The failure of HAART to cure the HIV-1/AIDS complex. Suggestions to add integrase inhibitors as complementary virostatics, and to replace their continuous long combination applications by short sequences differing by drug rotations.

While the intensive virostatic combinations applied according to the conventional models (such as HAART), based only on the attacks of two HIV-1 targets, retrotranscriptase and protease, and applied in a long and continuous fashion, a) are notably toxic, b) do not correct completely the abnormal immunologic parameters, and c) are followed by particularly severe and poorly sensitive relapses in case of discontinuation, we propose to the 'AIDS treatment headquarters' to include in their failing strategy the two original features which we have included in the treatment of a cohort of a dozen patients, treatment applied at all but one AIDS stage. We attack one more HIV-1 target than the conventional protocols do, by adding inhibitors of integrase; we apply the combinations of virostatics, comprising inhibitors of the three targets, in short sequences (of 3 weeks), between which the analogues are changed inside each series. The first patient of the cohort started his treatment 8.5 years ago, and the entries of the others into it have been at random and not randomized. All patients are alive today and in excellent condition.

Combinations of three or four HIV virostatics applied in short sequences which differ from each other by drug rotation. Preliminary results of the viral loads and CD4 numbers.

This paper presents the evolution during its follow-up of a virostatic combination study of the type I-II trial conducted on ten AIDS-related complex (ARC) or acquired immunodeficiency syndrome (AIDS) patients [1, 9, respectively]. Its concept is based on the following original notions: a) it is not the number of the virostatics applied to each patient at any phase which determines their effect; it is the number of affected virus targets which determines the effect. Thus, the so called "tritherapies", imposed by the "AIDS Command" to thousands of patients selected at random, to be compared to the same number of subjects receiving only "bi" or "monotherapies", might be beginning to face failure because they attack only two targets: retro-transcriptase and HIV1 protease. Having discovered, owing to our experimental screening, original HIV1 virostatics, acriflavine (ACF) and several ellipticine analogues among which we have used methyl-hydroxy-ellipticine (MHE), we are able to attack two virus targets unaffected by classical virostatics: ACF attacks DNA, from its integrated double branched stage to the provirus one, and MHE inhibits topoisomerase II. We experimentally combined these two agents with AZT, which inhibits retro-transcriptase, thus we realized a combination affecting three targets. This three agent combination was able to eradicate Friend's virus from infected mice. Clinically, combinations of three drugs affecting four targets (as they are selected among the ten virostatics available today) give a stronger result than three drug combinations affecting only three targets, because they were selected from the five virostatics which were the only ones available at the beginning of the present study. Five patients out of five who received the combinations of four virostatics chosen among the ten currently available (thus affecting four targets) from the beginning of their treatment to the present have all reduced their viral load (VL) and maintained it below the detectable level (< 200 RNA copies/mL then 20 copies/mL); b) as the toxicities of virostatics and as HIV1 resistances may happen as soon as 12 weeks of treatment, the combinations have been, in our study, applied in shorter (3 week) sequences, differing from each other due to drug rotation; c) neither toxicity nor resistance occurred; d) curiously, the CD4 numbers, even when they increased rapidly, has never attained their normal count, and their curve may be a Gombertzian one. This CD4 restoration limitation can be due to persisting virus, as indicated in some patients by small peaks which may appear on some VL plateaus, though they disappear without treatment change.

Hyponatremia in pediatric patients with HIV-1 infection.

