Heterosexual transmission of HIV-1 is associated with high plasma viral load levels and a positive viral isolation in the infected partner.

Risk factors for heterosexual HIV transmission are not fully understood. In fact, a proportion of people with sexual exposure to HIV remain uninfected despite multiple and continuous intercourse with HIV-infected partners. In this work, we have analyzed those virologic parameters potentially involved in the transmission of HIV through heterosexual contact. Thirty-eight couples with continuous unprotected sexual intercourse were included. HIV transmission occurred in 10 of 38 couples. No differences in clinical characteristics, exposure time, sexual practices, CD4 counts, or polymorphism in CCR5 were found between transmitter and nontransmitter groups. In contrast, virologic data were different between both groups; median values of viral load were 21.139 and 5.484 RNA copies/ml of plasma in the transmitter and nontransmitter groups, respectively, and a significant difference was found in mean viral load values (p = .03, Mann-Whitney test). Viral isolation was obtained in 90% of transmitters, but in only 44% of nontransmitter subjects (p = .02, Fisher's exact test). These data show that viral load levels and a positive viral isolation in culture must be considered as risk factors for heterosexual transmission of HIV-1.

Expression of IkappaBalpha in the nucleus of human peripheral blood T lymphocytes.

According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.

CCR5 genotype and HIV-1 infection in perinatally-exposed infants.

The CCR5 chemokine receptor is required by non-syncytium HIV-1 strains to infect target cells. A 32 base pair deletion (delta32) in the CCR5 gene causes a structural CCR5 modification that does not permit HIV-1 entry into cells. The rate of the CCR5 delta32 was investigated in 137 children born from HIV-infected mothers. Overall, five (10.6%) of 47 HIV-infected infants showed the defect in heterozygosis vs. eight (8.9%) of 90 uninfected children. No CCR5 delta32 homozygotes were found. Interestingly, among infected children, five (21.7%) of 23 showing a slow disease progression were heterozygous for the CCR5 delta32, meanwhile none of the 24 infants with rapid disease course had the deletion (P = 0.022). In conclusion, the CCR5 delta32 defect does not protect against vertical HIV-1 transmission, but is associated with a delayed disease progression in HIV-infected children.

Activation of blood T lymphocytes down-regulates CXCR4 expression and interferes with propagation of X4 HIV strains.

The chemokine receptor CXCR4 serves as a coreceptor for HIV-1 entry into CD4+ cells, in particular for strains emerging late in the infection. Cell surface expression of CXCR4 has, therefore, important implications for HIV-1 pathogenesis. Using blood lymphocytes cultured under various conditions, we studied the expression and regulation of CXCR4. Flow cytometry showed that only about 20% of freshly isolated lymphocytes expressed CXCR4 on the cell surface whereas in 80% of resting blood lymphocytes CXCR4 was located intracellularly. Within a few hours in culture, the intracellular CXCR4 was translocated to the surface and was expressed in the large majority of both naive and memory lymphocytes. A decrease in surface expression of CXCR4 was found when lymphocytes cultured overnight for maximal receptor expression were stimulated with phytohemagglutinin, anti-CD3 antibodies, phorbol 12-myristate 13-acetate and stromal cell-derived factor-1. The superantigen staphylococcal enterotoxin A, a more selective stimulus, induced a marked decrease in CXCR4 expression preferentially in cells positive for the CD25 activation marker. Confocal laser scanning microscopy demonstrated the presence of CXCR4 in the cytosol and on the surface of resting lymphocytes and also showed CXCR4 redistribution after activation. The number of cells infected by the X4 HIV strain NL4.3 paralleled the expression of CXCR4 in CD4+ T lymphocytes. Sustained reduction of CXCR4 cell surface expression upon activation with phytohemagglutinin correlated with a low number of CD4+ T lymphocytes expressing HIV p24 gag antigen. Our results indicate that activation of CD4+ T lymphocytes reduces surface expression of CXCR4 in part by receptor internalization and that cell activation-dependent CXCR4 down-regulation limits spread of infection by X4 viruses.

In vitro selective elimination of HIV-infected cells from peripheral blood in AIDS patients by the immunotoxin DAB389CD4.

