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1.  Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients. 
Journal of Clinical Microbiology  1994;32(5):1390-1394.
Our understanding of important stages in the pathogenesis of the human polyomavirus BK virus (BKV) and JC virus (JCV) infections is limited. In this context, nasopharyngeal aspirates from 201 children with respiratory diseases and saliva from 60 human immunodeficiency virus type 1-infected adults and 10 healthy adult controls were collected and analyzed for the presence of BKV and JCV DNA by PCR. Neither BKV nor JCV DNA was detected in the saliva specimens. We demonstrated BKV DNA, but no infectious BKV, in 2 of 201 nasopharyngeal aspirates. Each sample contained one unique rearranged noncoding control region variant of BKV. The results indicate that (i) BKV and JCV are not regularly associated with respiratory infections in children requiring hospitalization, (ii) nasopharyngeal cells are not an important site for primary replication of human polyomavirus BKV and JCV, and (iii) the salivary glands and oropharyngeal cells seem not to be involved in BKV and JCV persistence. We propose that for the polyomaviruses BKV and JCV the alimentary tract should be considered as a portal of entrance to the human organism.
PMCID: PMC263712  PMID: 8051277
2.  Functional Reorganization of Promyelocytic Leukemia Nuclear Bodies during BK Virus Infection 
mBio  2011;2(1):e00281-10.
BK virus (BKV) is the causative agent for polyomavirus-associated nephropathy, a severe disease found in renal transplant patients due to reactivation of a persistent BKV infection. BKV replication relies on the interactions of BKV with many nuclear components, and subnuclear structures such as promyelocytic leukemia nuclear bodies (PML-NBs) are known to play regulatory roles during a number of DNA virus infections. In this study, we investigated the relationship between PML-NBs and BKV during infection of primary human renal proximal tubule epithelial (RPTE) cells. While the levels of the major PML-NB protein components remained unchanged, BKV infection of RPTE cells resulted in dramatic alterations in both the number and the size of PML-NBs. Furthermore, two normally constitutive components of PML-NBs, Sp100 and hDaxx, became dispersed from PML-NBs. To define the viral factors responsible for this reorganization, we examined the cellular localization of the BKV large tumor antigen (TAg) and viral DNA. TAg colocalized with PML-NBs during early infection, while a number of BKV chromosomes were adjacent to PML-NBs during late infection. We demonstrated that TAg alone was not sufficient to reorganize PML-NBs and that active viral DNA replication is required. Knockdown of PML protein did not dramatically affect BKV growth in culture. BKV infection, however, was able to rescue the growth of an ICP0-null herpes simplex virus 1 mutant whose growth defect was partially due to its inability to disrupt PML-NBs. We hypothesize that the antiviral functions of PML-NBs are inactivated through reorganization during normal BKV infection.
BK virus (BKV) is a human pathogen that causes severe diseases, including polyomavirus-associated nephropathy in kidney transplant patients and hemorrhagic cystitis in bone marrow transplant recipients. How BKV replication is regulated and the effects of a lytic BKV infection on host cells at the molecular level are not well understood. Currently, there is no specific antiviral treatment for BKV-associated disease, and a better understanding of the complete life cycle of the virus is necessary. Here, we report the interplay between BKV and one of the regulatory structures in the host cell nucleus, promyelocytic leukemia nuclear bodies (PML-NBs). Our results show that BKV infection reorganizes PML-NBs as a strategy to inactivate the negative functions of PML-NBs.
PMCID: PMC3039439  PMID: 21304169
3.  Replication of Oral BK Virus in Human Salivary Gland Cells 
Journal of Virology  2014;88(1):559-573.
BK polyomavirus (BKPyV) is the most common viral pathogen among allograft patients. Increasing evidence links BKPyV to the human oral compartment and to HIV-associated salivary gland disease (HIVSGD). To date, few studies have analyzed orally derived BKPyV. This study aimed to characterize BKPyV isolated from throat wash (TW) samples from HIVSGD patients. The replication potential of HIVSGD-derived clinical isolates HIVSGD-1 and HIVSGD-2, both containing the noncoding control region (NCCR) architecture OPQPQQS, were assessed and compared to urine-derived virus. The BKPyV isolates displayed significant variation in replication potential. Whole-genome alignment of the two isolates revealed three nucleotide differences that were analyzed for a potential effect on the viral life cycle. Analysis revealed a negligible difference in NCCR promoter activity despite sequence variation and emphasized the importance of functional T antigen (Tag) for efficient replication. HIVSGD-1 encoded full-length Tag, underwent productive infection in both human salivary gland cells and kidney cells, and expressed viral DNA and Tag protein. Additionally, HIVSGD-1 generated DNase-resistant particles and by far surpassed the replication potential of the kidney-derived isolate in HSG cells. HIVSGD-2 encoded a truncated form of Tag and replicated much less efficiently. Quantitation of infectious virus, via the fluorescent forming unit assay, suggested that HIVSGD BKPyV had preferential tropism for salivary gland cells over kidney cells. Similarly, the results suggested that kidney-derived virus had preferential tropism for kidney cells over salivary gland cells. Evidence of HIVSGD-derived BKPyV oral tropism and adept viral replication in human salivary gland cells corroborated the potential link between HIVSGD pathogenesis and BKPyV.
PMCID: PMC3911688  PMID: 24173219
4.  Protection against Mycobacterium ulcerans Lesion Development by Exposure to Aquatic Insect Saliva 
PLoS Medicine  2007;4(2):e64.