Hyponatremia has been recognized as a complication in adults with acquired immunodeficiency syndrome (AIDS). We did a retrospective study evaluating the medical records of 86 children (age 4 months to 21 years) with human immunodeficiency virus (HIV-1) infection to determine the frequency and clinical associations of hyponatremia. Twenty-two children (26%) developed hyponatremia (serum sodium < 135 mEq/L; range 104 to 134 mEq/L; mean 130 mEq/L). Fourteen were male; 18 of the 22 patients were black and 4 were white. At the time of hyponatremia, the children frequently had comorbid associations, including 8 (35%) with AIDS encephalopathy; 3 (14%) with cardiomyopathy; 3 (14%) using diuretics; 1 (5%) using pentamidine; 3 (14%) with bacterial pneumonia; 2 (9%) requiring gastric lavage feedings; 2 (9%) with tuberculosis meningitis; 2 (9%) with gastroenteritis; 1 (5%) with infection caused by Mycobacterium avium-intracellulare; 1 (5%) each with brain tumor and tumor metastasis to brain. The cause of hyponatremia was attributed to syndrome of inappropriate antidiuretic hormone in 8 children; poor sodium intake and/or excessive diarrheal losses in 5; and the use of diuretics in 3 patients. Mild hyponatremia with no identifiable cause was found in 5 patients.

Differential reactivities of antibodies to HIV and HTLV-I in sera of suspected AIDS and ARC patients.

Sera collection from 255 clinically diagnosed AIDS and ARC patients were analyzed for antibodies to HIV and HTLV-I by Western blot and particle agglutination methods respectively. Antibodies to HIV were detected in 37.3% of the sera collected as compared to 5.5% for HTLV-I. Most (95%) of the HIV positive sera had dual reactivity to both HIV-I and HIV-2. Antibodies to HTLV-1 were more frequently detected in HIV positive sera (11.58%) than in HIV negative sera (1.88%). Conversely, antibodies to HIV were detected twice as frequently in HTLV-1 positive sera (78.6%) than in HTLV-1 negative sera (34.85%).

Langerhans' cells and lymphocytic infiltrate in AIDS-associated Kaposi's sarcoma. An immunohistochemical study.

The epidermal Langerhans' cells are dendritic cells of the skin capable of triggering cutaneous immune responses. They possess the membrane antigens required to this effect: class II histocompatibility antigen, CD1a and CD4; the latter acts as receptor for the human immunodeficiency virus. The skin is the organ primarily affected by Kaposi's sarcoma (KS). In epidemic KS, the local immunologic conditions of the skin are little known. We therefore studied 12 patients with AIDS-associated KS, evaluating the density and phenotypic expression in KS-affected and unaffected skin of the following antigens: CD1a, HLA-DR, CD4 in dendritic epidermal cells and dermis, and CD3, CD4 and CD8 in cells of the inflammatory infiltrate, using monoclonal antibodies applied to frozen sections with the avidin-biotin-peroxidase technique. Langerhans' cells in the AIDS-KS skin lesions were found to be decreased in number. This decrease was even more pronounced in the case of cells expressing HLA-DR antigen. A number of them were also revealed with CD4. The tumour lymphocytic infiltrate was almost exclusively composed of CD3+ CD8+ phenotype lymphocytes. The dermis also revealed CD4+ dendritic cells. The basal keratinocytes focally expressed HLA-DR. These phenotypical alterations of the Langerhans' cells and the local immunological imbalance observed may contribute to the growth and continuity of the KS lesions.

Analysis of natural killer (NK) cell subsets defined by the expression of two novel surface antigens (EB6 and GL183) in AIDS and AIDS-related conditions.

In this study we analyzed the expression of EB6 and GL183, which are part of P58 molecular family that represents the putative NK receptor for MHC class I molecules, in peripheral blood lymphocytes of 60 patients with HIV infection (20 asymptomatic HIV-seropositive individuals, 20 patients with constitutional symptoms, and 20 AIDS patients) and correlated it with the level of CD4+, CD56+ cells, and the NK cell activity in order to determine a possible relation with disease progression. The absolute number (but not the percentage) of CD56+, EB6+, and GL183+ cells was significantly reduced only in AIDS patients but not in the other AIDS-related clinical conditions. On the contrary, NK cell activity was reduced in all HIV-infected patients. In a 6-month follow-up, patients with constant clinical conditions and stable CD4+ cells level showed no significant difference, either in the percentage or absolute number of EB6+ and GL183+ cells. Interestingly, dual-color fluorescence indicates that GL183 and EB6 molecules (that in normal individuals are virtually absent on CD3- NK cells) are expressed in HIV-infected individuals not only in CD56+ cells but also in CD3+ cells. This may reflect a depletion of other T cell subsets or alternatively (less likely) a specific immune response. Our data indicate that the expression of EB6 and GL183 in T and NK cells from HIV-infected patients might be relevant in the course of the disease and for the disease-associated functional defect of NK cell activity.

