Phenotypic characteristics and tendency to apoptosis of peripheral blood mononuclear cells from HIV+ long term non progressors.

The aim of this study was to analyze (i) phenotype, (ii) in vitro spontaneous and induced apoptosis, (iii) glutathione (GSH) intracellular content and (iv) inhibitors of apoptosis of potential therapeutical use in peripheral blood mononuclear cells (PBMC) from HIV+ long term non progressors (LTNP), in comparison with progressors (HIV+P) and seronegative controls (HIV-). Three groups of subjects were studied: 15 HIV+P (patients losing >150 CD4+/year), 9 LTNP (subjects infected by HIV for at least 7 years without clinical and immunological signs of progression, with a mean of 898 CD4+/microL) and 18 HIV-. All subjects were living in a large community for former drug addicts, and were matched for age and sex. We used flow cytometry for analyzing PBMC phenotype and apoptosis; high performance liquid chromatography for measuring intracellular GSH content. PBMC phenotype of LTNP shared characteristics with those of both HIV- and HIV+P. Indeed, LTNP showed a normal number CD4+ cells (an inclusion criteria), but significantly increased numbers of CD8+ lymphocytes, activated T cells, CD19+, CD5+ B lymphocytes and CD57+ cells, as well as a decrease in CD19+, CD5- B lymphocytes and CD16+ cells. In LTNP, spontaneous apoptosis was similar to that of HIV- and significantly lower than that of HIV+P. Adding interleukin-2 (IL-2) or nicotinamide (NAM) significantly decreased spontaneous apoptosis in LTNP and HIV+P. Pokeweed mitogen-induced apoptosis was also similar in LTNP and HIV-, but significantly lower than that of HIV+P. In HIV+P, but also in LTNP, spontaneous apoptosis was inversely correlated to the absolute number and percentage of CD4+ cells and directly correlated to the number and percentage of activated T cells present in peripheral blood. GSH intracellular content was greatly decreased in PBMC from HIV+P and slightly, but significantly, reduced in LTNP. Adding 2-deoxy-D-ribose, an agent provoking apoptosis through GSH depletion, to quiescent PBMC resulted in similar levels of massive cell death in the three groups. This phenomenon was equally prevented in the three groups by N-acetyl-cysteine but not by IL-2. A complex immunological situation seems to occur in LTNP. Indeed, PBMC from LTNP are characterized by a normal in vitro tendency to undergo apoptosis despite the presence of a strong activation of their immune system, unexpectedly similar to that of HIV+P. Our data suggest that NAM and IL-2 are possible candidates for reducing spontaneous apoptosis in HIV infection.

Growth of human herpesvirus 6 in HEPG2 cells.

HepG2 cells, a well differentiated liver cell line, were shown to be permissive for both human herpesvirus 6 (HHV-6) A and B strains by three independent methods of analysis: detection of viral antigens, viral DNA sequences and infectious virus. HepG2 cell infection with HHV-6 resulted in functional damage as shown by the increased release in the culture medium of some hepatocyte markers. Cells surviving the acute infection were serially passaged without showing cytopathic effect, but, some months later, HHV-6 DNA was still present in the cells and virus induction with a phorbol ester was successful. A possible pathogenetic role of HHV-6 in liver diseases is discussed. Experiments of HepG2 infection with human herpesvirus 7 (HHV-7) were also carried out. The lack of an efficient virus replication suggested a difficulty for HHV-7 to infect hepatic cells.

Quantitation of HIV-1 p24 antibody expressed as relative binding capacity p24 antigen (p24 RBC) in infants born to HIV-1 seropositive mothers: correlation of this serological marker with the HIV-1 maternal antibody course and p24 antigenemia detection.

The antibody content to HIV-1 p24 Ag expressed as relative binding capacity to the target antigen (p24 RBC) was retrospectively quantified in serum samples from 20 HIV-1-uninfected infants born to HIV-1 seropositive mothers. p24 RBC values quantified at birth were included either in a low (0-20%) or high (80-100%) range of values, classified as group A (11 infants) and group B (9 infants), respectively. The course of maternal antibodies to HIV-1 antigens p17, p24, p31, gp41, p51, p66, gp120, and gp160 was studied in each group. A substantial difference in the amount and subsequently in the decline of maternal antibodies to gag proteins p17, p24, and p55 and to pol proteins p51 and p66 was observed in the two infant groups in contrast with a similar content and decline of the remaining antibodies. In 7 HIV-1-infected infants of whom 4 resembled infant group A and 3 infant group B for p24 RBC values, a relationship appeared between p24 antibody decline and p24 antigenemia detection.