Buruli ulcer is a severe human skin disease caused by Mycobacterium ulcerans. This disease is primarily diagnosed in West Africa with increasing incidence. Antimycobacterial drug therapy is relatively effective during the preulcerative stage of the disease, but surgical excision of lesions with skin grafting is often the ultimate treatment. The mode of transmission of this Mycobacterium species remains a matter of debate, and relevant interventions to prevent this disease lack (i) the proper understanding of the M. ulcerans life history traits in its natural aquatic ecosystem and (ii) immune signatures that could be correlates of protection. We previously set up a laboratory ecosystem with predatory aquatic insects of the family Naucoridae and laboratory mice and showed that (i) M. ulcerans-carrying aquatic insects can transmit the mycobacterium through bites and (ii) that their salivary glands are the only tissues hosting replicative M. ulcerans. Further investigation in natural settings revealed that 5%–10% of these aquatic insects captured in endemic areas have M. ulcerans–loaded salivary glands. In search of novel epidemiological features we noticed that individuals working close to aquatic environments inhabited by insect predators were less prone to developing Buruli ulcers than their relatives. Thus we set out to investigate whether those individuals might display any immune signatures of exposure to M. ulcerans-free insect predator bites, and whether those could correlate with protection.
Methods and Findings
We took a two-pronged approach in this study, first investigating whether the insect bites are protective in a mouse model, and subsequently looking for possibly protective immune signatures in humans. We found that, in contrast to control BALB/c mice, BALB/c mice exposed to Naucoris aquatic insect bites or sensitized to Naucoris salivary gland homogenates (SGHs) displayed no lesion at the site of inoculation of M. ulcerans coated with Naucoris SGH components. Then using human serum samples collected in a Buruli ulcer–endemic area (in the Republic of Benin, West Africa), we assayed sera collected from either ulcer-free individuals or patients with Buruli ulcers for the titre of IgGs that bind to insect predator SGH, focusing on those molecules otherwise shown to be retained by M. ulcerans colonies. IgG titres were lower in the Buruli ulcer patient group than in the ulcer-free group.
These data will help structure future investigations in Buruli ulcer–endemic areas, providing a rationale for research into human immune signatures of exposure to predatory aquatic insects, with special attention to those insect saliva molecules that bind to M. ulcerans.
Saliva from aquatic insects in areas where Buruli ulcer is endemic can protect mice against the disease's characteristic skin lesion and might play a role in natural immunity in humans.
Editors' Summary
Buruli ulcer disease is a severe skin infection caused by Mycobacterium ulcerans, a bacterium related to those that cause tuberculosis and leprosy. This poorly understood disease affects people living near slow-flowing or standing water in poor rural communities in tropical and subtropical countries. How people become infected with M. ulcerans is unclear but one possibility is that infected aquatic insects transmit it through their bites. The first sign of infection is usually a small painless swelling in the skin. Bacteria inside these swellings produce a toxin that damages nearby soft tissues until eventually the skin sloughs off to leave a large open sore. This usually heals but the resultant scar can limit limb movement. Consequently, 25% of people affected by Buruli ulcers—most of whom are children—are permanently disabled. If the disease is caught early, powerful antibiotics can prevent ulcer formation. But most patients do not seek help until the later stages when the only treatment is to cut out the infection and do a skin graft, a costly and lengthy treatment.
Why Was This Study Done?
There is currently no effective way to prevent Buruli ulcers. To develop an effective preventative strategy, researchers need to determine exactly how the infection is transmitted to people and what makes some individuals resistant to infection. Previous studies have indicated that 5%–10% of some aquatic insect predators that live in areas where Buruli ulcers occur have M. ulcerans in their salivary glands and that aquatic insects carrying M. ulcerans can transmit it to mice through bites. Furthermore, people working close to water inhabited by insect predators are less likely to develop Buruli ulcers than their relatives who do not work near water. In this study, therefore, the researchers investigated whether exposure to noninfected insect saliva provides some protection against M. ulcerans infection.
What Did the Researchers Do and Find?
The researchers let uninfected aquatic insects bite ten mice several times before exposing these mice and ten unbitten mice to M. ulcerans-infected water bugs. Only one pre-bitten mouse developed an M. ulcerans-containing lesion compared with eight control mice. Next, the researchers injected mice with insect salivary gland extracts before challenging them with “naked” M. ulcerans or bacteria coated with salivary gland extract. Most uninjected mice developed lesions when challenged with coated or naked M. ulcerans, as did experimental mice challenged with naked M. ulcerans. However, most experimental mice challenged with coated M. ulcerans remained lesion-free. In both experiments, the blood of the pre-bitten and extract-treated mice (but not the control mice) contained antibodies (immune system proteins that provide protection against infections and foreign proteins) to proteins in insect salivary gland extracts that stick to M. ulcerans. Finally, the researchers measured the blood concentration (the titer) of antibodies that bind insect salivary gland proteins in patients with Buruli ulcer and in healthy people living in the same area. People with high titers of these antibodies, they report, were less likely to have Buruli ulcers than those with low titers.
What Do These Findings Mean?
