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.

Shear wave elastography of parotid glands in pediatric patients with HIV infection.

OBJECTIVES: Parotid gland (PG) involvement is common among the patients with HIV infection. Shear wave elastography (SWE) is a noninvasive method used to measure the tissue stiffness of several organs including PG. The aim of this study was to evaluate the tissue stiffness values of PGs of HIV-infected children via SWE and compare the results with the counterparts of healthy subjects. MATERIALS AND METHODS: This single-center, prospective study included the PG examinations of 23 pediatric HIV patients and 40 healthy children via grayscale ultrasound and SWE. Independent sample T test and Mann-Whitney U test were used in statistical analysis. RESULTS: Stiffness of both PGs was significantly higher in patients' group when compared with control subjects. In addition, when the patients were separated into two groups according to the appearance of PG on grayscale ultrasound as homogeneous and heterogeneous, stiffness values were increased in the patients with homogeneous parenchymal appearance. No significant difference was achieved in terms of median CD4 and CD8 counts, HIV RNA levels or median duration of illnesses. CONCLUSIONS: PG examination of HIV-infected children via SWE reveals increased tissue stiffness when compared with healthy subjects. SWE can be used as an ultrasound-assisted noninvasive technique in this manner.

HIV-associated benign lymphoepithelial cysts of the parotid glands confirmed by HIV-1 p24 antigen immunostaining.

Approximately 1%-10% of patients with HIV infection have been reported to have salivary gland enlargement. Parotid swelling in patients with HIV is often associated with salivary gland disease, including benign lymphoepithelial cysts (BLECs). The presence of BLEC can serve as an indicator of HIV infection, and the diagnosis of HIV-associated BLEC is usually based on clinical course, HIV confirmatory blood testing, such as western blot or viral detection, and imaging studies, but not on biopsies or immunostaining. To exclude other diseases such as tuberculosis and malignant lymphoma and to further improve the diagnostic accuracy of BLEC, the detection of the HIV-1 p24 antigen by immunohistochemistry is a useful diagnostic method. We report a case of a 65-year-old Japanese man with swelling of the parotid glands and HIV-associated BLEC confirmed via HIV-1 p24 immunohistochemical staining.

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.

Immune reactivity after adenoviral-mediated aquaporin-1 cDNA transfer to human parotid glands.

OBJECTIVES: The purpose of this study was to examine the humoral and cellular immune reactivity to adenoviral vector (AdhAQP1) administration in the human parotid gland over the first 42 days of a clinical gene therapy trial. METHODS: Of eleven treated subjects, five were considered as positive responders (Baum et al, 2012). Herein, we measured serum neutralizing antibody titers, circulating cytotoxic lymphocytes, and lymphocyte proliferation in peripheral blood mononuclear cells. Additionally, after adenoviral vector stimulation of lymphocyte proliferation, we quantified secreted cytokine levels. RESULTS: Responders showed little to modest immune reactivity during the first 42 days following gene transfer. Additionally, baseline serum neutralizing antibody titers to serotype 5-adenovirus generally were not predictive of a subject's response to parotid gland administration of AdhAQP1. Cytokine profiling from activated peripheral blood mononuclear cells could not distinguish responders and non-responders. CONCLUSIONS: The data are the first to describe immune responses after adenoviral vector administration in a human parotid gland. Importantly, we found that modest (2-3 fold) changes in systemic cell-mediated immune reactivity did not preclude positive subject responses to gene transfer. However, changes beyond that level likely impeded the efficacy of gene transfer.

Activation of type I interferon signaling in the parotid and exorbital lachrymal glands during the acute phase of encephalomyocarditis (EMC) virus infection in mice.

