Resistance to Fas-mediated apoptosis of human T-cell lines expressing human T-lymphotropic virus type-2 (HTLV-2) Tax protein.

The susceptibility to Fas-mediated apoptosis was evaluated in seven T-cell lines (two infected with HTLV-2, one with HTLV-1, and four HTLV-free) as well as in Jurkat cells transfected with a Tax-2 expressing vector. Fas-mediated apoptosis was significantly reduced in the HTLV-1- and HTLV-2-infected lines in comparison with the HTLV-free lines regardless of the surface expression of Fas antigen (which was no different in the infected and uninfected cells). Fas-mediated apoptosis was also significantly inhibited in Jurkat cells transfected with the Tax-2 expressing vector without any modification in Fas expression. There was significantly more antiapoptotic Bcl-x(L) mRNA and protein in the transfected than in the untransfected Jurkat T cells. In conclusion, our results suggest that HTLV-2 is capable of inhibiting Fas-mediated apoptosis by means of a mechanism involving the tax-2 gene and probably the expression of bcl-x(L) messenger and protein.

Molecular epidemiology of TT virus in Italy and phylogenesis of viral isolates from subjects at different risk for parenteral exposure.

The prevalence and genotype distribution of human TT virus (TTV) in Italy were analysed in 593 subjects at different risk of parenteral infection who included blood donors, patients with chronic type C hepatitis (HCV), thalassemic patients, patients on haemodialysis, human immunodeficiency virus type 1 (HIV-1)-negative intravenous drug users (IVDUs), and HIV-1-infected subjects (IVDUs, heterosexual contacts and homosexual males). Plasma TTV-DNA was detected using nested PCR with primers deduced from the N22 region of the open reading frame 1 (ORF-1) and from the untranslated region (UTR) of the viral genome. Phylogenetic analysis of the sequences obtained from ORF-1 was also undertaken. A high prevalence of plasma TTV-DNA was observed using the UTR primers, with rates varying from 83-100% in the study groups. Using the N22 primers, HIV-1 positive IVDUs and homosexual males, haemodialysed patients and thalassemic patients had a significantly higher TTV prevalence (range: 23.0-86.1%) than blood donors, who displayed a high frequency of positivity (10.6%). Sequence analysis of 127 N22-positive isolates revealed that 42.5% were of type 1, 53.5% of type 2, 2.4% of type 3, and that two isolates (1.6%) were closely related to genotypes 1-2 but distinct from the other major genotypes. TTV-2 was significantly more prevalent in patients at high risk for parenteral infection and in HIV-1 positive homosexuals. In sequential samples from 15 TTV-infected subjects, N22 sequences were detectable persistently in 12 (80.0%) and UTR sequences persisted in all 15 patients over a mean period of 29.6 months. This data indicates that TTV is widespread in Italy in parenterally exposed subjects, and that the infection frequently persists.

A PCR-colorimetric microwell plate hybridization assay for detection of Mycobacterium tuberculosis and M. avium from culture samples and Ziehl-Neelsen-positive smears.

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

Human T-lymphotropic virus type 2 (HTLV-2) provirus in circulating cells of the monocyte/macrophage lineage in patients dually infected with human immunodeficiency virus type 1 and HTLV-2 and having predominantly sensory polyneuropathy.

We investigated the presence of human T-lymphotropic virus type 2 (HTLV-2) DNA in the peripheral blood mononuclear cell subsets obtained from 18 patients coinfected with human immunodeficiency virus type 1 and HTLV-2, 6 of whom also had predominantly sensory polyneuropathy (PSP). HTLV-2 DNA and RNA were found in CD8- and CD19-positive cells, and, for patients with PSP, in CD14-positive cells as well. Furthermore, the patients with PSP had higher proviral loads than those without PSP.

Prevalence of hepatitis G virus RNA in human immunodeficiency virus type 1-positive intravenous drug users.