OBJECTIVES: To study the antiviral efficacy of the recombinant immunotoxin DAB389CD4 against wild-type strains of HIV and to analyse its potential toxicity in non-infected peripheral blood mononuclear cells (PBMC). DESIGN AND METHODS: PBMC from HIV-seropositive patients were cultured in the presence of DAB389CD4. After 30 days in culture, viral load was assessed by quantification of RNA levels in supernatants and HIV-specific polymerase chain reaction (PCR) was performed for measuring proviral DNA as an indicator of remaining virus in cells. To study the toxicity of DAB389CD4, PBMC from healthy donors were isolated and cell viability and lymphocyte proliferation were assessed after immunotoxin treatment. RESULTS: DAB389CD4 presented a strong antiviral activity in five of the six primary isolates decreasing p24 production in cultures to undetectable levels and eliminating selectively HIV-infected cells as measured by HIV DNA-specific PCR. One viral isolate was resistant to DAB389CD4 treatment. The immunotoxin was active against both syncytial and non-syncytial HIV strains. DAB389CD4 was not toxic in non-infected PBMC as measured by different techniques: trypan blue exclusion, methyl thiazol tetrazolium oxidation, lymphocyte proliferation, and CD4 cell count. CONCLUSIONS: DAB389CD4 showed a strong antiviral and specific activity against primary HIV isolates by killing selectively HIV-infected cells without affecting non-infected cells. This antiviral effect produced the eradication of HIV in cultures and indicated the potential use of this drug as a new therapeutic tool in combination with antiretroviral drugs. This immunotoxin would be especially interesting in the context of the marginal populations of HIV-infected cells remaining after successful antiviral treatment.

Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes.

The role of NF-kappa B-dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF-kappa B subunit and the induction by phorbol esters of bona fide (p50-p65) NF-kappa B complexes. In parallel, we observed clear enhancer-dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B-responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF-kappa B-dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxic oil stimulates collagen synthesis acting at a pretranslational level in cultured fat-storing cells.

BACKGROUND/AIMS: The toxic oil syndrome appeared in Spain in 1981 as a result of ingestion of rapeseed oil denatured with aniline. Some patients developed scleroderma-like skin lesions and liver cirrhosis. Mechanisms of these fibrotic lesions are not known. The present study was designed to investigate the effect of toxic oils on collagen metabolism. METHODS: We measured the relative rate of collagen production, absolute rate of collagen synthesis, production, secretion, and degradation, proline transport, steady-state levels of procollagen alpha 1(l)-messenger RNA (mRNA) in cultured fat-storing cells, and chloramphenicol acetyltransferase activity in transfected cells. RESULTS: Toxic oils increased collagen synthesis, procollagen alpha 1(l)-mRNA levels, and chloramphenicol acetyltransferase activity in cultured fat-storing cells. Effect on collagen production correlated with lipid peroxide content in oils. Cycloheximide, alpha-tocopherol, and methylene blue prevented the increase in procollagen alpha 1(l)-mRNA. Oleylanilide and linoleylanilide, markers for toxic oils, reproduced the stimulatory effects of toxic oils on collagen production and procollagen alpha 1(l)-mRNA. CONCLUSIONS: Toxic oils increased collagen synthesis by acting on the promoter of procollagen alpha 1(l) gene, probably through lipid peroxides derived from acylanilides. We suggest that toxic oil may have stimulated procollagen gene expression through the formation of adducts of aldehydes with some transcription factor.

[Yield of detection of Clostridium difficile toxin versus stool culture in the study of nosocomial diarrhea]. / Rentabilidad de la detección de toxina de Clostridium difficile frente a coprocultivo para el estudio de la diarrea nosocomial.

BACKGROUND: The aim of the study was to determine whether the detection of Clostridium difficile toxin in stools may be more profitable than conventional stool cultures for the etiologic study of nosocomial diarrhea and to analyze what risk factors favor the development of nosocomial diarrhea by C. difficile. METHODS: The presence of enteropathogens and A and B toxins of C. difficile were investigated (by monoclonal antibody enzymoimmunoassay) in stools of patients with nosocomial diarrhea. A series of patients simultaneously admitted without diarrhea were selected as the control group. RESULTS: During a 6 month period 92 patients with nosocomial diarrhea and 82 controls without diarrhea were studied. The C. difficile toxin was detected in 8 of these 174 patients (4.6%). Eight point seven percent of the nosocomial diarrheas were related with C. difficile while only 1% were due to an enteropathogen (Salmonella enteritidis). C. difficile toxin was not detected in any patient who did not have diarrhea. In comparison with the patients with diarrhea due to other causes, the patients with diarrhea by C. difficile had more frequently received antibiotics over the previous 7 days (57 vs 88%) and had been hospitalized for a longer time (> or = 7 days) (58 vs 88%) (p < 0.05). CONCLUSIONS: In the author's institution infection by Clostridium difficile is the most frequent cause of nosocomial infectious diarrhea, especially in patients admitted for a prolonged time or who receive antibiotics. The routine investigation of enteropathogens in the cases of nosocomial diarrhea does not seem justified while the detection of the A and B toxins of C. difficile may be more profitable.

[Cytomegaloviruses in the bronchoalveolar lavage fluids of immunocompromised patients: microbiological results and clinical significance]. / Citomegalovirus en lavados broncoalveolares de pacientes inmunocomprometidos: resultados microbiológicos y significación clínica.