These findings suggest that exposure to aquatic insect saliva may provide some protection against M. ulcerans lesion development. However, the current results have several limitations. In particular they will only be relevant to human disease if M. ulcerans is normally transmitted by insect bites, and this has not been proven yet. Also, because the human study did not measure the overall immune status of the study participants, the people with Buruli ulcers may have had a general immune deficit rather than simply lacking antibodies against insect salivary gland proteins. However, if the human findings can be repeated and expanded, they suggest that low antibody titers to salivary gland proteins might identify those people who are most susceptible to M ulcerans infections and who would thus benefit most from regular tests for early signs of the disease. Finally, further work on the immune mechanism by which exposure to insect salivary gland proteins protects against M. ulcerans infections may help in the development of vaccines against Buruli ulcer disease.
Additional Information.
Please access these Web sites via the online version of this summary at
A related PLoS Medicine Perspective article by Manuel T. Silva and others discusses this study and others on insect-borne parasitic diseases
World Health Organization has information on Buruli ulcer disease
US Centers for Disease Control and Prevention has information on Buruli ulcer
The US Armed Forces Institute of Pathology Web site contains pages on Buruli ulcer
Leprosy Relief Emmaus Switzerland offers information on Buruli ulcer
Wikipedia contains pages on Buruli ulcer (note: Wikipedia is an online encyclopedia that anyone can edit)
PLoS Medicine has a detailed review article on Buruli ulcer by Paul D. R. Johnson and colleagues
PMCID: PMC1808094  PMID: 17326707
5.  Clinicopathological characteristics of immunoglobulin G4-related sialadenitis 
Immunoglobulin G4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition. Forty-two cases with immunoglobulin G4-related sialadenitis (IgG4-RS) confirmed by histopathological and immunohistochemical assessment were studied to clarify the clinicopathologic characteristics of the salivary glands involved in IgG4-RS, especially the relationship between the histopathologic features and function of salivary glands or serum levels of IgG4.
Clinical, serologic, imaging and histopathological data of these cases were analyzed. CT volumes of submandibular, parotid, and lacrimal glands were calculated. The saliva flow rate was measured. Scintigraphy with 99mTc-pertechnetate was undertaken in 31 cases, and the concentration index (CI) and secretion index (SI) was calculated. Relationships between fibrosis severity and salivary gland function or serum IgG4 levels were analyzed.
The first symptom was swelling of bilateral submandibular or lacrimal glands. Physical examination showed multiple bilateral major salivary glands (including sublingual and accessory parotid glands) and lacrimal glands were enlarged in IgG4 RS. Multiple enlarged cervical lymph nodes were noted in 30 patients. Saliva flow at rest was lower than normal in 34 cases; stimulated saliva flow was lower than normal in 15 cases. Secretory function was reduced more severely in the submandibular glands than in the parotid glands. Serum levels of IgG4 were elevated in 95.2% of cases and 78.6% patients had increased IgE levels. Serum IgG4 level was higher and saliva secretion lower as glandular fibrosis increased.
Prominent changes in the morphology, histology, immunohistochemistry and secretion of the major salivary glands of IgG4-RS patients were accompanied by involvement of the lacrimal glands and cervical lymph nodes. Elevated IgE, allergic history, eosinophil infiltration suggest allergic reactions as a potential pathogenesis of IgG4-RS. Severity of glandular fibrosis correlated with salivary function and serum levels of IgG4.
PMCID: PMC4508811  PMID: 26194097
6.  Polyomavirus Infection and Its Impact on Renal Function and Long-Term Outcomes after Lung Transplantation 
Transplantation  2009;88(3):360-366.
Polyomavirus infection causes nephropathy after kidney transplantation but has not been thoroughly investigated in nonrenal organ transplantation.
Ninety lung transplant recipients were enrolled and provided urine samples over 4.5 years. Samples were analyzed for BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) by conventional and quantitative real time polymerase chain reaction (PCR).
Fifty-nine (66%) patients had polyomavirus detected at least once, including 38 (42%) for BKV, 25 (28%) for JCV, and 6 (7%) for SV40. Frequency of virus shedding in serial urine samples by patients positive at least once varied significantly among viruses: JCV, 64%; BKV, 48%; SV40, 14%. Urinary viral loads for BKV (105.4copies/ml) and JCV (106.0 copies/ml) were higher than for SV40 (102.5 copies/ml; p=0.001 and p=0.0003, respectively). Polyomavirus infection was associated with a pre-transplant diagnosis of chronic obstructive pulmonary disease [odds ratio (OR) = 6.0; p=0.016], but was less common in patients with a history of acute rejection [OR=0.28; p=0.016]. SV40 infection was associated with sirolimus-based immunosuppression (p=0.037). Reduced survival was noted for patients with BKV infection (p=0.03). Patients with polyomavirus infection did not have worse renal function than those without infection, but in patients with BKV infection, creatinine clearances were lower at times when viral shedding was detected (p=0.038).
BKV and JCV were commonly detected in the urine of lung transplant recipients; SV40 was found at low frequency. No definite impact of polyomavirus infection on renal function was documented. BKV infection was associated with poorer survival.
PMCID: PMC2752200  PMID: 19667938
Polyomavirus; lung transplantation; kidney disease
7.  Aedes Mosquito Saliva Modulates Rift Valley Fever Virus Pathogenicity 
Rift Valley fever (RVF) is a severe mosquito-borne disease affecting humans and domestic ruminants. Mosquito saliva contains compounds that counteract the hemostatic, inflammatory, and immune responses of the host. Modulation of these defensive responses may facilitate virus infection. Indeed, Aedes mosquito saliva played a crucial role in the vector's capacity to effectively transfer arboviruses such as the Cache Valley and West Nile viruses. The role of mosquito saliva in the transmission of Rift Valley fever virus (RVFV) has not been investigated.