OBJECTIVE: The aim of this study was to evaluate the prevalence of the new human flavivirus hepatitis G virus (HGV) in Italian intravenous drug users (IDUs) and its interaction with human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). STUDY DESIGN/METHODS: Seventy-nine IDUs with different clinical stages of HIV-1 infection and 20 non-IDU patients with chronic HCV infection were included in the study. HGV RNA was detected by means of reverse transcription-polymerase chain reaction (RT-PCR) used for the amplification of two HGV-related sequences included in the 5'-noncoding (NCR) and NS5a regions. RESULTS: Eighteen (22.8%) of the 79 IDUs were positive for plasma HGV RNA; there was no difference in mean serum alanine aminotransferase (ALT) levels between the HGV-positive and HGV-negative patients. No significant correlation was observed between HGV and other viral markers (hepatitis B virus [HBV], HCV, human T-cell lymphotropic virus type II [HTLV-II]) or HCV genotype. The number of patients with symptomatic HIV-1 infection in whom HGV RNA was detected was significantly lower than the number of those who were asymptomatic (6 of 49 [12.2%] versus 12 of 30 [40%]; P = 0.004). The mean plasma HGV RNA titer was higher in the asymptomatic than in the symptomatic patients (4.6 versus 3.2 log PCR-amplified units in 1 mL of plasma sample [PU/mL]; P = 0.03). CONCLUSIONS: Our results show a considerable spread of HGV levels among Italian HIV-1-positive IDUs and do not indicate that HGV infection enhances liver impairment. We suggest that the greater prevalence of HGV RNA in IDUs with asymptomatic HIV-1 infection may reflect the relatively recent HGV infection in this population.

Patterns of in vitro anti-human immunodeficiency virus type 1 antibody production in long-term nonprogressors.

With the aim of evaluating the specific pattern of in vitro antibody production (IVAP) in human immunodeficiency virus type 1 (HIV-1)-infected long-term non-progressors (LTNPs), we tested 20 subjects who had remained asymptomatic for more than 8 years with a CD4+ cell count higher than 500/microliter and 59 patients at different stages of HIV-1 infection as controls. In cell cultures, IVAP was detected in 14 out of 20 LTNPs (70%), in 5 out of 6 recent seroconverters (83%), and in all the other control patients. Anti-p24 antibody production was significantly lower in LTNPs than in asymptomatic patients with a more recent infection. Recent seroconverters and patients with AIDS did not produce anti-p24 antibodies (P = 0.02). Anti-gp160 antibodies were produced by peripheral blood mononuclear cells from LTNPs in 12/20 cases. CD4+ cell count was significantly higher in IVAP-negative than in IVAP-positive LTNPs (P = 0.013), while the viral load was not significantly different. Specific anti-HIV-1 antibody production did not seem to be a correlate of long-term nonprogression.

Detection of hepatitis C virus RNA in CD19 peripheral blood mononuclear cells of chronically infected patients.

The presence of HCV RNA in peripheral blood mononuclear cells (PBMC) has been reported. To identify the cell populations carrying HCV RNA, the presence and amount of HCV RNA was investigated by limiting dilution nested reverse transcriptase-polymerase chain reaction (PCR) in PBMC subpopulations fractionated by automated cell sorting. Fifteen chronically HCV-infected patients were included in the study, 4 of whom also had mixed cryoglobulinemia. HCV RNA was present in the CD19 cells of all 15 patients, but only 5 (35.7%) of 14 and 5 (41.6%) of 12 showed HCV RNA in CD3 and CD14 cells, respectively (P < .001 by Fisher's test for each comparison). The median titer of HCV RNA was 1 PCR unit/380 CD19 cells, compared with median of 1 PCR unit/6600 PBMC as a whole. Titration was difficult in the CD3 and CD14 cells because of the frequent negativity of the first diluted sample. This study suggests that HCV RNA is selectively concentrated in B cells.

High prevalence of false-negative anti-HTLV type I/II enzyme-linked immunosorbent assay results in HIV type 1-positive patients.

