Immune defense is the primary function associated with the differentially expressed genes in the cochlea following acoustic trauma.

Our previous RNA-sequencing analysis of the rat cochlear genes identified multiple biological processes and molecular pathways in the cochlear response to acoustic overstimulation. However, the biological processes and molecular pathways that are common to other species have not been documented. The identification of these common stress processes is pivotal for a better understanding of the essential response of the cochlea to acoustic injury. Here, we compared the RNA-sequencing data collected from mice and rats that sustained a similar, but not identical, acoustic injury. The transcriptome analysis of cochlear genes identified the differentially expressed genes in the mouse and rat samples. Bioinformatics analysis revealed a marked similarity in the changes in the biological processes between the two species, although the differentially expressed genes did not overlap well. The common processes associated with the differentially expressed genes are primarily associated with immunity and inflammation, which include the immune response, response to wounding, the defense response, chemotaxis and inflammatory responses. Moreover, analysis of the molecular pathways showed considerable overlap between the two species. The common pathways include cytokine-cytokine receptor interactions, the chemokine signaling pathway, the Toll-like receptor signaling pathway, and the NOD-like receptor signaling pathway. Further analysis of the transcriptional regulators revealed common upstream regulators of the differentially expressed genes, and these upstream regulators are also functionally related to the immune and inflammatory responses. These results suggest that the immune and inflammatory responses are the essential responses to acoustic overstimulation in the cochlea.

Molecular profile of cochlear immunity in the resident cells of the organ of Corti.

BACKGROUND: The cochlea is the sensory organ of hearing. In the cochlea, the organ of Corti houses sensory cells that are susceptible to pathological insults. While the organ of Corti lacks immune cells, it does have the capacity for immune activity. We hypothesized that resident cells in the organ of Corti were responsible for the stress-induced immune response of the organ of Corti. This study profiled the molecular composition of the immune system in the organ of Corti and examined the immune response of non-immune epithelial cells to acoustic overstimulation. METHODS: Using high-throughput RNA-sequencing and qRT-PCR arrays, we identified immune- and inflammation-related genes in both the cochlear sensory epithelium and the organ of Corti. Using bioinformatics analyses, we cataloged the immune genes expressed. We then examined the response of these genes to acoustic overstimulation and determined how changes in immune gene expression were related to sensory cell damage. RESULTS: The RNA-sequencing analysis reveals robust expression of immune-related genes in the cochlear sensory epithelium. The qRT-PCR array analysis confirms that many of these genes are constitutively expressed in the resident cells of the organ of Corti. Bioinformatics analyses reveal that the genes expressed are linked to the Toll-like receptor signaling pathway. We demonstrate that expression of Toll-like receptor signaling genes is predominantly from the supporting cells in the organ of Corti cells. Importantly, our data demonstrate that these Toll-like receptor pathway genes are able to respond to acoustic trauma and that their expression changes are associated with sensory cell damage. CONCLUSION: The cochlear resident cells in the organ of Corti have immune capacity and participate in the cochlear immune response to acoustic overstimulation.

Myeloid-derived suppressor cells modulate immune responses independently of NADPH oxidase in the ovarian tumor microenvironment in mice.

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47 (phox-/-)) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47 (phox-/-) mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.

Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ß-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.

NADPH oxidase and Nrf2 regulate gastric aspiration-induced inflammation and acute lung injury.

Recruitment of neutrophils and release of reactive oxygen species are considered to be major pathogenic components driving acute lung injury (ALI). However, NADPH oxidase, the major source of reactive oxygen species in activated phagocytes, can paradoxically limit inflammation and injury. We hypothesized that NADPH oxidase protects against ALI by limiting neutrophilic inflammation and activating Nrf2, a transcriptional factor that induces antioxidative and cytoprotective pathways. Our objective was to delineate the roles of NADPH oxidase and Nrf2 in modulating acute lung inflammation and injury in clinically relevant models of acute gastric aspiration injury, a major cause of ALI. Acid aspiration caused increased ALI (as assessed by bronchoalveolar lavage fluid albumin concentration) in both NADPH oxidase-deficient mice and Nrf2(-/-) mice compared with wild-type mice. NADPH oxidase reduced airway neutrophil accumulation, but Nrf2 decreased ALI without affecting neutrophil recovery. Acid injury resulted in a 120-fold increase in mitochondrial DNA, a proinflammatory and injurious product of cellular necrosis, in cell-free bronchoalveolar lavage fluid. Pharmacologic activation of Nrf2 by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9 (11)-dien-28-oyl]imidazole limited aspiration-induced ALI in wild-type mice and reduced endothelial cell injury caused by mitochondrial extract-primed human neutrophils, leading to the conclusion that NADPH oxidase and Nrf2 have coordinated, but distinct, functions in modulating inflammation and injury. These results also point to Nrf2 as a therapeutic target to limit ALI by attenuating neutrophil-induced cellular injury.

