Stable expression of shRNA for the control of recombinant adenovirus replication.

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Stable expression of shRNA for the control of recombinant adenovirus replication

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Studying the toxicity of SLEnS-LAS micelles to collembolans and plants: Influence of ethylene oxide units in the head groups.

Mixed micelles of linear alkylbenzene sulfonic acid (LAS) and ether sulfate-based surfactants (SLEnS) can be added in household products and cleaning agents. SLEnS with higher ethylene oxide (EO) units in the head groups have economic and environmental advantages. This work aims to assess the influence of the number of EO units in the ecotoxicity of seven variants of SLEnS-LAS micelles (0-50 EO units) in soils. Ecotoxicological tests were carried out to assess emergence and growth of four plants species and reproduction of collembolans. Most of the variants inhibited plants growth at the highest concentrations (1237.5 µg SLEnS kg-1 of soildw). For reproduction, lower number of EO units resulted in EC50 from 924.2 (95 % CL: 760.7-1063.4) to 963.2 (95 % CL: 676.9-1249.6) µg SLEnS kg-1 of soildw, whereas for higher number of EO units (50 and 30) no inhibition was reported. Based on these results, we suggest that a higher number of EO units contribute to less hazardous formulations, confirming that different designs of surfactants may contribute to changes in the responses of terrestrial organisms. Therefore, we demonstrate that standardized ecotoxicological assays may contribute to more sustainable and effective formulations, when used upstream, prior to manufacture and marketing.

Nutritional shortage augments cisplatin-effects on murine melanoma cells.

Melanoma incidence increases every year worldwide and is responsible for 80% of skin cancer deaths. Due to its metastatic potential and resistance to almost any treatments such as chemo, radio, immune and targeted-therapy, the patients still have a poor prognosis, especially at metastatic stage. Considering that, it is crucial to find new therapeutic approaches to overcome melanoma resistance. Here we investigated the effect of cisplatin (CDDP), one of the chemotherapeutic agents used for melanoma treatment, in association with nutritional deprivation in murine melanoma cell lines. Cell death and autophagy were evaluated after the treatment with cisplatin, nutritional deprivation and its association using an in vitro model of murine melanocytes malignant transformation to metastatic melanoma. Our results showed that nutritional deprivation augmented cell death induced by cisplatin in melanoma cells, especially at the metastatic subtype, with slight effects on melanocytes. Mechanistic studies revealed that although autophagy was present at high levels in basal conditions in melanoma cells, was not essential for cell death process that involved mitochondrial damage, reactive oxygen species production and possible glycolysis inhibition. In conclusion, nutritional shortage in combination with chemotherapeutic drugs as cisplatin can be a valuable new therapeutic strategy to overcome melanoma resistance.

Diagnosis of Pneumocystis pneumonia: evaluation of four serologic biomarkers.

The diagnosis of Pneumocystis pneumonia (PCP) relies on microscopic visualization of Pneumocystis jirovecii organisms or DNA detection in pulmonary specimens. This study aimed to assess the usefulness of (1-3)-ß-d-glucan (BG), Krebs von den Lungen-6 antigen (KL-6), lactate dehydrogenase (LDH) and S-adenosyl methionine (SAM) as serologic biomarkers in the diagnosis of PCP. Serum levels of BG, KL-6, LDH and SAM were investigated in 145 Portuguese patients, 50 patients from the Netherlands, 25 Spanish patients and 40 Portuguese blood donors. Data on clinical presentation, chest imaging and gasometry tests were available. PCP cases were confirmed by microscopy and PCR techniques. A cost-effectiveness analysis was performed. BG was found to be the most reliable serologic biomarker for PCP diagnosis, followed by KL-6, LDH and SAM. The BG/KL-6 combination test was the most accurate serologic approach for PCP diagnosis, with 94.3% sensitivity and 89.6% specificity. Although less sensitive/specific than the reference standard classic methods based on bronchoalveolar lavage followed by microscopic or molecular detection of P. jirovecii organisms, the BG/KL-6 test may provide a less onerous procedure for PCP diagnosis, as it uses a minimally invasive and inexpensive specimen (blood), which may be also a major benefit for the patient's care. The BG/KL-6 combination test should be interpreted within the clinical context, and it may be used as a preliminary screening test in patients with primary suspicion of PCP, or as an alternative diagnostic procedure in patients with respiratory failure or in children, avoiding the associated risk of complications by the use of bronchoscopy.

