Biochemical characterization of P4-ATPase mutations identified in patients with progressive familial intrahepatic cholestasis.

Mutations in the P4-ATPase ATP8B1 cause the inherited liver disease progressive familial intrahepatic cholestasis. Several of these mutations are located in conserved regions of the transmembrane domain associated with substrate binding and transport. Assays for P4-ATPase-mediated transport in living yeast cells were developed and used to characterize the specificity and kinetic parameters of this transport. Progressive familial intrahepatic cholestasis mutations were introduced into the yeast plasma membrane P4-ATPase Dnf2p, and the effect of these mutations on its catalysis of phospholipid transport were determined. The results of these measurements have implications for the basis of the disease and for the mechanism of phospholipid transit through the enzyme during the reaction cycle.

Host-genetic-based outcome of co-infection by PCV2b and PRRSV in pigs.

Replication of porcine circovirus type 2 (PCV2), an important worldwide swine pathogen, has been demonstrated to be influenced by host genotype. Specifically, a missense DNA polymorphism (SYNGR2 p.Arg63Cys) within the SYNGR2 gene was demonstrated to contribute to variation in PCV2b viral load and subsequent immune response following infection. PCV2 is known to induce immunosuppression leading to an increase in susceptibility to subsequent infections with other viral pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). In order to assess the role of SYNGR2 p.Arg63Cys in co-infections, pigs homozygous for the favorable SYNGR2 p.63Cys (N = 30) and unfavorable SYNGR2 p.63Arg (N = 29) alleles were infected with PCV2b followed a week later by a challenge with PRRSV. A lower PCV2b viremia (P < 0.001) and PCV2-specific IgM antibodies (P < 0.005) were observed in SYNGR2 p.63Cys compared to SYNGR2 p.63Arg genotypes. No significant differences in PRRSV viremia and specific IgG antibodies were observed between SYNGR2 genotypes. Lung histology score, an indicator of disease severity, was lower in the pigs with SYNGR2 p.63Cys genotypes (P < 0.05). Variation in the lung histology scores within SYNGR2 genotypes suggests that additional factors, environmental and/or genetic, could be involved in disease severity.

Porcine circovirus type 2 (PCV2) is an important virus involved in the onset of a group of severe disease symptoms commonly known as porcine circovirus associated diseases (PCVAD). Vaccination options exist for PCV2, though the severity of PCVAD can be influenced by the presence of additional co-infecting pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), for which vaccination is still a challenge. Host genetic resistance is a potential avenue for solving this problem. Previously, a genetic polymorphism in the SYNGR2 gene was found to be associated with PCV2b viremia and immune response. The aim of this study was to determine the impact of this polymorphism in pigs experimentally co-infected with PCV2b and PRRSV. Pigs were weighed, and blood was collected at various days following infection to measure viremia and antibodies. Histological analysis was performed at the experiment completion to assess disease severity in lungs and lymph nodes. The results showed that variation within the SYNGR2 gene is involved in PCV2b disease progression including lung histology scores, but no evidence was seen in response to PRRSV infection.

Genetic diversity and detection of atypical porcine pestivirus infections.

Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previously reported quantitative polymerase chain reaction (qPCR)-based assays were unable to detect APPV in novel cases of congenital tremor originated from multiple farms from U.S. Midwest (MW). These assays targeted the viral polyprotein coding genes, which were shown to display substantial variation, with sequence identity ranging from 58.2% to 70.7% among 15 global APPV strains. In contrast, the 5'-untranslated region (5' UTR) was found to have a much higher degree of sequence conservation. In order to obtain the complete 5' UTR of the APPV strains originated from MW, the 5' end of the viral cDNA was obtained by using template switching approach followed by amplification and dideoxy sequencing. Eighty one percent of the 5' UTR was identical across 14 global and 5 MW strains with complete or relatively complete 5' UTR. Notably, some of the most highly conserved 5' UTR segments overlapped with potentially important regions of an internal ribosome entry site (IRES), suggesting their functional role in viral protein translation. A newly designed single qPCR assay, targeting 100% conserved 5' UTR regions across 19 strains, was able to detect APPV in samples of well documented cases of congenital tremor which originated from five MW farm sites (1-18 samples/site). As these fully conserved 5' UTR sequences may have functional importance, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