Using a murine model, we explored the potential for mosquitoes to impact the course of RVF disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva and salivary gland extract.
C57BL/6NRJ male mice were infected with the ZH548 strain of RVFV via intraperitoneal or intradermal route, or via bites from RVFV-exposed mosquitoes. The virus titers in mosquitoes and mouse organs were determined by plaque assays.
After intraperitoneal injection, RVFV infection primarily resulted in liver damage. In contrast, RVFV infection via intradermal injection caused both liver and neurological symptoms and this route best mimicked the natural infection by mosquitoes. Co-injections of RVFV with salivary gland extract or saliva via intradermal route increased the mortality rates of mice, as well as the virus titers measured in several organs and in the blood. Furthermore, the blood cell counts of infected mice were altered compared to those of uninfected mice.
Different routes of infection determine the pattern in which the virus spreads and the organs it targets. Aedes saliva significantly increases the pathogenicity of RVFV.
Author Summary
Rift Valley fever is an endemic and epidemic zoonosis in Africa and the Arabic Peninsula. In humans, in the most severe cases the viral infection causes fulminant hepatitis associated with haemorrhagic fever, permanent blindness or severe encephalitis. Despite the importance of vector transmission in the spread of arboviruses, few studies on the physiopathology of viral infection have considered the role of the arthropod in the efficiency of viral infection. Moreover, the route of virus inoculation and the presence of the vector's saliva can potentially affect virus pathogenicity. Our results show that saliva from Aedes mosquitoes increases Rift Valley fever pathogenicity. Importantly, our study also revealed that RVFV transmitted via mosquito bites spread differently than virus inoculated by other routes. These observations may have interesting repercussions given the role mosquitoes were shown to play in the transmission of RVFV in humans during the last outbreak of the disease in Saudi Arabia. Identification of salivary proteins able to increase RVFV virulence may pave the way to new approaches to prevent or cure the disease.
PMCID: PMC3681724  PMID: 23785528
8.  The M33 G Protein-Coupled Receptor Encoded by Murine Cytomegalovirus Is Dispensable for Hematogenous Dissemination but Is Required for Growth within the Salivary Gland 
Journal of Virology  2014;88(20):11811-11824.
Human cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of cytomegaloviruses, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. The MCMV genome, like the genomes of other beta- and gammaherpesviruses, encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the M33 GPCR carried by MCMV is an activator of CREB, NF-κB, and phospholipase C-β signaling pathways and has been implicated in aspects of pathogenesis in vivo, including persistence in the salivary glands of BALB/c mice. In this study, we used immunocompetent nonobese diabetic (NOD) and immunocompromised NOD-scid-gamma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus with an M33 deletion (ΔM33) can replicate in the salivary gland of immunocompromised animals, albeit with a 400-fold growth defect compared with the growth of wild-type virus. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the salivary gland. We conclude that the reduced replicative capacity of the ΔM33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since the salivary gland represents a site essential for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread.
IMPORTANCE Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lives. Under a number of circumstances, the virus can reactivate, leading to a variety of diseases in both adults and developing babies, and therefore, identifying the function of viral proteins is essential to understand how the virus spreads and causes disease. We aim to utilize animal models to study the function of an important class of viral proteins termed G protein-coupled receptors with the ultimate goal of developing inhibitors to these proteins that could one day be used to prevent viral spread.
PMCID: PMC4178735  PMID: 25100846
9.  Functional transplantation of salivary gland cells differentiated from mouse early ES cells in vitro 
Human Cell  2013;26(2):80-90.
Atrophy or hypofunction of the salivary gland because of aging or disease causes hyposalivation and has an effect on the quality of life of patients, for example not only dry mouth but deterioration in mastication/deglutition disorder and the status of oral hygiene. Currently conducted therapies for atrophy or hypofunction of the salivary gland in clinical practice are only symptomatic treatments with drugs and artificial saliva, and therefore it is preferable to establish a radical therapy. At this time, as a fundamental investigation, by co-culturing mouse early ES (mEES-6) cells with human salivary gland-derived fibroblasts (hSG-fibro), differentiation of mEES-6 cells to salivary gland cells has been attempted. Also, the possibility of cell engraftment was examined. After identifying the cells which were co-cultured with GFP-transfected mEES-6 cells and hSG-fibro, the cells were transplanted into the submandibular gland of SCID mice, and the degree of differentiation into tissues was examined. The possibility of tissue functional reconstitution from co-cultured cells in a three-dimensional culture system was examined. Our results confirmed that the co-cultured cells expressed salivary gland-related markers and had an ability to generate neo-tissues by transplantation in vivo. Moreover, the cells could reconstitute gland structures in a three-dimensional culture system. By co-culture with hSG-fibro, mEES-6 cells were successfully differentiated into salivary gland cells which were transplantable and have tissue neogenetic ability.
PMCID: PMC3676639  PMID: 23681939
Salivary gland; Cell-based therapy; Co-culture system; Cell differentiation; Regeneration
10.  Multiple components contribute to ability of saliva to inhibit influenza viruses 
Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied.
We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro.
Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study.
These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.
PMCID: PMC2848456  PMID: 19121065
histatins; innate immunity; MUC5B
11.  Induction of a Peptide with Activity against a Broad Spectrum of Pathogens in the Aedes aegypti Salivary Gland, following Infection with Dengue Virus 
PLoS Pathogens  2011;7(1):e1001252.
The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.