The present study was carried out to clarify the mechanisms of EMC virus-induced sialodacryoadenitis in mice during the acute phase infection focusing on the activation of type I interferon (IFN) signaling in the parotid and exorbital lachrymal glands. In the parotid gland, a few apoptotic acinar cells were detected at 2days post inoculation (DPI). The ratio of apoptotic acinar cells increased at 3 and 4DPI. On the other hand, in the exorbital lachrymal gland, apoptosis of acinar cells and infiltration of inflammatory cells mainly composed of mononuclear cells started at 3DPI, and prominent acinar cell damage developed at 4DPI. Viral RNA was detected at 3 and 4DPI in both glands and the expression level was higher in the exorbital lachrymal gland than in the parotid gland. The up-regulation of IFN-stimulated genes (ISGs), such as Irf7, Pkr and Oas, was quickly induced at 2DPI in the parotid gland, and this probably contributed to suppress viral replication and to eliminate affected cells by apoptosis. In the exorbital lachrymal gland, the expression levels of ISGs mRNAs were not elevated at 2DPI, suggesting no induction of an effective anti-viral response such as apoptosis at this time point. In the exorbital lachrymal gland, the mRNA expression of IFN beta and IFN alpha (type I IFNs) was weak- to strong-positive at 1DPI, and became negative at 2DPI. The weak- to strong-positive expression of IFNs at 1DPI is likely related to the abrupt viral replication and pathological changes in the exorbital lachrymal gland through activating the negative feedback regulation that depressed the IFN signaling cascade at 2DPI. In conclusion, the present study showed the changes in factors involved in the activation of type I IFN signaling cascade in the parotid and exorbital lachrymal glands and their differences between the two glands during the acute phase of EMC virus infection in mice.

Epstein-barr virus-associated extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT Lymphoma) arising in the parotid gland of a child with ataxia telangiectasia.

Hematologic malignancies, in particular T-cell lymphomas/leukemias, are prevalent in patients with ataxia telangiectasia (AT), with most reported cases being clinically aggressive and high grade. Epstein-Barr virus (EBV) is often associated with lymphoid proliferations/neoplasms arising in immunodeficient patients. Reports of low-grade B-cell neoplasms in the ataxia telangiectasia population are extremely rare. Here, we describe a case of EBV-associated extranodal marginal zone lymphoma (mucosa-associated lymphoid tissue lymphoma) of the parotid gland in a 16-year-old boy with AT. In addition, we review the literature of hematologic malignancies in the AT population as well as the occurrence of EBV in mucosa-associated lymphoid tissue lymphoma.

[STUDY OF SAFETY OF PAROTITIS VACCINE].

AIM: Monitoring of post-vaccinal complications in children immunized with a parotitis vaccine. MATERIALS AND METHODS: Observation of 198 945 children, immunized with 16 lots of parotitis vaccine with Leningrad-3 strain (L-3), was carried out for 3 years. Paired samples of sera and saliva were obtained from children, in whom adverse events were registered for 42 days after vaccination. Titers of specific IgM and IgG were determined in blood sera. Analysis of nucleotide sequences of genes F, SH and NH of RNA of parotitis virus was carried out from samples of blood and saliva. RESULTS: Intensive parameter of vaccine-associated aseptic meningitis under the conditions of the experiments was 0 for 100 000 immunized. Frequency of occurrence of post-vaccinal parotitis was 0.06% from the number of vaccinated--18 cases of vaccine-associated parotitis were registered and laboratory confirmed. A significant difference in specific activity was detected for 3 lots of the vaccine, that were associated with cases of development of parotitis, relative to that of 13 lots of vaccine, development of parotitis was not registered after administration of those. CONCLUSION: The study carried out confirmed low neurovirulence of the parotitis vaccine with the L-3 strain of parotitis virus, as well as a low degree of its reactogenicity. A relatively high immunization dose of the used vaccine could be one of the reasons of development of post-vaccinal complications in part of the immunized children.

Cystic lymphoid hyperplasia of the parotid gland as the initial manifestation of HIV infection.

