Human rheumatoid factor cross-idiotypes. IV. Studies on WA XId-positive IgM without rheumatoid factor activity provide evidence that the WA XId is not unique to rheumatoid factors and is distinct from the 17.109 and G6 XIds.

The WA cross-idiotype (XId) is the major XId among human monoclonal rheumatoid factors (mRF) and is almost always associated with the light (L) chain XId, 17.109, and the heavy (H) chain XId, G6. A cell line, 35G6, was cloned that bears the WA XId, but shows no reactivity with immunoglobulin G (IgG) and is negative for the 17.109 and G6 XIds. The 35G6 L chain appears to be derived from the same VKIII-JKI genes as most WA mRFs L chains. In contrast to the WA mRFs H chains in which VH1 genes are used, the 35G6 IgM expresses a VH3 gene. Sequence comparisons with other WA XId-positive mRF suggested several common structural features that may be related to the WA XId and differences that may relate to lack of IgG reactivity. Cells similar to 35G6 have previously been described in pokeweed mitogen-stimulated cell lines of peripheral blood lymphocytes from normal individuals and patients with rheumatoid arthritis and type II mixed cryoglobulinemia. These observations were confirmed, and in addition, it was shown that the majority of WA XId-positive cells in these cultures were negative for the 17.109 and G6 XIds. The presence of the WA XId in the absence of IgG reactivity suggests that the WA XId is more directly associated with an antigen specificity other than IgG, and its association with RF activity may be incidental. It is postulated that these WA XId-positive RF-negative antibodies may serve a physiologic role as natural antibodies to a pervasive pathogen, and that IgG reactivity is a consequence of somatic diversification accompanying proliferation of the WA XId-positive RF-negative cell.

Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle.

Concomitant with the onset of S phase, a series of thymidine kinase (TK) splicing intermediates as well as mature TK mRNA accumulates in the nucleus of BALB/c 3T3 cells. Most of the TK splicing intermediates are retained by oligo(dT)-cellulose chromatography, and, therefore, 3' end formation and polyadenylation probably precede the splicing of TK pre-mRNAs. We have further characterized the TK pre-mRNAs that are present in the nuclei of S-phase cells by using specific probes derived from each of the six TK intervening sequences. Based on the sizes of the pre-mRNAs and their patterns of hybridization with these intron probes, we propose a pathway for intron removal from nascent TK transcripts. Intron excision occurred by a preferred, but not necessarily obligatory, order which appears to have been conserved in mouse and Chinese hamster cells.

A competitive reverse transcription-polymerase chain reaction assay for quantitation of GB virus C/hepatitis G virus RNA that circumvents heteroduplex artifact.

The role of GB virus C (GBV-C)/hepatitis G virus (HGV) in hepatitis has been controversial. To investigate its possible pathogenicity and site(s) of replication, it is important to develop an accurate quantitative assay for both positive and negative strand GBV-C/HGV RNA. In this study, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for both positive and negative strand GBV-C/HGV RNA quantitation was developed. In developing the quantitative assay, heteroduplex formation was repeatedly observed. A heterologous competitor RNA with GBV-C/HGV primer-binding sequences was introduced, and heteroduplex artifact was circumvented successfully. Two-hundred thirty-seven serum specimens were screened by RT-PCR for GBV-C/HGV RNA. Two of the 62 patients infected with chronic hepatitis C virus (HCV) were found to be positive for GBV-C/HGV RNA. None of the 50 other patients with no evidence of HCV infection and none of the 125 normal individuals were positive for GBV-C/HGV RNA. The sensitivity of RT-PCR was 3000 gE/ml (30 gE in RT-PCR). Alternate methods for residual DNA removal and its detection in synthetic RNA were introduced. A RT control containing no primer before PCR is necessary to evaluate the trace amounts of template DNA remaining in synthesized RNA. The method will differentiate reliably between positive and negative strand RNAs up to a 10(4)-fold difference in titer. The positive and negative strand GBV-C/HGV RNAs were detected in one patient by RT-PCR and hybridization analysis, and the strand titer was determined by RT-PCR.

The association of hepatitis C virus infection with monoclonal rheumatoid factors bearing the WA cross-idiotype: implications for the etiopathogenesis and therapy of mixed cryoglobulinemia.

OBJECTIVE: To determine the prevalence of monoclonal rheumatoid factors (mRF) bearing the WA cross-idiotype (WA XId) in hepatitis C virus (HCV) positive type II mixed cryoglobulins, to review recent studies on the role of HCV in the cutaneous vasculitis lesions in patients with type II cryoglobulinemia and to discuss the implication of these studies for the etiopathogenesis and therapy of the disease. METHODS: Thirty type II cryoglobulins were tested for WA and PO XId and for HCV RNA: RESULTS: WA mRF were strongly, although not exclusively, associated with HCV in type II mixed cryoglobulinemia. CONCLUSION: These and other recent studies from our laboratory suggest that chronic HCV infection may be the stimulus for the production of WA mRF and that HCV may be directly involved in the pathogenesis of the cutaneous vasculitis in patients with type II cryoglobulinemia. The association of HCV infection with the disease provides a rationale for anti-viral therapy and for monitoring therapy by measuring the HCV level in both blood and liver.