We have evaluated the microbiologic output and clinical significance of the detection of cytomegalovirus in 111 bronchoalveolar lavage specimens from immunosuppressed patients with pneumonitis. The samples were simultaneously processed by conventional tube culture and the rapid shell-vial centrifugation culture assay. Cytomegalovirus was recovered from 30 specimens (27%). The rapid shell-vial procedure was more sensitive than the tube culture, but in two cases cytomegalovirus was isolated only in tube cultures. Cytomegalovirus was considered clinically significant in only 3 from 13 HIV positive patients. All culture positive, HIV negative patients received treatment with ganciclovir. However, ganciclovir was never used on culture negative, HIV negative patients and cytomegalovirus related morbi-mortality was not found in these patients. A prospective study is needed to conclude if a cytomegalovirus negative culture also has a treatment exclusion value in HIV positive patients.

Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients.

The polymerase chain reaction (PCR) and viral culture techniques were prospectively compared for the detection of cytomegalovirus (CMV) in blood samples from 24 liver transplant recipients. Nine patients had one or more episodes of viremia, seven of which were clinically symptomatic infections. All samples in which CMV was isolated by culture were positive by the PCR. However, the PCR result was also positive for one or more samples from 11 patients who never developed CMV-related symptoms. Although the PCR is a very sensitive technique for CMV detection in blood samples from liver transplant recipients, it is not useful as a marker of symptomatic CMV disease.

[Evaluation of the effect of the dilution of urine on the toxicity and isolation of cytomegalovirus using the rapid shell-vial centrifugation culture technique]. / Evalución del efecto de la dilución de la orina sobre la toxicidad y el aislamiento de citomegalovirus con la técnica rápida de cultivo-centrifugación en viales.

Working with highly passaged MRC-5 fibroblast monolayers in a shell-vial culture-centrifugation assay for rapid detection of cytomegalovirus (CMV), a direct toxic effect of the urine on the monolayer was observed in as many as 44% of cases when vials were inoculated with undiluted specimens. This toxicity was reduced to only 6% by inoculation of shell-vials with samples diluted 1:1 with viral transport medium. The recovery of CMV in shell-vials was not significantly affected by the dilution factor, since five cases detected in conventional culture in tubes and not in vials were compensated by seven cases detected in vials and not in tubes.

High-resolution microscopy of lung and intrathoracic tumors.

Effective chemotherapy is available for many types of lung tumors; however, to select the most appropriate regimen, precise classification is essential. The most important differentiation is that between small cell and non-small cell anaplastic carcinoma. It is also important to distinguish mediastinal neoplasms and neoplasms metastatic to lung from primary lung tumors. Processing tissue in methacrylate and epoxy resins results in excellent preservation of histologic detail and allows for maximum resolution under the light microscope. In addition, histochemical and immunochemical procedures can be carried out in these preparations, allowing the pathologist to more precisely classify the various tumors.

Cell population analysis of a heterogeneous blast cell crisis of chronic myelogenous leukemia.

A case of Philadelphia negative chronic myelogenous leukemia (CML) is described with the following features: 1) initial lymphoid blast crisis; followed by 2) a heterogeneous blast crisis including myeloid, monocytic, and lymphoid elements that could be distinguished by morphological, cytochemical, ultrastructural methods and by immunologic markers; and 3) clonal evolution as shown by methylcellulose cultures and ultrastructural studies with emergence of a relatively drug resistant subclone of leukemic cells. Determination of surface antigen phenotype in CML blast crisis thus provides clinically useful information for the structuring of treatment protocols. This study also confirms previous studies that lymphoid blast crisis of CML can occur in Ph'-cases and that the Philadelphia chromosome is probably a clonal marker only and its presence is not directly related to the subsequent clinical course of the disease.

Ultrastructural and Immunological methods in diagnostic pathology in a community hospital.

Five unusual tumors and one case of glomerulonephritis were examined by light microscopy, immunochemical methods, and electron microscopy in order to arrive at a correct diagnosis. We propose that these techniques can be used in community hospitals for selected cases in order to classify certain types of tumors, identify the primary site of some metastatic tumors, and help in the precise classification of some autoimmune diseases.

Thymoma immunological and ultrastructural characterization.

A 69-year-old man with a mediastinal mass was found to have atypical lymphoid cells in peripheral blood and bone marrow. Light and electron microscopic studies showed that the mediastinal mass was a thymoma. B- and T-cell differentiation and PHA responsiveness in the tumor and peripheral blood were consistent with T-cells. After excision of the tumor and a course of radiotherapy the B/T ratio and PHA responsiveness in peripheral blood returned to normal while the atypical lymphocytes disappeared from circulation.

Plasma-cell leukemia with unusual immunoglobulin abnormalities.

The cases of three patients who had clinical courses and hematologic data consistent with plasma-cell leukemia or a terminal leukemic phase of multiple myeloma are presented. A double Bence Jones protein was demonstrated in the urine of two of the patients. Immunoelectrophoresis and immunofluorescence studies demonstrated kappa light chains. In serum and urine of the third patient, no immunoglobulin abnormality was demonstrated. The three patients had rapidly fatal courses unresponsive to treatment.