Hepatitis C Virus Infection of Cultured Human Hepatoma Cells Causes Apoptosis and Pyroptosis in Both Infected and Bystander Cells.

Individuals infected with hepatitis C virus (HCV) are at high risk of developing progressive liver disease, including cirrhosis and hepatocellular carcinoma (HCC). How HCV infection causes liver destruction has been of significant interest for many years, and apoptosis has been proposed as one operative mechanism. In this study, we employed a tissue culture-adapted strain of HCV (JFH1T) to test effects of HCV infection on induction of programmed cell death (PCD) in Huh-7.5 cells. We found that HCV infection reduced the proliferation rate and induced caspase-3-mediated apoptosis in the infected cell population. However, in addition to apoptosis, we also observed infected cells undergoing caspase-1-mediated pyroptosis, which was induced by NLRP3 inflammasome activation. By co-culturing HCV-infected Huh-7.5 cells with an HCV-non-permissive cell line, we also demonstrated induction of both apoptosis and pyroptosis in uninfected cells. Bystander apoptosis, but not bystander pyroptosis, required cell-cell contact between infected and bystander cells. In summary, these findings provide new information on mechanisms of cell death in response to HCV infection. The observation that both apoptosis and pyroptosis can be induced in bystander cells extends our understanding of HCV-induced pathogenesis in the liver.

Distinct and overlapping genomic profiles and antiviral effects of Interferon-λ and -α on HCV-infected and noninfected hepatoma cells.

Recently, several SNPs in the region of the IL28B (IFN-λ) gene have been associated with spontaneous clearance of hepatitis C virus (HCV) and enhanced cure rates for IFN-alfa-based therapies, suggesting a potential correlation between IFN-λ and the ability to clear HCV. To understand the mechanism of IFN-λ's as compared to IFN-α's antiviral activity, we performed a comprehensive analysis of their anti-HCV effects, whole genome transcriptome profiling with validation, and signalling of IFN-α and IFN-λ using J6/JFH-1 and Huh7.5 cells in vitro. IFN-λ and IFN-α exhibited comparable anti-HCV activity and gene expression profiles in Huh7.5 cells. While the majority of genes induced by IFN-α and IFN-λ were similar, IFN-λ exhibits profound, but delayed kinetics of IFN-stimulated genes (ISG) induction, while IFN-α induced more rapid induction of ISGs. Furthermore, the increased induction of ISG expression by IFN-λ correlated with up-regulation of IFN-λ receptor (IL-28RA) expression and more prolonged activation of the Jak-STAT signalling pathway. The findings from our comparative analysis of IFN-α and IFN-λ in HCV-infected and noninfected cells support the clinical use of IFN-λ as a potential alternative to IFN-α in the treatment of chronic hepatitis C.

Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles.

Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding. Other entry-deficient mutants displayed normal E1E2 incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry.

Management of grade IV renal injury in children.

PURPOSE: Conservative nonsurgical management of major renal trauma in children is well established. However, when blunt trauma is accompanied by significant urinary extravasation, options are less than clearly defined. Endoscopic techniques, such as stents and percutaneous drainage, have not been widely used because of small caliber. We present our experience with endoscopic management of grade IV renal trauma. MATERIALS AND METHODS: From 1983 to 1996, 15 children satisfied the criteria for grade IV renal trauma. We retrospectively reviewed the charts to assess the mechanism of injury, associated injury, treatment, hospital stay and transfusion requirement. Patients were followed clinically with blood pressure and creatinine monitoring, and by radiograph with computerized tomography. RESULTS: Nine patients with isolated kidney injury were successfully treated with observation, 1 underwent early partial nephrectomy for persistent anemia and hypotension, and 5 had a urinoma, which was successfully treated with percutaneous drainage only in 2. The other 3 patients underwent cystoscopy and ureteral stent placement for high drainage output, leading to the resolution of urine leakage. In 1 patient who underwent percutaneous drainage only renovascular hypertension developed, requiring partial nephrectomy 3 months after the original injury. The remaining 13 patients had complete radiographic resolution of the injury and no evidence of hypertension. CONCLUSIONS: In the pediatric population grade IV blunt renal trauma usually resolves without intervention. When a symptomatic urinoma develops, percutaneous drainage, accompanied at times by ureteral stenting provides the complete resolution of persistent urine leakage.

Hydrophobic amino acids in the human immunodeficiency virus type 1 p2 and nucleocapsid proteins can contribute to the rescue of deleted viral RNA packaging signals.

An RNA fragment of 75 nucleotides, which is located between the primer binding site and the 5' major splice donor site in human immunodeficiency virus type 1, has been shown to participate in specific encapsidation of viral RNA. Compensation studies have identified two second-site mutations, namely, MP2 (a T12I substitution in p2) and MNC (a T24I substitution in the nucleocapsid [NC] protein) that were involved in the rescue of various deletions in the aforementioned RNA region (i.e., BH-D1, BH-D2, and BH-LD3). To study whether the MP2 and MNC point mutations exert their compensatory effects in a cis manner, production of Gag proteins was blocked by insertion of stop codons into LD3, LD3-MP2-MNC, and wild-type BH10 such that the constructs generated, i.e., LD3-DG, LD3-MP2-MNC-DG, and BH-DG, only provided RNA transcripts for packaging. The results of cotransfection experiments showed that the LD3-MP2-MNC-DG viral RNA was packaged as inefficiently as LD3-DG; in contrast, BH-DG was efficiently packaged. Therefore, nucleotide substitutions in MP2 and MNC did not act in a cis manner to correct the packaging deficits in LD3. Next, we deliberately changed the T12 in p2 or the T24 in the NC to each of 19 other amino acids. We found that amino acids with long hydrophobic side chains, i.e., V, L, I, and M, were favored at either position 12 in p2 or at position 24 in NC to compensate for the above-mentioned deletions. Further studies showed that only a few amino acids could not be used at these two sites by the wild-type virus due to decreased RNA levels in the virion or abnormal Gag protein processing. In this case, W, D, and E could not substitute for T12 in p2, and S, D, and N could not substitute for T24 in NC, without affecting viral infectivity. Therefore, the long hydrophobic side chains of V, L, I, and M are necessary for these amino acids to rescue the BH-D1, BH-D2, and BH-LD3 mutated viruses.