Herpesvirus DNA is frequently detected in liver tissue from hepatitis C patients.

BACKGROUND AND AIMS: Herpesviruses infect the liver and cause minor hepatitis. Our aim is to verify the presence of herpesviruses in the liver from hepatitis C patients and the possible influence of these viruses in the liver disease. METHODS: We searched for herpesvirus DNA in liver biopsies from patients with hepatitis C and from a control group without hepatitis by means of nested polymerase chain reaction. Serological investigations were carried out as well. RESULTS: Thirty-four liver specimens from hepatitis C patients were examined, 12 of which (35.3%) were positive for at least one herpesvirus DNA, whereas among the 19 control specimens only two were positive (10.5%; P = 0.049). Liver biopsies from seven patients, three with acute hepatitis of unknown origin, three with non-alcoholic steatohepatitis and one with autoimmune hepatitis were also investigated and three positive samples were found. CONCLUSIONS: The prevalence of herpesvirus DNA was found higher in patients with hepatitis C than in individuals without hepatitis. The influence of herpesviruses on the clinical course of hepatitis C is considered.

Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase.

Nalidixic acid and oxolinic acid, two antibacterial agents known to inhibit bacterial DNA gyrase, are shown to suppress the replication, as well as the cytopathic effect, of BK virus in Vero cell cultures. The inhibition of virus replication was detectable at day 4 post infection in cultures which had been continuously exposed to drugs at concentrations as low as 0.02 to 0.04 mM of nalidixic acid and 0.2 mM of oxolinic acid. These active concentrations are inferior to plasma levels attained in the course of clinical use of the drugs for antibacterial chemotherapy. Also, under these circumstances, no cytotoxicity occurred. The inhibition of development of cytopathology and of virus-induced cell death was demonstrable in cultures treated for 12 days with the drugs. Under these circumstances of prolonged action, oxolinic acid proved to be slightly cytotoxic in that virus inhibitory doses reduced the viability of normal cells. No alterations in the topological conformation of the viral genome or accumulation of end products of viral DNA replication were detected. However, accumulation of viral DNA form I at 48 h post infection suggests that the drugs act through a mechanism involving DNA topoisomerase.

Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation.

AIMS: To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. METHODS: The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. RESULTS: The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. CONCLUSIONS: The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.

Detection of Epstein-Barr virus DNA in cerebrospinal fluid from immunocompetent individuals with brain disorders.

Fifty four cerebrospinal fluid samples obtained from as many immunocomponent patients with disorders of the central nervous system were investigated for the presence of herpesvirus DNA by nested polymerase chain reaction in order to determine an etiological diagnosis. Four of these samples proved positive for the presence of Epstein-Barr virus DNA (7.4%). The result of this diagnostic study is reported to draw insiders' attention to the possible presence of EBV in cerebrospinal fluid from patients with central nervous system diseases.

SupT-1: a cell system suitable for an efficient propagation of both HHV-7 and HHV-6 variants A and B.

HHV-7 growth on Sup-T1, an immature T-cell line, was studied using different HHV-7 isolates obtained in our laboratory. Titration of viral yields showed that all the virus isolates propagate on this cell line more efficiently than in cord blood lymphocytes, the cells usually recommended for HHV-7 growth. The permissivity of Sup-T1 to HHV-6, whose ability to replicate in these cells was still unknown, was also investigated using two virus isolates representative of variants A and B respectively. Both isolates were able to propagate on Sup-T1 and viral titres were similar to those obtained in cord blood lymphocytes. As the efficient propagation of both HHV-7 and HHV-6 isolates in Sup-T1 cultures, these cells may replace more time consuming and expensive cord blood lymphocyte preparations for the propagation of both the viruses.

Primary infection by HHV-6 variant B associated with a fatal case of hemophagocytic syndrome.

The association of a human herpesvirus 6 primary infection with a fatal case of hemophagocytic syndrome in a 3 month old baby is described. The trigger mechanism of the virus for the clinical syndrome is discussed.

Epstein-barr virus DNA in the cerebrospinal fluid of patients with human immunodeficiency virus infection and central nervous system disorders.