We report the case of a patient who presented with cystic lymphoid hyperplasia of the right parotid gland as the index diagnosis of HIV infection. Histological examination of the excised parotid gland revealed a solid-cystic lymphoepithelial lesion with a non-keratinous squamous epithelium, which grew into the lymphoid component via anastomosing cords and islands. These anastomosing cords and islands contained variably abundant B cells, several subepithelial multinucleated histiocytes, salivary ducts infiltrated by small lymphocytes, and a dense lymphoid infiltrate containing lymphoid follicles with enlarged, irregular germinal centres.

Infection of mice, ferrets, and rhesus macaques with a clinical mumps virus isolate.

In recent years, many mumps outbreaks have occurred in vaccinated populations worldwide. The reasons for these outbreaks are not clear. Animal models are needed to investigate the causes of outbreaks and to understand the pathogenesis of mumps virus (MuV). In this study, we have examined the infection of three animal models with an isolate of mumps virus from a recent outbreak (MuV-IA). We have found that while both ferrets and mice generated humoral and cellular immune responses to MuV-IA infection, no obvious signs of illness were observed in these animals; rhesus macaques were the most susceptible to MuV-IA infection. Infection of rhesus macaques via both intranasal and intratracheal routes with MuV-IA led to the typical clinical signs of mumps 2 weeks to 4 weeks postinfection. However, none of the infected macaques showed any fever or neurologic signs during the experimental period. Mumps viral antigen was detected in parotid glands by immunohistochemistry (IHC). Rhesus macaques represent the best animal model for the study of mumps virus pathogenesis.

Lymphoepithelial carcinoma: a case of a rare parotid gland tumor.

A 29-year-old woman presented from another hospital with a 10-month history of an enlarging left-sided facial mass. Computed tomographic scan revealed a mass in the superficial lobe of the left parotid gland with left-sided cervical lymphadenopathy. The patient received a total left parotidectomy and a selective neck dissection. Histopathologic slides revealed lymphoepithelial carcinoma (LEC) that stained positive for cytokeratin, as well as Epstein-Barr virus (EBV). An LEC of the parotid is a rare salivary gland tumor accounting for less than 1% of all salivary gland tumors. As reaffirmed in our case, LEC is more common in women, occurs primarily in the parotid gland, and has an ethnic predilection. Histologic analysis reveals an infiltrative, poorly differentiated tumor nestled in a lymphoid stroma, with near 100% positivity for EBV in endemic areas. Complete resection of this poorly differentiated carcinoma followed by postoperative radiation is essential for local control.

Hantavirus-specific IgA in saliva and viral antigen in the parotid gland in patients with hemorrhagic fever with renal syndrome.

The Hantavirus genus comprises rodent borne, zoonotic viruses of the Bunyaviridae family that cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Rodent saliva contains infectious hantavirus and evidence suggests that hantavirus is also shed in human saliva, but person-to-person transmission is rare. In saliva, immunoglobulin (Ig) A is the predominant immunoglobulin class. Secretory IgA serves as an important first line of defence on epithelial surfaces and the binding of secretory IgA to pathogens can inhibit adherence of microorganisms to mucosal cells and neutralize viruses. This study investigated the presence and importance of salivary IgA in relation to viral antigen in the saliva by testing Puumala hantavirus (PUUV) specific IgA, and RNA in saliva in acutely ill patients with HFRS. In saliva samples, PUUV specific IgA was detected in 12 of 33 (36%) patients with HFRS and 20 (61%) were PUUV RNA positive. There was a statistically significant inverse association between the presence of salivary IgA antibodies and PUUV RNA in the saliva. PUUV-specific IgA in saliva was not found in a long-term follow-up, while PUUV IgA in serum was detected in three patients, 28-32 months after the initial study. Notably, both PUUV RNA and PUUV nucleocapsid antigen were detected in endothelial cells within the parotid gland of a deceased patient with HFRS.

AAV5-mediated gene transfer to the parotid glands of non-human primates.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.

Failure to detect active virus replication in mast cells at various tissue sites of HIV patients by immunohistochemistry.