Role for human immunodeficiency virus type 1 Tat protein in suppression of viral reverse transcriptase activity during late stages of viral replication.

We have examined the role of the human immunodeficiency virus type 1 (HIV-1) Tat protein in the regulation of reverse transcription. We show that a two-exon but not a one-exon form of Tat markedly suppressed cell-free reverse transcriptase (RT) activity. Conversely, viruses expressing two-exon Tat (pNL43 and pNL101) showed rapid replication kinetics and more efficient endogenous RT activity compared with viruses expressing one-exon Tat (pM1ex). The pM1ex virions, as well as pM1ex-infected cells, also contained higher levels of viral DNA than did either the pNL43 or pNL101 viruses, indicating that reverse transcription might have continued during later stages of viral replication in the absence of the second Tat exon. Moreover, degradation of viral genomic RNA was more apparent in the pM1ex virions. Accordingly, we propose that the two-exon Tat may help augment viral infectivity by suppressing the reverse transcription reaction during late stages of viral synthesis and by preventing the synthesis of potentially deleterious viral DNA products.

Deletion mutagenesis downstream of the 5' long terminal repeat of human immunodeficiency virus type 1 is compensated for by point mutations in both the U5 region and gag gene.

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5' long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA(3)(Lys) primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels.

Recruitment of multiple V beta genes in the TCR repertoire against a single pathogenic thyroglobulin epitope.

In autoimmune thyroid disease, the question whether thyroid-infiltrating, autoreactive T cells are derived from a polyclonal or oligoclonal subset has been the subject of considerable debate. In this report, we have examined the T-cell receptor (TCR) V beta profile of mouse clonal T cells responding to a single thyroiditogenic epitope, the As-restricted, 9mer mouse thyroglobulin (MTg) peptide (2496-04). In vitro recall assays based on lymph node cell (LNC) proliferation and cytokine release demonstrated that this peptide is a minimal T-cell epitope inducing a T-helper 1 (Th1) type of response in SJL hosts. A panel of cloned, interleukin-2 (IL-2)-secreting hybridomas was generated from this Th1 subset and their TCR-V beta gene utilization was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Ten clones derived from two independent fusions were found to utilize three V beta gene families (V beta 2, 4, and 17). To the extent that Tg or other thyroid autoantigens encompass multiple pathogenic epitopes it appears unlikely from these data that a restricted TCR-V beta chain usage will be a general characteristic of thyroiditogenic T cells.

Strength of clear corneal incisions in cadaver eyes.

PURPOSE: To determine the variation in strength of clear corneal incisions, as demonstrated by degree of incision leakage when challenged by increased intraocular pressure, in relationship to the incision width and length. SETTING: Mackool Eye Institute, Astoria, New York. METHODS: Clear corneal incisions 3.0 or 3.5 mm in width and from 1.0 to 3.5 mm in length (0.5 mm increments) were studied in human cadaver whole globes. Pressure was applied at the corneal apex or 8.0 mm posterior to the external wound margin to determine the strength of the incision with the application of external force. RESULTS: Clear corneal incisions of 3.0 or 3.5 mm in width and at least 2.0 mm in length demonstrated substantially greater resistance to incision failure than shorter incision lengths with both apical and posterior applied forces. CONCLUSION: Clear corneal incisions 2.0 mm or greater in length demonstrate resistance to leakage comparable to similarly constructed scleral tunnel incisions.

Effect of foldable intraocular lens insertion on incision width.

PURPOSE: To determine whether the surgical incision enlarges during insertion of foldable intraocular lenses (IOLs). SETTING: Mackool Eye Institute, Astoria, New York. METHODS: A variety of IOL insertion devices and foldable and injectable IOLs were inserted through 3.0 or 3.5 mm keratome incisions made in cadaver eyes. The external and internal incision widths were then measured. RESULTS: Each 3.0 mm incision was enlarged externally by 0.10 to 0.65 mm and internally by 0.50 to 0.75 mm by a variety of insertional devices and IOLs. One forceps-IOL combination required a 3.5 mm incision for lens insertion and resulted in a 0.4 mm enlargement of the internal incision. CONCLUSIONS: The use of a 3.5 mm incision and insertion devices that do not enlarge an incision of this size might be desirable.

Intracapsular posterior chamber intraocular lens insertion with posterior capsular tears or zonular instability.

A surgical technique is described for posterior chamber intraocular lens implantation within the capsular bag with a posterior capsular tear or weakened zonular support. Haptics are compressed before endocapsular insertion, minimizing capsular and zonular stress.

Extending the range of the surgical keratometer.

Interest in optical precision in ocular surgery has been increasing recently. The surgical keratometer is central to the practice of ocular surgery, and research is needed to increase the precision of surgical keratometry techniques. Animal corneas used for these purposes may be outside the range of the surgical keratometer. Extension of the lower range of the Terry keratometer is achieved by substituting a 200- or 250-mm-focal-length objective lens for the standard 175-mm lens on the Zeiss operating microscope. Conversion equations and graphs are presented that allow conversion of the values obtained with these lenses.