Adult Salivary Glands Research Articles

CSF1R-dependent macrophages in the salivary gland are essential for epithelial regeneration after radiation-induced injury.

The salivary glands often become damaged in individuals receiving radiotherapy for head and neck cancer, resulting in chronic dry mouth. This leads to detrimental effects on their health and quality of life, for which there is no regenerative therapy. Macrophages are the predominant immune cell in the salivary glands and are attractive therapeutic targets due to their unrivaled capacity to drive tissue repair. Yet, the nature and role of macrophages in salivary gland homeostasis and how they may contribute to tissue repair after injury are not well understood. Here, we show that at least two phenotypically and transcriptionally distinct CX3CR1+ macrophage populations are present in the adult salivary gland, which occupy anatomically distinct niches. CD11c+CD206-CD163- macrophages typically associate with gland epithelium, whereas CD11c-CD206+CD163+ macrophages associate with blood vessels and nerves. Using a suite of complementary fate mapping systems, we show that there are highly dynamic changes in the ontogeny and composition of salivary gland macrophages with age. Using an in vivo model of radiation-induced salivary gland injury combined with genetic or antibody-mediated depletion of macrophages, we demonstrate an essential role for macrophages in clearance of cells with DNA damage. Furthermore, we show that epithelial-associated macrophages are indispensable for effective tissue repair and gland function after radiation-induced injury, with their depletion resulting in reduced saliva production. Our data, therefore, provide a strong case for exploring the therapeutic potential of manipulating macrophages to promote tissue repair and thus minimize salivary gland dysfunction after radiotherapy.

Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway

BackgroundXerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation.MethodsObtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro.ResultshMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation.ConclusionsWe showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (Acari: Ixodidae).

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens.

Two Alimentary Canal Proteins, Fo-GN and Fo-Cyp1, Act in Western Flower Thrips, Frankliniella occidentalis TSWV Infection.

Tomato spotted wilt virus (TSWV) is a plant virus that causes massive economic damage to high-valued crops. This virus is transmitted by specific thrips, including the western flower thrips, Frankliniella occidentalis. TSWV is acquired by the young larvae during feeding on infected host plants. TSWV infects the gut epithelium through hypothetical receptor(s) and multiplies within the cells for subsequent horizontal transmission to other plant hosts via the salivary glands during feeding. Two alimentary canal proteins, glycoprotein (Fo-GN) and cyclophilin (Fo-Cyp1), are thought to be associated with the TSWV entry into the gut epithelium of F. occidentalis. Fo-GN possesses a chitin-binding domain, and its transcript was localized on the larval gut epithelium by fluorescence in situ hybridization (FISH) analysis. Phylogenetic analysis indicated that F. occidentalis encodes six cyclophilins, in which Fo-Cyp1 is closely related to a human cyclophilin A, an immune modulator. The Fo-Cyp1 transcript was also detected in the larval gut epithelium. Expression of these two genes was suppressed by feeding their cognate RNA interference (RNAi) to young larvae. The RNAi efficiencies were confirmed by the disappearance of the target gene transcripts from the gut epithelium by FISH analyses. The RNAi treatments directed to Fo-GN or Fo-Cyp1 prevented the typical TSWV titer increase after the virus feeding, compared to control RNAi treatment. Our immunofluorescence assay using a specific antibody to TSWV documented the reduction of TSWV in the larval gut and adult salivary gland after the RNAi treatments. These results support our hypothesis that the candidate proteins Fo-GN and Fo-Cyp1 act in TSWV entry and multiplication in F. occidentalis.

ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

Genetic Study of Elf5 and Ehf in the Mouse Salivary Gland

Salivary gland (SG) development, maturation, and homeostasis require coordinated roles of transcription factors (TFs) that dictate specific cell identities and fate. The ETS family of proteins are important transcriptional drivers of diverse cell lineages, tissue development, and differentiation programs and hence are also likely to play an important role in the SG. Here we have leveraged genomic and epigenomic data of the SG to examine the expression profile of ETS genes and identified 2 closely related paralogs, Elf5 and Ehf, that are highly expressed in distinct epithelial subpopulations. By using a well-defined mouse knockout model of Elf5, we show that Elf5, despite its enriched expression in the acinar cells, is functionally dispensable for maintaining the homeostatic state of the adult SG epithelium. The lack of a discernible phenotype of the Elf5-null SG might be due to possible functional redundancy with Ehf or other ETS factors. To probe this possibility and to examine the specific consequences of Ehf loss in the SG, we used CRISPR-Cas9 to generate mice in which the DNA-binding ETS domain of Ehf is disrupted due to an insertion mutation. We demonstrate that the Ehf mutant (EhfMut) mice exhibit a distinct cellular phenotype with decreased granular convoluted tubules that are accompanied by an increased accumulation of the intercalated Sox9-positive ductal cell population. Interestingly, the ductal phenotype of the EhfMut animals is highly pronounced in males, reaffirming the established sexual dimorphism of the SG that exists in rodents. Our results show that unlike Elf5, Ehf plays a nonredundant role in directing ductal cell differentiation of the SG and highlights the phenotypic subtlety in mutant mice of closely related TFs and the importance of careful consideration of cell type–specific studies.

Outcomes Following Proton Radiotherapy for Pediatric Salivary Gland Tumors

<h3>Purpose/Objective(s)</h3> In contrast to adult salivary gland tumors, our institutional guidelines in children utilize lower radiation doses, smaller target margins, and proton therapy to minimize radiation to the developing skull base anatomy. Furthermore, we do not routinely recommend prophylactic nodal irradiation or elective neck dissection. Previous series of children treated with adjuvant radiation therapy report 5-year local control ∼85%. Herein we report treatment outcomes using our pediatric approach. <h3>Materials/Methods</h3> Twenty-seven consecutive pediatric patients were treated with proton therapy between 2006 and 2020 for mucoepidermoid carcinoma (n= 13), acinic cell carcinoma (n= 5), adenoid cystic carcinoma (n= 4), pleomorphic adenoma (n= 2), myoepithelial carcinoma (n= 1), adenocarcinoma (n= 1), and sialoblastoma (n= 1) at our institution. The 8 patients with radiographic lymphadenopathy (cN1) underwent neck dissection prior to presentation (7/8 selective dissections involving <4 nodal levels). The most common indications for adjuvant radiotherapy were microscopic positive or close (<1 mm) margins (n=15), perineural invasion (PNI; n=9), high-grade histology (n=6), and gross residual disease (n=4). Radiotherapy was delivered in sequential plans using 2 clinical target volumes (CTV1, CTV2). Gross tumor volume (GTV=CTV2) included the residual tumor and tumor bed. CTV1 received 50.4 Gy and was defined as GTV + 1 cm and the at-risk neck if indicated. The dose to CTV2 depended on histology, grade, and margin status. The median total dose was 64.8 Gy (range, 61.2-70.2 Gy). Only 3 patients underwent prophylactic nodal irradiation. Toxicity data was graded based on CTCAE v3 or v4 using patient-reported and physician-reported outcomes according to our prospective outcome registry. <h3>Results</h3> The median follow-up was 5.5 years. Overall survival was 100% and local control was 96% with a single local recurrence seen in a patient with gross unresectable disease. There were no nodal recurrences. In terms of acute toxicity, 3 patients experienced mucositis requiring opioid pain medication and 2 patients developed grade 3 radiation dermatitis. The most serious late toxicity involved the ear or auditory system including 4 patients requiring hearing aids, 1 with chronic otitis media, 1 with osteonecrosis of the mastoid bone, and 1 with external ear canal stenosis. Four additional patients reported variable hearing loss not requiring hearing aids. Other serious late toxicity included 1 patient with persistent trismus, 1 with growth hormone deficiency, and 1 with excessive dental caries along the ipsilateral maxilla. No secondary malignancies nor toxicities grade ≥ 4 were seen. <h3>Conclusion</h3> An approach of conservative neck management and moderate-dose proton radiotherapy delivered to small volumes resulted in excellent disease control and limited toxicity in children with common salivary gland tumors at elevated risk of local recurrence.