Routine search for herpesvirus types 1-5 by nested polymerase chain reaction revealed Epstein-Barr virus (EBV) DNA in the cerebrospinal fluid (CSF) of ten out of seventy-nine patients with human immunodeficiency virus (HIV) infection and central nervous system (CNS) disorders not associated with the presence of primary CNS lymphomas. One out of the ten CSF samples was positive for EBV DNA only, six were also positive for microbial agents of recognised neurological pathogenicity while the remaining three samples had a high content of HIV p24 Ag. When six available CSF samples out of the ten EBV DNA positive specimens were investigated for an intrathecal EBV antibody response, all six samples proved EBV antibody-free. The concurrent detection of neurotropic infectious agents and the absence of EBV antibodies in the CSF contribute to the uncertainty on the role of EBV in the neurological illness of the patients studied. One hypothesis considered is that the presence of EBV DNA in the CSF of a large fraction of the ten patients under study is an incidental event associated with EBV reactivation in the host's peripheral blood monocytes, but not related to the genesis of neurological disorders.

Outbreak of aseptic meningitis by echo 4: prevalence of clinical cases among adults.

Twenty five cases of meningitis occurred in urban areas surrounding a city (Modena) in Northern Italy, in the period May-July 1999. When the patients were admitted to the Infectious Diseases Division of the University of Modena and Reggio Emilia Hospital and studied by virological and serological methods, the meningitis proved to have an enteroviral origin and enterovirus ECHO 4 type was responsible for all cases of illness. An epidemiological characteristic of the enteroviral meninigitis outbreak was the adult age in 23 out of the 25 patients (mean age 24.50 +/- 7.84 years). The monthly distribution of the aseptic meningitis cases was the following: five cases occurred in May, 13 in June and seven in July. The origin of the spread of the virus infection and the reason for its sudden end remained unknown. The unusual drop in temperature which occurred in the geographic area involved in the aseptic meningitis outbreak at the beginning of August could have interfered with the slowdown in virus circulation.

Electron microscope study on a transmissible cytotoxic factor isolated from cerebrospinal fluid of neurological patients: analysis of its structure and of its effects on various types of cell cultures.

AIMS: In previous studies, the present group showed that a factor, present in the cerebrospinal fluid of seven neuro-patients, was capable of inducing cell damages on cell cultures of epithelial cells (Vero), glial cells (DG54-MG) and human primary lymphocytes. The cytotoxicity, once induced, could be transmitted to fresh cell cultures using crude preparations obtained from the cytotoxic cell cultures. METHODS AND RESULTS: The present electron microscope study describes in detail the pathological changes occurring in the previously assayed cultured cell types, and for the first time in human fibroblasts, as a consequence of the treatment with crude cytotoxic preparation. It also demonstrates with reasonable certainty the absence of virus-like particles in both the treated cells and the partially purified cytotoxic fraction. Indeed high-resolution electron microscopy analysis shows that this fraction consists almost entirely of protein spheroids with a diameter of 8-12 nm. CONCLUSIONS: The possible significance of nanotubular aggregates, observed in particular in the glial and in the epithelial cells undergoing cytotoxic damage, is also discussed.

IgG antibodies to human herpesvirus-6 (HHV-6) in Italian people.

Sera from a sample of healthy Italian people were tested in an indirect immunofluorescence assay (IFA) for reactivity to a Human Herpesvirus-6 (HHV-6) strain called CV, isolated from a baby with exanthem subitum (Portolani et al., 1990). Seropositivity values of 83.78%, 92.68% and 63.64% were found in subjects aged 3 months-6 years, 6-18 years and over 18 years respectively. Sera from cordal blood, sera from subjects with evidence of active infection by Cytomegalovirus (CMV) or by Epstein-Barr virus (EBV), sera from seropositive adults to Human Immunodeficiency Virus type 1 (HIV) were also investigated for antibodies to the same HHV-6 strain. Values of antibody incidence and antibody content to HHV-6 in these groups of sera were generally higher than in the other groups. HHV-6 reinfections of both endogenous and exogenous origin and antibody cross-reactivity were considered among the reasons of this increase. HHV-6 seropositivity values in healthy Italian people and in people from different countries were also discussed in the light of the antigenic characteristics of the HHV-6 strains used and of the different test conditions.

Encephalomyeloradiculopathy associated with Epstein-Barr virus: primary infection or reactivation?

