Abstract: A collaborative project was established between the Alli Causai Foundation in Ambato, Ecuador, and the University of Genoa, Italy, to introduce the microscopic observation drug susceptibility (MODS) assay for the rapid identification of Mycobacterium tuberculosis in Ecuador. A total of 507 samples were evaluated during a 10-month period, and DNA was extracted from each isolate and sent to Genoa for confirmatory molecular analysis. M. tuberculosis was identified in 45 samples by MODS, and drug resistance was observed in approximately 21% of the isolates, with four multidrug-resistant strains detected in two patients.
Abstract: This is the first case of documented treatment failure of JCV nephritis, despite a reduction of immunosuppressive agents and the use of antiviral therapy. We consistently detected high levels of JCV viremia, which correlated with the progressive deterioration of the graft and caused the final loss of the kidney, suggesting that JCV plays an etiological role in the onset of severe nephropathy in kidney transplant patients.
Abstract: Cunninghamella bertholletiae infection occurs most frequently in neutropenic patients affected by haematological malignancies, is associated with an unfavourable outcome. We report a case of rhino-mastoidal fungal infection in a leukaemic patient. Bioptical tissue cultures yield the isolation of a mould with typical properties of Cunninghamella species. Liposomal amphotericin B (L-Amb) therapy combined with surgical intervention brought the lesion to recovery. Nevertheless, the patient died 14 days after bone marrow transplantation (BMT) from bacterial sepsis. Mastoiditis was documented at CT-scan. The conditioning regimen probably caused the reactivation of the Cunninghamella infection that led to the patient's fatal outcome; fungal hyphae were detected after autopsy of brain and lung tissue.
Abstract: Follow-up of chronically infected HCV patients is the primary clinical goal in therapy administration. In the absence of an HCV vaccine, the timely monitoring of HCV viral load combined with the information of the viral genotype could contribute to patient disease management. A LightCycler Real Time RT-PCR assay was developed and optimized allowing rapid and accurate quantification of HCV RNA over an extended dynamic range using a single human reference standard. A total of 5,096 plasma samples, collected over almost 5 years, were tested and HCV RNA was quantified in 2,435 samples with levels ranging from 5.7x10(1) to 2.52x10(9) IU/ml. The precision and reproducibility of the test are documented by various inter-assay parameters of the reference standard obtained in 409 RT-PCR runs. This Real Time RT-PCR protocol uses the LightCycler cDNA amplicons for direct sequence analysis and reduces the sequencing time to approximately 3 hours. Nearly all HCV genotypes were identified. Viral sequences showed a similarity level close to 100%, independently from the viral load, while the LightCycler melting temperature analysis did not correlate with HCV genotypes. All this makes the LightCycler Real Time RT-PCR protocol a suitable tool for the diagnosis and monitoring of HCV infections.
Abstract: We describe a case of primary cutaneous Absidia corymbifera infection in an AIDS patient with renal complications. The Sensititre YeastOne panel was adopted to determine antifungal susceptibility and liposomial amphotericin B was used which initially produced a significant clinical response.
Abstract: An inexpensive enzyme-linked immunosorbent assay method for human immunodeficiency virus type 1 quantitation, ultrasensitive p24 antigen assay (Up24), was compared with RNA viral load assay (VL). Up24 had 100% sensitivity of detection at a viral load of >/=30,000, with sensitivity of 46.4% at a viral load of <30,000 (232 specimens from 65 seropositive subjects). The assay was highly reproducible, with excellent correlation between duplicates and among three laboratories.
Abstract: Covert human immunodeficiency virus (HIV) replication was ongoing during the first 3 years of aviremia in 22 patients, as determined by detection of DNA containing two long terminal repeats (2 LTR DNA). Although total HIV DNA was detected in 60 2 LTR DNA-negative samples, the absence of 2 LTR DNA in 90% of patients following 7 to 8 years of highly active antiretroviral therapy suggests suppression of cryptic viral replication.