Interplay between apoptosis and autophagy, a challenging puzzle: new perspectives on antitumor chemotherapies.

Autophagy is a mechanism of protection against various forms of human diseases, such as cancer, in which autophagy seems to have an extremely complex role. In cancer, there is evidence that autophagy may be oncogenic in some contexts, whereas in others it clearly contributes to tumor suppression. In addition, studies have demonstrated the existence of a complex relationship between autophagy and cell death, determining whether a cell will live or die in response to anticancer therapies. Nevertheless, we still need to complete the autophagy-apoptosis puzzle in the tumor context to better address appropriate chemotherapy protocols with autophagy modulators. Generally, tumor cell resistance to anticancer induced-apoptosis can be overcome by autophagy inhibition. However, when an extensive autophagic stimulus is activated, autophagic cell death is observed. In this review, we discuss some details of autophagy and its relationship with tumor progression or suppression, as well as role of autophagy-apoptosis in cancer treatments.

Cateterismo posterior do plexo braquial em cão: estudo radiológico e avaliação de três protocolos anestésicos na duração do bloqueio motor e sensitivo / Posterior catheterization of the brachial plexus: radiological study and evaluation of three anesthetic protocols in motor and sensory block duration

Avaliou-se a eficiência do cateterismo posterior do plexo braquial em cães para promover bloqueio motor e sensitivo, por meio de três protocolos anestésicos. Foram utilizados nove cães, machos e fêmeas, sem distinção de raça e idade, com peso variando de 6 a 15kg, distribuídos em três grupos de três animais por grupo. Após a confirmação do correto posicionamento do cateter pela via posterior do plexo braquial por meio do exame radiográfico, foram aplicadas as medicações de acordo com os grupos. No grupo 1, a solução anestésica de bupivacaína 0,5% sem vasoconstrictor, na dose de 2mg.kg-1, foi usada isoladamente. No grupo 2, a solução anestésica de bupivacaína foi associada ao butorfanol na dose de 0,25mg.kg-1 . No grupo 3, o fentanil, na dose de 0,005mg.kg¹, foi associado à solução anestésica de bupivacaína. Não houve diferença estatística significante entre os grupos. Observou-se que a duração dos bloqueios motor e sensitivo foi clinicamente maior no grupo 2. O cateterismo posterior do plexo braquial permite a aplicação de fármacos mais próximos do plexo nervoso, promovendo analgesia complementar nos membros anteriores.

We evaluated the efficiency of the posterior brachial plexus catheterization in dogs to promote motor and sensory block using three anesthetic protocols. We used nine dogs, males and females, without distinction of race and age, weighing 6 to 15kg which were divided into three groups of three animals per group. After confirming the correct positioning of the catheter through the posterior brachial plexus through radiographic examination, the medications were administered according to the groups. In group 1 the anesthetic bupivacaine 0.5% without a vasoconstrictor dose of 2mg.kg-1 was used alone. In group 2 the anesthetic bupivacaine was associated with butorphanol at a dose of 0.25mg.kg-1. In group 3, the fentanyl dose was associated with 0.005mg.kg-1 anesthetic bupivacaine. There was no statistically significant difference between groups. It was observed that the duration of sensory and motor block was clinically higher in group 2. The posterior brachial plexus catheterization allows the application of drugs closer to the nerve plexus promoting additional analgesia in the forelimbs.

