Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1ß were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1ß immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

Systematic analysis of ACE2 and TMPRSS2 expression in salivary glands reveals underlying transmission mechanism caused by SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a global pandemic that has caused severe health threats and fatalities in almost all communities. Studies have detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a viral load that lasts for a long period. However, researchers are yet to establish whether SARS-CoV-2 can directly enter the salivary glands. Therefore, this study aimed to assess the presence of angiotensin-converting enzyme 2 (ACE2)/transmembrane serine proteases 2 (TMPRSS2) expression in salivary glands using publicly available databases. The distribution of ACE2 and TMPRSSs family in salivary gland tissue and other tissues was analyzed. The Genotype-Tissue Expression dataset was employed to explore the ACE2 and TMPRSS2 expression in various body organs and salivary glands in a healthy population. The single-cell sequencing data for salivary gland samples (including submandibular salivary gland and parotid gland) from mice were collected and analyzed. The components and proportions of salivary gland cells expressing the key protease TMPRSSs family were analyzed. Transcriptome data analysis showed that ACE2 and TMPRSS2 were expressed in salivary glands. The expression levels of ACE2 and TMPRSS2 were marginal without significant differences in different age groups or between men and women. Single-cell RNA sequence analysis indicated that TMPRSS2 was mainly expressed in salivary gland epithelial cells. We speculate that SARS-CoV-2 may be entered in salivary glands.

Salivary glands and human congenital cytomegalovirus infection: What happens in early fetal life?

Salivary glands are a site of human cytomegalovirus (CMV) replication, latency, and persistence. Prolonged secretion of virus in saliva for months following a primary infection contribute to horizontal transmission. In order to better understand the early effects of CMV on salivary glands and the mechanisms of viral persistent replication, submandibular glands of six CMV congenitally infected fetuses at 21 weeks gestation were studied. Three fetuses at the same gestational age from CMV-seronegative women were compared as negative controls. Tissue viral load and the type of inflammatory infiltrate were evaluated. Moreover, development and branching of salivary glands, the number of myoepithelial cells, cellular proliferation, and expression of secretory proteins of the saliva (Gross Cystic Disease Fluid Protein-15 and lysozyme) were studied. A low viral load and rare CMV-positive cells associated with T CD8 cytotoxic lymphocytes were observed. Branching was impaired with a decrease in terminal acinar structures, the number of myoepithelial cells, and cellular proliferation were reduced. In addition, a compromised secretion of defense proteins involved in the oral humoral immunity was observed. These findings suggest that CMV may affect salivary glands, impairing structure development and secretion of defense proteins, probably responsible for the prolonged viral shedding in saliva. J. Med. Virol. 89:318-323, 2017. © 2016 Wiley Periodicals, Inc.

In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins.

A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy.

Cytomegalovirus-induced salivary gland pathology: AREG, FGF8, TNF-α, and IL-6 signal dysregulation and neoplasia.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor originating in major and minor salivary glands (SGs). Although the precise multifactorial etiology of human SG-MEC is largely unknown, we have recently shown that cytomegalovirus (CMV) is an important component of MEC tumorigenesis. Despite the well-documented overexpression of the EGFR → ERK signaling pathway in SG-MEC, there has been limited to no clinical success with inhibition of this pathway. Using our previously characterized mouse model of CMV-induced SG dysplasia/neoplasia, we report that inhibitors of the EGFR → ERK pathway do not ameliorate or rescue well-established pathology, either singly or in combination, but they do inhibit the evolution of progressive pathogenesis ("disease tolerance") in the face of mounting CMV burden. Failure to rescue SG pathology, suggested a possible increase in the ligand levels of alternative pathways that share cell proliferation and survival effectors (e.g. ERK and PI3K). Here we present evidence of a highly significant upregulation of ligands for the EGFR, FGFR, IL-6R, and TNFR signaling pathways, all of which converge upon the Raf/MEK/ERK amplifier module. This explains our finding that even in the presence of the highest nontoxic dose of an ERK phosphorylation inhibitor, pERK is undiminished. Given the considerable pathway crosstalk, a deep understanding of subversion and dysregulation of the SG interactome by CMV is a priori quite daunting. Circumventing this dilemma, we present evidence that concurrent inhibition of ERK phosphorylation (U0126) and CMV replication (acyclovir) obviates progressive pathogenesis and results in complete SG rescue (tumor regression). These findings provide a mechanistic foundation for potential clinical trials that utilize similar concurrent treatment with extant FDA-approved drugs.