Abstract: Human immunodeficiency virus (HIV) RNA testing is the gold standard for monitoring antiretroviral therapy in HIV-infected patients. However, equipment and reagent costs preclude widespread use of the assay in resource-limited settings. The Perkin-Elmer Ultrasensitive p24 assay and the Cavidi Exavir Load assay both offer potentially simpler, less costly technologies for monitoring viral load. These assays were compared to the Roche Amplicor HIV-1 Monitor Test, v1.5, using panels of clinical samples (subtype B) from HIV-positive subjects and HIV-spiked samples (subtypes A, C, D, CRF_01AE, CRF_02AG, and F). The Ultrasensitive p24 assay detected 100% of the spiked samples with virus loads of >250,000 copies/ml and 61% of the clinical samples with virus loads of 219 to 288,850 copies/ml. Detection rates were improved substantially if an external lysis buffer was added to the procedure. The Cavidi assay detected 54 to 100% of spiked samples with virus loads >10,000 copies/ml and 68% of the clinical samples. These detection rates were also greatly improved with a newly implemented version of this kit. Coefficients of variation demonstrate good reproducibility for each of these kits. The results from the Cavidi v1.0, Cavidi v2.0, and Perkin-Elmer, and the Perkin-Elmer Plus external buffers all correlated well with the results from the Roche Monitor Test (r = 0.83 to 0.96, r = 0.84 to 0.99, r = 0.58 to 0.67, and r = 0.59 to 0.95, respectively). Thus, the use of these two assays for monitoring patients, together with less-frequent confirmation testing, offers a feasible alternative to frequent HIV RNA testing in resource-limited settings.
Abstract: Highly active antiretroviral therapy (HAART) induces a persistent reduction of the plasmatic viremia, contributing to decrease mortality and morbidity of infected people with human immunodeficiency virus (HIV). Thus, viral load (VL) is the reference method to evaluate therapy effectiveness. However, even in the presence of efficient HAART viral eradication was yet not achieved. In this study, we analyzed the presence of total HIV DNA (T-HIV DNA), non-integrated DNA with 2LTR (2LTR-HIV DNA) and HIV RNA in a group of 55 HIV-positive subjects from Rosario City, with different clinical stages, with and without HAART. All markers were evaluated by PCR assays optimized in our laboratory that included colorimetric detection in microplate. HIV RNA clinical sensitivity was compared with a reference test, bDNA, resulting in 74% and 64% respectively, with an 85% of agreement. Thus, our HIV RNA assay could be used to monitor patients under HAART and at risk of infection. The 2LTR-HIV DNA was 54% positive although it was absent in patients with high VL. This marker was considered a labile product therefore its presence was associated with recent infection. However, current evidences question its stability. Thus, its clinical significance should be reconsidered. The absence of 2LTR-HIV DNA in patients with detectable VL may relate to the heterogeneity of the sequence used for its detection. T-HIV DNA was present in 100% of the samples and could be a relevant remission marker when therapies that effectively eradicate the infection became available.
Abstract: Epigallocatechin-3-gallate (EGCG), one of the components of green tea, has been suggested to have antiviral activity. To determine the effects of EGCG on HIV infection, peripheral blood lymphocytes were incubated with either LAI/IIIB or Bal HIV strains and increasing concentrations of EGCG. EGCG strongly inhibited the replication of both virus strains as determined by reverse transcriptase and p24 assays on the cell supernatants.
Abstract: HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.
Abstract: A novel colorimetric assay was developed and validated for accurate quantitation of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMCs). We tested 318 sequential samples from 56 subjects, 53 of whom were undergoing dual or triple therapy. Patients were considered responders when viremia levels were below 5, 000 HIV RNA copies/ml. The mean DNA copy numbers for untreated and responder subjects were similar (72 and 75, respectively), while it was 4.54-fold higher for nonresponders (339). This report provides strong evidence that HIV DNA levels in PBMCs correlate with therapeutic efficacy and suggests that DNA quantitation is a useful tool to monitor the decay of the HIV reservoir toward disease remission, especially when viremia is undetectable.
Abstract: The performance in pediatric human immunodeficiency virus type 1 (HIV-1) infection of a signal-amplification boosted ELISA for HIV-1 p24 antigen in plasma after heat-mediated immune complex dissociation was prospectively compared with polymerase chain reaction-based procedures. Diagnostic sensitivity and specificity of the p24 antigen test were 100% and 99.2%, respectively. Quantification revealed RNA in 85.7% and p24 antigen in 87.4% of 230 samples from 25 infected children. Concentrations of these indices in individual samples correlated (P<.0001). Introduction or modification of antiretroviral treatment showed concordant responses of RNA and p24 antigen in 39 (90.7%) of 43 instances. The treatment-induced changes in concentrations of RNA were higher than those of p24 antigen in 11 instances. In 1 instance, however, the concentration change of p24 antigen was greater than that of RNA (P=. 002). Variation of RNA concentrations was more marked than that of p24 antigen (P=.002). The p24 antigen test was equivalent to PCR for diagnosing and monitoring pediatric HIV-1 infection.