Role of quiescent cells in the homeostatic maintenance of the adult submandibular salivary gland

SummaryStem/progenitor cells are required for maintenance of salivary gland (SG) function and serve as untapped reservoirs to create functional cells. Despite recent advancements in the identification of stem/progenitor pools, in the submandibular gland (SMG), a knowledge gap remains. Furthermore, the contribution to adult SMG homeostasis of stem/progenitor cells originating from embryonic development is unclear. Here, we employ an H2B-GFP embryonic and adult pulse-and-chase system to characterize potential SMG stem/progenitor cells (SGSCs) based on quiescence at different stages. Phenotypical profiling of quiescent cells in the SMG revealed that label-retaining cells (LRCs) of embryonic or adult origin co-localized with CK8+ ductal or vimentin + mesenchymal, but not with CK5+ or CK14 + stem/progenitor cells. These SMG LRCs failed to self-renew in vitro while non-label retaining cells displayed differentiation and long-term expansion potential as organoids. Collectively, our data suggest that an active cycling population of cells is responsible for SMG homeostasis with organoid forming potential.

A deeper insight into the sialome of male and female Ochlerotatus triseriatus mosquitoes

Over the last 20 years, advancements in sequencing technologies have highlighted the unique composition of the salivary glands of blood-feeding arthropods. Further biochemical and structural data demonstrated that salivary proteins can disrupt host hemostasis, inflammation and immunity, which favors pathogen transmission. Previously, a Sanger-based sialome of adult Ochlerotatus triseriatus female salivary glands was published based on 731 expressed sequence tag (ESTs). Here, we revisited O. triseriatus salivary gland contents using an Illumina-based sequencing approach of both male and female tissues. In the current data set, we report 10,317 DNA coding sequences classified into several functional classes. The translated transcripts also served as a reference database for proteomic analysis of O. triseriatus female saliva, in which unique peptides from 101 proteins were found. Finally, comparison of male and female libraries allowed for the identification of female-enriched transcripts that are potentially related to blood acquisition and virus transmission.

Engineering cryoelectrospun elastin-alginate scaffolds to serve as stromal extracellular matrices

Scaffold-based regenerative strategies that emulate physical, biochemical, and mechanical properties of the native extracellular matrix (ECM) of the region of interest can influence cell growth and function. Existing ECM-mimicking scaffolds, including nanofiber (NF) mats, sponges, hydrogels, and NF-hydrogel composites are unable to simultaneously mimic typical composition, topography, pore size, porosity, and viscoelastic properties of healthy soft-tissue ECM. In this work, we used cryoelectrospinning to fabricate 3D porous scaffolds with minimal fibrous backbone, pore size and mechanical properties similar to soft-tissue connective tissue ECM. We used salivary glands as our soft tissue model and found the decellularized adult salivary gland (DSG) matrix to have a fibrous backbone, 10–30 μm pores, 120 Pa indentation modulus, and ∼200 s relaxation half time. We used elastin and alginate as natural, compliant biomaterials and water as the solvent for cryoelectrospinning scaffolds to mimic the structure and viscoelasticity of the connective tissue ECM of the DSG. Process parameters were optimized to produce scaffolds with desirable topography and compliance similar to DSG, with a high yield of >100 scaffolds/run. Using water as solvent, rather than organic solvents, was critical to generate biocompatible scaffolds with desirable topography; further, it permitted a green chemistry fabrication process. Here, we demonstrate that cryoelectrospun scaffolds (CESs) support penetration of NIH 3T3 fibroblasts 250–450 µm into the scaffold, cell survival, and maintenance of a stromal cell phenotype. Thus, we demonstrate that elastin-alginate CESs mimic many structural and functional properties of ECM and have potential for future use in regenerative medicine applications.

