Cytomegalovirus infection in hematologic malignancy settings other than the allogeneic transplant.

Cytomegalovirus (CMV) infection in clinical settings other than the allogeneic transplant represents a poorly explored issue. Thus, we performed a comprehensive review of the medical literature about CMV infection in patients undergoing autologous hematopoietic stem cell transplant and in other nontransplant-related hematologic patients. In autologous hematopoietic stem cell transplant, a CMV reactivation is reported to occur in up to 41% of CMV seropositive patients, when a prospective monitoring of antigenemia and/or viremia by polymerase chain reaction was adopted. However, more contained frequencies, up to 12%, have been reported when the monitoring criteria were based on a clinically driven diagnostic strategy. The most relevant risk factors appear to be CD34 + selected autografts, total body irradiation, and prior treatment with Alemtuzumab, Fludarabine, or Bortezomib, respectively. Other possible risk factors (ie, prior treatment with Rituximab, T-cell lymphomas, and pretransplant HBcIgG seropositivity) are still debated. In nontransplant settings, the data are very heterogeneous; thus, CMV infection incidence and risk factors are more difficult to establish. Overall, the rate of CMV infection/reactivation ranges between 2 and 67%. High-dose steroids, advanced disease, poor performance status, and treatment with Alemtuzumab, Fludarabine, Bortezomib, and Rituximab appear as the most relevant, though putative, risk factors. Intravenous Ganciclovir represents the gold standard for first-line treatment of CMV infection in these patients. Oral Valganciclovir and Foscarnet are other possible options. Extensive prophylaxis and preemptive therapy are not generally recommended, with the exception of high-risk patients.

Evaluation of risk of symptomatic cytomegalovirus reactivation in myeloma patients treated with tandem autologous stem cell transplantation and novel agents: a single-institution study.

The introduction of proteasome inhibitors and/or immunomodulators in the treatment of myeloma has led to an increase in viral infections, particularly in the Herpesviridae family. Previous studies about the risk of cytomegalovirus (CMV) reactivation after autologous stem cell transplantation (ASCT) have examined the clinical outcome after the first ASCT; however, only 1 study to date has investigated the risk of CMV reactivation after a second transplantation. To address this issue, we performed a retrospective chart review on 78 consecutive myeloma patients (median age 56 years) who underwent a tandem non-CD34(+) selected ASCT after induction treatment with either conventional chemotherapy (n = 42) or with novel agents (n = 36), respectively. All subjects had been mobilized and conditioned with cyclophosphamide plus granulocyte colony-stimulating factor and melphalan alone, respectively. CMV DNA load in the blood has been determined by polymerase chain reaction in the case of a clinical suspicion of CMV reactivation; therefore, routine monitoring was not performed. Considering the outcome of both the first and the second transplantations, we observed a total of 13 episodes of symptomatic CMV reactivation (13/156, 8%), in 12 subjects (12/78, 15%), all successfully treated. Eight subjects experienced a CMV reactivation after the first ASCT (8/78, 10%); however, only 1 of them (1/8, 12%) experienced a CMV reactivation after the second transplantation. Conversely, 4 CMV reactivations (6%) were observed after the second transplantation in the group of 70 patients who did not experience a CMV reactivation after the first ASCT. No statistically significant difference was observed between first and second ASCT (8/78, 10% vs. 5/78, 6%; P = 0.767). Univariate analysis showed that a pre-transplant treatment with novel agents was the only baseline factor significantly associated with the occurrence of post-ASCT CMV symptomatic reactivation after the first transplant (odds ratio [OR]: 9.897; 95% confidence interval [CI]: 1.154-84.840; P = 0.021) but not after the second transplant (OR: 5.125; 95% CI: 0.546-48.119; P = 0.115). No end-organ disease or primary infection was documented. Our data suggest that second transplantation does not increase the risk of CMV reactivation in our patient population, when compared with the first one, and confirm the role of a pre-transplant treatment with novel agents as a risk factor for CMV symptomatic reactivation.