A recent report postulated that the mast cell population is a significant reservoir for persistent HIV infection. Our study attempted to validate this hypothesis by quantitatively comparing the distribution of mast cells and cells expressing the HIV protein p24 in HIV infected patients. Consecutive sections of paraffin-embedded human tissues from various tissue sites were subjected to immunohistochemistry with monoclonal antibodies to mast cell tryptase, viral protein p24, and other molecules. The sub-cellular distribution of these molecules was examined, to determine whether immunoreactivities to these molecules would be co-localized within the same cells. Our study revealed that, in two immediate adjacent sections immunostained for mast cell tryptase and p24, respectively, all or nearly all tryptase and p24 expressing cells were distributed at different areas. In the single section double immunostained for mast cell tryptase and p24, 5 (1.1%) of 460 large p24 expressing cell clusters encountered showed a single or few mast cells within or adjacent to p24 expressing cell clusters, but no distinct co-localization of these two proteins was observed. Similarly, no distinct co-localization was observed in any of over 500 isolated individual mast cells and p24 expressing cells. In contrast, macrophages were consistently intermixed with or adjacent to p24 expressing cells, and p24 immunostaining were seen in the cytoplasm of a subset of macrophages. These findings suggest that tissue mast cells do not show evidence for active virus replication by the techniques employed.

Otitis media observed in unexpected natural death of infants.

Intracranial complications due to otitis media such as brain abscess and leptomeningitis are well known as a cause of death. In recent years, encountering those serious intracranial complications in forensic medical practice is extremely rare. However, we rarely encounter autopsy cases with otitis media of which pathological damage is mild or moderate. We investigated 11 autopsy cases (6 cases of pneumonia and 5 cases of SIDS) in unexpected natural death of infants under one year old, and found 3 cases with otitis media. The tympanic cavity was investigated by chiseling a petrosal part of the base of the skull. In the case of otitis media, serous and mucous exudate containing leucocytes examined microscopically was observed. Otitis media, as such, was not a cause of death in our cases presented. Background factors causing otitis media seems to be not only functional insufficiency of the auditory tube but also other delicate constitution, hidden dysfunction or immature function in immune system, which could be easily infected. Of 3 cases of otitis media, cytomegalovirus infection was observed in 2 cases simultaneously. In our department, we have little opportunity to encounter autopsy cases of infant under one year old. If many infant cases could be investigated, many autopsy cases with otitis media might be encountered in unexpected infant deaths.

Characterization of large mumps outbreak among vaccinated Palestinian refugees.

During a large mumps virus (MuV) outbreak which occurred in the Palestinian refugee camps of the West Bank, 68.1% (2,636/3,871) of the cases were vaccinated with one dose of trivalent measles, mumps, and rubella (MMR) vaccine. Attack rates by camp ranged from less than 1 case per 1,000 people in the population to 43/1,000 (overall, 11/1,000). The outbreak lasted from December 2003 to June 2005, with two peaks, one from April to May 2004 and the other from March to April 2005. To control the outbreak, a mass MMR vaccination campaign was conducted in May 2005. Evaluation of the immune status of cases (n=59) and healthy controls (n=51) revealed high levels of mumps immunoglobulin G (IgG) and a low MuV-specific IgM in clinical cases indicative of a booster immune response. This suggested a secondary rather than a primary infection due to the insufficient protection conferred by the single vaccine dose included in the vaccination program. This prediction was further confirmed by the low seroprevalence (68.6%) found in the healthy control group, which was below the threshold level required for MuV herd immunity. Mumps diagnosis was established mainly by reverse transcription-PCR in clinical samples obtained within 48 h from the onset of disease. Of the parotid fluids and nasopharyngeal aspirates analyzed, 92% were positive for MuV RNA, while only 33% of the urine samples were positive. Phylogenetic analysis of the MuV SH gene identified the outbreak strain as the H genotype, which has been in circulation worldwide at least since 1989.

Lymphoepithelial cyst of the parotid gland: its possible histopathogenesis based on clinicopathologic analysis of 64 cases.

