HIV expression is induced in dying cells.

Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. gamma rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that gamma-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.

HIV recombination: what is the impact on antiretroviral therapy?

Retroviral recombination is a potential mechanism for the development of multiply drug resistant viral strains but the impact on the clinical outcomes of antiretroviral therapy in HIV-infected patients is unclear. Recombination can favour resistance by combining single-point mutations into a multiply resistant genome but can also hinder resistance by breaking up associations between mutations. Previous analyses, based on population genetic models, have suggested that whether recombination is favoured or hindered depends on the fitness interactions between loci, or epistasis. In this paper, a mathematical model is developed that includes viral dynamics during therapy and shows that population dynamics interact non-trivially with population genetics. The outcome of therapy depends critically on the changes to the frequency of cell co-infection and I review the evidence available. Where recombination does have an effect on therapy, it is always to slow or even halt the emergence of multiply resistant strains. I also find that for patients newly infected with multiply resistant strains, recombination can act to prevent reversion to wild-type virus. The analysis suggests that treatment targeted at multiple parts of the viral life-cycle may be less prone to drug resistance due to the genetic barrier caused by recombination but that, once selected, mutants resistant to such regimens may be better able to persist in the population.

Cyclobutane pyrimidine dimers in UV-DNA induce release of soluble mediators that activate the human immunodeficiency virus promoter.

Ultraviolet (UV) irradiation of human cells induced expression of a stably maintained fusion gene consisting of the human immunodeficiency virus long terminal repeat promoter controlling the bacterial chloramphenicol acetyltransferase gene. Two experiments demonstrated that DNA damage can initiate induction: UV induction was greater in DNA repair-deficient cells from a xeroderma pigmentosum patient than in repair-proficient cells, and transfection of UV-irradiated DNA into unirradiated cells activated gene expression. Increased repair of cyclobutane pyrimidine dimers by T4 endonuclease V abrogated viral gene activation, suggesting that dimers in DNA are one signal leading to increased gene expression. This signal was spread from UV-irradiated cells to unirradiated cells by co-cultivation, implicating the release of soluble factors. Irradiation of cells from DNA repair-deficiency diseases resulted in greater release of soluble factors than irradiation of cells from unaffected individuals. These results suggest that UV-induced cyclobutane pyrimidine dimers can activate the human immunodeficiency virus promoter at least in part by a signal-transduction pathway that includes secretion of soluble mediators.

Human immunodeficiency virus and the skin: selected controversies.

Acquired immunodeficiency syndrome was first recognized as a new disease in 1981 because of the unusual association of Kaposi's sarcoma and Pneumocystis carinii pneumonia in young men. The skin remains one of the most important clinical markers for acquired immunodeficiency syndrome, now recognized as the end stage of infection with the human immunodeficiency virus (HIV). Indeed, an urticarial viral exanthem appearing during seroconversion may allow early identification of newly infected individuals who might benefit from administration of antiviral therapy during plasma viremia. The "asymptomatic HIV infection" is often accompanied by multiple skin complaints, which commonly include xerosis, pruritus, psoriasis/seborrheic dermatitis, and pruritic papular eruptions, the cause of which remains controversial. Psoriasis and Kaposi's sarcoma lesions share features including angiogenesis, dermal dendrocytes infected with HIV, and epidermal hyperproliferation, and are manifested by mice transgenic for HIV provirus or Tat-ltr. Changes in the immune system including T-cell function, antigen response, and shifting cytokine expression as well as a propensity for autoimmune reactions must underlie the skin immunodysfunction occurring in the setting of HIV infection. One of the most unsettling controversies suggested by in vitro data is that ultraviolet light, an effective therapy for HIV-related skin disorders, may actually activate the virus.

Inactivation of the human immunodeficiency virus by hypericin: evidence for photochemical alterations of p24 and a block in uncoating.