Immunologic biomarkers for clinical and therapeutic management of psoriasis.

BACKGROUND: The therapeutic management of psoriasis includes conventional treatments as well as the new generation of highly effective TNF-α inhibitors. However, psoriasis has proven to be a complex therapeutic challenge and treatment failures are not uncommon. Thus, laboratory biomarkers of disease progression/therapeutic efficacy may greatly help in the clinical management of psoriasis. AIMS: To identify laboratory biomarkers for clinical management and therapeutic monitoring of psoriasis. METHODS: An observational study performed on 59 patients, presenting moderate to severe psoriasis, undergoing treatment with anti-TNF-α agents (etanercept, adalimumab, and infliximab). Soluble and cellular immune/inflammatory parameters were assessed at baseline and after 12 and 24 weeks of treatment. RESULTS: Clinical efficacy was achieved in 88% of the subjects at 12 weeks, reaching 90% after 24 weeks. IL-6 and IL-22, which were elevated at baseline, were significantly reduced, in association with a significant decrease of CLA+ T cells and an increase of Treg lymphocytes. T, B, and NK cell subsets and T cell response to recall antigens did not show any evidence of immune suppression. CONCLUSIONS: Immune/inflammatory parameters including IL-6 and IL-22, CLA+ T cells, and Treg lymphocytes may prove to be valuable laboratory tools for the clinical and therapeutic monitoring of psoriasis.

Continuous increase in HIV-1 incidence after the year 2000 among men who have sex with men in Rome: insights from a 25-year retrospective cohort study.

To assess trends in HIV-1 incidence and risk factors for seroconversion among men who have sex with men (MSM) resident in Rome, Italy, a retrospective longitudinal cohort study was conducted over 25 years. Incidence rates and trends were modelled using Poisson regression and risk factors were assessed by multivariate Cox models. Of 1,862 HIV-1-negative individuals, 347 seroconverted during follow-up. HIV-1 incidence rates increased from 5.2/100 persons/year (p/y) in 1986 (95% confidence interval (CI): 2.3­11.5) to 9.2/00 p/y in 1992 (95% CI: 6.4­13.0), decreased to 1.3/100 p/y in 2001 and increased until 2009 (11.7/100 p/y; 95% CI: 7.4­18.6). The risk of HIV-1 seroconversion increased during the study period in younger MSM (incidence rate ratio (IRR) = 17.18; 95% CI: 9.74­30.32 in 16­32 year-olds and IRR = 5.09; 95% CI: 2.92­8.87 in 33­41 year-olds) and in those who acquired syphilis (IRR = 7.71; 95% CI: 5.00­11.88). In contrast, the risk of seroconversion decreased among highly educated MSM (IRR = 0.54; 95% CI: 0.35­0.82) and those without Italian citizenship (IRR = 0.45; 95% CI: 0.28­0.71). The HIV epidemic in MSM living in Rome continues to expand. Targeted prevention programmes against sexually transmitted infections to enhance knowledge transfer and behavioural skills are urgently required.

Effective treatment of Kaposi's sarcoma by electrochemotherapy and intravenous bleomycin administration.

The present prospective study was aimed at evaluating the long-term efficacy of local electrochemotherapy (ECT) with the intravenous administration of bleomycin, on disease progression and viral activity in classic Kaposi's sarcoma (cKS), a vascular tumor related to human herpes virus-8 infection. Eighteen patients affected by isolate or multiple cutaneous lesions, refractory to conventional treatments, although in the absence of visceral involvement, were enrolled in a study. Follow-up visits were performed after 4 weeks and every 6 months for up to 48 months. A more extensive exploration of the immunologic status as well as of virological parameters was performed in nine patients. The results showed a significant clinical improvement in all patients after 4 weeks. A complete regression was observed in 12 patients after the first ECT, while four patients required a second treatment on the residual lesions after 4 weeks from the first intervention. The positive outcome persisted during the subsequent clinical control visits. Two patients, that showed rapidly evolving did not improve and relapsed despite a second round of ECT treatment. Effective treatment was associated with the reduction of viral load to undetectable levels. These data support the conduct of larger studies directed at validating the efficacy of ECT as a first-line therapy for cKS.