Impairment of locomotor activity induced by the novel N-acylhydrazone derivatives LASSBio-785 and LASSBio-786 in mice

The N-acylhydrazone (NAH) analogues N-methyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-785) and N-benzyl 2-thienylidene 3,4-benzoylhydrazine (LASSBio-786) were prepared from 2-thienylidene 3,4-methylenedioxybenzoylhydrazine (LASSBio-294). The ability of LASSBio-785 and LASSBio-786 to decrease central nervous system activity was investigated in male Swiss mice. LASSBio-785 or LASSBio-786 (30 mg/kg, ip) reduced locomotor activity from 209 ± 26 (control) to 140 ± 18 (P < 0.05) or 146 ± 15 crossings/min (P < 0.05), respectively. LASSBio-785 (15 or 30 mg/kg, iv) also reduced locomotor activity from 200 ± 15 to 116 ± 29 (P < 0.05) or 60 ± 16 crossings/min (P < 0.01), respectively. Likewise, LASSBio-786 (15 or 30 mg/kg, iv) reduced locomotor activity from 200 ± 15 to 127 ± 10 (P < 0.01) or 96 ± 14 crossings/min (P < 0.01), respectively. Pretreatment with flumazenil (20 mg/kg, ip) prevented the locomotor impairment induced by NAH analogues (15 mg/kg, iv), providing evidence that the benzodiazepine (BDZ) receptor is involved. This finding was supported by the structural similarity of NAH analogues to midazolam. However, LASSBio-785 showed weak binding to the BDZ receptor. LASSBio-785 or LASSBio-786 (30 mg/kg, ip, n = 10) increased pentobarbital-induced sleeping time from 42 ± 5 (DMSO) to 66 ± 6 (P < 0.05) or 75 ± 4 min (P < 0.05), respectively. The dose required to achieve 50% hypnosis (HD50) following iv injection of LASSBio-785 or LASSBio-786 was 15.8 or 9.5 mg/kg, respectively. These data suggest that both NAH analogues might be useful for the development of new neuroactive drugs for the treatment of insomnia or for use in conjunction with general anesthesia.

Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells.

Interleukin-7 (IL-7) activates phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, proliferation and growth of T-cell acute lymphoblastic leukemia (T-ALL) cells. Reactive oxygen species (ROS) can be upregulated by growth factors and are known to regulate proliferation and viability. Here, we show that IL-7 upregulates ROS in T-ALL cells in a manner that is dependent on PI3K/Akt/mTOR pathway activity and that relies on both NADPH oxidase and mitochondrial respiratory chain. Conversely, IL-7-induced activation of PI3K signaling pathway requires mitochondrial respiration and ROS. We have previously shown that IL-7-mediated activation of PI3K pathway drives the upregulation of the glucose transporter Glut1, promoting glucose uptake in T-ALL cells. Using phloretin to inhibit Glut function, we demonstrate that glucose uptake is mandatory for ROS upregulation in IL-7-treated T-ALL cells, suggesting that IL-7 stimulation leads to increased ROS via PI3K pathway activation and consequent upregulation of Glut1 and glucose uptake. Overall, our data reveal the existence of a critical crosstalk between PI3K/Akt signaling pathway and ROS that is essential for IL-7-mediated T-ALL cell survival, and that may constitute a novel target for therapeutic intervention.

Intravenous administration of azumolene to reverse malignant hyperthermia in swine.

BACKGROUND: The efficacy of intravenous (IV) administration of azumolene (Az), an analogue 30-fold more soluble than dantrolene, on pigs susceptible to malignant hyperthermia (MH) is incompletely understood. OBJECTIVE: To evaluate efficacy of Az on MH crisis in pigs. ANIMALS: Eight normal (MHN) and 7 susceptible to MH (MHS) pigs (Landrace × Large White × Pietran). METHODS: Prospective, laboratory trial. Hypermetabolic crisis was observed in MHS pigs, but not in MHN pigs, after a combined administration of inhaled halothane (1.5%) and IV injection of succinylcholine (SCh; 2.5 mg/kg). Susceptibility was confirmed using a caffeine and halothane contracture test. Az was administered 15 minutes after administration of SCh. RESULTS: Respiratory acidosis (pH 7.16 ± 0.02; Pco(2) , 46.2 ± 9.1 mmHg, HCO(3) , 22.5 ± 2.3 mmol/L), fever (38.2 ± 1.1°C), cardiac arrhythmias, and muscle contracture were observed in MHS pigs. MHS pigs (n = 5) treated with Az (2 mg/kg IV) survived the crisis with attenuation of signs (pH 7.30 ± 0.10; Pco(2) , 36.3 ± 4.5 mmHg; HCO(3) , 22.9 ± 2.3 mmol/L) and recovery of normal muscle tone and cardiac rhythm. CONCLUSIONS AND CLINICAL IMPORTANCE: Az represents a possible substitute for dantrolene to reverse MH crisis in susceptible pigs.