Differential expression of tumor necrosis factor alpha and interleukin 1 beta compared with interleukin 6 in monocytes from human immunodeficiency virus-positive individuals measured by polymerase chain reaction.

Expression of tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6) was evaluated in unstimulated peripheral blood monocytes obtained from human immunodeficiency virus-positive (HIV+) individuals using a reverse transcription-polymerase chain reaction (RT-PCR) method. In all, 40 subjects were included--13 asymptomatic, 11 with ARC, seven with AIDS, and nine HIV- controls. Of the asymptomatic individuals, 85% were positive for TNF alpha and IL-1 beta compared with only 27% of the ARC and 42% of the AIDS patients. Expression of IL-6 message was observed in lesser proportions, with no significant differences among disease states. Quantitation of IL-1 beta and TNF alpha mRNA from the positive samples fell into two categories, low responders (six of 17), with < 5,000 copies of IL-1 beta and TNF alpha mRNA, and high responders (11 of 17), with > 5,000 copies per 10 pg of total cellular RNA. There was no correlation of mRNA detection or concentration with CD4+ cell number or beta 2-microglobulin levels. However, the levels of mRNA, but not its presence alone, were positively correlated with neopterin levels. The data show differential cytokine regulation in monocytes, observed as an increase in the expression of TNF alpha and IL-1 beta compared with IL-6 in HIV+ patients. Our report also emphasizes the utility of an RT-PCR system in analyzing multiple cytokine transcript levels in small amounts of clinical materials.

Pathology of lymph nodes in human immunodeficiency virus (HIV) infection.

The actual knowledge concerning the main physiological and histological data of AIDS are presented. The pictures are of lymph node biopsies from patients with AIDS, admitted in the Clinic of Hematology, Fundeni Hospital, Bucharest.

Persistent generalized lymphadenopathy and non-Hodgkin's lymphoma in AIDS: association with Rochalimaea henselae infection.

Cat scratch disease, which is caused by infection with Rochalimaea henselae, is often manifested as lymphadenopathy. R. henselae has also been isolated from human immunodeficiency virus (HIV)-positive patients with bacillary angiomatosis. In order to determine the frequency of R. henselae-reactive antibodies in HIV-positive patients with persistent generalized lymphadenopathy (PGL) or non-Hodgkin's lymphoma (NHL), we tested a total of 124 HIV-positive patients for R. henselae-reactive immunoglobulin G (IgG), IgM, and IgA antibodies by an enzyme immunoassay procedure using whole R. henselae antigen. Of the patients, 7 had PGL, 17 had NHL, and 100 were HIV stage IV (Centers for Disease Control criteria). A total of 86% of PGL patients (6 of 7) were positive for R. henselae antibodies (three were positive for IgG, IgA, and IgM, one was positive for IgG and IgA only, and two were positive for IgG only). A total of 29% of NHL patients (5 of 17) were positive for R. henselae antibodies (two were positive for IgG, IgA, and IgM and three were positive for IgG only). Only 5% of HIV Stage IV patients without adenopathy (5 of 100) were positive for R. henselae-reactive IgG, IgA, and IgM. The high prevalence of R. henselae-reactive antibodies in HIV-positive PGL and NHL patients suggests that R. henselae is a potential etiologic agent or cofactor in these patients.

Two-site enzyme immunoassay of CD4 and CD8 molecules on the surface of T lymphocytes from healthy subjects and HIV-1-infected patients.

A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.

Characteristics of IgA antibodies against HIV-1 in sera and saliva from HIV-seropositive individuals in different clinical stages.