Primary xenografts of human prostate tissue as a model to study angiogenesis induced by reactive stroma.

Characterization of the mechanism(s) of androgen-driven human angiogenesis could have significant implications for modeling new forms of anti-angiogenic therapies for CaP and for developing targeted adjuvant therapies to improve efficacy of androgen-deprivation therapy. However, models of angiogenesis by human endothelial cells localized within an intact human prostate tissue architecture are until now extremely limited. This report characterizes the burst of angiogenesis by endogenous human blood vessels in primary xenografts of fresh surgical specimens of benign prostate or prostate cancer (CaP) tissue that occurs between Days 6-14 after transplantation into SCID mice pre-implanted with testosterone pellets. The wave of human angiogenesis was preceded by androgen-mediated up-regulation of VEGF-A expression in the stromal compartment. The neo-vessel network anastomosed to the host mouse vascular system between Days 6-10 post-transplantation, the angiogenic response ceased by Day 15, and by Day 30 the vasculature had matured and stabilized, as indicated by a lack of leakage of serum components into the interstitial tissue space and by association of nascent endothelial cells with mural cells/pericytes. The angiogenic wave was concurrent with the appearance of a reactive stroma phenotype, as determined by staining for α-SMA, Vimentin, Tenascin, Calponin, Desmin and Masson's trichrome, but the reactive stroma phenotype appeared to be largely independent of androgen availability. Transplantation-induced angiogenesis by endogenous human endothelial cells present in primary xenografts of benign and malignant human prostate tissue was preceded by induction of androgen-driven expression of VEGF by the prostate stroma, and was concurrent with and the appearance of a reactive stroma phenotype. Androgen-modulated expression of VEGF-A appeared to be a causal regulator of angiogenesis, and possibly of stromal activation, in human prostate xenografts.

Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense.

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.

Nrf2 regulates chronic lung inflammation and B-cell responses to nontypeable Haemophilus influenzae.

Nrf2 is a leucine zipper transcription factor that protects against oxidant-induced injury. Nontypeable Haemophilus influenzae is responsible for frequent disease exacerbations in patients with chronic obstructive pulmonary disease and is responsible for causing otitis media in young children. We hypothesized that Nrf2 would limit inflammatory responses to nontypeable H. influenzae. The objective of this study was to assess the role of Nrf2 in chronic lung inflammation and regulation of immune responses to nontypeable H. influenzae in mice. Wild-type (C57BL/6) mice and Nrf2(-/-) mice were instilled by oropharyngeal aspiration of 1 × 10(6) colony-forming units of live, nontypeable H. influenzae (NTHI) twice a week for 4 to 16 consecutive weeks to generate a chronic inflammatory milieu within the lungs that models chronic bronchitis. Nrf2(-/-) mice had increased lymphocytic airway inflammation compared with WT mice after NTHI challenge. Although the extent of NTHI-induced peribronchovascular inflammation did not significantly differ between the genotypes, plasma cell infiltration was significantly more abundant in Nrf2(-/-) mice. Most strikingly, Nrf2(-/-) mice generated significantly enhanced and persistent levels of serum antibodies against P6, a key outer membrane protein of NTHI. Lung dendritic cells from Nrf2(-/-) mice challenged with NTHI had increased activation markers compared with dendritic cells from similarly treated WT mice. Nrf2 regulates NTHI-induced airway inflammation characterized by lymphocytic and plasma cell infiltration and the activation of lung dendritic cells and B-cell responses in mice. Nrf2 may be a potential therapeutic target in limiting the bacterial infection-induced airway inflammation that drives exacerbations of chronic obstructive pulmonary disease.

Androgen deprivation induces rapid involution and recovery of human prostate vasculature.

The response of the prostate tissue microenvironment to androgen deprivation (AD) represents a critical component in the treatment of benign prostatic hyperplasia and prostate cancer (CaP). Primary xenografts of human benign and CaP tissue transplanted to immunocompromized SCID mice were used to characterize the response of the prostate vasculature during the initial 14 days of AD. Microvessel density and vascular lumen diameter in the prostate xenografts decreased rapidly after AD, reached a nadir on days 2-4, and recovered between days 4 and 14. The number of apoptotic endothelial cells peaked on day 2 after AD and decreased to precastration levels over days 4-7. Leakage of vascular contents in the interstitial space was apparent between days 1 and 3 after AD; however, the vascular permeability barrier reestablished between days 7 and 14. Expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor-2, and basic fibroblast growth factor protein increased in endothelial cells between days 2 and 4 after AD, which preceded vascular recovery and appeared to be a direct and specific response of the endothelial cells to AD. Lack of comparable upregulation of these genes in primary cultures of human prostate endothelial cells in response to AD suggests a role for paracrine signaling mediated through stromal or epithelial cells. VEGF-A expression by prostate endothelial cells appears to represent a key facilitator of the vascular rebound in human prostate tissue induced by removal of circulating testicular androgens.