Following attachment and entry of human immunodeficiency virus (HIV) into a host cell, the HIV genomic RNA is reverse transcribed to cDNA. This step may be inhibited by hypericin, a compound that induces alterations of the retroviral capsid. Incubation of HIV with hypericin rendered the virus noninfectious. The replication of HIV was blocked early; HIV cDNA could not be detected in cells challenged with hypericin-treated HIV. Hypericin did not inhibit the binding of recombinant gp120 to CD4+ cells, nor did hypericin inhibit syncytium formation. However, reverse transcriptase activity could not be released from hypericin-treated virions. Western blot analysis revealed altered mobility of the HIV major capsid protein (p24) following hypericin treatment. Hypericin-treated recombinant HIV p24 exhibited similar altered mobility. The inactivation of HIV infectivity and the alterations in p24 mobility required hypericin incubations in the presence of visible light. Collectively, these data suggest that photochemical alterations of the HIV capsid may contribute to the hypericin-mediated inactivation of HIV. Such alterations may inhibit the release of RT activity from treated HIV, and prevent uncoating and subsequent reverse transcription of the HIV genome within a target cell.

Eliminating PCR contamination: is UV irradiation the answer?

The sensitivity of the polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for the detection of human cytomegalovirus (CMV) and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. The sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, the ultimate sensitivity of a PCR system for the amplification of an early gene promotor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worthwhile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system.

Effect of low-dose gamma radiation of HIV replication in human peripheral blood mononuclear cells.

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of gamma-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. With this in mind, we have studied the effects of gamma-radiation on HIV replication in mononuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy gamma-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting.

Role of the p38 and MEK-1/2/p42/44 MAP kinase pathways in the differential activation of human immunodeficiency virus gene expression by ultraviolet and ionizing radiation.

Ultraviolet (UV) radiation is a potent activator of human immunodeficiency virus (HIV) gene expression in a HeLa cell clone having stably integrated copies of an HIV cat (cat gene under control of the HIV promoter) reporter construct, whereas ionizing radiation is ineffective. UV-activated HIV gene expression is completely blocked by the specific p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 and by expression of a kinase-inactive p38 mutant that interferes with normal p38 function, suggesting that this stress-activated protein kinase plays an important role in UV-mediated transcriptional activation of HIV. In support of these findings, we show here that Western blot analysis demonstrated rapid and significant activation of p38 MAP kinase by UV. On the other hand, gamma-radiation activated p38 MAP kinase very poorly in HeLa cells at both low and high doses at times (5-30 min) when UV radiation was effective. UV radiation also activated HIV gene expression (< or = 9-fold) in 1G5 Jurkat T-cells stably transfected with a luciferase reporter gene under control of the HIV promoter. In these cells, gamma-radiation stimulated HIV gene expression but to a lesser extent (< or = 3-fold) and with different kinetics than after UV radiation, and this response was obliterated by the incubation of cells with the mitogen-activated protein kinase/Erk kinase (MEK)-1/2 inhibitor PD98059. This result suggests that in these cells signaling in response to gamma-radiation is transduced through the MEK-1/2/p42/44 MAP kinase pathway to increase HIV gene expression. All combined, these results suggest that activation of p38 MAP kinase is necessary for efficient HIV gene expression triggered by DNA damaging agents, and, in a cell type-specific manner, activation of the MEK-1/2/p42/44 MAP kinase pathway is important for triggering a response to gamma-radiation. Thus, it appears as if UV signaling leading to HIV gene expression requires the p38 MAP kinase pathway whereas activation by gamma-radiation requires the MEK-1/2/p42/44 MAP kinase pathway.

Medical UV exposures and HIV activation.