High prevalence of latent tuberculosis infection in autoimmune disorders such as psoriasis and in chronic respiratory diseases, including lung cancer.

The early diagnosis and treatment of individuals harboring M. tuberculosis is key to ensuring the effectiveness of health programs aimed at the elimination of tuberculosis (TB). Monitoring for TB also has other important health care implications for the related immune pathology caused by the chronic inflammatory response to M. tuberculosis. Moreover, the recent introduction of biologic therapies for the treatment of several immune-mediated inflammatory diseases has shown unexpected high frequencies of reactivation of latent TB. The present cross-sectional study is aimed at estimating the prevalence of latent tuberculosis infection (LTBI) in different groups of subjects, either undergoing a routine program of screening for TB or a clinical monitoring of autoimmune or lung disorders, by analyzing their immune response in vitro to a pool of different M. tuberculosis antigens through an IFN-gamma-release assay (IGRA). We consecutively tested 1,644 subjects including health care workers (931), healthy immigrants from different countries (93), patients with a diagnosis of psoriasis (405), patients with lung inflammatory disease (60) or lung neoplasia (32) and a group of HIV-1 infected Italian subjects (120). The prevalence of IGRAs positive responses among health care workers was 8.9 percent. In comparison, significantly higher frequencies were found in healthy immigrant subjects (33.3%), similar to those found in inflammatory broncho-pneumopathies (34.5%) or lung cancer (29.6%). Interestingly, an unexpected high prevalence was also found in patients affected by psoriasis (18.0%), while HIV-infected subjects had values comparable to those of health care workers (10.8%). An age cut-off was determined and applied for each group by receiver operating characteristic (ROC) curves in order to perform the statistical analysis among age-comparable groups. Multivariate analysis showed that the age and clinical conditions such as having a diagnosis of psoriasis or a lung inflammatory disease were independent risk factors for developing an IGRA positive response. This study highlights an unprecedented high prevalence of IGRA positive responses among patients affected by psoriasis and emphasizes the need for a preliminary assessment of LTBI before the administration of any biologic therapy based on cytokine antagonists such as anti-TNF-alpha. Moreover, screening for LTBI should be routinely performed in the presence of a chronic pulmonary disease.

Release of palladium from biomechanical prostheses in body fluids can induce or support PD-specific IFNgamma T cell responses and the clinical setting of a palladium hypersensitivity.

The increased use of Palladium (Pd) for biomedical applications, which has more than doubled in the last ten years, appears to be associated with an increased frequency of adverse reactions to Pd. The aim of this study is to investigate the relationship between the implant of a biomechanical apparatus containing Pd and the setting of a hypersensitivity to Pd by determining the levels of the metal released in biological fluids, assessing the effects of Pd on peripheral blood mononuclear cell (PBMC) cytokine production and exploring the clinical setting of skin sensitization. Of a total of 3,093 subjects examined in 2006, sensitization to Pd alone or in association with nickel (Ni) was observed in 1.6% and 13.03% of the individuals, respectively. Of these, a group of six subjects positive to Pd and negative to Ni at patch testing were selected on the basis of the oral clinical symptoms in order to measure both the levels of Pd in biological fluids and the degradation of the dental prostheses. Specific Pd measurements were carried out on salivary fluid, urine and serum samples by High Resolution Inductively Coupled Plasma-Mass Spectrometry. In addition, the degradation of the dental prostheses was assessed by both a leaching test and an analysis of the micro morphology of orthodontic prostheses. The induction of IFN-gamma production by Pd was assessed in PBMC by the ELISpot assay. Skin sensitization to Pd was evaluated by patch testing and clinical examination. Ten healthy subjects were comparatively tested as controls. We found a specific induction of an IFN-gamma response by Pd in PBMC collected from all the subjects positive to Pd at patch testing. On the contrary, control subjects did not show any response to Pd as assessed by IFN-gamma ELISpot assay or by skin testing. Remarkably, the levels of Pd in all biological samples (saliva, sera, urine) were significantly higher in Pd-sensitized patients than in those collected from controls, reaching the highest concentrations in the urine. The leaching studies gave additional evidence that the dental appliances can release measurable levels of Pd in saliva. Oral clinical symptoms in patients with Pd dental prostheses were associated with measurable levels of Pd in the biological fluids, the induction of Pd-specific IFN-gamma responses in PBMC and the clinical evidence of skin sensitization to Pd. These data suggest that dental appliances may represent an active source of Pd in the body, and this, in turn, can favour the clinical setting of a hypersensitivity to this metal.