Role of NADPH oxidase in host defense against aspergillosis.

NADPH oxidase plays a critical role in antimicrobial host defense, as evident in chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal diseases. Invasive aspergillosis and other moulds are the major cause of mortality in CGD. We also learn from CGD patients that NADPH oxidase plays an important role in regulating inflammation; CGD patients are prone to developing inflammatory diseases such as inflammatory bowel disease, obstructive granulomata of the genitourinary tract, and hypersensitivity pneumonitis. Indeed, the NADPH oxidase plays an essential role in calibrating innate and T-cell responses to control the growth of inhaled fungi while protecting against excessive and injurious inflammation. Knowledge gained on the mechanisms by which NADPH oxidase kills fungi and regulates inflammation may lead to new therapeutics for CGD and will have broad relevance to understanding host-pathogen interactions between mammals and ubiquitous moulds to which we are continually exposed.

NADPH oxidase limits innate immune responses in the lungs in mice.

BACKGROUND: Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47(phox-/-) mice and gp91(phox)-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-kappaB activation, and elevated downstream pro-inflammatory cytokines (TNF-alpha, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-kappaB activation. CONCLUSIONS/SIGNIFICANCE: These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.

Cyclosporin A, tacrolimus and sirolimus are potent inhibitors of the human breast cancer resistance protein (ABCG2) and reverse resistance to mitoxantrone and topotecan.

PURPOSE: Several studies have demonstrated significant interactions between immunosuppressants (e.g., cyclosporin A) and chemotherapeutic drugs that are BCRP substrates (e.g., irinotecan), resulting in increased bioavailability and reduced clearance of these agents. One possible mechanism underlying this observation is that the immunosuppressants modulate the pharmacokinetics of these drugs by inhibiting BCRP. Therefore, the aim of this study was to determine whether the immunosuppressants cyclosporin A, tacrolimus and sirolimus are inhibitors and/or substrates of BCRP. METHODS: First, the effect of the immunosuppressants on BCRP efflux activity in BCRP-expressing HEK cells was measured by flow cytometry. RESULTS: Cyclosporin A, tacrolimus and sirolimus significantly inhibited BCRP-mediated efflux of pheophorbide A, mitoxantrone and BODIPY-prazosin. The EC(50) values of cyclosporin A, tacrolimus and sirolimus for inhibition of BCRP-mediated pheophorbide A efflux were 4.3 +/- 1.9 microM, 3.6 +/- 1.8 microM and 1.9 +/- 0.4 microM, respectively. Cyclosporin A, tacrolimus and sirolimus also effectively reversed resistance of HEK cells to topotecan and mitoxantrone conferred by BCRP. When direct efflux of cyclosporin A, tacrolimus and sirolimus was measured, these compounds were found not to be transported by BCRP. Consistent with this finding, BCRP did not confer resistance to the immunosuppressants in HEK cells. CONCLUSION: These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs.

Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo cells.

The breast cancer resistance protein (BCRP) is abundant in the placenta and protects the fetus by limiting placental drug penetration. We hypothesize that pregnancy-specific hormones regulate BCRP expression. Hence, we examined the effects of progesterone (P4) and 17beta-estradiol (E2) on BCRP expression in the human placental BeWo cells. P4 and E2 significantly increased and decreased BCRP protein and mRNA, respectively. Likewise, treatment with P4 and E2 increased and decreased, respectively, fumitremorgin C-inhibitable mitoxantrone efflux activity of BeWo cells. Reduction in BCRP expression by E2 was abrogated by the estrogen receptor (ER) antagonist ICI-182,780. However, the progesterone receptor (PR) antagonist RU-486 had no effect on P4-mediated induction of BCRP. P4 together with E2 further increased BCRP protein and mRNA compared with P4 treatment alone. This combined effect on BCRP expression was abolished by RU-486, ICI-182,780, or both. Further analysis revealed that E2 significantly decreased ER beta mRNA and strongly induced PR(B) mRNA in a dose-dependent manner but had no effect on PR(A) and ER alpha. P4 alone had no significant effect on mRNA of ER alpha, ER beta, PR(A), and PR(B). E2 in combination with P4 increased PR(B) mRNA, but the level of induction was significantly reduced compared with E2 treatment alone. Taken together, these results indicate that E2 by itself likely downregulates BCRP expression through an ER, possibly ER beta. P4 alone upregulates BCRP expression via a mechanism other than PR. P4 in combination with E2 further increases BCRP expression, presumably via a nonclassical PR- and/or E2-mediated synthesis of PR(B).