IgA antibodies were analysed in sera and saliva from 40 HIV-1 seropositive individuals. The level of total IgA in serum was elevated according to the progress of the disease. IgA antibodies against p24 and gp160 were detected in the asymptomatic phase of infection. However, they declined in the symptomatic phases in contrast with IgG antibodies. Interestingly, three patients in the symptomatic phase who showed high levels of IgA antibodies were all in relatively good clinical condition. The IgG and IgA antibodies in saliva declined in the symptomatic phase. The level of IgG anti-p24 antibodies in saliva correlated with that in serum, suggesting that IgG anti-p24 antibodies in saliva originated from those in the serum. These results indicate that IgA antibodies are regulated independently from IgG antibodies and that the mucosal immune system is impaired early in the symptomatic phase of HIV infection, which starts with mucosal impairment. Detection of IgA antibodies may be useful for prognosis of the disease in HIV-infected individuals. The results indicate also that treatment for the impaired IgA mucosal immune system should be taken into consideration.

Use of the recombinant chimera proteins, LacZ-Env and Gag-Env, for immunological studies on HIV-1 infection.

To use Env proteins as antigens for detection of the human immunodeficiency virus type-1 (HIV-1) antibodies, we attempted to overexpress the Env proteins in Escherichia coli. To study the epitopes in the Env proteins recognized by the sera of HIV carriers, various regions of the proviral DNA encoding the Env region were fused to the 3' end of the lacZ gene. The immunoblotting analysis of the LacZ-Env(512-611) and LacZ-Env(721-826) proteins with the 41 positive sera revealed that the former and the latter immunologically reacted with 100 and 78% of the sera, respectively. To avoid rare false-positive reactions due to the LacZ moiety of the fusion protein, we attempted to express the Env(512-611) alone or Gag-Env(512-611) under the control of bacteriophage T7 promoter. Although we could express only a low level of the Env(512-611) peptide in E. coli, we succeeded in producing large amounts of the Gag(121-406)-Env(512-611) and Gag(308-406)-Env(512-611) proteins as chimeric proteins. Both of these chimera proteins strongly reacted with the 41 positive sera. We purified these proteins and analyzed the immunological reactivity by dot blot with the 60 positive sera and the 84 normal sera. As little as 20 ng of the dotted proteins was enough for the reaction with the positive sera, whereas as much as 320 ng of them did not show false-positive reactions with the normal sera. We obtained highly purified Gag-Env proteins with highly specific seroreactivity, which should be useful for diagnosis and prognosis.

A pilot study of sequential therapy with zidovudine plus acyclovir, dideoxyinosine, and dideoxycytidine in patients with severe human immunodeficiency virus infection.

A pilot study was initiated to explore a sequential combination antiretroviral regimen in 21 patients with AIDS or advanced human immunodeficiency virus (HIV) infection, who had received little or no prior anti-HIV therapy. The mean entry CD4 cell count was 184/mm3. Patients received 3-week cycles consisting of zidovudine plus acyclovir, dideoxyinosine, and dideoxycytidine for 1 week each. Overall, the regimen was well tolerated for up to 3 years. The principal toxicities were anemia, nausea, and vomiting; 1 patient developed retinal lesions. The mean CD4 cell count reached a peak of 64 cells/mm3 above baseline at week 8 (P = .005 compared to baseline) and remained above baseline for > 40 weeks. Patients also gained weight and had decreases in serum HIV p24 antigen. Eight patients developed opportunistic infections or tumors. Only 4 patients died during 3 years of follow-up. This regimen appears to be generally tolerable and to have anti-HIV activity. Additional studies will be needed, however, to learn how to best combine the available agents in patients with HIV infection.

Zidovudine in persons with asymptomatic HIV infection and CD4+ cell counts greater than 400 per cubic millimeter. The European-Australian Collaborative Group.