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal.

AIMS: Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal. METHODS AND RESULTS: The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46.3%) were positive for Cryptosporidium and 67 (38.3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21.7%) and 9 (5.1%) water samples, respectively. C. parvum was the most common species (78.9%), followed by C. hominis (13.2%), C. andersoni (5.3%), and C. muris (2.6%). Subtype IdA15 was identified in all C. hominis-positive water samples. Subtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified. CONCLUSIONS: The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.

Central nervous system AIDS--related diseases.

The neurological complications of HIV contribute importantly to patient morbidity and mortality. Major common AIDS-related CNS diseases are ADC, metabolic encephalopaties, CMV encephalitis, TE, PCNSL, PML, criptococcal meningitis, and aseptic meningitis. After HAART, declines in incidence and improved outcome of several HIV-1 related opportunistic infections, including CNS-ADIs have been reported. The differential diagnosis of CNS complications of AIDS is routinely established according to temporal evolution, clinical data, and neuroradiological imaging. Combining neuroradiological imaging with the new CSF PCR tests may improve the diagnostic accuracy of some CNS-ADIs without the need of stereotactic brain biopsy, that may become limited to the situations where data remain conflicting.

DNA-lipid systems. A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection

Non-Hodgkin lymphoma in HIV-infected patients in the era of highly active antiretroviral therapy.

This study was designed to assess the influence of highly active antiretroviral therapy (HAART) on non-Hodgkin lymphoma (NHL) among patients infected with human immunodeficiency virus (HIV). Within EuroSIDA, a multicenter observational cohort of more than 8500 patients from across Europe, the incidences of NHL and subtypes (Burkitt, immunoblastic, primary brain lymphoma [PBL], and other/unknown histology) were determined according to calendar time of follow-up, and for those who initiated HAART (> or =3 drugs) also time on HAART. Potential predictive factors of NHL were evaluated in Cox proportional hazard models. Over 26 764 person-years of prospective follow-up (PYF) from May 1994 to December 2000, the incidence of NHL decreased from 1.99 (95% confidence interval, 1.51-2.47) before September 1995 to 0.30 (0.19-0.42) cases/100 (PYF) after March 1999 (P <.001). The incidence of all subtypes of NHL decreased significantly and most pronouncedly for PBL. Among patients who started HAART, the incidence of NHL decreased from 0.88 (0.60-1.16) within the first 12 months after starting HAART to 0.45 (0.31-0.60) cases/100 PYF after more than 24 months (P =.004). In an adjusted Cox model for patients on HAART, the latest CD4 cell count and plasma viral load were both significantly associated with diagnosis of NHL; the relative hazard was 1.39 (range, 1.14-1.69) per 50% lower CD4 cell count, and 1.51 (range, 1.21-1.88) per 1 log higher plasma viral load. In conclusion, the incidence of NHL among HIV-infected patients has decreased significantly after the introduction of HAART, and the decline was most pronounced for PBL. After starting HAART, patients with insufficient immunologic and virologic responses were at highest risk of NHL.

Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state.

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.

AIDS across Europe, 1994-98: the EuroSIDA study.

