Rob Striker

Vitamin D and the anti-viral state

Vitamin D has long been recognized as essential to the skeletal system. Newer evidence suggests that it also plays a major role regulating the immune system, perhaps including immune responses to viral infection. Interventional and observational epidemiological studies provide evidence that vitamin D deficiency may confer increased risk of influenza and respiratory tract infection. Vitamin D deficiency is also prevalent among patients with HIV infection. Cell culture experiments support the thesis that vitamin D has direct anti-viral effects particularly against enveloped viruses. Though vitamin Ds anti-viral mechanism has not been fully established, it may be linked to vitamin Ds ability to up-regulate the anti-microbial peptides LL-37 and human beta defensin 2. Additional studies are necessary to fully elucidate the efficacy and mechanism of vitamin D as an anti-viral agent.

Sensitivity of hepatitis C virus to cyclosporine A depends on nonstructural proteins NS5A and NS5B

HCV reoccurs after liver transplantation and increases mortality. Cyclosporine, but not tacrolimus, has potent antiviral effects against HCV replication in cell culture. To determine the conditions, if any, under which HCV is susceptible to cyclosporine in vivo, we selected for cyclosporine‐resistant mutant HCV in vitro. The resulting mutations were mapped to x‐ray crystallographic structures and sequence databases. Mutations selected by cyclosporine were clustered in the nonstructural (NS) proteins NS5A and NS5B. Different sets of mutations in NS5A, paired with the same 2 NS5B mutations, conferred different levels of cyclosporine resistance when engineered back into the HCV replicon. Mutations in NS5B are structurally consistent with a proposed model of regulation of RNA binding by cyclophilin B (CyPB). These mutations also highlight a natural polymorphism between different HCV genotypes that correlates with the variation in response to cyclosporine A (CsA) noted in some clinical trials. Replicons engineered to have mutations in only NS5A (P ≤ 0.0001) or only NS5B (P = 0.002) suggest that while both NS5A or NS5B variants alter cyclosporine susceptibility, NS5A has the largest effect. Conclusion: Preexisting sequence variation could alter the effect of cyclosporine on HCV in vivo. (HEPATOLOGY 2007.)

Cyclosporine inhibits a direct interaction between cyclophilins and hepatitis C NS5A.

Background Hepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations. Methodology/Principal Findings We examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture. Conclusions/Significance Collectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.

Addition of ceftaroline to daptomycin after emergence of daptomycin-nonsusceptible Staphylococcus aureus during therapy improves antibacterial activity.

ABSTRACT Antistaphylococcal beta-lactams enhance daptomycin activity and have been used successfully in combination for refractory methicillin-resistant Staphylococcus aureus (MRSA) infections. Ceftaroline possesses MRSA activity, but it is unknown if it improves the daptomycin potency comparably to other beta-lactams. We report a complex patient case of endocarditis who was treated with daptomycin in combination with ceftaroline, which resulted in clearance of a daptomycin-nonsusceptible strain. An in vitro pharmacokinetic/pharmacodynamic model of renal failure was used to simulate the development of daptomycin resistance and evaluate the microbiologic effects of daptomycin plus ceftaroline treatment. Combination therapy with daptomycin and ceftaroline restored daptomycin sensitivity in vivo and resulted in clearance of persistent blood cultures. Daptomycin susceptibility in vitro was increased in the presence of either ceftaroline or oxacillin. Daptomycin at 6 mg/kg of body weight every 48 h was bactericidal in the model but resulted in regrowth and daptomycin resistance (MIC, 2 to 4 μg/ml) with continued monotherapy. The addition of ceftaroline at 200 mg every 12 h after the emergence of daptomycin resistance enhanced bacterial killing. Importantly, daptomycin plus ceftaroline as the initial combination therapy produced rapid and sustained bactericidal activity and prevented daptomycin resistance. Both in vivo- and in vitro-derived daptomycin resistance resulted in bacteria with more fluid cell membranes. After ceftaroline was added in the model, fluidity was restored to the level of the initial in vivo isolate. Daptomycin-resistant isolates required high daptomycin exposures (at least 10 mg/kg) to optimize cell membrane damage with daptomycin alone. Ceftaroline combined with daptomycin was effective in eliminating daptomycin-resistant MRSA, and these results further justify the potential use of daptomycin plus beta-lactam therapy for these refractory infections.

Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli

The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human‐specific PapG adhesin. Three classes of PapG adhesins exist with different binding specificities to Galα4Gal‐containing glycolipids. The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination. The minimum binding epitope was confirmed as the Galα4Gal moiety, but parts of the GalNAcβ and glucose residues, which flank the Galα4Gal in globoside (GbO4), were also shown to be important for strong binding. Furthermore, the same five hydroxyl groups of Galα4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human‐specific PapG in binding the GbO4 that dominates In the human kidney. Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E. coli to human kidney sections. Knowledge of the molecular details of the PapG‐GbO4 interaction will make it possible to design antiadherence therapeutics.

