Discontinuation of primary prophylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis in human immunodeficiency virus type I-infected patients: the changes in opportunistic prophylaxis study.

A multicenter open, randomized, controlled trial was conducted to determine whether primary prophylaxis for Pneumocystis carinii pneumonia and toxoplasmic encephalitis can be discontinued in patients infected with human immunodeficiency virus type 1 (HIV-1) whose CD4+ T cell counts have increased to >200 cells/mm3 (and who have remained at this level for at least 3 months) as a result of highly active antiretroviral therapy (HAART). Patients were randomized to either the discontinuation arm (i.e., those who discontinued prophylaxis; n=355) or to the continuation arm (n=353); the 2 arms of the study were similar in terms of demographic, clinical, and immunovirologic characteristics. During the median follow-ups of 6.4 months (discontinuation arm) and 6.1 months (continuation arm) and with a total of 419 patient-years, no patient developed P. carinii pneumonia or toxoplasmic encephalitis. The results of this study strongly indicate that primary prophylaxis for P. carinii pneumonia and toxoplasmic encephalitis can be safely discontinued in patients whose CD4+ T cell counts increase to >200 cells/mm3 during HAART.

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.

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.

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.

A randomized, double-blind trial on the use of a triple combination including nevirapine, a nonnucleoside reverse transcriptase HIV inhibitor, in antiretroviral-naive patients with advanced disease.

The immunologic and virologic activity of nevirapine in combination with two nucleosides (zidovudine [ZDV] and didanosine [ddI]) was evaluated in antiretroviral-naive patients with a CD4 count <200/mm3 or clinical AIDS. In all, 68 patients were enrolled in a 48-week double-blind, placebo-controlled trial. A group of 32 patients received ZDV + ddI + nevirapine, and 36 patients received ZDV + ddI. Primary efficacy parameters were the activity on HIV-1 RNA and on peripheral blood CD4+ cells, with differences between groups analyzed by the Wilcoxon's nonparametric two-sample test. Baseline RNA was high in both treatment groups (median values, 5.8 and 5.7 log10). RNA and CD4 responses were significantly higher with the triple combination (median RNA reductions, 2.69 versus 1.05 log10 at 24 weeks and 1.97 versus 1.20 log10 at 48 weeks; median CD4 increases, 81 versus 64 cells/mm3 at 24 weeks and 101 versus 27 cells/mm3 at 48 weeks). This study demonstrates that a triple combination of ZDV + ddI + nevirapine used as first-line regimen in antiretroviral-naive patients can induce sustained virologic and immunologic response in patients with low CD4 count or a previous diagnosis of AIDS.

Apoptotic features of peripheral blood granulocytes and monocytes during primary, acute HIV infection.

Apoptosis plays a major role during HIV infection, including the primary, acute HIV syndrome (AHS), during which such phenomenon is massive. We asked whether apoptosis involved not only peripheral blood lymphocytes, but also monocytes (PBM) and granulocytes (PBG). Thus, we studied cells from different patients during the acute phase of the viral syndrome. The CD95 molecule was expressed at high density on the PBM and PBG surface during AHS. Culturing PBG for a few hours resulted in a significant membrane expression of phosphatidylserine, consistent with apoptosis. However, cells maintained for hours plasma membrane integrity and showed no relevant changes in mitochondrial membrane potential. The overexpression of CD95 was not associated with high plasmatic levels of sCD95 and, together with apoptosis and its related markers decreased after a few weeks of highly active antiretroviral therapy. During AHS, a deregulation of the CD95 system occurs in monocytes and granulocytes, is related to a high propensity of PBG to undergo apoptosis, and may contribute to the pathogenesis of the disease. Antiretroviral treatment resulted not only in a decrease of virus production, but also in a reduced PBG tendency to undergo spontaneous apoptosis. Even if the mechanism(s) responsible for this phenomenon remains to be elucidated, our data suggest a possible (indirect?) action of antiretroviral therapies on PBG and PBM which could explain, at least partially, the rescue of natural immunity and the reduced use of granulocyte-colony stimulating factor during such treatments.

[AIDS splenomegaly and related iron problems]. / Splenomegalia da AIDS e problematiche siderotiche connesse.

Spleens collected from 85 consecutive autopsies of AIDS patients (mean age 37 years) were studied. Splenomegaly, observed in 59 cases (69.4%), does not statistically correlate with life style and blood transfusions. Eleven very large spleens (over 890 g) were associated with opportunistic infections (i.e.: mycobacteria, true fungi and rochalimaea). The histological pattern was characterized by marked lymphoid depletion of the white pulp and--in 67 cases (89.4%)--packing of the pulp cords by macrophages engulfed of brown pigment which was strongly positive to the Perls reaction for ferric iron. The contemporary presence of Perls positive and p24 immunoreactive material was diffusely observed in the cytoplasm of splenic macrophages, also positive to the alkaline tetrazolium reaction. The same was observed in the cytoplasm of monocytes/macrophages of lung and brain (in 5 out of 5 patients with splenomegaly > 600 g, randomly selected). We believe that our findings deal with the formation of a haemoglobin--p24 complex and are in keeping with recent data which suggest the formation of disulphide bonds between viral proteins and haemoglobin.