BACKGROUND: The clinical presentation of HIV-1 related diseases could have changed after the introduction of highly active antiretroviral treatment (HAART). We aimed to assess changes over time in the incidence of ADIs overall and within CD4 lymphocyte count strata, the relationship with treatment and degree of immunodeficiency at diagnosis of ADIs. METHODS: We did a prospective observational multicentre study of over 7300 patients in 52 European HIV-1 outpatient clinics. Incidence rates per 100 patient-years of observation were calculated. FINDINGS: In total, we recorded 1667 new ADIs; the incidence of ADIs declined from 30.7 per 100 patient-years of observation during 1994 (95% CI 28.0-33.4) to 2.5 per 100 patient-years of observation during 1998 (95% CI 2.0-3.0, p<0.0001, test for trend). Median CD4 lymphocyte count at diagnosis of a new ADI increased from 28 cells/microL to 125 cells/microL between 1994 and 1998 (p<0.0001), yet a steep decline in the rate of ADIs was seen after stratification by latest CD4 lymphocyte count within each year (< or = 50, 51-200, and > 200 cells/microL). Patients on HAART had a lower rate of ADIs than patients not on this treatment within each CD4 lymphocyte count strata. The proportion of ADIs attributable to cytomegalovirus retinitis and Mycobacterium avium complex declined over time (p=0.0058 and 0.0022, respectively), whereas the proportion of diagnoses attributable to non-Hodgkin lymphoma has increased (p<0.0001). In 1994, less than 4% of ADIs were non-Hodgkin lymphoma, in 1998 the proportion was almost 16%. This condition has become one of the most common ADIs in patients on HAART. INTERPRETATION: Our findings lend support to the idea that treatment regimens can lower the incidence of ADIs. The immediate risk of an ADI for a given CD4 lymphocyte count has declined over time and is lower among patients on HAART. Long-term follow-up of patients on combination treatment is essential to monitor the incidence of new and emerging diagnoses.

Bulk cytokine production versus frequency of cytokine-producing cells in HIV1 infection before and during HAART.

Cytokine imbalances play a major role in HIV immunopathogenesis. This study analyzes simultaneously the frequency of cytokine-producing cells at the single cell level by flow cytometry and the disturbances in cytokine secretion assessed by ELISA in a cohort of asymptomatic HIV1 patients in different stages of CD4 depletion and during antiretroviral therapy (HAART). Early in the disease, there is an increased frequency of IFN-gamma(+) lymphocytes and bulk IFN-gamma production, in parallel with a reduced proportion of IL4(+) cells and IL4 secreted. The two IL4 measurements are significantly correlated. No such correlation was found for IFN-gamma, which is consistent with a large variation in the amount of IFN-gamma released per individual cell. Moreover, HAART was associated with a reduction to normal levels in the bulk IFN-gamma secretion concomitant with a persistency of the overexpanded IFN-gamma(+) cell subset in the peripheral blood. This study emphasizes the importance of using a conjoint approach to assess the cytokine network in trials of antiretroviral and/or immune-based therapies to avoid missing significant effects which are possibly relevant in the clinical setting.

The peripheral serotonergic system and platelet aggregation in cyclosporin A-induced hypertensive rats.

Cyclosporin A plays an important role in preventing rejection in allograft transplant recipients. However, the therapeutic use of cyclosporin A is associated with increased incidence of thromboembolic complications and drug-related hypertension. In order to study the mechanisms by which cyclosporin A induces these abnormal pathophysiological situations, we have assessed the platelet serotonin contents and whole blood platelet aggregation in control rats as well as in rats treated (orally) with 30 and 5 mg/kg/day of cyclosporin A, after 2 and 7 weeks of treatment. These doses correspond respectively to CsA "peak" and "trough" concentrations achieved in human blood in clinical practice (immediately following an intake of a daily dose of CsA and when the blood concentration stabilizes, respectively). Both trough and peak doses caused an increase in blood pressure after 2 and 7 weeks. Platelet serotonin content decreased in the cyclosporin-treated groups, in contrast with the control. Collagen-induced whole blood platelet aggregation increased drastically for the peak concentration-treated group, while adenosine 5'-diphosphate-induced platelet aggregation did not reach statistical significance. Finally, in vitro platelet thromboxane A2 generation increased in cyclosporin A concentrations when platelets were stimulated with either collagen or adenosine 5'-diphosphate. In conclusion, both tested cyclosporin A concentrations induced important changes in platelet serotonin and thromboxane content and aggregation, factors which may play a decisive role in the development and/or maintenance of hypertension and thrombotic complications.