Mutations in agr do not persist in natural populations of methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus organisms vary in the function of the staphylococcal virulence regulator gene agr. To test for a relationship between agr and transmission in S. aureus, we determined the prevalence and genetic basis of agr dysfunction among nosocomial methicillin-resistant S. aureus (MRSA) in an area of MRSA endemicity. Identical inactivating agr mutations were not detected in epidemiologically unlinked clones within or between hospitals. Additionally, most agr mutants had single mutations, indicating that they were short lived. Collectively, the results suggest that agr dysfunction is adaptive for survival in the infected host but that it may be counteradaptive outside infected host tissues.

Human Herpesvirus-8 Infection Associated with Kaposi Sarcoma, Multicentric Castleman's Disease, and Plasmablastic Microlymphoma in a Man with AIDS: A Case Report with Review of Pathophysiologic Processes.

Kaposi sarcoma (KS), multicentric Castleman's disease (MCD), and plasmablastic microlymphoma, are all linked to human herpesvirus-8 (HHV-8) infection and HIV-induced immunodeficiency. Herein, we describe the case of a Kenyan man diagnosed with HIV in 2000. He deferred highly active antiretroviral therapy (HAART) and remained in good health until his CD4+ count declined in 2006. He was hospitalized with bacterial pneumonia in 2008, after which he agreed to take HAART but did so sporadically. In 2010, he was hospitalized with fever, lymphadenopathy, pancytopenia, and an elevated HHV-8 viral load. A lymph node biopsy showed findings consistent with KS, MCD, and plasmablastic microlymphoma. Eight months after starting liposomal doxorubicin, Rituximab, and a new HAART regimen, he has improved clinically, and his HIV and HHV-8 viral loads are suppressed. These three conditions, found in the same lymph node, underscore the inflammatory and malignant potential of HHV-8, particularly in the milieu of HIV-induced immunodeficiency.

Liposome-mediated transfer of nitric oxide synthase gene improves renal function in ureteral obstruction in rats.

BACKGROUND: The protective effect of nitric oxide has been demonstrated in several renal disease models. We augmented renal nitric oxide production by transfer of the inducible nitric oxide synthase (iNOS) gene into rat kidney in controls and in unilateral ureteral obstruction (UUO). METHODS: The human iNOS gene was inserted into a pcDNA 3.1-backbone plasmid with the FLAG epitope (FLAG-iNOS). In vitro, transduction of FLAG-iNOS was confirmed by Western blot and Griess reaction. In vivo, we transfected either FLAG-iNOS or control plasmid (CMV-LacZ), using cationic liposomes. Urinary nitric oxide metabolites and immunohistochemistry confirmed iNOS transduction. Renal function was also assessed. RESULTS: In vitro, increased iNOS expression was demonstrated in human embryonic kidney (HEK293) cells, along with increased release of nitric oxide metabolites, NO(2)/NO(3). In vivo, FLAG-iNOS was detected by polymerase chain reaction (PCR) up to 35 days after the transfection. Urine collection documented increased urinary NO(2)/NO(3). Immunohistochemistry localized iNOS to collecting ducts, distal tubules, and glomerulus of the injected kidney. Renal function measured up to 21 days after transfection in control animals was not significantly different between the two groups. In contrast, renal function after 24 hours of UUO was significantly improved in FLAG-iNOS-treated animals. CONCLUSION: This study demonstrates the feasibility of liposome-mediated iNOS gene transfer into the kidney. Furthermore, the improvement of renal function in UUO demonstrates that the transfected iNOS gene is active and suggests that decreased iNOS activity contributes to the decreased renal function in UUO. This iNOS construct may have therapeutic utility in the pathophysiologic sequelae of UUO and other renal diseases.