Author Summary
Dengue viruses (DENV) are generally maintained in a cycle which requires horizontal transmission via their arthropod vector, Ae. aegypti, to the vertebrate host. One important consequence of this process is the interference of the virus with the immune systems of both the mosquito and its host. While infection of humans causes disease, the presence of DENV in mosquitoes gives rise to life-long and persistent infection with active viral replication in the salivary glands. In the present study, we have evaluated the mosquito's immune response following DENV infection by analyzing the gene expression profile of infected and uninfected salivary glands. The results show that DENV infection activates signaling pathways and induces the expression of gene products that are involved in the innate immune response to DENV infection, and in particular a putative antibacterial cecropin-like peptide. The immature and mature forms of this peptide were found to be active against a variety of pathogens including DENV and Chikungunya viruses, as well as the Leishmania parasite. This study is the first to establish a comparative analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We demonstrate that certain DENV-induced peptides possess broad-spectrum anti-pathogenic activity and may have therapeutic potential in the treatment of human infectious disease.
PMCID: PMC3020927  PMID: 21249175
12.  In Vitro and In Vivo Characterization of a Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame M43 
Journal of Virology  2000;74(20):9488-9497.
We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.
PMCID: PMC112378  PMID: 11000218
13.  Permeability of Oral Tissues to Blood-borne Coxsackievirus B-1 
Applied Microbiology  1967;15(2):296-299.
The ability of coxsackievirus B-1 to pass the barriers of the circulatory system into whole saliva has been shown previously. In this investigation, the major salivary glands and the oral mucosa were studied, and their role as participants in the excretion of coxsackievirus B-1 during viremia was evaluated. The effect of the salivary-gland stimulant pilocarpine nitrate on both the salivary flow rate and the recovery of virus during viremia was determined. A comparison was made between the amount of virus recovered from whole saliva during viremia in animals deficient in one or both of the major salivary-gland pairs and animals with a complete complement of salivary glands. The salivary glands in other animals were cannulated, and pure glandular secretions were collected during viremia and assayed for the presence of virus The amount of virus passing from the capillaries of the oral mucosa to the surface was also determined to evaluate this route as a possible site for the excretion of virus into saliva during viremia. The major salivary glands did not excrete appreciable quantities of virus during viremia. The submaxillary-gland secretions did not contain virus, and the parotid-gland secretions showed virus only at extremely high blood virus levels. Either removal of the major salivary glands or decreased salivary flow rates increased the concentration of virus in whole saliva. This observation suggested that the production of saliva by the major salivary glands tends to dilute the virus in the oral cavity. A 0.88-cm2 sample of the oral mucosa excreted significantly large amounts of virus during viremia and suggested that the passage of virus through the oral mucosa was the major route for the excretion of virus into saliva during viremia.
PMCID: PMC546894  PMID: 6029830
14.  Frequent Detection of Polyomaviruses in Stool Samples from Hospitalized Children 
The Journal of infectious diseases  2005;192(4):658-664.
Infection with BK virus (BKV) generally occurs early during life, but its mode of transmission has not been clearly defined. We tested the hypothesis that polyomavirus shedding in stool may be a source of BKV exposure.
Pediatric stool and rectal swab samples were tested for the presence of polyomavirus DNA by a polymerase chain reaction (PCR) assay that could detect a conserved region in the large T antigen gene of BKV, JC virus (JCV), and simian virus 40 (SV40). The specific viruses detected by this assay were confirmed by DNA sequence analysis of the PCR amplicons.
Of 120 samples collected from 99 patients, 54 (45.0%) were positive for polyomavirus DNA. Of the 99 patients, 46 (46.5%) had at least 1 positive sample, with 38 (38.4%) positive for BKV and 8 (8.1%) positive for SV40. JCV was not detected. There was no association between polyomavirus fecal shedding and age, sex, race/ ethnicity, immune status, or symptoms of gastrointestinal disease in the children studied. The BKV strains detected displayed polymorphisms in the T antigen sequence.
Polyomaviruses are frequently present in stool samples from hospitalized children. These findings suggest that fecal-oral transmission of BKV may play a role in the ubiquity of infection.
PMCID: PMC4010313  PMID: 16028135
15.  Modulation of Sodium/Iodide Symporter Expression in the Salivary Gland 
Thyroid  2013;23(8):1029-1036.
Physiologic iodide-uptake, mediated by the sodium/iodide symporter (NIS), in the salivary gland confers its susceptibility to radioactive iodine–induced damage following 131I treatment of thyroid cancer. Subsequent quality of life for thyroid cancer survivors can be decreased due to recurrent sialoadenitis and persistent xerostomia. NIS expression at the three principal salivary duct components in various pathological conditions was examined to better our understanding of NIS modulation in the salivary gland.
NIS expression was evaluated by immunohistochemistry in human salivary gland tissue microarrays constructed of normal, inflamed, and neoplastic salivary tissue cores. Cumulative 123I radioactivity reflecting the combination of NIS activity with clearance of saliva secretion in submandibular and parotid salivary glands was evaluated by single-photon emission computed tomography/computed tomography imaging 24 hours after 123I administration in 50 thyroid cancer patients.