Aldehyde dehydrogenase 3A1 deficiency leads to mitochondrial dysfunction and impacts salivary gland stem cell phenotype.

Adult salivary stem/progenitor cells (SSPC) have an intrinsic property to self-renew in order to maintain tissue architecture and homeostasis. Adult salivary glands have been documented to harbor SSPC, which have been shown to play a vital role in the regeneration of the glandular structures postradiation damage. We have previously demonstrated that activation of aldehyde dehydrogenase 3A1 (ALDH3A1) after radiation reduced aldehyde accumulation in SSPC, leading to less apoptosis and improved salivary function. We subsequently found that sustained pharmacological ALDH3A1 activation is critical to enhance regeneration of murine submandibular gland after radiation damage. Further investigation shows that ALDH3A1 function is crucial for SSPC self-renewal and survival even in the absence of radiation stress. Salivary glands from Aldh3a1–/– mice have fewer acinar structures than wildtype mice. ALDH3A1 deletion or pharmacological inhibition in SSPC leads to a decrease in mitochondrial DNA copy number, lower expression of mitochondrial specific genes and proteins, structural abnormalities, lower membrane potential, and reduced cellular respiration. Loss or inhibition of ALDH3A1 also elevates ROS levels, depletes glutathione pool, and accumulates ALDH3A1 substrate 4-hydroxynonenal (4-HNE, a lipid peroxidation product), leading to decreased survival of murine SSPC that can be rescued by treatment with 4-HNE specific carbonyl scavengers. Our data indicate that ALDH3A1 activity protects mitochondrial function and is important for the regeneration activity of SSPC. This knowledge will help to guide our translational strategy of applying ALDH3A1 activators in the clinic to prevent radiation-related hyposalivation in head and neck cancer patients.

Indications for surgical treatment of major salivary glands pathologies with epidemiology analysis in adults - cohort study of 1173 cases.

The review of indications for surgical treatment of major salivary glands in adults hospitalized in the Department of Otorhinolaryngology, Head and Neck Surgery, Medical University of Warsaw. The retrospective analysis was based on the 1173 postoperative histopathological examinations of the salivary glands collected over a period of 10 years (2010-2020). Analysis included histopathological diagnosis, localization of lesions, multifocality, complete resection, lymph node involvement, as well as demographical data of sex and age. Over half (61.38%) of all indications for surgical treatment of the salivary glands were benign tumors (n = 720) with the most common pleomorphic adenoma, which accounted for 33.5% of all cases (n = 393). The next most frequent group of diagnoses were non-neoplastic diseases of the salivary glands, 24.98% of all cases (n = 293). Malignant neoplasms of the salivary glands accounted for 13.64% of all diagnoses (n = 160). Salivary gland diseases slightly predominated among the female sex, with a particularly pronounced predominance in pleomorphic adenoma. Men, on the other hand, were treated more often for malignant neoplasms. The mean age of the patients was the lowest in the group of non-neoplastic diseases of the salivary glands. The mean age of patients with malignant neoplasms was significantly higher than in other pathologies. The largest tumors size was identified for malignant neoplasms. Diseases of the salivary glands treated surgically were most often located in the parotid gland, with the exception of non-neoplastic diseases, which most often involved the submandibular gland. Surgical management in pathologies of the salivary glands applies to all types of lesions, both neoplastic and non-neoplastic diseases. Patients with particular diseases are characterized by a different structure of age, sex and location of changes.

Application of the Milan System for Reporting Salivary Gland Cytopathology in pediatric patients: An international, multi-institutional study.