Abstract: A communication system for the automation of the follow up of AIDS patients set up by DIST at the Molecular Virology Unit in the Advanced Biotechnology Centre of Genova and at the Department of Internal Medicine of the Medical School of Genova is presented. This system includes a distributed database to store both clinical and virological data and a set of procedures to transfer patient data with a complete respect of requirements about completeness and privacy.
Abstract: This study addresses the limited range of quantification with colorimetric assays (ELISA) starting from the analysis of color production in a reference external curve. An automatic ELISA management software, designated Quanti-Kin Detection System (QKDS) is described, which retains the sensitivity of the end-point reading and extends the dynamic range up to five logarithms with mathematical interpretation of color production. The QKDS software is a generic system suitable for different types of ELISA with substrate incubation at room temperature, does not require dedicated instruments, performs accurate quantification (including assay quality control) and has a user friendly interface. Specific applications were developed for three types of analytes: antibodies, viral antigens and nucleic acids. Data are presented on three representative QKDS applications to HIV antibodies, p24 antigen and proviral DNA kits. The precision of quantification is strictly correlated with the precision of the kit; however, for almost all samples with known analyte amount, the error percentage was below 10%, only for two cases in quantification of HIV proviral DNA the error percentage was around 25%. The necessity for a wide quantification range has been demonstrated by measuring clinical samples, which showed a distribution in all possible quantification ranges for all kits.
Abstract: The present work aims to obtain groups of patients with similar profiles of p24 antigen concentration and of CD4+ cell counts. These two markers were chosen because their evaluation represents a significant step in the clinical follow up of HIV-1 infected subjects. The classifications were obtained by a Kohonen neural net trained in three ways: with p24 antigen profiles only, with CD4+ cell count profiles only and with both sets of profiles. The results show that the clustering fashion of the two parameters closely resembles the clustering fashion of CD4+ only rather than the one of p24Ag, both with reference to cluster formation and with reference to distance among clusters.
Abstract: The fluctuations of HIV-1 p24 antigen concentration have been monitored in the follow-up of 118 subjects in different clinical stages and compared to their CD4 cell count; 104 patients received antiretroviral therapy. Persistent (65%) or sporadic (28%) antigenaemia has been detected in most patients in different clinical stages. The variations of the p24 Ag level are significantly correlated with the CD4 cell count and therapy administration (P = 0.0001). In patients with relatively conserved immune function (CDC II and III), antiretroviral therapy shows the best efficacy and can be efficiently monitored by p24 and CD4 surrogate markers. The data here suggest that although the informative value of p24 Ag is not representative of an AIDS-defining event, it can be used as a short-term and relatively inexpensive virological marker of antiviral activity in vivo, to support the routine management of patients.
Abstract: The aim of the present work is to obtain groups of patients with similar profiles of p24 antigen concentration and of CD4+ cell counts. These two markers were chosen because their evaluation represents a significant step in the clinical follow up of HIV-1 infected subjects. The detection methods for p24 antigen concentration and for CD4+ cell counts are well assessed and guarantee easy reproducibility of data obtained in different laboratories. A set of observations with the same time intervals were derived from a continuous function obtained for each patient by a back-propagation neural net trained on the raw data from the patient. The classifications were obtained by a Kohonen neural net trained in three ways: with p24 antigen profiles only, with CD4+ cell count profiles only and with both sets of profiles. The results show that the clustering fashion of the two parameters closely resembles the clustering fashion of CD4+ only, rather than that of p24Ag, both with reference to cluster formation and with reference to distances between clusters.
Abstract: The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection.
Abstract: The performance and clinical relevance of a qualitative PCR-based assay for the detection of HIV-1 DNA sequences in peripheral blood mononuclear cells (PBMCs) was evaluated by two different laboratories. Four hundred and one samples were obtained from 397 individuals from different risk populations. All blood donors tested had negative results; positive signals were obtained from all infected patients. HIV-1 DNA was detected in 3 of 17 infants born to seropositive mothers; Western blot indeterminate blood donors and exposed health-care workers had negative results. Our results demonstrate that this PCR assay provides both sensitive and specific results and is suitable for testing large numbers of samples and for rapid identification of HIV-1 infection.
