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author:("bondo, A")
1.  Rotavirus Replication in the Cholangiocyte Mediates the Temporal Dependence of Murine Biliary Atresia 
PLoS ONE  2013;8(7):e69069.
Biliary atresia (BA) is a neonatal disease that results in obliteration of the biliary tree. The murine model of BA, which mirrors the human disease, is based upon infection of newborn mice with rhesus rotavirus (RRV), leading to an obstructive cholangiopathy. The purpose of this study was to characterize the temporal relationship between viral infection and the induction of this model. BALB/c mice were infected with RRV on day of life (DOL) 0, 3, 5, and 7. Groups were characterized as early-infection (infection by DOL 3) or late-infection (infection after DOL 5). Early RRV infection induced symptoms in 95% of pups with a mortality rate of 80%. In contrast, late infection caused symptoms in only 50% of mice, and 100% of pups survived. The clinical findings correlated with histological analysis of extrahepatic biliary trees, cytokine expression, and viral titers. Primary murine cholangiocytes isolated, cultured, and infected with RRV yielded higher titers of infectious virus in those harvested from DOL 2 versus DOL 9 mice. Less interferon alpha and beta was produced in DOL 2 versus DOL 9 RRV infected primary cholangiocytes. Injection of BALB/c interferon alpha/beta receptor knockout (IFN-αβR−/−) pups at DOL 7 showed increased symptoms (79%) and mortality (46%) when compared to late infected wild type mice. In conclusion, the degree of injury sustained by relatively immature cholangiocytes due to more robust RRV replication correlated with more severe clinical manifestations of cholangiopathy and higher mortality. Interferon alpha production by cholangiocytes appears to play a regulatory role. These findings confirm a temporal dependence of RRV infection in murine BA and begin to define a pathophysiologic role of the maturing cholangiocyte.
PMCID: PMC3700947  PMID: 23844248
2.  The Rhesus Rotavirus Gene Encoding VP4 Is a Major Determinant in the Pathogenesis of Biliary Atresia in Newborn Mice▿† 
Journal of Virology  2011;85(17):9069-9077.
Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene “loss-of-function” reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene “gain-of-function” reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the “loss-of-function” reassortant RT(VP4), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the “gain-of-function” reassortant TR(VP4) resulted in murine BA with 88% mortality. Compared with those for RRV, RT(VP4) binding and titers in cholangiocytes were significantly attenuated, while TR(VP4) binding and titers were significantly increased over those for TUCH. Reassortants RT(VP3) and TR(VP3) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.
PMCID: PMC3165802  PMID: 21697466
3.  Toward the development of a stable, freeze-dried formulation of Helicobacter pylori killed whole cell vaccine adjuvanted with a novel mutant of E. coli heat-labile toxin 
Vaccine  2009;28(5):1404.
No vaccine exists for the prevention of infection with the ubiquitous gastric pathogen Helicobacter pylori, and drug therapy for the infection is complicated by poor patient compliance, the high cost of treatment, and ineffectiveness against drug resistant strains. A new medical advancement is required to reduce the incidence of peptic ulcer disease and stomach cancer, two conditions caused by infection with H. pylori. Clinical trials have been performed with a formalin-inactivated Helicobacter pylori Whole Cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of E. coli heat-labile toxin. Following the initial dose of this vaccine, some subjects experienced gastrointestinal side effects. To reduce side effects and potentially further increase the amount of adjuvant that can safely be administered with the HWC vaccine, experiments were performed with a form of LT that carried two mutations in the A subunit, a substitution of G for R at position 192, and A for L at position 211. The double-mutant LT (dmLT) adjuvant stimulated immune responses as effectively as the single mutant LT in mice. Additionally, following a challenge infection, the dmLT-adjuvanted vaccine was as effective as single mutant LT in reducing gastric urease levels (diagnostic for H. pylori infection), and H. pylori colonization in the stomach as assessed by quantitative analysis of stomach homogenates. A lyophilized formulation of HWC was developed to improve stability and to potentially reduce reliance on cold chain maintenance. It was observed that a dmLT-adjuvanted lyophilized vaccine was equally as protective in the mouse model as the liquid formulation as assessed by gastric urease analysis and analysis of stomach homogenates for viable H. pylori. No readily detectable effect of tonicity or moisture content was observed for the lyophilized vaccine within the formulation limits evaluated. In an accelerated stability study performed at 37°C the lyophilized vaccine remained equally as protective as vaccine stored at 2–8°C. The formulation selected for clinical development consisted of 2.5×1010 formalin-inactivated cells per ml in 6.5% trehalose, 0.5% mannitol, and 10 mM citrate buffer at pH 6.8.