NIS is highly expressed in the basolateral membranes of the majority of striated ducts, yet weakly expressed in few intercalated and excretory duct cells. The ratio of 123I accumulation between parotid and submandibular glands is 2.38±0.19. However, the corresponding ratio of 123I accumulation normalized by volume of interest is 1.19±0.06. The percentage of NIS-positive striated duct cells in submandibular salivary glands was statistically greater than in parotid salivary glands, suggesting a higher clearance rate of saliva secretion in submandibular salivary glands. NIS expression in striated ducts was heterogeneously decreased or absent in sialoadenitis. Most ductal salivary gland tumors did not express NIS. However, Warthin's tumors of striated duct origin exhibited consistent and intense NIS staining, corresponding with radioactive iodine uptake.
NIS expression is tightly modulated during the transition of intercalated to striated ducts and striated to excretory ducts in salivary ductal cells. NIS expression in salivary glands is decreased during inflammation and tumor formation. Further investigation may identify molecular targets and/or pharmacologic agents that allow selective inhibition of NIS expression/activity in salivary glands during radioactive iodine treatment.
PMCID: PMC3752512  PMID: 23441638
16.  Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients 
PLoS Pathogens  2012;8(4):e1002650.
BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy.
Author Summary
Serological studies have shown that nearly all humans are chronically infected with BK polyomavirus (BKV). The infection isn't usually associated with noticeable symptoms. However, opportunistic replication of BKV in therapeutically immunosuppressed kidney transplant recipients (KTRs) can lead to dysfunction or loss of the engrafted kidney. BKV associated nephropathy can occur even in KTRs with high levels of anti-BKV antibodies that might be expected to neutralize the virus. In this report we provide a possible explanation: we show there are at least two BKV genotypes, which are distinct serotypes with respect to antibody-mediated neutralization. Using a novel neutralization-based approach, we found that about half of 108 KTRs did not have detectable levels of antibodies capable of neutralizing BKV genotype IV (BKV-IV) at the time of transplantation. Of these initially BKV-IV naïve KTRs, about half experienced acute BKV-IV specific seroconversion during the first year after transplantation. This likely reflects a de novo BKV-IV infection arising from the engrafted kidney. In a pilot study, we show that recombinant BKV-IV VLPs can induce high levels of BKV-IV-neutralizing antibodies in vaccinated animals. Our results suggest that administration of a BKV VLP-based vaccine to prospective KTRs might protect against the development of opportunistic BKV replication.
PMCID: PMC3325208  PMID: 22511874
17.  Immunity to Distinct Sand Fly Salivary Proteins Primes the Anti-Leishmania Immune Response towards Protection or Exacerbation of Disease 
Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects biologically active salivary components that favorably change the environment at the feeding site. Exposure to bites or to salivary proteins results in immunity specific to these components. Mice immunized with Phlebotomus papatasi salivary gland homogenate (SGH) or pre-exposed to uninfected bites were protected against Leishmania major infection delivered by needle inoculation with SGH or by infected sand fly bites. Immunization with individual salivary proteins of two sand fly species protected mice from L. major infection. Here, we analyze the immune response to distinct salivary proteins from P. papatasi that produced contrasting outcomes of L. major infection.
Methodology/Principal Findings
DNA immunization with distinct DTH-inducing salivary proteins from P. papatasi modulates L. major infection. PpSP15-immunized mice (PpSP15-mice) show lasting protection while PpSP44-immunized mice (PpSP44-mice) aggravate the infection, suggesting that immunization with these distinct molecules alters the course of anti-Leishmania immunity. Two weeks post-infection, 31.5% of CD4+ T cells produced IFN-γ in PpSP15-mice compared to 7.1% in PpSP44-mice. Moreover, IL-4-producing cells were 3-fold higher in PpSP44-mice. At an earlier time point of two hours after challenge with SGH and L. major, the expression profile of PpSP15-mice showed over 3-fold higher IFN-γ and IL-12-Rβ2 and 20-fold lower IL-4 expression relative to PpSP44-mice, suggesting that salivary proteins differentially prime anti-Leishmania immunity. This immune response is inducible by sand fly bites where PpSP15-mice showed a 3-fold higher IFN-γ and a 5-fold lower IL-4 expression compared with PpSP44-mice.
Immunization with two salivary proteins from P. papatasi, PpSP15 and PpSP44, produced distinct immune profiles that correlated with resistance or susceptibility to Leishmania infection. The demonstration for the first time that immunity to a defined salivary protein (PpSP44) results in disease enhancement stresses the importance of the proper selection of vector-based vaccine candidates.
Author Summary
In vector-borne diseases, the role of vectors has been overlooked in the search for vaccines. Nonetheless, there is a body of evidence showing the importance of salivary proteins of vectors in pathogen transmission. Leishmaniasis is a neglected vector-borne disease transmitted by sand flies. Pre-exposure to sand fly saliva or immunization with a salivary protein protected mice against cutaneous leishmaniasis. Using DNA immunization we investigated the immune response induced by abundant proteins within the saliva of the sand fly Phlebotomus papatasi. We found that one salivary protein protected while another exacerbated L. major infection, suggesting that the type of immune response induced by specific salivary proteins can prime and direct anti-Leishmania immunity. This stresses the importance of the proper selection of vector-based vaccine candidates. This work validates the powerful protection that can be acquired through vaccination with the appropriate salivary molecule and more importantly, shows that this protective immune response is efficiently recalled by sand fly bites, the natural route of transmission.
PMCID: PMC2291569  PMID: 18414648
18.  Spatial mapping of gene expression in the salivary glands of the dengue vector mosquito, aedes aegypti 
Aedes aegypti mosquitoes are the main vectors of dengue viruses to humans. Understanding their biology and interactions with the pathogen are prerequisites for development of dengue transmission control strategies. Mosquito salivary glands are organs involved directly in pathogen transmission to vertebrate hosts. Information on the spatial distribution of gene expression in these organs is expected to assist in the development of novel disease control strategies, including those that entail the release of transgenic mosquitoes with impaired vector competence.