Abstract: Since there have been a few reports of pediatric HIV-2 infection. We therefore investigated the perinatal transmission of HIV-2 in 147 malnourished and 164 well-nourished children attending a health center in the northern part of Guinea Bissau. Specific HIV-2 antibodies were detected in 17 mothers and in 2 malnourished children, one of them with pediatric AIDS. This study demonstrates that mother to child transmission of HIV-2 infection occurs in Guinea Bissau and suggests that there is an increased likelihood of detecting HIV-2 infection in malnourished children. The high seroprevalence of HIV-2 in a rural population without known risk factors may represent a hidden threat to mother/child health.
Abstract: We present a qualitative model of the interaction between the HIV-1 virus and the human cell, based on qualitative process theory. This model takes into account a previous qualitative model of the cell growth. The model presented here can be regarded as a first step for setting up a comprehensive model of the HIV-1 virus-cell interaction in which the possible points where a drug can attack the virus are evident. The first simulation trials indicate that the presented model reproduces (even though in a simplified way) the features of the real behaviour that have been considered in the modelling phase. Although our simulation is limited in the knowledge it expresses, it still gives a stimulating opportunity for the evaluation of the criteria chosen as discriminant in the interaction between virus and cell.
Abstract: The detection of HIV-1 proviral DNA in children born to seropositive mothers was studied using the polymerase chain reaction with either a radioactive electrophoretic method or a noval procedure that employs colorimetric microwell visualization. Peripheral blood mononuclear cell lysates from 18 HIV-1 infected children and 28 uninfected subjects were assayed for a 142 bp fragment of DNA from the gag region of HIV-1 using the primer pair SK145-431. Detection of amplified DNA was carried out by hybridization with a radiolabeled SK102 probe, or with a tagged SK102 probe permitting colorimetric detection. The radioactive detection procedure demonstrated 100% specificity and correlated with the serological results. The assay was more sensitive than the p24 antigen test, but two false negative results were obtained. One was from a sample taken at 2 weeks, an age at which undetectable provirus levels were reported in almost all HIV-1 infected newborns. The second was probably due to a low copy number of proviral DNA, as positive results were obtained in all other (6) samples from this child. Comparative analysis in a limited number of specimens of radioactive and colorimetric detection following PCR revealed 100% specificity and comparable sensitivity with 4 discordant results. The results show that PCR is the best method for early diagnosis of HIV-1 infection in pediatric subjects. The study also demonstrated the value of a colorimetric detection method for PCR products. This colorimetric microwell plate procedure may prove a useful technique in routine diagnosis of HIV-1 infection in children.
Abstract: The availability of a small laboratory animal model suitable for the evaluation of methods for prevention and treatment of human immunodeficiency virus type 1 infection would be a valuable resource for AIDS research. Here we describe the infection of a strain of domestic rabbits by intraperitoneal inoculation with cell-free human immunodeficiency virus type 1. Evidence of infection includes the presence of an immune response that has persisted for almost 3 years and the detection of an reisolation of infectious virus from peripheral blood mononuclear cells (PBMCs) and other tissues during the first 2 years. Typical viral proteins, DNA and RNA patterns, were observed in rabbit PBMCs and in cells infected by cocultivation with rabbit PBMCs. While a number of possible pathological changes were evaluated in infected rabbits, the presence of changes in lymph node structure similar to those reported in infected humans merits further investigation.
Abstract: OBJECTIVE: To evaluate an acid pretreatment method designed to dissociate HIV p24 antigen from immune complexes in serum. DESIGN: Patient sera and sera containing experimental immune complexes were quantified for p24 antigen before and after immune complex dissociation (ICD). The clinical application of ICD was assessed in 1328 serum and plasma samples collected from HIV-infected patients. METHODS: Immune complexes were created artificially by mixing purified p24 antigen with antibody-positive sera or a standardized concentration of human antibody to p24. ICD was achieved by incubation of samples with an equal volume of Glycine HCl for 90 min at 37 degrees C followed by neutralization with Tris NaOH. Samples were quantified for p24 antigen using a commercial enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: ICD resulted in significant release of purified antigen from simulated immune complexes in antibody-positive sera. Variation in antigen sequestration and dissociation was related to anti-gag antibody titers. ICD resulted in complete recovery of 500 pg of antigen complexed with human anti-p24 antibody at concentrations up to 2.5 U/ml. In seropositive patients, the mean level of serum antigen was 3.5-fold higher after ICD, and an additional 21% were antigen-positive. CONCLUSIONS: Pretreatment greatly improved antigen detection in HIV-antibody-positive sera by effectively dissociating immune complexes without compromising reactivity of the antigen itself. The treatment also facilitated routine monitoring of patients by revealing fluctuations in serum antigen that were indistinguishable or poorly defined in untreated sera.