Aminopeptidases highly specific for glutamyl residues from Neisseria meningitidis and Moraxella urethralis.

Cell-associated glutamyl aminopeptidase activity was detected in several strains of Neisseria meningitidis and Moraxella urethralis grown in liquid culture. Enzymatic activity was released from washed cells by ultrasonic treatment and monitored fluorometrically by measuring the release of aryl groups from 17 different aminoacyl-beta-naphthylamides. Substrates containing a glutamyl moiety were readily hydrolyzed by both N. meningitidis and M. urethralis. Glutamyl aminopeptidase activity was partially purified from crude sonicates by means of ion-exchange and gel chromatography, and samples were examined by polyacrylamide gel electrophoresis. Kinetic and pH studies were performed to partially characterize activities. The molecular weight of the M. urethralis enzyme was approximately 88,000, whereas the apparent molecular weight of the N. meningitidis enzyme was shown to be in excess of 200,000. M. urethralis produced two glutamyl aminopeptidases, one specific for a gamma-glutamyl moiety, the other specific for an alpha-glutamyl moiety. In contrast, N. meningitidis produced a single glutamyl aminopeptidase which hydrolyzed alpha- and gamma-glutamyl-substituted beta-naphthylamides.

Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.

The posttranscriptional regulatory mechanism(s) underlying thymidine kinase (TK) mRNA accumulation was investigated in BALB/c 3T3 cells during their progression from G0 into S phase of the cell cycle. Very little TK mRNA could be detected in either the nuclear or the cytoplasmic compartment from cells harvested in G0 or G1. At the onset of S phase, however, the level of nuclear TK mRNA precursors and mature TK mRNAs increased dramatically. The high molecular weight TK heterogeneous nuclear RNA species detected in the nuclei of S-phase cells were polyadenylylated and hybridized to intron sequences derived from the TK gene. A series of high molecular weight precursors could be chased to lower molecular weight species in the presence of actinomycin D, suggesting an ordered removal of intron sequences with the kinetics of a precursor-product relationship. These results demonstrate a striking change in the nuclear posttranscriptional processing of TK heterogeneous nuclear RNA at the G1-S boundary and, furthermore, define a model system for the examination of RNA-processing events in vivo.

Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.

Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene.

Hepatitis C virus but not GB virus C/hepatitis G virus has a role in type II cryoglobulinemia.

OBJECTIVE: Hepatitis C virus (HCV) infection is associated with type II cryoglobulinemia. HCV is specifically concentrated in type II cryoglobulins and has been implicated in the cutaneous vasculitis associated with the disease. In contrast to HCV, a role for hepatitis G virus (HGV) in type II cryoglobulinemia has not been defined, although prevalences as high as 43% of HGV infections in type II cryoglobulinemia have also been reported. METHODS: We studied 34 patients with type II and 29 patients with type III cryoglobulinemia associated with HCV infection, 6 patients with essential mixed cryoglobulinemia (EMC; all with type II), 50 hospital control patients, and 125 normal individuals. Serum HCV and HGV RNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). In coinfected sera, HCV and HGV were quantitated by competitive RT-PCR assays. One coinfected patient was studied longitudinally for 6 years. RESULTS: Two (5.9%) of 34 patients with HCV-infected type II cryoglobulinemia, none of 29 patients with type III cryoglobulinemia, and none of 6 patients with EMC were positive for HGV RNA, for an overall prevalence of 3.0% in mixed cryoglobulinemia. None of the control populations were positive for HGV. No statistical difference was seen between the prevalence in patients with type II cryoglobulinemia and the other populations studied. In coinfected sera, HCV, but not HGV, was concentrated in cryoglobulins, and HCV, but not HGV, correlated with cryoglobulinemia in a longitudinal study. CONCLUSION: There is a low prevalence of coinfection with HGV in patients with mixed cryoglobulinemia and HCV infection in the United States. HCV is selectively precipitated by type II cryoglobulins in coinfected sera. HGV infection does not appear to have a role in mixed cryoglobulinemia.

Detection of widespread hepatocyte infection in chronic hepatitis C.

The controversial question of the extent of hepatocyte infection in chronic hepatitis C was re-examined in both chimpanzees and humans using a newly modified in situ hybridization (ISH) method for detecting hepatitis C virus (HCV) RNA. The specificity of the methodology for distinguishing positive- and negative-strand synthetic HCV RNA was at least six magnitudes greater than the reverse-transcription polymerase chain reaction (RT-PCR) assay for HCV. The sensitivity of the methodology as determined by cell culture assay was 14 +/- 2 genomic equivalents (gE) of HCV positive strand per cell, which was three magnitudes less sensitive than RT-PCR quantitation of HCV. In contrast to previous studies in both humans and chimpanzees with chronic hepatitis C, a high percentage of hepatocytes positive for both positive- and negative-strand HCV RNA was found in most specimens studied. In humans, the extent of hepatocyte infection varied with histological activity index (HAI). In the two chimpanzees studied, the liver biopsies showed minimal histological disease activity, but high percentages of hepatocytes were HCV-positive by ISH that correlated with hepatocyte ultrastructural changes associated with HCV infection. Hepatocyte infection was confirmed by RNA extraction and RT-PCR techniques for detecting HCV RNA that minimize the false detection of negative strands. In both human and chimpanzee liver biopsies showing minimal HAI, the hepatocyte concentration of HCV was estimated to be very low. These findings suggested the hypothesis that persistent infection in the liver may be caused in part by low-level HCV replication. The theoretical and clinical implications of these findings are discussed.