This paper presents the first attempt to evaluate the potential of clinical UV exposures to induce the human immunodeficiency (HIV) promoter and, thus, to upregulate HIV growth in those skin cells that are directly affected by the exposure. Using the data for HIV promoter activation in vitro, we computed UVB and psoralen plus UVA (PUVA) doses that produce 50% of the maximal promoter activation (AD50). Then, using (a) literature data for UV transmittance in the human skin, (b) a composite action spectrum for HIV promoter and pyrimidine dimer induction by UVB and (c) an action spectrum for DNA synthesis inhibition by PUVA, we estimated the distribution of medical UVB and PUVA doses in the skin. This allowed us to estimate how deep into the skin the HIV-activating doses might penetrate in an initial and an advanced stage of UVB or PUVA therapy. Such analysis was done for normal type II skin and for single exposures. The results allow us to predict where in the skin the HIV promoter may be induced by selected small and large therapeutic UVB or PUVA doses. To accommodate changes in skin topography due to disease and UV therapy, our considerations would require further refinements. For UVB we found that, when the incident dose on the surface of the skin is 500 J/m2 (290-320 nm) (initial stage of the therapy), the dose producing 50% of the maximal HIV promoter activation (ADUVB50) is limited to the stratum corneum. However, with an incident dose of 5000 J/m2 (an advanced stage of the therapy), ADUVB50) may be delivered as far as the living cells of the epidermis and even to some parts of the upper dermis. For PUVA we found that, when the incident UVA doses are 25 or 100 kJ/m2 (320-400 nm) (an initial and an advanced stage of therapy, respectively), and the 8-methoxypsoralen concentration in the blood is 0.1 microgram/mL (the desired level), the combined doses to the mid epidermis (and some areas of the upper dermis) are well below the 50% HIV promoter-activating PUVA dose (ADPUVA50). Only under the worst scenario conditions, i.e. an exceptionally high drug concentration in the patient's tissues and localization of HIV in the nearest proximity to the skin surface, would the combined PUVA dose expected during photochemotherapy exceed ADPUVA50. These results suggest that the probability of HIV activation in the epidermis by direct mechanisms is higher for UVB than for PUVA treatment. However, complexities of the UV-inducible HIV activation and immunomodulatory phenomena are such that our results by themselves should not be taken as an indication that UVB therapy carries a higher risk than PUVA therapy when administered to HIV-infected patients.

Activation of the human immunodeficiency virus promoter by UVA radiation in combination with psoralens or angelicins.

The effects of mono- and bifunctional furocoumarins plus UVA radiation (PUVA and related treatments) on the human immunodeficiency virus-1 (HIV-1) promoter were studied using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV-1 promoter. The experiments were performed with three psoralens (5-methoxypsoralen, 5-MOP; 8-methoxypsoralen, 8-MOP; and 4'-aminomethyl-4,8,5'-trimethylpsoralen, AMT) and four angelicins (angelicin; 4,5'-dimethylangelicin, 4,5'-DMA; 6,4'-dimethylangelicin, 6,4'-DMA; and 4,6,4'-trimethylangelicin, TMA). The drugs alone and UVA radiation alone showed no effect on the HIV promoter. However, when the cells were incubated with the furocoumarins at 0.1-40 micrograms/mL and then irradiated, the HIV promoter was activated in distinct fluence ranges, i.e. (1) no promoter activity was discernible at low fluences (e.g. at 0.1 microgram/mL of 8-MOP up to 100 kJ/m2), (2) as the fluence was increased, the promoter activity increased to reach a maximum (10-50-fold with respect to the unexposed samples), and (3) as the fluence was further increased, the promoter activity decreased. Similar (although shifted on the fluence scale) patterns were observed with either > 340-nm UVA radiation or with UVA radiation contaminated with a small amount of UVB radiation (typical for PUVA lamps). The effective fluences were inversely related to the drug concentration. Experiments with 5-MOP and 8-MOP indicated reciprocity of the drug concentration and radiation fluence. The HIV promoter response patterns were similar for monofunctional angelicins and bifunctional psoralens.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFkappaB blocking agents.

To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6-10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFkappaB) in UVB-inducible HIV activation, two types of blockers, NFkappaB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFkappaB.

Comparison of the effects of different antiviral treatments on the antioxidant systems of stroma-free hemoglobin.

The effect of virus inactivation by 1,9-dimethylmethylene blue (DMMB) phototreatment, methylene blue (MB) phototreatment or heat on the activities of antioxidant systems of stroma-free hemoglobin (SFH) was studied. DMMB photoinactivated human immunodeficiency virus by > 3.69 log10 under conditions that inactivated 3.33 log10 of vesicular stomatitis virus (VSV). Under conditions which inactivated VSV by 6.10 log10 (1.37 J/cm2 irradiation and 2 microM DMMB), there was little change in the methemoglobin (Met-Hb) formation, concentration of reduced glutathione (GSH), or superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPX) activities. However, the activity of glutathione reductase (GR) was decreased by 77%. Under conditions that inactivated VSV by 5.69 log10 (1.37 J/cm2 irradiation and 24 microM MB) there was little effect of MB phototreatment on SOD, CAT, GPX and GSH activities. However, GR activity was decreased by 74% and Met-Hb content reached 3.98%. Under conditions that inactivated VSV by more than 6.20 log10 (60 degrees C for 2 min), virucidal heat treatment resulted in 27% Met-Hb formation and decreased GPX activity by 43%. No significant decline in SOD, CAT or GR activities or GSH concentration was observed. These results suggest that, compared with heat treatment and MB phototreatment, virucidal DMMB treatment preserves not only the oxidative state of hemoglobin but also the antioxidant systems against superoxide and hydrogen peroxide, although the reduced GR activity may limit the quenching capacity of antioxidants in DMMB-treated SFH.