Abstract: OBJECTIVE: To evaluate changes in serum HIV p24-antigen levels in a subset of patients who participated in a European/Australian double-blind, placebo-controlled trial evaluating the efficacy of zidovudine (250 mg every 6 h) alone or in combination with acyclovir (800 mg every 6 h) in patients with AIDS, AIDS-related complex (ARC) or Kaposi's sarcoma (KS). DESIGN: Double-blind, placebo-controlled randomized clinical trial of less than or equal to 6 months' therapy. SETTING: Samples were obtained from patients attending teaching hospital outpatient clinics in seven European countries and Australia. SUBJECTS: One hundred and ninety-seven HIV-infected patients (60 with AIDS and 137 with ARC or KS). MAIN OUTCOME MEASURES: Serum HIV p24-antigen levels measured using the Abbott HIV solid-phase enzyme immunoassay. RESULTS: Of 76 ARC/KS patients who were initially HIV p24-antigen-positive, one out of 25 randomized to placebo, eight out of 23 to zidovudine and 11 out of 28 to the zidovudine/acyclovir combination became antigen-negative. The proportion of patients who became antigen-negative was significantly higher in both the zidovudine group (P = 0.016) and the zidovudine/acyclovir group (P = 0.004), compared with the placebo group. There were no statistical differences between the zidovudine and the zidovudine/acyclovir groups. During the trial p24-antigen levels in the zidovudine-treated patients reached their minimum after 4-8 weeks of therapy, and tended to increase gradually thereafter. Disease progression occurred irrespective of whether p24-antigen levels declined during therapy. No association between p24-antigen responses to therapy and baseline disease stage, Karnofsky score or baseline CD4 cell count was detectable. CONCLUSION: Acyclovir does not potentiate the effect of zidovudine on p24-antigen levels. Change in antigen level in response to antiviral therapy needs further investigation before it is used as a surrogate marker for clinical efficacy of antiviral therapy.
Abstract: Described are the results of an international collaborative study to evaluate the use of whole blood samples spotted on filter-paper (BSP) for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). BSP samples were collected from 40 patients at risk for HIV-1 infection and tested blindly using commercially available HIV antibody test kits, either specifically manufactured or modified for this purpose. Parallel serum samples were also collected, and the antibody reactivity was defined and confirmed by Western blot. The results demonstrate that recovery of antibodies from BSP samples after elution can be comparable to that from serum. Some kits can be easily adapted to test BSP samples, while others cannot. At present, detection of HIV antibodies in BSP samples should therefore be carried out using kits specifically manufactured for this purpose or by the development of a modified protocol using a panel of BSP and their corresponding serum specimens.
Abstract: OBJECTIVE: To evaluate the prevalence of antibodies to HIV-1/2 and HTLV-I/II in 1305 transfusion-dependent beta-thalassemics treated in 36 centres in Italy. DESIGN: Patient serum samples were collected during 1990 and tested in Milan. METHODS: Sera were screened using an enzyme-linked immunosorbent assay (ELISA) containing viral lysate antigens from HIV-1 and HIV-2, and a particle agglutination assay for the detection of antibodies to HTLV-I and HTLV-II. Repeatedly reactive samples were examined by Western blot (WB) assays containing recombinant and viral lysate antigens. Differential diagnosis was finally made by ELISA based on synthetic peptides. RESULTS: Samples from 36 of the 1305 patients (2.76%) contained anti-HIV-1 antibodies. In four patients seroconversion occurred after the implementation of anti-HIV-1 screening in blood donors in Italy (1985). Of the 36 HIV-1-antibody-positive samples, four were HIV-2 [corrected] WB indeterminate. These four samples were negative in assays based on specific synthetic peptides, suggesting cross-reactivity. Anti-HTLV-I antibodies were found in two patients from Sicily and one from Apulia, both southern Italian regions. Anti-HTLV-II antibodies were detected in another patient from Sicily. CONCLUSIONS: Antibodies to HIV-1, HIV-2, HTLV-I and HTLV-II were detected in 2.76, 0, 0.23 and 0.08% of patients, respectively. The residual risk of HIV-1 infection through blood transfusion after the implementation of anti-HIV-1 screening in blood donors in Italy was approximately 1:50,000 blood units; this is based on an approximate number of 200,000 blood units administered to our group of patients during 1986-1990 and the occurrence of four new anti-HIV-1 seroconversions. Seroconversions to HTLV-I/II suggest that these viruses are present in Italian blood donors.