BACKGROUND: Zidovudine therapy is of benefit in the treatment of symptomatic and asymptomatic human immunodeficiency virus (HIV) infection in persons with CD4+ cell counts of less than 500 per cubic millimeter. The efficacy, safety, and duration of benefit of zidovudine in those with 500 or more CD4+ cells per cubic millimeter are uncertain. METHODS: In a double-blind, placebo-controlled trial, 993 patients with asymptomatic HIV infection and CD4+ cell counts above 400 per cubic millimeter were randomly assigned to receive zidovudine (500 mg twice daily) or placebo for three years. The primary end point was progression of disease, as defined by the development of Centers for Disease Control and Prevention (CDC) group IV disease (including recurrent oral candidiasis, hairy leukoplakia, or progressive diarrhea) or two CD4+ cell counts below 350 per cubic millimeter. This outcome measure was changed from the original end point of the acquired immunodeficiency syndrome (AIDS) or advanced AIDS-related complex to reflect changes in recommendations for management. The study was terminated after the first interim analysis. RESULTS: Disease progression was significantly less frequent in the zidovudine group (relative risk, 0.56; 95 percent confidence interval, 0.43 to 0.75; P < 0.001 by the log-rank test). The probability of disease progression at two years was 0.19 with zidovudine, as compared with 0.34 with placebo (95 percent confidence interval for the difference, -0.21 to -0.08). Progression to CDC group IV disease was reduced by half in the zidovudine recipients (relative risk, 0.49; P = 0.049) and decline in CD4+ cell counts to below 350 per cubic millimeter was reduced by 40 percent (relative risk, 0.60; P < 0.001). The inclusion of early HIV disease events (oral candidiasis, oral hairy leukoplakia, and herpes zoster) as end points confirmed the effects of zidovudine on the progression of clinical disease (relative risk, 0.55; 95 percent confidence interval, 0.37 to 0.84; P = 0.004). The median duration of treatment was 94 weeks. Severe hematologic or clinical side effects were rare. CONCLUSIONS: Treatment with zidovudine benefits HIV-infected persons with CD4+ cell counts above 400 per cubic millimeter. Despite the use of doses larger than those now generally prescribed, zidovudine was well tolerated for up to three years by most of our patients.

Tissue infiltration in a CD8 lymphocytosis syndrome associated with human immunodeficiency virus-1 infection has the phenotypic appearance of an antigenically driven response.

HIV-1 infection may initiate to an HLA-associated response designated diffuse infiltrative lymphocytosis syndrome, characterized by increased numbers of circulating CD8 T cells that infiltrate salivary glands, lungs, gastrointestinal tract, and kidneys. Since this response could either be an antigenically driven process induced by HIV-1 or a lymphoproliferation of cells with neoplastic or unusual features, we sought to define the phenotype of the cellular populations, the nature of tissue derangement, and the tissue localization of virus in diffuse infiltrative lymphocytosis syndrome. Circulating CD8 T cells were greatly increased while CD4 T cell numbers remained in the range found in asymptomatic seropositive persons. The majority of CD8 and CD4 T cells in both blood and tissues had the memory phenotype of CD29+ (beta 1 integrin) and CD11a+/CD18 (beta 2 integrin) expression, but lacked markers of recent activation. A proportion of the circulating CD8 T cells also expressed CD57 (Leu 7) but not other markers of natural killer cells. HIV-encoded proteins were identified in tissue macrophages located in periacinar areas of the salivary glands. CD54 (intercellular adhesion molecule-1), a ligand for the CD11a integrin, was strongly expressed on postcapillary venule endothelium within lymphoid foci, and HLA-DR molecules were found on limited regions of ductular epithelium adjacent to lymphoid aggregates. These findings suggest that (a) the visceral lymphocytic infiltration in diffuse infiltrative lymphocytosis syndrome is an antigen-driven, and MHC-determined, host immune response to an element associated with HIV-1 infection, and (b) that the specific adhesive molecule interactions mediating the cellular influx, as well as the subsequent tissue damage, reflect altered patterns of gene expression in tissues undergoing an immune response.

90K protein: a new predictor marker of disease progression in human immunodeficiency virus infection.