We report here the hybridization in situ patterns of 30 transcripts expressed in the salivary glands of adult Ae. aegypti females. Distinct spatial accumulation patterns were identified. The products of twelve genes are localized exclusively in the proximal-lateral lobes. Among these, three accumulate preferentially in the most anterior portion of the proximal-lateral lobe. This pattern revealed a salivary gland cell type previously undescribed in Ae. aegypti, which was validated by transmission electron microscopy. Five distinct gene products accumulate in the distal-lateral lobes and another five localize in the medial lobe. Seven transcripts are found in the distal-lateral and medial lobes. The transcriptional product of one gene accumulates in proximal- and distal-lateral lobes. Seven genes analyzed by quantitative PCR are expressed constitutively. The most abundant salivary gland transcripts are those localized within the proximal-lateral lobes, while previous work has shown that the distal-lateral lobes are the most active in protein synthesis. This incongruity suggests a role for translational regulation in mosquito saliva production.
Transgenic mosquitoes with reduced vector competence have been proposed as tools for the control of dengue virus transmission. Expression of anti-dengue effector molecules in the distal-lateral lobes of Ae. aegypti salivary glands has been shown to reduce prevalence and mean intensities of viral infection. We anticipate greater efficiency of viral suppression if effector genes are expressed in all lobes of the salivary glands. Based on our data, a minimum of two promoters is necessary to drive the expression of one or more anti-dengue genes in all cells of the female salivary glands.
PMCID: PMC3043528  PMID: 21205315
Virus research  2010;149(2):190-196.
The major capsid protein, VP1, of the human Polyomavirus BK (BKV) is structurally divided into five outer loops, referred to as BC, DE, EF, GH, and HI. The BC loop includes a short region, named the BKV subtyping region, spanning nucleotides 1744–1812 and characterized by non-synonymous nucleotide polymorphisms that have been used to classify different strains of BKV into four subtypes. The aim of this study was to determine if the nucleotide changes clustered within the BKV subtyping region may influence the in vitro growth efficiency of the virus. We therefore infected the African Green Monkey kidney cell line Vero with four different viral strains (named BKV I, II, III, and IV) that contained the nucleotide sequences of the BKV subtypes within the same genomic background. Infected cells were followed for 59 days and viral replication was assessed at different time points by Quantitative Real Time PCR (Q-PCR). BKV I, II, and IV were successfully propagated over time in Vero cells, whereas BKV III viral loads progressively decreased during the infection course, demonstrating that the non-synonymous nucleotide polymorphisms of subtype III confer a strong disadvantage for viral replication. Since subtype III differs from all the other subtypes at position 68 of the VP1, where Leu is replaced by Gln, we created viral strains bearing Gln at this position together with the polymorphisms of subtypes I, II, IV and tested their growth in Vero cells. Our results demonstrate that this amino acid substitution does not lower the replication efficiency of subtypes I, II, and IV. In conclusion, this study provides further insights to the importance of the BC loop of BKV in the virus life cycle. In addition, given the effect of the amino acid substitutions of the four BKV subtypes on infectious spread of the virus, our results suggest the need to investigate their potential association with BKV related complications.
PMCID: PMC2873212  PMID: 20138933
BK virus; subtype; BC loop; subtyping region
20.  Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia 
Human Gene Therapy  2013;24(6):604-612.
Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (1011 vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p<0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection against radiation assists in preserving submandibular gland function. AAV2/9-TLK1B treatment could prove beneficial in attenuating xerostomia in patients with head and neck cancer undergoing radiotherapy.
Shanmugam and colleagues demonstrate that a single instillation of recombinant AAV9 encoding Tousled-like kinase 1B (TLK1B) in rat submandibular glands leads to a complete amelioration of salivary dysfunction caused by fractionated radiation. They suggest that this treatment modality may be beneficial in attenuating xerostomia in headand-neck cancer patients undergoing radiotherapy.
PMCID: PMC3689188  PMID: 23614651
21.  A Focused Salivary Gland Infection with attenuated MCMV: An Animal Model with Prevention of Pathology Associated with Systemic MCMV Infection1, 2 
While the salivary gland has been recognized as an important effector site of the common mucosal immune system, a useful model for studying anti-viral salivary gland immune responses in vivo and for exploring the role of the salivary gland within the common mucosal system has been lacking. Murine cytomegalovirus (MCMV) is a beta-herpesvirus that displays a strong tropism for the salivary gland and produces significant morbidity in susceptible mice when introduced by intraperitoneal (i.p.) inoculation. This study tested the hypothesis that MCMV morbidity and pathology could be reduced by injecting the virus directly the submandibular salivary gland (intraglandular (i.g.)), using either in vivo derived MCMV or the less virulent, tissue culture-derived MCMV (tcMCMV). Peak salivary gland viral titers were completely unaffected by infection route (i.p vs. i.g.) after inoculation with either MCMV or tcMCMV. However, i.g. tcMCMV inoculation reduced viremia in all systemic tissues tested compared to i.p. inoculation. Further, systemic organ pathology observed in the liver and spleen after i.p. inoculation with either MCMV or tcMCMV was completely eliminated by i.g. inoculation with tcMCMV. Cellular infiltrates in the salivary glands, after i.p. or i.g. inoculation were composed of both B and T cells, indicating the potential for a local immune response to occur in the salivary gland. These results demonstrate that a focused MCMV infection of the salivary gland without systemic organ pathology is possible using i.g. delivery of tcMCMV.