Ultraviolet irradiation and cytokines as regulators of HIV latency and expression.

The ability of the human immunodeficiency virus (HIV) to persist and replicate in human CD4+ T lymphocytes and mononuclear phagocytes is under the control of both virally encoded proteins and a variety of host-related factors. Ultraviolet (UV) light has been shown to induce transcription and expression of HIV. Both DNA damage and repair and DNA damage/repair-independent pathways caused by UV irradiation lead to expression of proviral HIV genomes via activation of the cellular transcription factor NF-kappa B. Transgenic mice that contain either long terminal repeat (LTR)-reporter genes or HIV genomes, either full length or deleted in the gag-pol region, express RNA and proteins at the epidermal level, particularly after UV irradiation. Furthermore, UV-triggered release of soluble factors capable of inducing expression of HIV in non-irradiated cells has been observed. Among other host factors, the functional network of pro-inflammatory and immunoregulatory cytokines has been demonstrated to act as a potent regulator of HIV replication, at least in different in vitro systems of infection.

Effects of gamma irradiation on the human immunodeficiency virus. A study in frozen human bone-patellar ligament-bone grafts obtained from infected cadavera.

We studied the effects of several different doses of gamma radiation, ranging from 20,000 to 40,000 gray (2.0 to 4.0 megarad), with respect to the inactivation of the human immunodeficiency virus in fresh-frozen, whole bone-patellar ligament-bone grafts. Although the International Atomic Energy Agency has recommended the use of 25,000 gray of gamma radiation for the sterilization of medical products, the dose required for the inactivation of the human immunodeficiency virus in frozen allografts has not been established. Using one of the most sensitive and specific tests for the detection of the human immunodeficiency virus, the polymerase-chain-reaction test, we found that doses of 20,000 or 25,000 gray of gamma radiation did not destroy the genes of the human immunodeficiency virus effectively; DNA of the virus was detectable in the DNA of bone-marrow tissue obtained from grafts treated with these doses. However, DNA of the human immunodeficiency virus was not detectable in the grafts treated with 30,000 or 40,000 gray of gamma radiation. We conclude that a dose of 30,000 gray of gamma radiation or more is necessary for the sterilization of a fresh-frozen bone-patellar ligament-bone allograft, so that it can be used for reconstructive procedures without the risk of transmission of the virus to the recipient.

Potential risks of ultraviolet radiation in HIV infection.

The hazards of ultraviolet radiation (UVR) include immunosuppression, activation of human immunodeficiency virus (HIV) type 1 expression, and photocarcinogenesis particularly in immunosuppressed individuals. Fifty-eight male homosexuals positive for HIV antibody and 61 controls not at risk for HIV infection answered a questionnaire on their attitudes and exposure to natural and artificial sources of UVR. Controls were matched for sex but were not from an at-risk group for HIV infection. Mean ages were similar for both groups. HIV seropositives had greater recreational UVR exposure than controls: 12/58 versus 4/61 had regular use of a sunbed (P less than 0.05), and experienced 11.6 weeks versus 9.5 weeks of prolonged natural UVR exposure (P = 0.056) over a four-year period. One reason for this difference may be the misconception present in two-thirds of the HIV seropositive group that a suntan would improve their health and the outcome of their HIV infection. Those with HIV infection must be made aware that there is a potential for further immunosuppression and viral activation from UVR and they should be advised to avoid undue recreational exposure.

Destruction of human immunodeficiency-infected cells by ferrofluid particles manipulated by an external magnetic field: mechanical disruption and selective introduction of cytotoxic or antiretroviral substances into target cells.