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.

Relevance of clinical and laboratory findings in the diagnosis of cytomegalovirus encephalitis in patients with AIDS.

A retrospective evaluation was conducted in patients with AIDS and an autopsy diagnosis of cytomegalovirus (CMV) encephalitis to determine the relevance of clinical and laboratory findings in establishing a diagnosis. On autopsy of 100 patients, CMV encephalitis was diagnosed in 13 patients; eight had periventricular CMV encephalitis, four micronodular CMV encephalitis, and one both conditions. Seven patients had had a CMV infection previously (6 cases of retinitis, 1 case of colitis), and at the onset of encephalitis all of them were receiving a maintenance dose of ganciclovir. Examination of the CSF showed specific changes in patients with periventricular encephalitis. CT revealed no characteristic findings, while MRI showed an increased signal intensity on T2 weighted images. CMV DNA amplification by nested PCR was performed in nine patients with CMV encephalitis; PCR was positive in eight patients whose CSF was collected during CMV encephalitis, and negative in one patient whose CSF was collected six months before death. In conclusion, some clinical findings suggest a presumptive diagnosis, especially of periventricular encephalitis, and nested PCR appears to be a reliable and rapid technique for making an antemortem diagnosis.

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.

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.

A randomized trial (ISS 901) of switching to didanosine versus continued zidovudine after the diagnosis of AIDS.

Although the efficacy of switching from zidovudine (AZT) to didanosine (ddI) has already been evaluated in controlled studies, prior investigations were not specifically designed to evaluate this issue in patients with acquired immune deficiency syndrome (AIDS). This open, randomized, multicenter study (ISS 901) was designed to evaluate the clinical benefit in patients with AIDS of switching to ddI after 6-18 months of AZT and no major intolerance. Patients were randomized to continue AZT, maintaining the current dosage at randomization (n = 79), or to receive ddI (n = 80) at the dosage of 375 mg and 250 mg b.i.d. for body weight > 60 and < or = 60 kg, respectively. Primary efficacy measures were survival and time to new AIDS-defining events, analyzed by the intent-to-treat approach. The two groups were comparable for baseline characteristics, follow-up (15 months), and time spent on allocated treatment. At the end of the study, 104 patients (48 AZT, 56 ddI) had died and 90 had at least one new AIDS-defining event (44 AZT, 46 ddI). Kaplan-Meier estimates of survival and of time to first new AIDS-defining event showed no differences between the treatment groups. No differences were detected in other efficacy measurements (p24 antigenemia, CD4+ count, Karnofsky score, and body weight), occurrence of severe toxicities, and treatment modifications. Pancreatitis occurred only in ddI-treated patients (6%). In our population of patients with advanced disease, switching from AZT to ddI did not produce apparent benefits, suggesting that application of this strategy earlier in the course of human immunodeficiency virus disease should be considered.

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.

Massive activation of immune cells with an intact T cell repertoire in acute human immunodeficiency virus syndrome.

In 8 patients with symptomatic, acute primary infection with human immunodeficiency virus (HIV), a dramatic and persistent decrease in CD4+ lymphocytes was seen, accompanied by a marked increase in activated/memory CD8+ T cells (CD38+, CD45R0+, HLA-DR+, with high amounts of cell adhesion molecules), which represented most circulating lymphocytes, but no gross alterations in V beta T cell repertoire. Extremely high plasma levels of proinflammatory cytokines were observed. Three patients were followed for 2-3 years: The number of CD4+ cells, extremely low at first, increased significantly in a few months but decreased rapidly after a short stable period. Cytotoxic T lymphocytes bearing markers of immunologic activation/memory could play an important role in the earliest phases of the disease. It remains to be established how such a dramatic onset could determine the rapid progression of the infection that seems characteristic of patients with acute HIV syndrome.

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.

Non-typhoid Salmonella subdural empyema in a patient with AIDS.

In AIDS patients, non-typhoid salmonella metastatic abscesses in lung and brain due to bacteremia have been described previously. Here we present a case in which a group B Salmonella, serotype Copenhagen, caused right parietal subdural empyema. The etiologic diagnosis was based on culture of pus obtained from the lesion. The patient was treated for bacterial meningitis and made a good recovery. He is at present reasonably well and is taking ciprofloxacin as prophylaxis against salmonella relapse.

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.

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.