CD81 expression on CD19+ peripheral blood lymphocytes is associated with chronic HCV disease and increased risk for HCV infection: a putative role for inflammatory cytokines.

The level of CD81 cell surface expression, a cellular co-receptor for hepatitis C virus (HCV), is critical for productive HCV infection of host cells. In addition, the cross-linking of HCV-E2 protein to CD81 can alter the function of T and B lymphocytes as well as that of NK cells by interfering with the activation signalling pathway. The down-regulation of CD81 expression on peripheral blood lymphocytes (PBL) has been associated to effective therapy of HCV infection. The aim of the present study is to quantitatively assess the levels of CD81 expression in PBL from HCV-infected patients compared to subjects at high risk for HCV infection such as HIV-infected individuals or patients with Porphyria Cutanea Tarda (PCT). The expression of CD81 was quantified by flow-cytometry using Phycoerythrin-labelled standard beads. Determination of CD81 was performed on CD3+ and CD19+ lymphocytes from 34 healthy controls, 51 patients with HCV infection and different clinical outcomes [these included HCV-RNA-negative subjects (8), patients with chronic active hepatitis (16), recipients of liver transplantation under immunosuppressive therapy (12), a subgroup with concomitant HIV infection (9) or concomitant PCT (6)]. In addition, 60 HIV-infected subjects and 4 patients with PCT were studied. The putative role of inflammatory cytokines in modulating CD81 was explored in vitro by assessing the effect of IL-6 or IFN-gamma on cultured human hepatocytes. A significant increase of the CD81 expression was found on CD19+ lymphocytes in association with either HIV or HCV infection, as compared to the control group. Immunosuppressive therapy with FK506, subsequent to liver transplantation, restored CD81 expression at normal levels. Data gathered in vitro using the WRL 68 hepatocytic cell line confirmed that inflammatory cytokines can up-regulate CD81 expression in liver cell inclusion. Our data suggest that CD81 up-regulation can increase the risk of HCV infection, particularly in HIV-infected subjects. In addition, the results strongly suggest that the cytokines released by activated lymphocytes at sites of inflammation may play a part in up-regulating CD81 expression.

Inflammatory cytokines and biofilm production sustain Staphylococcus aureus outgrowth and persistence: a pivotal interplay in the pathogenesis of Atopic Dermatitis.

Individuals with Atopic dermatitis (AD) are highly susceptible to Staphylococcus aureus colonization. However, the mechanisms driving this process as well as the impact of S. aureus in AD pathogenesis are still incompletely understood. In this study, we analysed the role of biofilm in sustaining S. aureus chronic persistence and its impact on AD severity. Further we explored whether key inflammatory cytokines overexpressed in AD might provide a selective advantage to S. aureus. Results show that the strength of biofilm production by S. aureus correlated with the severity of the skin lesion, being significantly higher (P < 0.01) in patients with a more severe form of the disease as compared to those individuals with mild AD. Additionally, interleukin (IL)-ß and interferon γ (IFN-γ), but not interleukin (IL)-6, induced a concentration-dependent increase of S. aureus growth. This effect was not observed with coagulase-negative staphylococci isolated from the skin of AD patients. These findings indicate that inflammatory cytokines such as IL1-ß and IFN-γ, can selectively promote S. aureus outgrowth, thus subverting the composition of the healthy skin microbiome. Moreover, biofilm production by S. aureus plays a relevant role in further supporting chronic colonization and disease severity, while providing an increased tolerance to antimicrobials.

