three-dimensional preparations of SIKVAV-enriched laminin-1 showed the presence of pseudocystic spaces. Pseudocysts are hallmarks of adenoid cystic carcinoma in vivo. We hypothesized that these pseudocystic spaces could be due to the protease-inducing/activating role of SIKVAV. Thus, we studied the presence of matrix metalloproteinases (MMPs) in CAC2 cells treated either by laminin-1 or by SIKVAV-enriched laminin-1. Immunohistochemistry and zymography suggested that SIKVAV enhanced the secretion of MMP-2 and MMP-9 in CAC2 cells. We propose that SIKVAV induces pseudocystic formation probably through the secretion of MMPs 2 and 9.

Use of cow-side progesterone tests to improve reproductive performance of high-producing dairy cows.

This study evaluated the effectiveness of the cow-side ELISA milk progesterone test in improving postpartum reproductive performance in the Dordt College dairy herd. Cows that produced more than 18,500 lb of milk per lactation were assigned to the high production group (40 cows), while cows that produced less than 18,500 lb of milk (42 cows) were assigned to the low production group. Twenty-one cows in the high production group and 19 cows in the low production group received no ELISA testing (untreated controls), while the remaining cows in each group were evaluated by ELISA test every 7 d beginning on Day 27 post partum (treated cows). A sequence of 2 high progesterone tests and 1 low test indicated the cows were cycling normally. Cows that had low milk progesterone levels (<5 ng/ml) for 3 consecutive tests were assumed to have follicular cysts and were treated with 2 ml GnRH (Cystorelin, 50 mug/ml). Cows that had 3 consecutive high tests (>5 ng/ml) were assumed to have persistent corpora lutea (CL) and were treated with 5 ml PGF(2)alpha (Lutalyse, 5 mg/ml). In both the high and low production groups, treated cows had higher (P < 0.08) pregnancy rates by Day 210 than the untreated controls (63.2 vs 38.1% and 56.5 vs 42.1%, respectively). The days open were reduced (P < 0.05) for the treated animals by 41.6 d compared with the controls. The treated cows produced a net savings of $70.42 (US) per cow assuming a $3.00 savings/day open.

Influence of different ratios of corn and corn silage, housing systems and seasons on the performance of feedlot steers.

Observations on the effects of season, housing and diet were made on 916 steers in three winter and two summer trials. Diets consisted of corn grain and corn silage, in balanced rations, fed ad libitum with energy ratios of 25:75 (Diet 1), 55:45 (Diet 2) and 85:15 (Diet 3). There were three types of housing systems: outside lots without access to overhead shelter (NS), outside lots with access to overhead shelter (OS) and an open-front confinement building (C). Average ambient temperatures and precipitation for winter and summer trials were -1.0 and 15.3 C and 4.67 and 10.81 cm/mo, respectively. Steers gained more (P less than .05) in summer than in winter. Within housing system, OS and NS steers gained faster and consumed more dry matter (DM) and energy (P less than .05) than C steers; C-fed steers were less (P less than .05) efficient (kg feed DM/kg gain) than OS steers. Steers fed Diet 1 had lower (P less than .05) average daily gain (ADG) than those fed Diets 2 and 3. Steers on Diet 3 consumed less DM (P less than .05) than those on Diets 1 and 2. Estimated metabolizable energy intake (MEI) was significantly less for cattle fed Diet 1 than those fed Diets 2 or 3. Diet 3 was more (P less than .05) efficient than Diet 1. Season x diet (P less than .10) and season x housing (P less than .10) interactions were found for daily DM intake and MEI. This resulted in greater cattle growth rate responses to higher grain diets in summer than in winter and more pronounced adverse effects of confinement rearing in summer than in winter. No evidence was found of other two-way or three-way interactions for any of the performance characteristics studied. These results indicate that in addition to important singular effects of season, housing and diet, important interactions of these factors also exist.