A high frequency of false-negative anti-HTLV-I/II ELISA results has been reported by several authors. To verify the possible underestimate of the prevalence of HTLV-II infection in subjects infected by HIV-1, we used the PCR to investigate the presence of HTLV DNA in peripheral blood mononuclear cells (PBMCs) collected from a group of 67 HIV-1-positive anti-HTLV-I/II ELISA-negative individuals; the study population included 31 patients with HIV-1-related peripheral neuropathy (PN), 15 with non-Hodgkin lymphoma (NHL), and 23 without PN or NHL. Two subjects had both PN and NHL. All of the patients who were positive at PCR were investigated for the presence of serum anti-HTLV-I/II antibodies by means of Western blot (WB). Eighteen (26.9%) of the 67 anti-HTLV-I/II ELISA-negative patients had HTLV DNA in their PBMCs and WB-detectable serum antibodies directed against one or more HTLV antigens. The individuals affected by predominantly sensory polyneuropathy (PSP) had a significantly higher prevalence of HTLV DNA than the others. All of the patients in whom HTLV-I/HTLV-II discrimination was successful had HTLV-II, with the exception of one patient who was infected by HTLV-I. The present study confirms the possibility of HTLV infection in the absence of ELISA-detectable serum anti-HTLV-I/II antibodies, especially in the particular setting of HIV-1-infected individuals. Moreover, the fact that the prevalence of HTLV DNA was significantly higher in the subjects affected by predominantly sensory polyneuropathy further supports the possibility of an association between HIV-1-related PSP and HTLV-II.

HTLV infection in ELISA-negative blood donors.

Human T lymphotropic viruses (HTLVs) have a limited spread in the general populations of Western countries. Consequently, the transfusional risk for HTLV is consider to be low in Italy and the screening for anti-HTLV-I/II antibodies has not yet been introduced. In 1992, 1087 blood donors attending a transfusional center in northern Italy underwent anti-HTLV-I/II screening carried out by means of two different ELISA tests. Eleven individuals who were negative at the first test were borderline at the second, eight of them showing reactivity to Western blot (WB). Polymerase chain reaction (PCR) for the detection of HTLV DNA, subsequently performed on the peripheral blood mononuclear cells of these 11 subjects, was positive in the same 8 WB-reactive donors. Five of them were infected by HTLV-II, and three by HTLV-1. Our results confirm that the sensitivity of the ELISA tests actually used for the detection of HTLV-I/II antibodies is low, and that HTLV-infected blood donors may be frequently undetected. Moreover, in our study population, the prevalence of HTLV infection (0.73%) was greater than that which might be expected from the existing seroepidemiological data in Italy.

High prevalence of human T cell lymphotropic virus type II infection in patients affected by human immunodeficiency virus type 1--associated predominantly sensory polyneuropathy.

The etiopathogenesis of peripheral neuropathy (PN) that frequently affects human immunodeficiency virus (HIV) type 1-positive patients remains undefined. Forty-seven HIV-1-positive patients with PN (8 with inflammatory demyelinating polyneuropathy and 39 with predominantly sensory polyneuropathy [PSP]) and 266 controls with symptomatic HIV-1 infection without PN were screened for antibodies to human T cell lymphotropic virus (HTLV) types I and II. The prevalence of antibodies to HTLV-II was significantly higher in patients with PSP than in controls (30.8% vs. 8.3%; P < .001). All seropositive patients with PN had HTLV-II DNA in their peripheral blood mononuclear cells by polymerase chain reaction (PCR) analysis. PCR analysis of tissues from 1 patient with PSP who died during the study showed HTLV-II proviral sequences in the femoral nerve and basal nuclei. These results support the hypothesis that HTLV-II represents an etiologic factor in the pathogenesis of a considerable proportion of PSP in patients infected with HIV-1.

HCV genotypes in bone marrow and peripheral blood mononuclear cells of patients with mixed cryoglobulinemia.

OBJECTIVE: The association of the hepatitis C virus (HCV) with the cryoglobulinemic syndrome is well known, but its pathogenetic mechanism still remains to be clarified. HCV-RNA has been found in the peripheral blood mononuclear cells (PBMC) of infected subjects. We investigated the presence of the HCV genome in bone marrow cells (BMC), and the distribution of different HCV genotypes in individuals with mixed cryoglobulinemia (MC) and in noncryoglobulinemic controls. METHODS: 15 anti-HCV positive subjects with MC, 7 non-cryoglobulinemic patients with type C chronic active hepatitis (CAH) and 2 anti-HCV negative controls were studied. HCV-RNA was detected by nested PCR of the highly conserved 5'-NCR sequence. HCV typing was carried out by means of the hybridization of the same amplified region with specific probes. RESULTS: HCV-RNA was present in the PBMC of a large proportion of the MC patients and controls without any significant differences. On the contrary, HCV-RNA was present in the bone marrow cells of all the patients with MC and in 43% of the CAH controls. The HCV 1b and 2a genotypes seem to be the most prevalent among MC patients. Nevertheless, the patients with type II MC had a very high prevalence of the 2a genotype (77%). CONCLUSION: The results suggest that the presence of HCV-RNA in bone marrow cells may be correlated to the pathogenetic mechanism of MC. Other studies are needed to confirm the frequent association of HCV genotype 2 with MC.