Pediatric salivary gland fine-needle aspiration (FNA) is uncommon with a higher frequency of inflammatory lesions and a small proportion of malignancies. This international, multi-institutional cohort evaluated the application of the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) and the risk of malignancy (ROM) for each diagnostic category. Pediatric (0- to 21-year-old) salivary gland FNA specimens from 22 international institutions of 7 countries, including the United States, England, Italy, Greece, Finland, Brazil, and France, were retrospectively assigned to an MSRSGC diagnostic category as follows: nondiagnostic, nonneoplastic, atypia of undetermined significance (AUS), benign neoplasm, salivary gland neoplasm of uncertain malignant potential (SUMP), suspicious for malignancy (SM), or malignant. Cytology-histology correlation was performed where available, and the ROM was calculated for each MSRSGC diagnostic category. The cohort of 477 aspirates was reclassified according to the MSRSGC as follows: nondiagnostic, 10.3%; nonneoplastic, 34.6%; AUS, 5.2%; benign neoplasm, 27.5%; SUMP, 7.5%; SM, 2.5%; and malignant, 12.4%. Histopathologic follow-up was available for 237 cases (49.7%). The ROMs were as follows: nondiagnostic, 5.9%; nonneoplastic, 9.1%; AUS, 35.7%; benign neoplasm, 3.3%; SUMP, 31.8%; SM, 100%; and malignant, 100%. Mucoepidermoid carcinoma was the most common malignancy (18 of 237; 7.6%), and it was followed by acinic cell carcinoma (16 of 237; 6.8%). Pleomorphic adenoma was the most common benign neoplasm (95 of 237; 40.1%). The MSRSGC can be reliably applied to pediatric salivary gland FNA. The ROM of each MSRSGC category in pediatric salivary gland FNA is relatively similar to the ROM of each category in adult salivary gland FNA, although the reported rates for the different MSRSGC categories are variable across institutions.

Multiple stimulation with spheroids comprising salivary gland and adipose-derived stem cells enhances regeneration of radiation-damaged salivary glands

Tissue engineering approaches using a single stem cell type have been unsuccessful in achieving regeneration of radiation-damaged salivary glands (SGs). Therefore, we combined adult SG stem cells (SGSCs) and adipose-derived stem cells (ASCs) in optimized spheroids to enhance the regeneration of damaged SGs in mice. SGSC/ASC spheroids survived longer and secreted more vascular endothelial growth factor (VEGF) than did SGSC spheroids. To enhance cell viability and differentiation, SGSC/ASC spheroids were stimulated with the Wnt/β-catenin signaling activator CHIR99021 and retinoic acid inhibitor BMS431 to maintain stemness. FGF7/10 induced the differentiation of multiple stimulated SGSC/ASC spheroids. SGSC/ASC spheroids exhibited improved viability, calcium channel function, differentiation, and VEGF secretion after multiple, sequential stimulation. The 21-day survival rate of multiply stimulated SGSC/ASC spheroids was two-fold that of non-treated SGSC/ASC spheroids. Optimized SGSC/ASC spheroids enhanced the in vivo regeneration and functional recovery of SG tissue in mice by promoting neovascularization. Genes related to VEGF, such as Vegfa and Vegfb, were highly expressed 2∼6 times for in vitro and 1.5 times for in vivo experiment in the multiply stimulated SGSC/ASC spheroids. Therefore, SGSC/ASC spheroids that have undergone multiple stimulation may be useful for healing radiation-damaged SGs.

Carcinoma de células acinares de glándulas salivales: estudio clínico e histopatológico de 12 casos

Objective:Acinic cell carcinoma (CCA) is the third malignant epithelial tumor of the salivary glands in adults; low-grade tumor of malignancy, composed of neoplastic cells with serous acinar differentiation. The objective of this work was to analyze 12 cases of CCA according to their location, clinical characteristics, histological and immunohistochemical pattern and cell types, following the latest classification of the World Health Organization. Methods: The study included 12 cases of CCA from the files of salivary tumor biopsies of our work team, corresponding to the period 1997-2020. A numerical code was used to identify the samples, preserving the identity of the patients. Histological sections of the paraffin-embedded biopsies were evaluated with H/E, PAS and Toluidine blue and immunostained with the monoclonal antibodies pancytokeratin AE1 / AE3, Ki67, MUC-1 and mammaglobin. Results: The most frequent histologic pattern was the solid type as a single pattern or integrated with other patterns of lesser development, with almost exclusive location in the parotid gland and more frequent in women. Cells like normal acinar serocytes predominated in the solid growth pattern. The most frequent cell type in the microcystic patternwas the nonspecific glandular cell together with a lower proportion of acinar and intercalated duct-like cells. The papillary-cystic pattern was lined by nonspecific glandular cells. No clear cells found. With Ki67 a low cell proliferation was demonstrated in all the cases studied. Cell labeling for MUC-1 was grade 1 positive (less than 10% immunoreactive cells) and negative for mammaglobin.Conclusions: Patient follow-up is a priority because CCA tends to recur and metastasize and its behavior can become aggressive. We must deepen the study of its proliferative capacity as a treatment and prognosis tool, especially with immunohistochemistry and standardized molecular biology methods.

