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.

Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome.

OBJECTIVE: To study alterations of mitochondrial membrane potential (delta psi) and the propensity to undergo apoptosis in peripheral blood lymphocytes (PBL) from subjects with acute HIV syndrome; and to evaluate possible modulations of these phenomena by antioxidants that can be used in therapy, such as N-acetyl-cysteine (NAC), nicotinamide (NAM), or L-acetyl-carnitine (LAC). METHODS: Mitochondrial function and the tendency of PBL to undergo spontaneous apoptosis were studied on freshly collected PBL from patients with symptomatic, acute HIV-1 primary infection, which were cultured for different durations in the presence of absence of NAC. NAM or LAC. By a cytofluorimetric method allowing analysis of delta psi in intact cells, we studied the function of these organelles under the different conditions. PBL apoptosis was evaluated by the classic cytofluorimetric method of propidium iodide staining, capable of revealing the typical DNA hypodiploid peak. RESULTS: Significant delta psi alterations and tendency to undergo apoptosis were present in PBL from the subjects we studied. Indeed, when cultured even for a few hours in the absence of any stimulus, a consistent number of cells died. However, the presence of even different levels of NAC, NAM or LAC was able to rescue most of them from apoptosis. Both a fall in delta psi and apoptosis were evident in PBL collected in the earliest phases of the syndrome (before seroconversion), and changed significantly after a few days. A significant correlation was found between spontaneous apoptosis and tumour necrosis factor (TNF)-alpha or p24 plasma levels, as well as between apoptosis and the percentages of circulating CD4+ or CD8+ T cells. CONCLUSIONS: PBL from patients with acute HIV syndrome are characterized by both significant mitochondrial alterations and a dramatic tendency to undergo apoptosis. The use of NAC, NAM or LAC seems to rescue cells through a protective effect on mitochondria, a well-known target for the action of TNF-alpha and for reactive oxygen species, the production of which is strongly induced by this cytokine. Thus, our data could provide the rationale for the use of such agents in addition to antiviral drugs in primary infection.

Lack of selective V beta deletion in CD4+ or CD8+ T lymphocytes and functional integrity of T-cell repertoire during acute HIV syndrome.

OBJECTIVE: To study the V beta T-cell repertoire in peripheral blood lymphocytes (PBL) during acute HIV syndrome by using several anti-V beta monoclonal antibodies (MAb) and to analyse its functionality by stimulating PBL with superantigens (SAg) such as Staphylococcus aureus enterotoxins. METHODS: Cytofluorimetric analysis of V beta T-cell-receptor expression was performed on PBL from eight patients with symptomatic, acute HIV-1 primary infection, showing a dramatic decrease of CD4+ PBL accompanied by a marked increase in activated/memory CD8+ T cells, and on 12 age- and sex-matched healthy controls. PBL were then isolated, stimulated with different SAg, anti-CD3 MAb or phytohaemagglutinin and cultured for 3 days. PBL capability to progress through cell cycle was studied by the classic cytofluorimetric method of bromodeoxyuridine incorporation and DNA staining with propidium iodide. RESULTS: Despite the presence of a few expansions of some V beta families among CD8+ T lymphocytes, no gross alterations in T-cell repertoire were present in patients with acute HIV syndrome. Its functionality was maintained overall, as PBL responsiveness to SAg was well preserved. Interestingly, all CD8+ T cells, although bearing different V beta T-cell receptors, expressed marked signs of activation, i.e., CD45R0, CD38 and major histocompatibility complex class II molecules, and also high amounts of CD11a and CD18. CONCLUSIONS: Our data suggest, at least in the early phases and in the acute form of the infection, that HIV is not likely to act as a SAg. However, further studies are needed to analyse other sites, such as lymph nodes, where HIV could exert other, significant effects, and to study the expression of other V beta families than those investigated here.

Deregulation of the CD95/CD95L system in lymphocytes from patients with primary acute HIV infection.