Use of whole blood specimens for routine clinical quantitation of hepatitis C virus RNA does not increase assay sensitivity.

The measurement of hepatitis C virus (HCV) RNA levels in the blood has, in the last few years, become a critical component in the therapy of patients with HCV infections. Initially, extraction methods for serum and plasma were used, but a newer method that uses Catrimox-14 as the extraction agent for whole blood has been reported. Because the whole blood extraction method may yield higher virus levels if significant levels of virus are present in the white blood cells (WBC), the method was evaluated for use in our clinical diagnostic laboratory despite its higher reagent costs and more time-consuming methodology. RNA was simultaneously extracted from 39 clinical samples by four different methods: Catrimox-14-Trizol extraction from whole blood, Trizol extraction from whole blood, Trizol extraction from serum, and a commercial serum extraction method, the EZNA total RNA kit. In addition, in an effort to quantitate the amount of HCV RNA virus in the WBC, Trizol extraction from isolated WBC was also performed. Quantitative results for samples from which RNA was extracted by all four methods were essentially the same; the Catrimox-14-Trizol method did not yield increased virus levels. Insignificant levels of virus were found in the WBC. The results did not demonstrate a clinical usefulness for the Catrimox-14-Trizol method.

Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.

Endocytosis of the Flaviviridae viruses, hepatitis C virus, GB virus C/hepatitis G virus, and bovine viral diarrheal virus (BVDV) was shown to be mediated by low density lipoprotein (LDL) receptors on cultured cells by several lines of evidence: by the demonstration that endocytosis of these virus correlated with LDL receptor activity, by complete inhibition of detectable endocytosis by anti-LDL receptor antibody, by inhibition with anti-apolipoprotein E and -apolipoprotein B antibodies, by chemical methods abrogating lipoprotein/LDL receptor interactions, and by inhibition with the endocytosis inhibitor phenylarsine oxide. Confirmatory evidence was provided by the lack of detectable LDL receptor on cells known to be resistant to BVDV infection. Endocytosis via the LDL receptor was shown to be mediated by complexing of the virus to very low density lipoprotein or LDL but not high density lipoprotein. Studies using LDL receptor-deficient cells or a cytolytic BVDV system indicated that the LDL receptor may be the main but not exclusive means of cell entry of these viruses. Studies on other types of viruses indicated that this mechanism may not be exclusive to Flaviviridae but may be used by viruses that associate with lipoprotein in the blood. These findings provide evidence that the family of LDL receptors may serve as viral receptors.

Predictors of long-term response to high-dose interferon therapy in type II cryoglobulinemia associated with hepatitis C virus infection.

We have prospectively studied patients with type II cryoglobulinemia since 1985 to assess the efficacy of treatment with interferon-alpha at cumulative doses ranging from 234 to 849 MU. In the present study we retrospectively evaluated in this cohort parameters associated with complete response to therapy in 31 consecutive patients with type II cryoglobulinemia associated with hepatitis C virus (HCV) infection. Prevalence of complete response of cryoglobulinemia (disappearance of symptoms and signs of vasculitis and decrease of cryocrit below 10% of the initial value) was 62%, with a median response duration of 33 months and a range of 3 to 100 months. Three patients were putatively cured, as they remained in complete remission for more than 5 years off therapy. Eighteen patients (58%) had liver disease evidenced by histopathology and/or raised transaminase levels. Prevalence of normalization of transaminase levels was 100%, with a median response duration of 36 months. Relapse of hypertransaminasemia occurred in 100% and 8% of patients receiving less than or greater than 621 MU, respectively. By logistic regression analysis, the only pretherapy parameter that associated significantly (P = .0393) with complete response of cryoglobulinemia was the solitary anti-C22 (HCV core) antibody pattern, which was observed in 29% of patients. Association with older age and low cryocrit approached statistical significance (P = . 06), while no significant correlations were found with serum IgM levels, duration of disease, HCV genotype, NS5a gene mutations, liver histology, HLA-DR phenotype, or WA cross-idiotype. Complete responses were also associated, on univariate statistical analysis, with low pretherapy HCV viremia. Responses were accompanied by decrease of viremia, of anti-HCV antibody levels and cryocrit. The usefulness of a high dose regimen is underscored by the higher rates of sustained responses of cryoglobulinemia and transaminase levels compared with previous studies.