'Candidatus Mycoplasma haematohydrochoerus', a novel hemoplasma species in capybaras (Hydrochoerus hydrochaeris) from Brazil.

Three different species of hemoplasmas have been described in rodents, Mycoplasma coccoides, 'Candidatus Mycoplasma haemomuris' and 'Candidatus Mycoplasma haemosphiggurus'. Additionally, potentially novel hemoplasma species have been detected in wild rodents from Brazil, including capybaras (Hydrochoerus hydrochaeris). Capybaras are the largest rodent in the world and are well adapted to live within close proximity to humans, which increases the risk to spread of zoonotic pathogens. Herein, we investigate the occurrence and genetic diversity of hemoplasmas infecting free-ranging capybaras from southern Brazil. Blood samples and ticks from 17 capybaras were collected. Packed cell volume and total plasma protein were measured, DNA was extracted, and further screened by species-specific and pan-hemoplasma PCR assays targeting the 16S rRNA gene of hemoplasmas. Sixteen out of 17 (94.12%; 95% CI: 73.02-98.95%) were anemic. Only one young female was hypoproteinemic. All capybaras were infested by adults and nymphs of Amblyomma dubitatum ticks. Using the PCR assay targeting the 16S rRNA gene of M. coccoides, 13/17 (76.47%; 95% CI: 52.74-90.44%) capybaras were positive for hemoplasmas. When DNA samples were tested by the pan-hemoplasma PCR, 16/17 (94.12%; 95% CI: 73.02-98.95%) animals were positive. One out of 11 (9.09%) adult ticks salivary glands tested positive for hemoplasma by the pan-hemoplasma PCR assay. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a novel hemotropic Mycoplasma sp. previously reported in capybaras from Brazil. Additionally, sequencing and phylogenetic analysis of the 23S rRNA gene from three hemoplasma-positive capybaras samples from a previous study performed in midwestern Brazil also confirm our findings. Based on phylogenetic and Neighbor-Net network analysis of the 16S rRNA and 23S rRNA genes, the name 'Candidatus Mycoplasma haematohydrochoerus' is proposed for this novel organism.

Loss of Hs3st3a1 or Hs3st3b1 enzymes alters heparan sulfate to reduce epithelial morphogenesis and adult salivary gland function

Heparan sulfate 3-O-sulfotransferases generate highly sulfated but rare 3-O-sulfated heparan sulfate (HS) epitopes on cell surfaces and in the extracellular matrix. Previous ex vivo experiments suggested functional redundancy exists among the family of seven enzymes but that Hs3st3a1 and Hs3st3b1 sulfated HS increases epithelial FGFR signaling and morphogenesis. Single-cell RNAseq analysis of control SMGs identifies increased expression of Hs3st3a1 and Hs3st3b1 in endbud and myoepithelial cells, both of which are progenitor cells during development and regeneration. To analyze their in vivo functions, we generated both Hs3st3a1−/- and Hs3st3b1−/- single knockout mice, which are viable and fertile. Salivary glands from both mice have impaired fetal epithelial morphogenesis when cultured with FGF10. Hs3st3b1−/- mice have reduced intact SMG branching morphogenesis and reduced 3-O-sulfated HS in the basement membrane. Analysis of HS biosynthetic enzyme transcription highlighted some compensatory changes in sulfotransferases expression early in development. The overall glycosaminoglycan composition of adult control and KO mice were similar, although HS disaccharide analysis showed increased N- and non-sulfated disaccharides in Hs3st3a1−/− HS. Analysis of adult KO gland function revealed normal secretory innervation, but without stimulation there was an increase in frequency of drinking behavior in both KO mice, suggesting basal salivary hypofunction, possibly due to myoepithelial dysfunction. Understanding how 3-O-sulfation regulates myoepithelial progenitor function will be important to manipulate HS-binding growth factors to enhance tissue function and regeneration.