OBJECTIVE: To analyze the role of CD95/CD95 ligand (CD95L) expression and functionality in peripheral blood lymphocytes (PBL) during primary, acute HIV syndrome (AHS) and in the subsequent period. PATIENTS: Twelve patients were studied during the acute phase of the viral infection and most were followed for some months. METHODS: Cell culture and cytotoxicity assays based upon 51Cr release and flow cytometry were used to evaluate cell killing via CD95 molecule, flow cytometry to assess surface antigens, enzyme-linked immunosorbent assay (ELISA) for the determination of soluble CD95 and CD95L plasma levels, quantitative competitive (QC) reverse transcription polymerase chain reaction (RT-PCR) with an original RNA competitor for the analysis of CD95L mRNA expression and QC RT-PCR for determining plasma viral load. RESULTS: The analysis of PBL during this phase revealed that almost all cells, including CD8 T cells with a virgin phenotype, B lymphocytes and natural killer cells displayed CD95 molecules on the plasma membrane. Activation of CD95 on the surface of isolated lymphocytes by anti-CD95 monoclonal antibodies or binding to CD95L induced rapid apoptosis. However, CD95L mRNA was not expressed in PBL from these patients and was poorly inducible. Soluble CD95 was found in the plasma of all patients, but only in a few at high levels, even some months after seroconversion. In contrast, soluble CD95L was detected in only one patient, this occurring after the symptomatic period. For 10 of the 12 patients, expression of CD95 on the cell membrane or in the plasma did not correlate with the plasma viral load, which varied widely from patient to patient. Further, plasma levels of soluble CD95 were not altered by decreased lymphocyte activation or by efficient antiretroviral therapy. CONCLUSIONS: In patients experiencing an acute, primary HIV infection, a prolonged deregulation of the CD95/CD95L system may exist, which is probably not entirely related to virus production but may contribute to the pathogenesis of the disease. The hypothesis can be put forward that a complex balance exists between proapoptotic events (increase in CD95 expression), probably triggered by the host as a method to limit viral production, and antiapoptotic events (decrease in CD95L expression) probably triggered by the virus as a way to increase its production and survival.

Detection of HIV-1 in epidermal Langerhans cells of HIV-infected patients using the polymerase chain reaction.

Langerhans cells (LC) are bone marrow-derived, HLA-DR+, CD1a+, dendritic antigen-presenting cells found in stratified squamous epithelia. Within resident epidermal cells (EC), LC are the only cells expressing the CD4 antigen and are, therefore, a possible target for human immunodeficiency virus (HIV) infection. To date, conflicting results have been reported on the in vivo infection of LC by HIV. The aim of the present study was to investigate the presence of HIV-1 proviral DNA in epidermal LC of HIV-1-infected patients. EC suspensions were prepared from clinically normal skin of nine seropositive patients. Purified LC and LC-depleted EC were obtained by immunomagnetic separation and analyzed for the presence of HIV-1 proviral DNA by the polymerase chain reaction using primer pairs from different conserved regions (env and gag) of the HIV-1 genome. HIV-1 proviral DNA was detected in LC from seven of nine patients. LC-depleted EC fractions from the same nine patients were all negative, with the exception of one case. Altogether these results demonstrate that epidermal LC are infected by HIV-1 and constitute the only resident cell type in the epidermis harboring the virus. Further studies are, however, needed to demonstrate HIV replication in LC and to elucidate the functional role of LC in this infection.

Differential down-regulation of CD95 or CD95L in chronically HIV-infected cells of monocytic or lymphocytic origin: cellular studies and molecular analysis by quantitative competitive RT-PCR.

We analysed the expression of CD95/CD95L in two widely used models for studying the cellular effects of chronic infection with human immunodeficiency virus type 1 (HIV-1), i.e. ACH-2 cells, derived from the lymphocytic cell line A301, and U1, derived from monocytic U937 cells. A301 and ACH-2 mounted the same amount of plasma membrane CD95, while U1 had a consistent decrease in CD95 expression. Using different antibodies, we failed to detect the plasma membrane form of its ligand, CD95L, but we could see the intracellular presence of that molecule in A301 cells and, to a lesser extent, in ACH-2 cells, but not in U937 or U1 cells. To confirm the cytofluorimetric data and quantify the expression of CD95L at the RNA level, we developed a quantitative competitive RT-PCR assay. The HUT78 cell line had about 50,000 copies mRNA/1000 cells, three times more after induction with a phorbol ester and ionomycin. ACH-2 expressed about 400- (basal) or 10- (induced) fold less CD95L mRNA than the parental cell line A301; U937 and U1 were below the limit of detection. In cells of lymphoid origin (ACH-2) chronic HIV infection inhibits the expression of CD95L, the phenomenon occurring at the transcriptional level. In cells of monocytic origin (U1) the infection decreases the plasma membrane expression of CD95. This suggests that HIV could trigger different anti-apoptotic strategies which likely depend upon the cell line which is infected. In monocytic cells which act as a viral reservoir, the expression of the molecule whose binding triggers apoptosis decreases, while in lymphoid cells, capable of exerting cytotoxicity, the expression of a molecule which induces apoptosis is reduced.

Heterosexual transmission of the human immunodeficiency virus: a seroepidemiological study.