Small molecule inhibitors of the host cell COX/AREG/EGFR/ERK pathway attenuate cytomegalovirus-induced pathogenesis.

As with other herpesviruses, human cytomegalovirus (hCMV) has the ability to establish lifelong persistence and latent infection following primary exposure, salivary glands (SMGs) being the primary site of both. In the immunocompromised patient, hCMV is a common cause of opportunistic infections, and subsequent morbidity and mortality. Elucidating the molecular pathogenesis of CMV-induced disease is critical to the development of more effective and safer drug therapies. In the present study, we used a novel mouse postnatal SMG organ culture model of mCMV-induced dysplasia to investigate a candidate signaling network suggested by our prior studies (COX-2/AREG/EGFR/ERK). The objective was to employ small molecule inhibitors to target several key steps in the autocrine loop, and in this way ameliorate pathology. Our results indicate that upregulation of ERK phosphorylation is necessary for initial mCMV-induced pathogenesis, and that ErbB receptor family phosphorylation and downstream signaling are highly relevant targets for drug discovery.

Sialadenitis: A Possible Early Manifestation of COVID-19.

Acute sialadenitis may be caused by viruses, including coronaviruses. Although there are anecdotal reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) salivary gland infections, there have been no well-documented cases of sialadenitis in patients with COVID-19 described in the literature. We report a case of parotitis and submandibular gland sialadenitis, as well as an isolated case of parotitis, in two patients with concurrent SARS-CoV-2 infections. Computed tomography imaging demonstrated parotid and submandibular gland enlargement with heterogenous enhancement and attenuation, consistent with sialadenitis. Medical management was sufficient for successful resolution of the acute sialadenitis. Laryngoscope, 130:2595-2597, 2020.

Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals.

The ability to dynamically image cellular and subcellular structures in a live animal and to target genes to a specific cell population in a living tissue provides a unique tool to address many biological questions in the proper physiological context. Here, we describe a powerful approach that is based on the use of rat submandibular salivary glands, which offer the possibility to easily perform intravital imaging and deliver molecules from the oral cavity, and plasmid DNA, which offers the advantage of rapid manipulations. We show that, under different experimental conditions, a reporter molecule can be rapidly expressed in specific compartments of the glands: 1) in the intercalated ducts, when plasmid DNA is administered alone, and 2) in granular ducts, striated ducts, and, to a lesser extent, acini, when plasmid DNA is mixed with replication-deficient adenovirus subtype 5 particles. Remarkably, we also found that gene expression can be directed to acinar cells when plasmid DNA is administered during isoproterenol-stimulated exocytosis, suggesting a novel mechanism of plasmid internalization regulated by compensatory endocytosis. Finally, as a practical application of these strategies, we show how the expression of fluorescently tagged molecules enables the study of the dynamics of various organelles in live animals at a resolution comparable to that achieved in cell cultures.

Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Bilateral isolated submandibular gland mumps.

Isolated submandibular swellings pose a diagnostic challenge to the practising otolaryngologist. We report an unusual case of mumps isolated to bilateral submandibular glands. We discuss the case and the literature surrounding this condition and remind clinicians that mumps should be considered as a diagnosis in the presence of submandibular gland swelling in the absence of typical parotid swelling associated with mumps. Early consideration of this differential diagnosis, serological testing and a multidisciplinary approach may help to clinch the diagnosis earlier and prevent spread of the virus.

Cytomegalovirus-induced embryopathology: mouse submandibular salivary gland epithelial-mesenchymal ontogeny as a model.