BCRP transports dipyridamole and is inhibited by calcium channel blockers.

PURPOSE: We investigated whether dipyridamole and various calcium channel blockers are inhibitors and/or substrates of breast cancer resistance protein (BCRP). METHODS: The effect of dipyridamole and the calcium channel blockers on mitoxantrone efflux by BCRP-overexpressing human embryonic kidney (HEK) cells was determined by flow cytometry. The ability of some of these compounds to reverse BCRP-mediated mitoxantrone resistance was measured by cytotoxicity assays. Transport studies were performed using radiolabeled compounds. RESULTS: Dipyridamole, nicardipine, nitrendipine, and nimodipine effectively inhibited BCRP-mediated mitoxantrone efflux; however, bepridil, diltiazem, and verapamil had no significant effect. Nifedipine is a much weaker BCRP inhibitor compared with other dihydropyridines tested. Nicardipine and dipyridamole were the most potent BCRP inhibitors among the compounds tested with IC50 values of 4.8 +/- 1.3 and 6.4 +/- 0.9 microM, respectively. Nicardipine and dipyridamole also effectively reversed BCRP-mediated mitoxantrone resistance in HEK cells. [3H]Nitrendipine was found not to be transported by BCRP. However, the transport of [3H]dipyridamole by BCRP was observed in both HEK and Madin-Darby canine kidney cells stably expressing the transporter, and this transport was completely abolished by fumitremorgin C, a known BCRP inhibitor. CONCLUSIONS: Dipyridamole and several dihydropyridines are effective BCRP inhibitors, but bepridil, diltiazem, and verapamil are not. We also identified a new BCRP substrate, dipyridamole.

Functional analysis of the human variants of breast cancer resistance protein: I206L, N590Y, and D620N.

Previous studies have shown that the V12M and Q141K variants of breast cancer resistance protein (BCRP) can affect expression and function of the transporter. In this study, the effects of the I206L, N590Y, and D620N variants on protein expression, plasma membrane localization, and transport activity of BCRP were investigated. Wild-type BCRP and the three variants were stably expressed in human embryonic kidney (HEK) cells. Confocal microscopy analysis showed that the three variants were predominantly routed to the plasma membrane of HEK cells. The expression level of I206L in the plasma membrane was approximately 45% of that of wild-type protein, whereas the N590Y and D620N levels were increased approximately 3.6-fold and 2.4-fold, respectively, as determined by immunoblotting. All three variants transported mitoxantrone, pheophorbide a, and BODIPY FL-prazosin. After normalization for differences in BCRP expression, I206L, N590Y, and D620N exhibited approximately 2-fold, 0.3-fold, and 0.5-fold wild-type efflux activities, respectively. The variants also conferred resistance to mitoxantrone and topotecan. Mitoxantrone and topotecan resistance by I206L and N590Y was approximately 2-fold and 0.3-fold of the wild-type BCRP resistance levels, respectively. Although D620N conferred a topotecan resistance similar to that of the wild-type protein, its level of mitoxantrone resistance was decreased by 50%. After normalization to BCRP expression levels, ATPase activities of I206L were not significantly different from those of wild-type protein, whereas N590Y and D620N exhibited approximately 30% and 50% of wild-type ATPase activities, respectively. These results suggest that I206L has the lowest protein expression and the highest activity, whereas N590Y and D620N display higher expression and lower activity, relative to wild-type BCRP.

Prevalence of, and antigenic variation in, serotype G10 rotaviruses and detection of serotype G3 strains in diarrheic calves: implications for the origin of G10P11 or P11 type reassortant asymptomatic strains in newborn children in India.

Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of the VP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with the VP7 from several animal and animal rotavirus-related human G3 strains (Simian SA11; equine ERV316 and FI-14; canine CU-1 and K9; porcine 4F; Feline Cat2 and human HCR3, YO and AU1). Partial nucleotide sequence analysis of the NSP1 gene of J63 showed greatest nt sequence homology (95.9%) to the NSP1 gene allele of the Indian G8 strain, isolated from a diarrheic child, which is likely to have been transmitted directly from cattle and 92.6% homology to that of the bovine G8 strain A5-10 suggesting the likely origin of J63 by gene reassortment between a bovine G8 strain and a G3 animal strain. Prevalence of G10P11 strains in cattle and G10P11 or P11 type reassortant strains in asymptomatic neonates as well as detection of G8P[1] strains in diarrheic children support our hypothesis for bidirectional transmission of rotaviruses between humans and cattle and origin of novel strains catalyzed by the age-old traditions and socio-economic conditions in India.