PMCID: PMC2814929  PMID: 19897067
Helicobacter pylori; vaccine; adjuvant
4.  Prevention of the murine model of biliary atresia following live rotavirus vaccination of dams 
Journal of pediatric surgery  2009;44(8):1479-1490.
Biliary atresia (BA) is a neonatal disease that results in the obliteration of the biliary tree. The murine model of biliary atresia (BA) has been established where rhesus rotavirus (RRV) infection of newborn mice leads to an obstructive cholangiopathy. We determined whether maternal, post-conception rotavirus vaccination could prevent the murine model of biliary atresia.
Materials and Methods
Female mice were mated and injected intraperitoneally with one of the following materials: purified rotavirus strains RRV or Wa, high or low dose Rotateq® (a pentavalent rotavirus vaccine (PRV)), purified recombinant viral antigens of rotavirus (VP6) or influenza (NP), or saline. B-cell-deficient females also underwent post-conception PRV injection. Maternal vaccination with PRV improves survival of pups infected with RRV.
Maternal vaccination with PRV improves survival of pups infected with RRV. Serum rotavirus IgG, but not IgA, levels were increased in pups delivered from dams who received RRV, Wa, PRV, or VP6, but in the case of the Wa, PRV, and VP6 groups, these antibodies were not neutralizing. Post-conception injection of high dose PRV did not improve survival of pups born to B-cell deficient dams.
Maternal vaccination against RRV can prevent the rotavirus-induced murine model of biliary atresia in newborn mouse pups.
PMCID: PMC2748872  PMID: 19635292
Biliary atresia; rotavirus; Rotateq®; maternal immunization
5.  Cholangiocyte secretion of chemokines in experimental biliary atresia 
Journal of pediatric surgery  2009;44(3):500-507.
Biliary atresia (BA) is a disease of the newborn which results in obstruction of the biliary tree. The cause of BA remains unknown; however, recent studies using the murine model of biliary atresia have found that rotavirus infection of the biliary epithelial cell (cholangiocyte) triggers an inflammatory response. We hypothesized that rotavirus infection of cholangiocytes results in the release of chemokines, important mediators of the host immune response.
In vivo, Balb/c pups were injected with rhesus rotavirus (RRV) or saline, and, their extrahepatic bile ducts were microdissected 2,5, 7, and 14 days after injection. Next, an immortalized cholangiocyte cell line (mCl) was incubated with RRV or serum free media. Qualitative and quantitative chemokine assement was performed using ELISA, PCR, and immunohistchemistry.
In vivo, increased levels of the chemokines MIP-2, MCP-1, KC and RANTES were found in RRV-infected murine bile ducts. In vitro, infected mCl cells produced increasing amounts of these same chemokines in relation to dose and time.
These novel results suggest that chemokine expression by RRV-infected cholangiocytes may trigger a host inflammatory process that causes bile duct obstruction. Understanding how viral infection initiates this response may shed light on the pathogenesis of biliary atresia.
PMCID: PMC2730110  PMID: 19302848
6.  Granulomatous Pneumocystis carinii pneumonia in patients with malignancy 
Thorax  2002;57(5):435-437.
Background: A review was undertaken of the clinical features and results of diagnostic tests in non-HIV infected patients who developed granulomatous Pneumocystis carinii pneumonia (PCP).
Methods: A retrospective review was performed of the charts and radiographs of patients with a granulomatous reaction to P carinii identified from computerised pathology records at Memorial Sloan Kettering Cancer Center, a university affiliated tertiary care hospital.
Results: Three cases were identified; the incidence of granulomatous PCP was 3%. All patients had risk factors for PCP and had received high dose corticosteroids which had been stopped. Two patients had received chemotherapy. Presentation was insidious with only mild symptoms; only one patient had fever. Chest radiographs showed a reticulonodular pattern. Bronchoscopy was negative for PCP in all cases and open lung biopsy was necessary.
Conclusion: A granulomatous pathological reaction to PCP occurs rarely in patients with malignancy. In these cases the clinical presentation may be atypical and bronchoscopy can be non-diagnostic.
PMCID: PMC1746334  PMID: 11978921

Results 1-6 (6)