Immune-derived cytokines in the nervous system: epigenetic instructive signals or neuropathogenic mediators?

The investigation of the effects of inflammatory cytokines (IC) on the growth and differentiation of neural cells has provided new insights on the role of such soluble mediators in nervous system development and/or plastic remodeling as well as in the pathogenesis of inflammatory neurodegenerative disorders, which are characterized by chronic IC dysregulation in the central nervous system (CNS). Thus, the study of the interaction between CNS and immune-derived soluble signals in physiological or pathological conditions is of increasing interest. This review first discusses experimental evidence supporting the instructive/permissive role of immune-derived cytokines on CNS development and plasticity. Next, we focus on human neurological disease states such as multiple sclerosis and the neurodegeneration associated to the acquired immune deficiency syndrome in which different inflammatory cytokines have been proposed as potential neuropathogenic mediators.

Plasma viraemia in seronegative HIV-1-infected individuals.

It has been reported that the period of latency between HIV-1 infection and the production of antibodies against the virus is sometimes prolonged for greater than 6 months. However, the data supporting this are still controversial and it is not known whether these individuals are actually infectious, especially through body fluids. We have performed a prospective study of 65 high-risk HIV-1-antibody-negative individuals who were followed-up for a period of at least 1 year. Twelve of these individuals were shown by polymerase chain reaction (PCR) to be carriers of HIV-1 proviral sequences. The virus was isolated from lymphocytes in five out of 10 PCR-positive subjects and from cell-free plasma in two. Our data indicate that in some cases delayed seroconversions may be associated with productive infection, suggesting that mechanism(s) other than viral latency may be responsible for the absence of antibody responses to HIV-1 proteins.

Basic fibroblast growth factor supports human olfactory neurogenesis by autocrine/paracrine mechanisms.

Throughout life, olfactory sensory neurons are renewed from a population of dividing stem cells. Little is known about the molecular mechanisms that regulate the activation, self-renewal and differentiation of olfactory neuronal precursors; however, evidence indicates that soluble mediators may play a central role in olfactory neurogenesis. To identify molecules that regulate olfactory self-renewal and differentiation, we have recently established, cloned and propagated in vitro primary long-term cell cultures from the human fetal olfactory neuroepithelium. Here we show that primary human olfactory neuroblasts synthesize and release biologically active basic fibroblast growth factor which, in turn, supports neuroblast growth by autocrine/paracrine mechanisms. The growth-promoting activity of basic fibroblast growth factor is dose dependent and is accompanied by morphological changes of the cells and by an increase in the expression of neuronal-related genes. These observations indicate that endogenous basic fibroblast growth factor participates in controlling olfactory self-renewal and suggest that this cytokine represents a key regulatory element of olfactory neurogenesis.

Distribution of the natural killer-related receptor for HLA-C during highly active antiretroviral therapy for human immunodeficiency virus infection.

Receptors interacting with Major Histocompatibility Complex class I molecules have been initially found on the surface of human natural killer (NK) cells, where they deliver inhibitory signals to the lysis, being thus defined killer inhibitory receptors (KIR). Subsequently, they were detected also on the surface of T-CD8(+) lymphocytes and are particularly expanded during human immunodeficiency virus (HIV) infection, where they downregulate HIV-specific cytolysis. The expression of KIR recognizing human leukocyte antigen-C alleles was assessed in HIV-infected patients, undergoing highly active antiretroviral therapy (HAART). To this end, the combined expression of CD16/CD56, of CD3 and CD8 as well as of KIR (CD158a and CD158b) surface molecules was analyzed on peripheral blood mononuclear cells by monoclonal antibodies, and flow cytometry. An increase of CD3(+)CD8(+)CD158b(+) cells was found after 6 months of HAART. This finding may have implications for the regulation of T-cell mediated cytolysis during HAART.