Submagnetic domain magnetic fluid particles of approximately 10 nm average diameter complexed with CD4 or monoclonal antibody and then injected into the patient, will localize to the cell membrane of the target cell. These ferrofluid particles will interact with an externally applied rotating magnetic field of rapidly changing polarity. Under these conditions, the ferrofluid particles will be drawn into a circular path and an axial spin will be induced as each particle aligns itself with the magnetic force lines. A portion of these magnetic fluid particles will be drawn into the target cell membrane and into the cytoplasm causing brief perforations of the cell membrane of the target cells. If enough mechanical damage is done to the plasma membrane or to the intracellular structures, cell lysis may result, but in any case the brief disruptions of the target cell membrane can be used to selectively introduce membrane impermeant cytotoxic or antiretroviral substances into the target cell while relatively sparing normal cells.

[A new method of bone sterilization]. / Eine neue Methode der Knochensterilisation.

Bone transplantation is associated with two major problems: the sterility in particular with respect to HIV, and the osteoinductivity of the transplant. We describe a procedure which sterilizes the graft while keeping the osteoinductive potential of the bone. After harvesting the material the bone is immediately deep frozen (-80 degrees C), and then freeze dried. To sterilize the bone it is treated with gamma-radiation at a dose of 2.5 Mrad in an argon atmosphere. All grafts are subsequently stored in argon at -80 degrees C.

Manifestaciones clínico radiológicas en pacientes con coinfección tuberculosis pulmonar y VIH/sida / Clinical-radiologic manifestations inpatients with pulmonary tuberculosis and HIV/AIDS coinfection

Introducción: la infección por Mycobacterium tuberculosis es considerada la coinfección más común en personas VIH positivas. Objetivo: describir las principales características según variables sociodemográficas y clínicas seleccionadas de los pacientes VIH/sida con Tb pulmonar e identificar los hallazgos radiológicos más frecuentes para contribuir a un diagnóstico más temprano y disminuir la mortalidad por esta coinfección. Métodos: se realizó un estudio de casos clínicos. Se incluyeron 120 pacientes con VIH/sida y cultivo de esputo positivo de Mycobacterium tuberculosis, atendidos en el Hospital del Instituto de Medicina Tropical Pedro Kourí, en el periodo comprendido entre enero de 2004 y diciembre del 2010. Resultados: de los casos estudiados el 92,5 por ciento eran masculinos; 48,3 por ciento con color de piel blanca, la media de edad fue 35,6 años. La tuberculosis fue definitoria de sida en el 25,8 por ciento de los casos. La media del conteo de linfocitos T CD4+ fue 193,91 cel/µL. Las manifestaciones clínicas más frecuentes fueron fiebre, tos y pérdida de peso. Predominó el patrón radiológico primario, con infiltrados en lóbulos inferiores, infiltrados con derrame pleural e infiltrados extensos. El 25 por ciento tenían Rx de tórax normales. Conclusiones: la coinfección se presentó independientemente de los niveles de linfocitos T CD4. Las radiografías de tórax sin alteraciones no excluyeron el diagnóstico de tuberculosis en pacientes VIH/sida(AU)

Introduction: Mycobacterium tuberculosis infection is considered the most common coinfection in HIV-positive people. Objective: To describe the main characteristics according to the sociodemographic and clinical variables chosen from HIV/AIDS patients with pulmonary tuberculosis and to identify the most frequent radiological findings, in order to contribute to an earlier diagnosis and to reduce the mortality due to this coinfection. Methods: A clinical case study was carried out. A total of 120 patients with HIV/AIDS and sputum culture positive for Mycobacterium tuberculosis, treated at the Hospital of Pedro Kourí Institute of Tropical Medicine (IPK), and who were admitted between January 2004 and December 2010. Results: Out of the cases studied, 92.5 percent were male, 48.3 percent had white skin, and their mean age was 35.6 years. Tuberculosis was a defining condition for AIDS in 25.8 percent of the cases. The mean CD4+ T-lymphocyte count was 193.91 cells/?L. The most frequent clinical manifestations were fever, cough and weight loss. There was a predominance of the primary radiological pattern, with infiltrates in the lower lobes, infiltrates with pleural effusion, and extensive infiltrates. 25 percent percent had normal chest X-rays. Conclusions: Coinfection occurred independently of CD4+ T-lymphocyte levels. Unchanged chest radiographs did not exclude the diagnosis of tuberculosis in HIV/AIDS patients(AU)