3D Organoid Culture From Adult Salivary Gland Tissues as an ex vivo Modeling of Salivary Gland Morphogenesis.

Lumen formation of salivary glands has been investigated using in vivo or ex vivo rudiment culture models. In this study, we used a three-dimensional (3D) salivary gland organoid culture system and demonstrated that lumen formation could be recapitulated in mouse SMG organoids. In our organoid culture system, lumen formation was induced by vasoactive intestinal peptide and accelerated by treatment with RA. Furthermore, lumen formation was observed in branching duct-like structure when cultured in combination of fibroblast growth factors (FGF) in the presence of retinoic acid (RA). We suggest RA signaling-mediated regulation of VIPR1 and KRT7 as the underlying mechanism for lumen formation, rather than apoptosis in the organoid culture system. Collectively, our results support a fundamental role for RA in lumen formation and demonstrate the feasibility of 3D organoid culture as a tool for studying salivary gland morphogenesis.

TAZ inhibits acinar cell differentiation but promotes immature ductal cell proliferation in adult mouse salivary glands.

There are currently no treatments for salivary gland diseases, making it vital to understand signaling mechanisms operating in acinar and ductal cells so as to develop regenerative therapies. To date, little work has focused on elucidating the signaling cascades controlling the differentiation of these cell types in adult mammals. To analyze the function of the Hippo-TAZ/YAP1 pathway in adult mouse salivary glands, we generated adMOB1DKO mice in which both MOB1A and MOB1B were TAM-inducibly deleted when the animals were adults. Three weeks after TAM treatment, adMOB1DKO mice exhibited smaller submandibular glands (SMGs) than controls with a decreased number of acinar cells and an increased number of immature dysplastic ductal cells. The mutants suffered from reduced saliva production accompanied by mild inflammatory cell infiltration and fibrosis in SMGs, similar to the Sjogren's syndrome. MOB1-deficient acinar cells showed normal proliferation and apoptosis but decreased differentiation, leading to an increase in acinar/ductal bilineage progenitor cells. These changes were TAZ-dependent but YAP1-independent. Biochemically, MOB1-deficient salivary epithelial cells showed activation of the TAZ/YAP1 and β-catenin in ductal cells, but reduced SOX2 and SOX10 expression in acinar cells. Thus, Hippo-TAZ signaling is critical for proper ductal and acinar cell differentiation and function in adult mice.

A U.S. isolate of Theileria orientalis, Ikeda genotype, is transmitted to cattle by the invasive Asian longhorned tick, Haemaphysalis longicornis

BackgroundTheileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T.orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia–Pacific region, where it affects 25% of Australian cattle, T.orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T.orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H.longicornis ticks were identified on cattle in the T.orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T.orientalis Ikeda. A strain of T.orientalis from a previous U.S. outbreak was not transmissible by H.longicornis; however, H.longicornis is the primary tick vector of T.orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H.longicornis ticks in the U.S. are competent vectors of T.orientalis Ikeda.MethodsNymphal H.longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T.orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T.orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T.orientalis Ikeda infection via blood smear cytology and PCR.ResultsAfter acquisition feeding on a VA-U.S.-T.orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T.orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T.orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4–0.9%.ConclusionsOur findings demonstrate that U.S. H.longicornis ticks are competent vectors of the VA-U.S.-T.orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures.Graphical