Early reduction of the over-expression of CD40L, OX40 and Fas on T cells in HIV-1 infection during triple anti-retroviral therapy: possible implications for lymphocyte traffic and functional recovery.

Fas, CD40L and OX40 are members of the tumour necrosis factor (TNF) receptor superfamily with critical roles in T cell activation and death, B cell function, dendritic cell maturation and leucocyte traffic regulation. The aim of this study was to evaluate the effects of anti-retroviral therapy (HAART) on CD40L, OX40 and Fas expression on freshly isolated peripheral blood T cells by three-colour flow cytometry and compare them with lymphoproliferative responses, peripheral blood cell counts and viral load. Fourteen asymptomatic HIV-1+ patients treated with Lamivudine, Stavudine and Nelfinavir were prospectively investigated sequentially for 48 weeks. At baseline, patients exhibited significantly enhanced proportions and counts of CD40L+ and OX40+ cells within the CD4 subset which were corrected by weeks 8-16 of HAART. Interestingly, in the five patients showing viral load rebound during therapy in spite of increasing CD4 counts, the reduction of the levels of these costimulatory molecules was similarly maintained. Therapy induced a decrease in the over-expression of Fas, particularly in the CD4 subset where normal levels were reached at week 8. This reduction occurred in parallel with the major recovery of lymphoproliferative responses. Higher basal levels and lower reduction of Fas were associated with suboptimal suppression of viraemia. In conclusion, this previously undescribed increased expression of CD40L and OX40 may play a role in the HIV-associated pan-immune activation and represent a possible target for immunointervention, as suggested for several immunologically mediated diseases. Moreover, HAART induced an early correction of the over-expression of Fas, CD40L and OX40 in CD4 T cells which could be involved in the recovery of the cell traffic disturbances and in the T cell renewal capacity.

Prevalence of cryptosporidiosis in AIDS patients with diarrhoea in Santa Maria Hospital, Lisbon.

We report the findings of a longitudinal observational study on HIV-infected patients grouped by presumed transmission group, who had diarrhoea. The purpose of this study was to assess the prevalence of and factors associated with Cryptosporidium infection on these patients. Modifiied formol-ether concentration followed by modified Ziehl-Neelsen and phenol-auramine/carbol-fuchsin staining techniques were used to identify Cryptosporidium from 465 patients. Cryptosporidiosis was reported in 36/465 (8% and 95% confidence interval 6, 10) patients. Of the positive patients 30 (83%) were men and 6 (17%) women. Prevalence of infection was higher among HIV-seropositive patients whose exposure category was through sexual contact (69%) than among patients in other HIV exposure categories (9%, Standard Z test, P < 0.001). Median CD4+ cell count/mm3 was 120 (range 3-600). Besides diarrhoea, the main clinical manifestations were fever and weight loss in 14 (39%) and 26 (72%) patients, respectively. Cryptosporidium infection was considered to be the first AIDS defining disease in 31% of the patients followed by tuberculosis in 19%, Pneumocystis carinii pneumonia in 14%, Salmonella sepsis in 6%, isosporiasis in 3%, toxoplasmic encephalitis in 3%, leishmaniasis in 3% and Kaposi's sarcoma in 3% of the patients. There was no significant difference (P = 0.82) in survival times for those given folate antagonists to treat other opportunistic infections. The decrease in prevalence of cryptosporidiosis observed from 1994 until May 1997 is not statistically significant (P = 0.11). Most cases of cryptosporidial infection in AIDS patients in Lisbon occurred in those whose HIV infection was assumed to have been acquired by the sexual route (hetero-, homo- and bisexual), with few cases occurring in drug-abusers.