PMCID: PMC3506192  PMID: 17320076
rodent; viral; MCMV; mucosa; spleen and lymph nodes; salivary gland; antibodies; mucosal immunity
22.  Natural Killer Cells Regulate Murine Cytomegalovirus-Induced Sialadenitis and Salivary Gland Disease 
Journal of Virology  2012;86(4):2132-2142.
The transmission of herpesviruses depends on viral shedding at mucosal surfaces. The salivary gland represents a major site of persistent viral replication for many viruses, including cytomegalovirus. We established a mouse model of salivary gland dysfunction after acute viral infection and investigated the cellular requirements for the loss of secretion. Murine cytomegalovirus (MCMV) infection severely impaired saliva secretion independently of salivary gland virus levels. Lymphocytes or circulating monocytes/macrophages were not required for secretory dysfunction. Dysfunction occurred before glandular inflammation, suggesting that a soluble mediator initiated the disruption of acinar cell function. Despite genetic differences in innate resistance to MCMV, NK cells protected the host against acinar atrophy and the loss of secretions under conditions of an exceedingly low virus inoculum. NK cells also modulated the type of glandular inflammation after infection, as they prevented an influx of Siglec-F+ polymorphonuclear leukocytes (PMNs). Therefore, beyond their recognized role in controlling MCMV replication, NK cells preserve organ integrity and function and regulate the innate inflammatory response within the gland.
PMCID: PMC3302420  PMID: 22156514
23.  Salivary Gland Hypofunction Induced by Activation of Innate Immunity is Dependent on Type I Interferon Signaling 
Activation of innate immunity through polyinosinic:polycytidylic acid (poly(I:C)) causes acute salivary gland hypofunction. Since a major consequence of poly(I:C) treatment is type I interferon (IFN) production, this study was undertaken to investigate their role in salivary gland dysfunction.
Different strains of mice, deficient either in interferon alpha receptor (IFNAR1−/−), or IL-6−/−, or IL-10−/−, or EBI3−/− were treated with poly(I:C). Salivary gland function was determined by measuring pilocarpine induced saliva volume. Gene expression levels were measured by real time PCR. Ca2+ mobilization studies were done using ex-vivo acinar cells.
A single injection of poly(I:C) rapidly induced salivary gland hypofunction in wild type B6 mice (41% drop in saliva volumes compared to PBS treated mice). In contrast, the loss of function in poly(I:C) treated IFNAR−/− mice was only 9.6%. Gene expression analysis showed reduced levels of Il-6, Il-10 and Il-27 in submandibular glands of poly(I:C) treated IFNAR−/− mice. While salivary gland dysfunction in poly(I:C) treated IL-10−/− and EBI3−/− mice was comparable to wild type mice, the IL-6−/− mice were more resistant, with only a 21 % drop in function. Pilocarpine induced Ca2+ flux was significantly suppressed in acinar cells obtained from poly(I:C) treated wild type mice.
Our data demonstrates that a combined action of type I IFNs and IL-6 contributes towards salivary gland hypofunction. This happens through interference with Ca2+ mobilization within acinar cells. Thus, in acute viral infections and diseases like Sjögren’s syndrome, elevated levels of type I IFNs and IL-6 can directly affect glandular function.
PMCID: PMC3443546  PMID: 22672212
Xerostomia; Sjogren’s syndrome; Innate Immunity; Interferon
Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögren’s syndrome (SS) patients. Since viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function.
Female NZB/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid (poly(I:C)). TLR3 expression within submandibular glands was studied by immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time PCR. Pilocarpine induced saliva volume was used as an index of glandular function.
Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules and ducts. Poly(I:C) treatment rapidly upregulated the mRNA levels of type I IFN and inflammatory cytokines in the submandibular glands. By one week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the PBS treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped.
Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.
PMCID: PMC2763314  PMID: 19192049
Inflammatory Cytokines; Poly(I:C); Salivary Gland; Sjögren’s Syndrome TLR3
25.  Functional salivary gland regeneration by transplantation of a bioengineered organ germ 
Nature Communications  2013;4:2498.
Salivary gland hypofunction, also known as xerostomia, occurs as a result of radiation therapy for head cancer, Sjögren’s syndrome or aging, and can cause a variety of critical oral health issues, including dental decay, bacterial infection, mastication dysfunction, swallowing dysfunction and reduced quality of life. Here we demonstrate the full functional regeneration of a salivary gland that reproduces the morphogenesis induced by reciprocal epithelial and mesenchymal interactions through the orthotopic transplantation of a bioengineered salivary gland germ as a regenerative organ replacement therapy. The bioengineered germ develops into a mature gland through acinar formations with a myoepithelium and innervation. The bioengineered submandibular gland produces saliva in response to the administration of pilocarpine and gustatory stimulation by citrate, protects against oral bacterial infection and restores normal swallowing in a salivary gland-defective mouse model. This study thus provides a proof-of-concept for bioengineered salivary gland regeneration as a potential treatment of xerostomia.
Salivary gland dysfunction as a result of diseases or ageing reduces the quality of life and causes various oral health problems. Here the authors show that the salivary gland function of mice can be recovered by orthotopic transplantation of a bioengineered salivary gland germ.
PMCID: PMC3806330  PMID: 24084982

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