INTRODUCTION: Encephalomyeloradiculopathy (EMR) is a new syndrome, characterized by extensive involvement of the nervous system at different levels, including brain, medulla and spinal roots. We describe a patient presenting with prodromal febrile illness, followed by a wide infection of the nervous system with transverse myelitis and less severe meningitis, encephalitis and polyradiculopathy. The patient was treated with high-dose corticosteroids, antibiotics and acyclovir; in spite of therapy his condition improved very slowly, with severe neurological sequelae. MATERIAL AND METHODS: Antiviral antibodies were searched for in serum and cerebrospinal fluid (CSF) by commercially available ELISA kits. Viral investigations were performed by cell culture isolation and search for viral antigens, and genomic nucleic acids were investigated by polymerase chain reaction (PCR). RESULTS: Virological and serological studies evidenced a primary infection by cytomegalovirus (CMV), possibly responsible for the prodromal illness, persisting in the course of the disease. PCR performed in the peripheral blood mononuclear cells (PBMCs), DNA collected early and in the CSF drawn 30 days after the onset of the disease showed Epstein-Barr virus (EBV) DNA. The serum panel of EBV antibodies was typical of an intercurrent virus reactivation, more than of a primary infection. CONCLUSION: EBV is known to be highly infectious for the nervous system, in this case of EMR the presence of DNA sequences in the PBMCs and CSF suggests that EBV plays a role in the development of this newly described syndrome.

Antibodies to papillomavirus genus-antigens in women with genital warts.

Thirty-three sera from women symptom-free for papillomatous disease and 27 sera from women with flat or exophytic genital warts were examined for the presence of IgG and IgM antibodies to papillomavirus (PV) genus-antigens. For this purpose, sera were challenged with genus-antigens extracted from both human and bovine purified virions of PVs, in a micro solid-phase enzyme-linked immunosorbent assay (ELISA). The assay performed with PV genus-antigens of human origin showed that in women with genital warts, IgG antibodies were present in a percentage of 70.37% and IgM antibodies in a percentage of 40.74%; in apparently uninfected women, IgG and IgM antibodies were present in a percentage of 54.54% and 24.24% respectively. When sera were challenged with PV genus-antigens of bovine origin for IgG antibody class, positivity was 70.37% in women with genital disease and 45.45% in symptom-free women. IgG and IgM antibody response in women with and without papillomatous genital lesions is discussed.

Search for herpesvirus DNA in cerebrospinal fluid of HIV patients with brain disorders: prevalence of cytomegalovirus DNA findings.

A study was carried out to search for the presence of the seven human herpesvirus DNAs in cerebrospinal fluid from 52 human immunodeficiency virus-infected patients with brain disorders. Cytomegalovirus DNA was the most prevalent with 12 positive samples; Epstein-Barr virus and varicellazoster DNAs were detected in three and two samples, respectively, while no sample was positive for the DNA of the other herpesviruses.

Epstein-Barr virus DNA in cerebrospinal fluid from an immunocompetent man with herpes simplex virus encephalitis.

Herpes simplex virus 1 meningo-encephalitis was ascertained in a 63-year-old immunocompetent man. To determine the duration of the persistence of herpesvirus DNA in the central nervous system, the cerebrospinal fluid was periodically monitored by polymerase chain reaction for 53 days. In addition to HSV-1, Epstein-Barr virus DNA was detected in the cerebrospinal fluid 9 days after disease onset. The possible meaning of the Epstein-Barr virus DNA finding is discussed.

Search for human herpesvirus 6 and human cytomegalovirus in bronchoalveolar lavage from patients with human immunodeficiency virus-1 and respiratory disorders.

Virus isolation and viral DNA detection by the polymerase chain reaction were used to investigate the presence of human herpesvirus 6 (HHV-6) and human cytomegalovirus (HCMV) in bronchoalveolar lavage from 34 human immunodeficiency virus-1 (HIV-1)-infected patients with respiratory disorders. The aim was to assess the presence of reactivated HHV-6 in lung tissues for a subsequent evaluation of the frequency of virus involvement in respiratory clinical manifestations in the course of HIV-1 infection. Bronchoalveolar lavage samples were tested for the presence of HCMV, as a routine investigation within a protocol monitoring opportunistic infections in symptomatic HIV-1 patients. Whereas HCMV DNA was detected by the polymerase chain reaction in 12 bronchoalveolar lavage specimens, 10 of which were also positive for virus isolation, all samples were negative for HHV-6 by both virological procedures. The HHV-6 DNA finding in bronchoalveolar lavage from an HIV-1-seronegative patient with renal carcinoma, investigated accidentally together with the bronchoalveolar lavage specimens from HIV-1 seropositive patients, stressed the HHV-6 polymerase chain reaction-negative results in the bronchoalveolar lavage samples under study. It is concluded that the lung may be a target organ for HCMV infection in HIV-1-seropositive patients affected by respiratory symptoms but that this does not seem to be the case for HHV-6.