Evidence for separation of HCV subtype 1a into two distinct clades.

Summary.  The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error‐prone RNA‐dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole‐genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star‐like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a‐specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain‐specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

ABSTRACT Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naïve patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Donor‐Derived Trypanosoma cruzi Infection in Solid Organ Recipients in the United States, 2001–2011

Although Trypanosoma cruzi, the parasite that causes Chagas disease, can be transmitted via organ transplantation, liver and kidney transplantation from infected donors may be feasible. We describe the outcomes of 32 transplant recipients who received organs from 14 T. cruzi seropositive donors in the United States from 2001 to 2011. Transmission was confirmed in 9 recipients from 6 donors, including 3 of 4 (75%) heart transplant recipients, 2 of 10 (20%) liver recipients and 2 of 15 (13%) kidney recipients. Recommended monitoring posttransplant consisted of regular testing by PCR, hemoculture, and serology. Thirteen recipients had no or incomplete monitoring; transmission was confirmed in five of these recipients. Four of the five recipients had symptomatic disease and all four died although death was directly related to Chagas disease in only one. Nineteen recipients had partial or complete monitoring for T. cruzi infection with weekly testing by PCR, hemoculture and serology; transmission was confirmed in 4 of 19 recipients with no cases of symptomatic disease. Our results suggest that liver and kidney transplantation from T. cruzi seropositive donors may be feasible when the recommended monitoring schedule for T. cruzi infection is followed and prompt therapy with benznidazole can be administered.

Genetic, biochemical, and structural studies of biogenesis of adhesive pili in bacteria

Publisher Summary This chapter discusses how the molecular details of pilus biogenesis, including the roles of pilus subunits and accessory proteins, have been studied using a powerful blend of genetics, biochemistry, carbohydrate chemistry, X-ray crystallography, and high-resolution electron microscopy techniques; and focuses on the P-pilus system as a model for a detailed analysis of postsecretional assembly. Pathogenic organisms appear to have an abundant repertoire of adhesive organelles, probably only a fraction of which have been identified. The different architectures of pill represent a variety of strategies that these pathogens have evolved to adhere effectively to host tissues. Although the minor and major pilus subunits have a diversity of function, interestingly, they are incorporated into adhesive structures using common mechanisms of assembly. The ongoing efforts to identify, isolate, and analyze products of pilin gene clusters will undoubtedly lead to the discovery of additional, highly related members of the immunoglobulin-like chaperone family and the outer membrane usher family, and the discovery of a seemingly diverse group of minor pilin subunits. The sequence comparisons of the pilus chaperone family have highlighted highly conserved amino acid residues which may be critical to chaperone function. Site-directed mutations in these residues have indicated that residues within the cleft are critical to the ability of the chaperone to bind pilus subunits.

Comparison of hepatitis C virus treatment between incarcerated and community patients

The prevalence of chronic hepatitis C virus (HCV) infection among incarcerated individuals in the United States is estimated to be between 12% and 31%. HCV treatment during incarceration is an attractive option because of improved access to health care and directly observed therapy. We compared incarcerated and nonincarcerated HCV‐infected patients evaluated for treatment at a single academic center between January 1, 2002 and December 31, 2007. During this period, 521 nonincarcerated and 388 incarcerated patients were evaluated for HCV treatment. Three hundred and nineteen (61.2%) nonincarcerated patients and 234 (60.3%) incarcerated patients underwent treatment with pegylated interferon and ribavirin. Incarcerated patients were more likely to be male, African‐American race, and have a history of alcohol or intravenous drug use. Treated incarcerated patients were less likely to have genotype 1 virus and were less likely to have undergone previous treatment. There was a similar prevalence of coinfection with human immunodeficiency virus (HIV) in both groups. A sustained viral response (SVR) was achieved in 97 (42.9%) incarcerated patients, compared to 115 (38.0%) nonincarcerated patients (P = 0.304). Both groups had a similar proportion of patients that completed a full treatment course. Stepwise logistic regression was conducted, and the final model included full treatment course, non‐genotype 1 virus, younger age at treatment start, and negative HIV status. Incarceration status was not a significant predictor when added to this model (P = 0.075). Conclusion: In a cohort of HCV‐infected patients managed in an academic medical center ambulatory clinic, incarcerated patients were as likely to be treated for HCV and as likely to achieve an SVR as nonincarcerated patients. (HEPATOLOGY 2012)