The transmission of the human immunodeficiency virus (HIV) was studied in 647 subjects who presented no apparent risk factors for the infection other than having had promiscuous heterosexual relations, heterosexual relations with people with an elevated risk of infection, or heterosexual relations with people infected by human immunodeficiency virus. Thirty subjects were found to be seropositive for anti-human immunodeficiency virus antibodies. The elevated risk factors included being the habitual partner of a person at risk of infection or of a person who was infected by human immunodeficiency virus, or being the partner of a patient with acquired immunodeficiency syndrome. The transmission of the virus was verified in 13 of 284 subjects (4.57%) who had had heterosexual intercourse three or more times with persons at risk and in 16 of 101 subjects (15.84%) who had had heterosexual intercourse three or more times with persons who were seropositive for human immunodeficiency virus antibodies. No significant correlation between human immunodeficiency virus infection and a history of sexually transmitted infections, nor between human immunodeficiency virus infection and female subjects was found. These data suggest that the epidemic of acquired immunodeficiency syndrome can also spread through heterosexual relations, even if the possibility of becoming infected in this way seems at the moment limited to particular risk behaviors.

Isolation of mumps virus from vaginal secretions in association with oophoritis.

Mumps virus was isolated from the cervico-vaginal secretions of a 31-year-old woman presenting with mild signs of oophoritis accompanied by xantholeucorrhoea. The serological finding of mumps-specific IgM antibodies was further evidence confirming the casual link between mumps virus and ovarian pathology. Rarely, ovarian involvement may be the only manifestation of mumps infection. The importance of this diagnosis and its relationship to the possible development of secondary oligomenorrhoea and early menopause is discussed.

Primary hepatic actinomycosis.

A case of primary hepatic actinomycosis is reported. Multiple liver abscesses were diagnosed in a febrile 60-year-old-man by means of non-invasive techniques such as ultrasonography, CT and radioisotope scans. The aetiology was confirmed by the unusual finding of Actinomyces israelii in anaerobic blood cultures. Other sites were not involved. Early penicillin therapy enabled surgery to be avoided.

Acanthamoeba meningoencephalitis in a patient with acquired immunodeficiency syndrome.

Several cases of Acanthamoeba encephalitis (ie, granulomatous amebic encephalitis) have been reported in patients with acquired immunodeficiency syndrome from the United States. To our knowledge, none so far has been reported from Europe, and this is the first case of amebic meningoencephalitis due to Acanthamoeba in a patient with acquired immunodeficiency syndrome from Italy. The patient was a 24-year-old, human immunodeficiency virus-positive heterosexual man with a 6-year history of intravenous drug use. He was admitted to the hospital because of severe headache, confusion, nuchal rigidity, jaundice, and ascites. He died 5 days later. At autopsy, the brain showed extensive hemorrhagic necrosis with numerous trophic and cyst forms of Acanthamoeba. The amebas were identified as Acanthamoeba divionensis by the indirect immunofluorescence test.

Oral hairy leukoplakia in AIDS patients: an ultrastructural study.

Hairy leukoplakia is a specific oral lesion associated with the opportunistic development of Epstein-Barr virus in the oral epithelium. It is now considered to be an early sign of HIV-induced immunosuppression. Four cases of oral hairy leukoplakia (OHL) from the lateral borders of the tongue of male AIDS patients were investigated by transmission electron microscopy. At the ultrastructural level, herpes-like viral particles were detected in the oral lesions of all cases. Indirect immunofluorescence performed on two cases showed the presence of EBV antigens in the nuclei and the cytoplasm of the infected epithelial cells. None of the specimens contained ultrastructural evidence of human papillomaviruses.

Polymerase chain reaction in the early diagnosis of HIV-1 infection in high risk subjects.

We investigated the presence of the HIV-1 infection using the polymerase chain reaction (PCR) test in seronegative sexual partners of HIV-infected subjects and in children born to seropositive mothers. By using PCR assay, no HIV-1 DNA was detected in 32 female partners of HIV positive patients including three pregnant women who were also studied during pregnancy and after delivery. HIV-1 DNA was found in 12 out of 38 children born to seropositive mothers; five of them also had detectable serum HIV-1 p24 Ag levels. On the whole, our data stress the importance of using a very sensitive technique, i.e. PCR, for the early diagnosis of HIV-1 infection.

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.

[Tuberculous lymphadenitis. Apropos of a non-typical histological picture]. / Linfoadenite tubercolare. A proposito di un quadro istologico non tipico.

The case is presented of a young Somali with acute lymphadenitis. Whereas histological examination indicated abscessing granulomatous lymphadenitis, bacteriological studies revealed the presence of M. tuberculosis in the pus taken from the lymph node. Hence the aetiological diagnosis contradicted the morphological picture.

[Vaginitis caused by Eikenella corrodens]. / Vaginite da Eikenella corrodens.

The Authors report a case of vaginitis caused by Eikenella corrodens (E.C.) and point out it is very rare all over the world (probably the first case reported in Italy). The E.C. was the only one isolated from the vaginal specimen. They emphasized the importance of bacteria isolation in subacute or relapsing vaginitis and relevant sensibility to antibiotic assays. These are generally neglected but still able to give unexpected results then allowing a target therapy.