Effects of placing cattle on feed at two-month intervals and housing on feedlot performance and carcass grades.

A 5-yr study was conducted involving the placement of yearling steers on feed at 2-mo intervals under three different housing systems. A total of 3,571 steers (180 pens) initially averaging 318 kg was used. Evaluations were made for DM intake, ADG, feed efficiency (FE), carcass quality (QG), and yield grades (YG). Cattle were assigned to either an open lot with overhead shelter (S), an open lot without overhead shelter (NS), or an open-front confinement building (C). Each treatment consisted of two lots of 20 steers each per interval per trial. Corn grain provided 85% of the energy; the remainder was supplied by corn silage and protein supplement. Cattle were fed 140 to 180 d to achieve an average slaughter weight of 500 kg. The main effects of year (Y), month (M), and housing (H) affected DM intake, ADG, FE, and final live weight (P less than .01). The interactions for Y x M, M x H and Y x M x H affected ADG (P less than .05). Month and H affected hot carcass weight (P less than .01). Year affected YG, and year and month affected QG (P less than .01). Month effects on DM intake and ADG indicated that cattle started in May had the highest intake and ADG (P less than .05) and that cattle started in November had the lowest (P less than .05) DMI and ADG. Month effects on FE indicated that cattle were most efficient when placed on feed during March, May, and July (5.82, 5.72, and 5.66 kg DM/kg gain; P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Factors affecting in-transit shrink and liver abscesses in fed steers.

Live weight loss (shrink) and liver conditions (abscesses) were determined on 3,570 crossbred steers of predominantly British breeding over a 5-yr period. Starting in November 1979, steers were placed on feed at bimonthly intervals and provided one of three housing treatments: no shelter, access to overhead shelter or confinement. All steers were implanted with Synovex during the first 3 yr and Compudose the last 2 yr and fed a diet consisting of high-moisture corn grain, which provided 85% of the energy, and corn silage, along with a protein, vitamin and mineral supplement. Cattle were processed into beef after a feeding period of approximately 160 d. Year affected shrink (P less than .001), and month on feed and housing type tended to alter shrink. Cattle marketed during summer and fall and those outside without overhead shelter tended to shrink more. Year, month on feed and housing type affected liver condition (P less than .01). Cattle started on feed in November and January and cattle housed in confinement or outside without overhead shelter had higher incidences of liver abscesses and slower average daily gains (P less than .01). Daily gains for steers with normal and abnormal livers were not different (P greater than .19) for any month started on feed or housing treatment. These results indicate that under the conditions of this study a 2 or 3% weight loss should be expected during the marketing of finished steers and a 16% incidence of liver abscesses should be anticipated, with some modification of the latter due to time of year and housing. The presence of liver abscesses at the time of processing steers into beef did not reduce feedlot performance.

Some epidemiological and scanning electron microscopic features of crazing of the dental enamel of Polynesians.

The prevalence of enamel crazing was determined from the maxillary incisors and canines of 1,109 Tongan residents aged 5-20 years, 1,417 Cook Island residents aged 6-20 years, 520 French Polynesian residents aged 10-15 years, and 92 New Zealand-born and -resident Polynesians aged 11-18 years. Crazing occurred in 22 percent of Tongans, 7 percent of Cook Islanders, but not in French Polynesians or New Zealand-born Polynesians. Cracks become clinically apparent at about 8 years of age; were more common in males than females; and became more common with increasing age. The central incisors were the most commonly affected of the maxillary anterior teeth. Cross-sections of teeth with crazing viewed by light and scanning electron microscopy appeared similar to other stained cracks. The indications are that crazing is a post-eruptive change caused by trauma from local environmental factors resulting in stress fractures. The significance of crazing on enamel structure and strength is unknown, but it is probably minor without long-term disadvantages. The prevalence of crazing may prove useful in anthropology as an indicator of the use of teeth as tools and in assessing the "modernisation" of a population.