Sixty-four cases of lymphoepithelial cysts of the parotid gland, the largest scale collection in the literature, were clinicopathologically analyzed for their possible pathogenesis. All 64 cases were unilateral, 27 left and 37 right. There were 28 male and 36 female patients with a ratio of 1:1.3. The mean age of the patients was 52.0 years, and their average duration of symptoms was 29.3 months. The mean longest diameter of the cysts was 3.0 cm. Histologically, lymphoepithelial cysts were classified into 3 subtypes: type I, a cystic dilation of ducts within parotid glands (9 cases, 14.1%); type II, partially demarcated cystic lesions with lymphoid stroma (27, 42.2%); type III, well-encapsulated cystic lesions with lymphoid stroma containing lymph follicular structures (28, 43.8%). Based on immunohistochemical results for lymphocyte/macrophage (CD20/CD45RO/IgG4), cell cycle (Ki-67), and lymphatic (D2-40) markers, the lymphoid stroma was shown to have neither the usual lymph follicular distributions of T/B cells nor lymph sinus structures. No viral infection was confirmed. The results seemed to indicate that the lymphoid stroma were induced along with the growth of the cystic dilatation of ducts within sialadenitis, which were neither induced by Epstein-Barr virus nor HIV infections, and that the formation of lymphoepithelial cysts was completed by demarcation, which should have been a kind of granulation tissue reaction, from the parotid parenchyma but did not arise from intraparotid lymph nodes.

Multinucleated giant cells in HIV-associated benign lymphoepithelial cyst-like lesions of the parotid gland on FNAC.

Benign lymphoepithelial cyst-like lesion (BLEL), a previously rare lesion of the parotid gland consisting of marked lymphoid hyperplasia with accompanying squamous-lined cysts, has been described in patients with the acquired immune deficiency syndrome (AIDS) or AIDS risk factors. Histologically, these cysts are lined by a squamous or cuboidal epithelium. The lumen contains a pale homogenous material with foamy macrophages and lymphocytes with the cyst wall having germinal centers and a dense infiltrate of lymphoid cells. On FNAC, the aspirates are mostly cystic with the presence of reactive lymphoid tissue, numerous histiocytes, and metaplastic cell clusters. Multinucleated giant cells (MGCs) are also rarely seen in such lesions. We report a case of HIV-associated BLEL with numerous large sized multinucleated giant cells.

Detection of hepatitis B surface antigen, hepatitis B core antigen, and hepatitis B virus DNA in parotid tissues.

OBJECTIVE: To examine the presence of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and hepatitis B virus (HBV) DNA in parotid tissues from patients with positive serum HBV markers. METHODS: HBsAg and HBcAg were examined in parotid biopsy tissues from patients with suspected parotid tumor and positive serum HBV markers by immunocytochemistry, and HBV DNA was detected in parotid tissues by PCR. RESULTS: Among the 22 patients with a parotid tumor, only one was pathologically confirmed as a neoplasm; all others were benign. HBsAg and HBcAg were present in parotid cells with positive rates of 45.5% (10/22) and 40.9% (9/22), respectively, with an overall positive rate of 54.5% (12/22). Of the 22 cases with serum markers of HBV infection, seven (31.8%) had both HBsAg and HBcAg in the parotid cells. HBV DNA was present in seven of the 12 samples in which hepatitis B antigen was detected (58.3%). CONCLUSIONS: HBV in saliva might originate from the infected salivary glands and the infectious saliva could transmit HBV.

Lymphoepithelial carcinoma of the parotid gland: is an association with Epstein-Barr virus possible in non-endemic areas?

Lymphoepithelial carcinoma (LEC) is a rare histological type of cancer of the salivary glands. Here is reported a case of LEC of the parotid gland that developed in a Caucasian female, whose serology was positive for Epstein-Barr virus antibody. The patient underwent surgical treatment and postoperative radiotherapy. Because of the relatively limited clinical data concerning LEC of the salivary glands compared to other more common histological types, the clinical course, optimal treatment and prognosis have not been extensively studied. The aim of this report was to summarize all the key points, following a comprehensive literature review.