BACKGROUND: Human studies suggest, and mouse models clearly demonstrate, that cytomegalovirus (CMV) is dysmorphic to early organ and tissue development. CMV has a particular tropism for embryonic salivary gland and other head mesenchyme. CMV has evolved to co-opt cell signaling networks so to optimize replication and survival, to the detriment of infected tissues. It has been postulated that mesenchymal infection is the critical step in disrupting organogenesis. If so, organogenesis dependent on epithelial-mesenchymal interactions would be particularly vulnerable. In this study, we chose to model the vulnerability by investigating the cell and molecular pathogenesis of CMV infected mouse embryonic submandibular salivary glands (SMGs). RESULTS: We infected E15 SMG explants with mouse CMV (mCMV). Active infection for up to 12 days in vitro results in a remarkable cell and molecular pathology characterized by atypical ductal epithelial hyperplasia, apparent epitheliomesenchymal transformation, oncocytic-like stromal metaplasia, beta-catenin nuclear localization, and upregulation of Nfkb2, Relb, Il6, Stat3, and Cox2. Rescue with an antiviral nucleoside analogue indicates that mCMV replication is necessary to initiate and maintain SMG dysmorphogenesis. CONCLUSION: mCMV infection of embryonic mouse explants results in dysplasia, metaplasia, and, possibly, anaplasia. The molecular pathogenesis appears to center around the activation of canonical and, perhaps more importantly, noncanonical NFkappaB. Further, COX-2 and IL-6 are important downstream effectors of embryopathology. At the cellular level, there appears to be a consequential interplay between the transformed SMG cells and the surrounding extracellular matrix, resulting in the nuclear translocation of beta-catenin. From these studies, a tentative framework has emerged within which additional studies may be planned and performed.

Difference in replication of low-passage MCMV HaNa1 in BALB/c, C57BL/6 and NOD mice and role of different branches of immunity in susceptibility.

Currently, murine cytomegalovirus (MCMV) infections have been studied extensively in inbred mice via intraperitoneal route with highly passaged strains. However, the question how a low-passage MCMV replicates in inbred mice via a natural route remained unanswered. Here, different inbred mice (BALB/c, C57BL/6 and NOD) were inoculated oronasally with a low-passage MCMV strain, HaNa1. Viral replication was evaluated by virus titration and quantitative real-time PCR, and antibody response was assessed by immunoperoxidase cell monolayer assay (IPMA). In BALB/c mice, virus persisted in nasal mucosa (from 3 dpi) and submandibular glands (from 7 dpi) until the end of experiment (49 dpi). In C57BL/6 mice, infectious virus was only detected in nasal mucosa from 3 dpi until 21 dpi; viral genome was still detectable in nasal mucosa until 49 dpi. Although infectious virus was not detected in submandibular glands of C57BL/6 mice, viral genome was detected from 7 dpi until 49 dpi. NOD mice appeared to be even more resistant with absence of any productive infection; viral genome was detected at low levels in nasal mucosa. We demonstrated that there was a strong correlation between on the one hand degree of productive replication and on the other hand the time of first appearance and titer of MCMV-specific IgG antibody. The deficiency of functional T and B cells and interleukin-2 (IL-2) common-γ chain (γc) did not increase the susceptibility to MCMV by the use of NOD.SCID and NSG mice. In addition, using monocytic cells from different inbred mice we found patterns of resistance similar to those seen in vivo, as assessed by viral antigen expression. Taken together, these results demonstrated that upon oronasal inoculation low-passage MCMV HaNa1 replication clearly differs between different inbred mice (BALB/c>C57BL/6>NOD); resistance in vivo to MCMV is partly due to less susceptibility of host target cells and is independent of T, B cells and γc signaling cytokine-dependent NK cell activities.

Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells.

Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the alpha-subunit of Gq (Ad-EF1-dnG alpha q) and the other expressing the wild-type alpha-subunit of Gq (Ad-EF1-wtG alpha q). We used an adenovirus expressing green fluorescent protein (Ad-EF1-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca2+]i) or the carbachol responses in these cells. We further show that Ad-EF1-dnG alpha q inhibits the increase in [Ca2+]i produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EF1-wtG alpha q, which indicates that in both HSG and HT29 cells, the increase in [Ca2+]i produced by muscarinic receptor activation is largely mediated by activation of Gq. Neither virus affected the resting level of [Ca2+]i in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.