Viral gene transfer to developing mouse salivary glands.

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.

Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis.

The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. However, the mechanisms by which epithelial progenitor cells are maintained during organogenesis are poorly understood. We report that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number and morphogenesis of keratin 5-positive epithelial progenitor cells. These effects were rescued with an acetylcholine analog. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and epidermal growth factor receptor, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Parasympathetic innervation maintained the epithelial progenitor cell population in an undifferentiated state, which was required for organogenesis. This mechanism for epithelial progenitor cell maintenance may be targeted for organ repair or regeneration.

Environmental factors affecting feed intake of steers in different housing systems in the summer.

A total of 188 yearling steers of predominantly Angus and Hereford breeds, with mean body weight of 299 kg, were used in this study, which started on 8 April and finished on 3 October, to assess the effects of environmental factors on feed intake of steers in various housing systems. Housing consisted of outside lots with access to overhead shelter, outside lots with no overhead shelter and a cold confinement building. Ad libitum corn, 2.27 kg of 35% dry matter whole plant sorghum silage and 0.68 kg of a 61% protein-vitamin-mineral supplement was offered. Feed that was not consumed was measured to determine feed intake. The temperature data were recorded by hygro-thermographs. Hourly temperatures and humidity were used to develop weather variables. Regression analysis was used and weather variables were regressed on dry matter intake (DMI). When addition of a new variable did not improve R (2) more than one unit, then the number of variables in the model was truncated. Cattle in confinement had lower DMI than those in open lots and those in open lots with access to an overhead shelter (P < 0.05). Cattle in outside lots with access to overhead shelter had similar DMI compared to those in open lots (P = 0.065). Effect of heat was predominantly displayed in August in the three housing systems. In terms of explaining variation in DMI, in outside lots with access to overhead shelter, average and daytime temperatures were important factors, whereas in open lots, nocturnal, peak and average temperatures were important factors. In confinement buildings, the previous day's temperature and humidity index were the most important factors explaining variation in DMI. Results show the effect of housing and weather variables on DMI in summer and when considering these results, cattle producers wishing to improve cattle feedlot performance should consider housing conditions providing less stress or more comfort.

Association between increased CCL2 (MCP-1) expression in lesions and persistence of disease activity in giant-cell arteritis.

OBJECTIVE: Patients with giant-cell arteritis (GCA) usually respond dramatically to corticosteroid treatment. However, recurrences are frequent and corticosteroid requirements are highly variable among patients. The aim of our study was to identify genes potentially involved in disease persistence. METHODS: Gene expression was explored with cDNA arrays in temporal artery biopsies from six GCA patients with relapsing disease and six patients who easily achieved sustained remission. Differentially expressed genes of interest were subsequently analysed by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry in temporal artery biopsies from 35 patients with biopsy-proven GCA and nine controls. RESULTS: CCL2 (MCP-1) was up-regulated in temporal artery samples from relapsing individuals. In the extended series of patients, CCL2 mRNA concentration in lesions was significantly higher than in controls (31 +/- 15.6 vs 0.44 +/- 0.10, P = 0.0001). In addition, CCL2 was more abundant in patients who experienced two or more relapses during the first year compared with those who endured sustained remission (127 +/- 82 vs 11 +/- 5.5, P = 0.0233) and correlated with the cumulated prednisolone dose (R = 0.533, P = 0.0024). CCL2 mRNA concentration correlated with IL-1beta (R = 0.45, P = 0.02), tumour necrosis factor-alpha (TNF-alpha) (R = 0.47, P = 0.013) and IL-6 (R = 0.52, P = 0.0053) mRNA. However, circulating CCL2 determined by ELISA was decreased in patients with strong systemic inflammatory response, suggesting that reduction in circulating CCL2 may reinforce the local gradient in lesions. CONCLUSION: Increased CCL2 (MCP-1) expression in lesions is associated with persistence of disease activity in GCA.

Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly.

The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development.