Release of natural killer cytotoxic factor in patients with lymphoproliferative disease of granular lymphocytes.

To further investigate the mechanisms accounting for defective natural killer (NK) activity observed in the majority of patients with lymphoproliferative disease of granular lymphocytes (LDGL), we have studied the generation of natural killer cytotoxic factor (NKCF) from peripheral blood lymphocytes recovered from twelve LDGL patients. On the basis of their cytotoxic activity against the K-562 target cells, cases under study were separated in two groups. Cells from five patients, referred to as NK+, were found to exhibit high levels of NK-cell mediated cytotoxicity; cells from seven patients, referred to as NK-, despite their ability to bind to the K-562 targets, displayed a defective NK function. The coculture of cells from NK+ patients with NK sensitive K-562 cells triggered a significantly higher production of NKCF with respect to NK- patients (p less than 0.001 at 1:2 dilution). Using phytohemagglutinin (PHA) as the NKCF stimulator, differences between the groups were not significant, due to a recovery of NKCF generation in NK- patients. Furthermore, a consistent lectin (PHA) dependent cellular cytotoxicity (LDCC) was exhibited by both groups of patients. The addition of gamma-interferon (IFN-gamma) or interleukin-2 (IL-2) during NKCF generation did not significantly modify the production of this factor. Our data point out that (a) NKCF is involved in the lytic activity of LDGL patients' cells, (b) a constitutional impairment in the generation of NKCF is not present in NK- patients, since recovery of lytic function occurs after PHA stimulation, and (c) IL-2 and IFN-gamma do not play a relevant role in triggering NKCF production when added during the generation of this factor. These studies help to clarify the factors involved in the cytotoxic machinery of LDGL patients' cells, suggesting that the defect of the cytotoxic function observed in some LDGL patients could lie at the level of target recognition.

Role of immune-derived diffusible mediators in AIDS-associated neurological disorders.

Neurologic abnormalities are common in HIV-1 infected patients and often represent the dominant clinical manifestation of pediatric AIDS. Although the neurological dysfunction has been directly related to CNS invasion by HIV-1, the pathogenesis of neurologic disorders remains unclear. This review will first discuss the spectrum of potential interactions between HIV-1 and neural (neuronal and glial) cells, in the face of experimental data. Next, we will focus on the role of immune-derived cytokines and other soluble compounds which have been proposed to act as neurotoxic mediators and appear to play a role in the pathogenesis of AIDS-associated neurodegeneration.

Parallel conduction of the phase I preventive and therapeutic trials based on the Tat vaccine candidate.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 microg, 15 microg or 30 microg doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug-treated individuals (http://www.hiv1tat-vaccines.info/).

HIV-1 gene expression and replication in neuronal and glial cell lines with immature phenotype: effects of nerve growth factor.

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.

Proviral sequences detection of human immunodeficiency virus in seronegative subjects by polymerase chain reaction.

Human immunodeficiency virus type 1 (HIV-1) can be isolated from lymphocytes and tissues of symptomatic and asymptomatic seropositive subjects. However, in some individuals, virus isolation is not always positive, especially in asymptomatics. In this paper we report the results of HIV-1 DNA detection by means of polymerase chain reaction (PCR), a new technique that permits the amplification of specific DNA sequences. PCR was carried out to amplify two highly conserved env regions on samples from 20 normal individuals used as controls, 20 seropositive patients at different stages of HIV disease and 25 seronegative individuals at high risk for infection, such as sexual partners of seropositive patients and intravenous drug addicts. Eighteen out of 20 seropositive subjects were positive by PCR while among seronegatives HIV DNA was detected in 7/25 individuals. Virus isolation was positive only in 2/7. These subjects, followed for HIV antibody production for a period of 10-12 months, remained seronegative except one case who seroconverted after a few weeks. Long latency of HIV infection without detectable antibodies seems prevalent in these subjects. PCR assay represents a useful technique for identifying proviral sequences in seronegative high-risk individuals, to confirm the infection during its early phases and during the follow-up of patients with HIV disease.