Abstract: The principal neutralizing epitope of the human immunodeficiency virus type 1 (HIV-1) lies between two invariant cysteines in the third variable region (V3) of the viral envelope (gp120), and its amino acid sequence varies among different HIV-1 isolates. HIV-2 carries an analogous amino acid sequence between two cysteines of the V3 regions, but its functional similarity with the HIV-1 principal neutralizing epitope is uncertain. We studied the degree of genetic variation of the HIV-2 V3 region in fresh blood samples from 12 HIV-2-seropositive individuals from Guinea-Bissau. Polymerase chain reaction was used to amplify viral fragments of 465 bp containing the V3 region from cellular DNA. Nucleotide sequence analysis of the entire envelope fragment from each patient revealed that the degree of variation among field isolates of HIV-2 is comparable to that observed in the analogous region of HIV-1. Most of the HIV-2 isolates studied were highly related, suggesting the existence of a limited number of different viral strains in the cohort studied. Thus, the HIV-2 and HIV-1 V3 regions vary to a similar degree and may also have analogous functions.
Abstract: We have defined continuous native epitopes of HIV proteins by using a systematic epitope-scanning technology. We have demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 that is immunoreactive with all studied HIV-1 antibody-positive sera. The corresponding region in HIV-2 gp34 behaves similarly. There is a clear difference, however, between HIV type 1 and type 2 transmembrane proteins in the number of highly immunoreactive regions, when presented properly as synthetic antigens in solid-phase EIA, can provide tests unusually suitable for early and reliable diagnosis of HIV-1 and HIV-2 infections and for type-specific distinction of the two types of HIV infections.
Abstract: Spontaneous and induced transmission of oncogenic retroviruses from parents to offspring is well documented in mammals, as exemplified by murine leukaemia viruses. In addition, it is now common knowledge that transgenic mice, carrying viral and cellular transforming genes, can show unusual tumour incidences and can transmit this character to successive generations. The present report deals with the occurrence of tumours in rodents exposed directly in utero to murine sarcoma viruses (MSV). Attention was focused on whether MSV are involved in the etiopathogenetic processes leading to early-life and adult-type neoplasms in mice. According to our data, this is a likely possibility, since, for example, Kirsten MSV (Ki-MSV) induced stage-specific tumours in CD-1 mice, including lung tumours and skin papillomas, following intraembryonal injections on gestation days 8 and 10, respectively. When the injections were given in the second half of pregnancy, however, only capillary angiomas and other vascular malformations of the brain, as well as mesenchymal sarcomas, were induced in the newborn. Harvey MSV (Ha-MSV), a close relative of Ki-MSV, and Moloney MSV (Mo-MSV) were much less dependent on the stage of embryogenesis, and mesenchymal sarcomas were frequently detected in prenatally infected animals. Other MSV viruses, including 3611, J-2 and 4070A, were active carcinogens in a way totally independent of the embryonal stage exposed, and tumours in the progeny ranged from mesenchymal sarcomas to lymphoblastomas and rhabdomyosarcomas, respectively. We next looked at the possibility that spontaneous tumours of CD-1 mice might result from the interaction of certain chemical carcinogens with developmentally expressed components of the Ki-MSV virus. The experiments conducted thus far have provided dubious results. An example is given by experiments with N-ethyl-N-nitrosourea (ENU). This compound is a powerful transplacental carcinogen in CD-1 mice, where it induces lung tumours. It was expected that by exposing developing mice to Ki-MSV on day 8 of embryogenesis and to ENU late in pregnancy or in newborns, a great increase in the incidence of lung tumours would have appeared in the offspring. However, this did not happen, and the agents behaved as if different cellular targets were involved. Understanding this difference could be very informative and useful in identifying neoplastic factors in mammalian embryos.