HCV-RNA detection using different PCR methods in sera, cryoglobulins and peripheral blood mononuclear cells of patients with mixed cryoglobulinemia.

OBJECTIVE: The hepatitis C virus (HCV) is frequently associated with mixed cryoglobulinemia (MC), and a number of authors have reported the presence of anti-HCV antibodies and HCV-RNA in the blood of MC patients. The presence of the HCV genome in the blood cells of individuals infected by HCV may correlate with the etiopathology of MC. We investigated the presence of HCV-related sequences in the sera, cryoglobulins and peripheral blood mononuclear cells (PBMC) of patients with MC and of individuals with type C chronic active hepatitis (CAH). METHODS: 39 patients with MC, 11 non-cryoglobulinemic HCV-positive individuals with CAH, and 2 anti-HCV negative controls were included in the study. The presence of HCV-RNA was detected by nested RT-PCR and by a commercial kit. The PCR was performed by amplifying the 5'-non coding region (5'-NCR) of HCV. RESULTS: HCV-RNA was detected in the sera and cryoglobulins of about 90% of the patients; the commercial kit showed a higher sensitivity than nested PCR. One MC patient showed HCV-RNA only in the cryoglobulins. HCV-RNA was present in the PBMC of 14 of the 20 (70%) MC patients analyzed. No differences in serum and PBMC HCV-RNA positivity were found between MC patients and controls. CONCLUSION: Our results confirm the spread of HCV infection among patients with MC. HCV-RNA is present in the serum, cryoglobulins and PBMC of a large proportion of MC patients. The prevalence of HCV-RNA in the PBMC of MC patients and controls did not differ significantly; this may suggest a tropism of HCV for PBMC regardless of the presence of cryoglobulinemia.

Frequent detection of antibodies against HTLV antigens in patients with AIDS-related non-Hodgkin lymphoma.

We studied the prevalence of anti-HTLV-I/II antibodies in 22 patients with AIDS-related non-Hodgkin lymphoma (NHL), 453 HIV-1-infected patients without lymphoma (194 of whom were diagnosed as having AIDS), and 6 HIV-1-positive and 75 HIV-1-negative patients with Hodgkin lymphoma. The frequency of serological reactivity against HTLV antigens was significantly higher in the AIDS patients with lymphoma than in those without (8 of 22, 36.4% vs. 20 of 194, 10.3%-p = 0.0027). One of the HIV-1-positive and none of the HIV-1-negative patients with Hodgkin lymphoma showed anti-HTLV-I/II reactivity. Four of the eight seropositive NHL patients showed antibodies directed against HTLV-II recombinant antigens when tested for serological discrimination in a Western blot assay. A PCR study of PBMCs from the only patient with NHL still alive at the time of the study showed HTLV-II-specific sequences in the genomic DNA. These data suggest that HTLV-II or a closely homologous retrovirus infects a high proportion of patients with AIDS-associated NHL.

Hepatitis C virus RNA in the bone marrow of patients with mixed cryoglobulinemia and in subjects with noncryoglobulinemic chronic hepatitis type C.

Hepatitis C virus (HCV) infection is associated with most mixed cryoglobulinemia (MC) syndromes. In this study, HCV RNA was detected in the peripheral blood mononuclear cells of 11 (73.3%) of 15 patients with MC and in 5 (71.4%) of 7 noncryoglobulinemic patients with chronic hepatitis type C. All patients with cryoglobulinemia and 3 (42.8%) of the 7 without cryoglobulinemia (P < .05) had HCV RNA in bone marrow cells. Subjects in both groups with HCV-positive bone marrow also had HCV RNA in serum. The majority of patients with MC syndromes were infected with HCV subtypes 1b and 2a. Two patients with MC had different genotypes in serum and cells. Further studies are needed to determine which bone marrow cell population is preferentially infected by HCV and to determine if this phenomenon is involved in inducing the production of cryoglobulins.

A nucleosome assembly factor is a constituent of simian virus 40 minichromosomes.

Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.