Immunohistochemical assessment of fractalkine, inflammatory cells, and human herpesvirus 7 in human salivary glands.

Human fractalkine (CX3CL1), a delta-chemokine, is implicated in the mediation of multiple cell functions. In addition to serving as a chemotactic factor for mononuclear cell subtypes, membrane-bound fractalkine may promote viral infection by interacting with virions that encode putative fractalkine-binding proteins. Fractalkine expression in normal epithelial tissues studied to date is either constitutive or is upregulated with inflammation. In salivary glands, the expression of fractalkine is unknown. Moreover, salivary glands are a major site for the persistent and productive infection by human herpesvirus (HHV)-7, which encodes two putative fractalkine-binding gene products, U12 and U51. Surprisingly, the cellular distribution of HHV-7 in major salivary glands has not been explored. We therefore determined by immunohistochemistry the cellular localization of fractalkine in three different salivary glands: parotid, submandibular, and labial glands. Fractalkine expression was highly variable, ranging from high to undetectable levels. We further examined the association of fractalkine with inflammatory cell infiltration or HHV-7 infection of salivary epithelial cells. Inflammatory cells were always adjacent to epithelial cells expressing fractalkine, consistent with a function of fractalkine in inflammatory cell recruitment and/or retention in salivary glands. In contrast, HHV-7-infected epithelial cells did not always express fractalkine, suggesting that fractalkine may not be an absolute requirement for viral entry.

Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections.

Non-isotopic in situ hybridization with a novel phenytoin (PHE)-labeled probe was developed. The mixture of cloned cytomegalovirus (CMV) DNA fragments was labeled by random primer technique using PHE-11(spacer)-dUTP, instead of dTTP. The tissue sections were treated with 0.2 N HCl and with proteinase K (1 microgram/ml), and then heated at 70 degrees C in the presence of 50 or 75% formamide. The sections were hybridized with PHE-labeled probe at 37 degrees C overnight. The hybridization signal was visualized by alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate (BCIP)/4-nitroblue tetazolium (NBT) system. Strong hybridization signals were detected in sections of the small intestine and the placenta, even when denatured in the presence of 50% formamide. In the case of small intestine, CMV DNA was also detected in the endothelial cells of the mucosa where apparent infected cell was not observed histologically. In the sections of the submaxillary gland, the lung, the adrenal gland and the ovary, hybridization signal was not detected when denatured in the presence of 50% formamide, but detected after denaturation with 75% formamide. Thus, in situ hybridization with the novel PHE-labeled probe is applicable to conventional formalin-fixed, paraffin-embedded tissue sections.

Efferent connections of the lamina terminalis, the preoptic area and the insular cortex to submandibular and sublingual gland of the rat traced with pseudorabies virus.

Neurones situated in the lamina terminalis (organum vasculosum of the lamina terminalis, median preoptic nucleus and subfornical organ) as well as within medial and lateral parts of the preoptic area and in the insular cortex become transneuronally labelled following pseudorabies virus injections into the submandibular or the sublingual gland. These neurones are efferently connected to a chain of central neurones directed to secretory or vascular tissue of the submandibular or the sublingual gland. By varying the postinoculation time a stepwise infection of different forebrain nuclei was registered, with the hypothalamic paraventricular nucleus and the lateral hypothalamic area being the first forebrain structures labelled. Such early infected neurones within these hypothalamic nuclei are in all likelihood third order neurones regulating salivary secretion and might have functioned as relays transmitting virus to other forebrain structures. The above mentioned forebrain areas together with several other hypothalamic nuclei as well as the bed nucleus of the stria terminalis, the central nucleus of the amygdala and the substantia innominata, seem to be the widespread anatomical basis for the central regulation of salivary gland function.

Cytomegalovirus in the principal submandibular gland of the little brown bat, Myotis lucifugus.