A 90,000-Da molecular mass tumor-associated protein has recently been identified in the sera of patients infected by HIV. In this study, we have evaluated the serum levels of 90K for its ability to predict the progression to ARC or AIDS retrospectively in 49 HIV-seropositive subjects who were initially symptom-free. 90K levels were higher in those HIV-seropositive subjects who progressed to ARC or AIDS than in those who had not progressed both at entry into the study and at the latest visit. CD4+ cell number was not different in the two groups at entry but was lower in the progressors at the latest visit. Evaluation of the patterns of change over time showed that 90K increased and CD4+ cells decreased more in progressors than in nonprogressors. During the 3 years preceding the onset of ARC or AIDS, 90K increased regularly while CD4+ cell decrease was later. Elevated levels of 90K (p = 0.007) and lower numbers of CD4+ cells (p = 0.001) were significantly associated with a higher cumulative incidence of ARC or AIDS. These findings suggest that 90K is an early indicator of progression to ARC and AIDS.

Increased expression of interferon-gamma in hyperplastic lymph nodes from HIV-infected patients.

Polyclonal B cell activation is characteristic of HIV infection and occurs in the presence of severe CD4+ lymphocyte depletion. In contrast, CD4+ lymphocytes are the dominant T cell in the reactive lymphoid tissues of patients not infected with HIV. In this study, lymph node biopsies from eight HIV-infected patients with persistent generalized lymphadenopathy syndrome (PGL) were assessed for IL-1 beta, IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) gene expression using the polymerase chain reaction (PCR). The cytokine gene expression of two cases of reactive adenopathy in patients not infected with HIV was assessed for comparison. IFN-gamma was expressed much more strongly in the PGL samples than in control reactive lymphoid tissues, whereas the other cytokines were expressed to a similar extent in both types of tissues. IFN-gamma may have an important role in maintaining the adenopathy of HIV-infected patients. Expression of cytokines such as IL-2, IL-4 and IL-10 in HIV nodes may be adequate to allow the recruitment of naive B cells to the reactive process.

Didanosine in the treatment of AIDS and AIDS-related complex: a critical appraisal of the dose and frequency of administration.

In the summer of 1988, dose-finding phase 1 trials of the efficacy and safety of didanosine were begun by investigators at the National Cancer Institute (NCI), by the AIDS Clinical Trials Group (ACTG) at New York University School of Medicine and the University of Rochester School of Medicine, and by researchers at Boston City Hospital (BCH). The schedules of drug administration studied included once daily (BCH), twice daily (ACTG and NCI), and three times daily (NCI). The total daily dose studied ranged from 0.8 to 66 mg/(kg.d). Significant toxicities developed in more than half of the patients receiving doses of > or = 12 mg/(kg.d). Only 5% of patients given < 10 mg/(kg.d) developed grade 3 or 4 toxicities. Although CD4+ lymphocyte counts increased at doses as low as 1.6 mg/(kg.d), a minimal effective dose could not be determined. An analysis of data on serum levels of p24 antigen suggests that a dose of > or = 5 mg/(kg.d) is superior to lower doses in terms of activity against human immunodeficiency virus. A dose given on a once-daily schedule may exhibit less antiretroviral activity than the same dose given on a twice-daily schedule. Long-term follow-up of the phase 1 trial of the once-daily regimen at BCH has further documented the safety of didanosine given at doses of < 12 mg/(kg.d) for extended periods. A review of these studies suggests that a twice-daily schedule of didanosine administration at a daily dose of 5-10 mg/(kg.d) is safe and is associated with significant antiretroviral activity in vivo.

Cognitive performance in HIV-1 infection: relationship to severity of disease and brain atrophy.

We examined cognitive performance in 72 HIV-1 infected patients and 34 controls. None of the patients had opportunistic infections or unusual neoplasms of the central nervous system (CNS). Factors other than HIV-1 known to cause cognitive decline were excluded from both groups. Cognitive functioning analysed with special emphasis on the severity of HIV infection was related to neuroradiological and immunological findings. In patients with AIDS-related complex (CDC IVa) or AIDS (CDC IVc,d), a deterioration of memory as well as cognitive speed and flexibility was detected. Furthermore, memory deficits were associated with central cerebral and infratentorial atrophy in those patients, while no association was found between cognitive deficits and immunological abnormalities. Patients at CDC stages II or III showed slight association between altered cognitive speed and flexibility and elevated leukocyte count, suggesting a subclinical CNS disease already at early stages of HIV infection.