Abstract: An important requirement for the development of a vaccine against the human immunodeficiency virus (HIV-1), the causative agent of AIDS, is a readily available animal model that would allow possible immunogens to be evaluated. The only species to have been infected with HIV-1 so far is the chimpanzee. However, the scarcity of this animal and its designation as an endangered species place severe restrictions on its use as an animal model. Attempts to infect mice, rats, hamsters, guinea-pigs, musk shrews, and rabbits with HIV-1 or infected cells have all been unsuccessful. We now report that the intraperitoneal inoculation of rabbits with HIV-1 or chronically infected H9 cells consistently induces a persistent infection.
Abstract: The suitability of collecting whole blood specimens on filter paper disks for HIV antibody assay was evaluated. ELISA and Western blot assay results were in complete agreement for serum and blood spot disk samples. Sensitivity of the two methods was tested using diluted whole blood and sera from HIV-seropositive individuals. Results demonstrate that ELISA and Western blot assays performed on punched-out disks of the blood-impregnated papers had the same sensitivities as those obtained with serum samples. This study suggests that whole blood collection on filter paper can be effectively substituted for serum sampling in HIV antibody screening programs.
Abstract: Since HIV-2 infection has been identified in some European countries, we investigated whether HIV-2 infection is present in groups of Italian subjects at risk for AIDS. Our results clearly indicate that the parallel Western blot assay for HIV-1 and HIV-2 antibodies can detect HIV-2 infection, which is presently not epidemic in Italy. Careful examination of the serological data is mandatory before announcing the detection of HIV-2-infected patients.
Abstract: In the present study both responsiveness and stimulatory capacity in autologous mixed lymphocyte reactions (AMLRs) of non-T/T and T/T type, as well as in allogeneic mixed lymphocyte reaction (MLR), were evaluated in 30 intravenous drug abusers (IDAs) infected by the human immunodeficiency virus (HIV) and in 10 HIV-negative IDAs. The production of interleukin 2 (IL2), and the expression of HLA Class II antigens and IL2 receptors by PHA-activated T lymphocytes were also evaluated. A severe impairment of both responsiveness and stimulatory capacity in MLR and AMLRs was found in the HIV-positive IDAs and not in the HIV-negative IDAs. The HIV-positive IDAs showed also a defective expression of HLA Class II antigens, whereas the IL2 production and the IL2 receptor expression were in the normal range. The present data are consistent with similar observations in male homosexuals with AIDS-related complex and confirm that the HIV infection induces a broad spectrum of immunological abnormalities leading to a progressive derangement of the immunocompetence.
Abstract: The effect of Coumermycin (CA1) on the replication of different ecotropic retroviruses was studied. Most strains showed reductions in infectivity of less than ten fold, while two strains demonstrated a marked sensitivity to the drug. The isolation of one resistant strain could suggest a specific CA1 antiviral activity.
Abstract: The Moloney (MoMSV) and Kirsten (KiMSV) strains of murine sarcoma viruses are known to induce mesenchymal sarcomas upon infection of newborn rodents. To determine their activity in mouse embryos, 11- to 15-day-pregnant CD-1 mice were laparotomized, and the single implants were inoculated into the abdominal portion of the embryonal body with an average of 15 and 1500 focus-forming particles/g of body weight of the MoMSV and KiMSV viruses, respectively. Another group of less than 1-day-old pups was given a comparable amount of either virus. Tumors appeared in the young within the first few weeks of life with incidences and histological types dependent on the gestational day and the viral strain inoculated. Mixed mesenchymal sarcomas at or near the site of inoculation and vascular tumors of the brain were by far the most frequent neoplasms observed in the newborn. With MoMSV there was an increased incidence of sarcomas with advancing age at treatment, being 0% at 11 days of pregnancy and 96% in newborn (P for trend, less than 0.025). By contrast, KiMSV caused an incidence of sarcomas below 20% throughout (P for trend, greater than 0.05). Brain tumors were identified in the several MoMSV and KiMSV groups, with a peak value of 43% following the inoculation of both viruses into 13- and 15-day-old embryos, respectively. While the total incidence of these tumors was significantly different from controls, no positive trend by day of treatment was found among the MoMSV and KiMSV viruses (P less than 0.05). The tumors were mainly capillary angiomas, but a few cavernous angiomas were also detected. In addition, eight pups which were given injections of both viruses at developmental Days 11 to 13 had tumors of the choroid plexus. In many instances, newborn pups were affected by multiple vascular abnormalities of the brain, including capillary telangiectases and multiple hemorrhagic areas. No such lesions nor tumors at any site were found among the control animals. The present results are important not only because of the evidence that Swiss embryos respond selectively to the carcinogenic effects by murine sarcoma viruses, but also because they offer the opportunity to dissect directly in vivo the mechanisms underlying the stage-related sensitivity of prenatal mice to oncogenic retroviruses.