In the course of a large scale study of salivary gland ultrastructure in chiropterans, enlarged cells infected with numerous virus particles were encountered in some acinar cells in the principal submandibular glands of two of 34 little brown bats (Myotis lucifugus). The characteristic morphology of the viruses, together with the cytomegaly that they induced, led to their identification as cytomegalovirus (CMV). In a reversal of the situation in other animal species, bat virus particles within cytoplasmic vacuoles lacked capsomeres, whereas the latter were prominent in particles free in the cytosol. The generally accepted schemes for CMV production cannot explain this seemingly aberrant morphology. This report extends to four the number of mammalian orders in which CMV has been documented by means of electron microscopy as occurring in salivary glands.

In vivo use of adenoviral vectors: effects on salivary gland structure.

Recently there has been considerable progress in the development of in vivo gene transfer technology. By this means, new genetic information may be introduced directly to cells, while the cells remain in their natural milieu. The ability to express exogenous proteins makes it possible to explore the functions of native or altered proteins and thereby develop new insights into cell function and dysfunction. We have demonstrated that the major salivary glands are efficiently infected by recombinant adenovirus vectors. These vectors are capable of expressing transgenes in both acinar and ductal cell types. Recently, we have developed vectors that contain cell-specific promoters so that proteins may be expressed in selected subpopulations of salivary cells. Early generations of adenoviral vectors elicited potent immune responses in vivo. However, modified vectors and adjunctive measures have improved the safety of gene transfer to salivary glands. Future studies will aim to increase the duration of adenovirus-based gene expression and to produce vector systems that are not toxic to the host.

[Exocrinopathy resembling Sjögren's syndrome induced by a murine retrovirus].

Recently, retrovirus is suggested to participate in the pathogenesis of Sjögren's syndrome (SjS). During the course of our study on mice infected with LP-BM5 murine leukemia virus, we found SjS-like systemic exocrinopathy in those mice. This virus is known to induce murine acquired immunodeficiency syndrome (MAIDS) in sensitive strains of mice. Most cells infiltrating into the glands were composed of CD3+ T cells (CD4-dominant), Mac-1+ cells and also B220+ cells. The virus genome was detected in the submandibular glands by immunohistochemistry or by PCR and the retroviral particles were also detected by electron microscopy. This mice might become an animal model for SjS and provide us with valuable informations for analysing the mechanism of how a retrovirus could induce SjS.

The "Vampirome": Transcriptome and proteome analysis of the principal and accessory submaxillary glands of the vampire bat Desmodus rotundus, a vector of human rabies.

Vampire bats are notorious for being the sole mammals that strictly feed on fresh blood for their survival. While their saliva has been historically associated with anticoagulants, only one antihemostatic (plasminogen activator) has been molecularly and functionally characterized. Here, RNAs from both principal and accessory submaxillary (submandibular) salivary glands of Desmodus rotundus were extracted, and ~200 million reads were sequenced by Illumina. The principal gland was enriched with plasminogen activators with fibrinolytic properties, members of lipocalin and secretoglobin families, which bind prohemostatic prostaglandins, and endonucleases, which cleave neutrophil-derived procoagulant NETs. Anticoagulant (tissue factor pathway inhibitor, TFPI), vasodilators (PACAP and C-natriuretic peptide), and metalloproteases (ADAMTS-1) were also abundantly expressed. Members of the TSG-6 (anti-inflammatory), antigen 5/CRISP, and CCL28-like (antimicrobial) protein families were also sequenced. Apyrases (which remove platelet agonist ADP), phosphatases (which degrade procoagulant polyphosphates), and sphingomyelinase were found at lower transcriptional levels. Accessory glands were enriched with antimicrobials (lysozyme, defensin, lactotransferrin) and protease inhibitors (TIL-domain, cystatin, Kazal). Mucins, heme-oxygenase, and IgG chains were present in both glands. Proteome analysis by nano LC-MS/MS confirmed that several transcripts are expressed in the glands. The database presented herein is accessible online at http://exon.niaid.nih.gov/transcriptome/D_rotundus/Supplemental-web.xlsx. These results reveal that bat saliva emerges as a novel source of modulators of vascular biology. BIOLOGICAL SIGNIFICANCE: Vampire bat saliva emerges as a novel source of antihemostatics which modulate several aspects of vascular biology.