Abstract: We have characterized various biologic, immunologic and growth properties of several cell lines established from a spontaneous rat sarcoma that was discovered more than 60 yr ago. The tumors consisted of mixed cell types with no detectable host cellular immune response. Cultures derived from single-cell clones of the parental cell line were non-invasive but highly tumorigenic even in adult rats. The cultured cells spontaneously released replication-competent endogenous rat type C virus which did not carry a transforming gene in its genome. Since normal cells from the same rat strain did not produce a retrovirus, it is possible that production of the endogenous retrovirus may have triggered specific cellular changes necessary for the oncogene expression and development of this tumor.
Abstract: Mechanisms of retrovirus induced transformation have been intensively and successfully investigated in recent years. The most important consequence of the developed techniques and the information obtained is the demonstration of the presence in the normal invertebrate and vertebrate, including human, cells of genes capable of inducing neoplastic transformation. These cellular genes have been identified by hybridization of normal cellular DNA with retroviral transforming sequences and by the demonstration of the biological activity of cellular DNA from different tumors in mouse cell transfection assays. These genomic sequences have been called proto-oncogenes to distinguish them from their retroviral equivalents, to recall their cellular origin and to differentiate them from their activated oncogenic counterparts. The different mechanisms involved in the activation of proto-oncogenes include point mutation, deletion, promoter insertion, translocation and amplification. Recent evidence suggest that transformation is a multistep process in which different oncogenes may play different roles.
Abstract: The effect of coumermycin A1 activity on the infection and replication of murine type C retroviruses was studied in vitro. The infectivity of five prototype ecotropic retroviruses was reduced by 50 to 94%, with viral titres decreased up to seven-fold. These values were substantiated by progeny production studies. Similar results were obtained with five strains of xenotropic retroviruses. Delayed inhibition of growth kinetics in mouse SC-1 cells was observed with 7.5 and 10 mg/l of coumermycin A1. This effect was markedly reduced after three cycles of freezing and thawing of the drug. Changes in the absorption spectra of coumermycin A1 were observed after eight cycles of freezing and thawing.
Abstract: Three xenotropic retroviruses have been biologically cloned from cells cultured from the kidney of a 3-month-old NZB female mouse. They were obtained by first cocultivating the kidney cells for several weeks with mink, dog, and human cells and then cloning them by endpoint dilution. The cloned viruses differ in their infectivity and replicative ability in a variety of heterologous cell lines. The mink cell line-derived virus (X-NZB/K-Mlc) reaches titers in culture of over 10(8) infectious viruses/ml, and is produced in high titer within 24 hr after infection of mink lung cells. The human and dog cell-derived NZB viruses (X-NZB/K-Huc and X-NZB/K-Dgc) grow to lower titers and are similar in many respects. They differ in their relative ability to replicate in dog and human cells and to transform mink S+L- cells. Peptide mapping studies indicate that the X-NZB/K-Mlc virus has a unique p15(E) protein which distinguishes it from the other two cloned NZB viruses. These results lend further support to the observation that several types of xenotropic virus are present in a mouse strain and that more than one virus can be expressed by one organ of a particular mouse.
Abstract: A sensitive micro in situ fluorescent focus assay (mISFA), which utilizes cell monolayers fixed on a plastic surface, was developed for the detection of NZB xenotropic type C retroviruses grown in mammalian cell lines. The mISFA technique is an easier, more rapid and less expensive assay than other biological tests used to detect these viruses; it gives a more precise viral titer and is slightly more sensitive. This technique allows extension of host-range investigations to include any growing cell cultures and is applicable to various virus-host cell systems.
Abstract: By co-cultivation procedures, infectious xenotropic type C viruses have been recovered from kidney cells of several strains of mice. They have host-range patterns which place them into separate subgroups. In cells cultivated from one NZB kidney, two biologically different xenotropic type C retroviruses were found. One, X-NZB/K-1, infects and replicates well in human and mink fibroblast cells but does not induce foci in mink S + L - cells with good efficiency. The other, X-NZB/K-2, infects and replicates well in mink but not human fibroblast cells, and induces foci readily in mink S + L - cells. Cross-infection studies indicate that these viruses, classified as xenotropic by host range and envelope properties, are genetically stable.
