Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor.

Cysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients' plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted - apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients' plasma VWF. The in vitro expression of the E244Lfs*211-VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

A Lys49-PLA2 myotoxin of Bothrops asper triggers a rapid death of macrophages that involves autocrine purinergic receptor signaling.

Lys49-PLA(2) myotoxins, an important component of various viperid snake venoms, are a class of PLA(2)-homolog proteins deprived of catalytic activity. Similar to enzymatically active PLA(2) (Asp49) and to other classes of myotoxins, they cause severe myonecrosis. Moreover, these toxins are used as tools to study skeletal muscle repair and regeneration, a process that can be very limited after snakebites. In this work, the cytotoxic effect of different myotoxins, Bothrops asper Lys49 and Asp49-PLA(2), Notechis scutatus notexin and Naja mossambica cardiotoxin, was evaluated on macrophages, cells that have a key role in muscle regeneration. Only the Lys49-myotoxin was found to trigger a rapid asynchronous death of mouse peritoneal macrophages and macrophagic cell lines through a process that involves ATP release, ATP-induced ATP release and that is inhibited by various purinergic receptor antagonists. ATP leakage is induced also at sublytical doses of the Lys49-myotoxin, it involves Ca(2+) release from intracellular stores, and is reduced by inhibitors of VSOR and the maxi-anion channel. The toxin-induced cell death is different from that caused by high concentration of ATP and appears to be linked to localized purinergic signaling. Based on present findings, a mechanism of cell death is proposed that can be extended to other cytolytic proteins and peptides.

New insights on culture and calcium signalling in neurons and astrocytes from epileptic patients.

Primary brain cell cultures are a useful tool for understanding the physiopathology of epilepsy and for searching new potential antiepileptic drugs. These cell types are usually prepared from murine species and few human models have been described. The main goal of this study is the establishment of experimental conditions to isolate and culture neurons and astrocytes from human brain and to test its functionality. The tissues came from antiepileptic drug-resistant epileptic patients undergoing surgery. Human neurons and astrocytes were isolated following an enzymatic and mechanical dissociation protocol. Cultures were viable for 3-6 weeks. Cytological characterization was performed by immunocytochemistry using specific antibodies against both neuron (anti-NeuN) and astrocyte (anti-GFAP) protein markers. In order to test their viability and functionality, cells were loaded with the fluorescent calcium probe fura-2 and variations in cytosolic calcium concentrations ([Ca2+]c) were measured by cell imaging. [Ca2+]c increases were evoked upon cell stimulation with high K+ (KCl 75 mM), glutamate (500 µM) or bicuculline (100 µM). Interestingly, spontaneous [Ca2+]c transients were also observed in some neuron-like cells. A novel unreported finding in this study has been the incorporation of human serum that was critical for cell functionality. The setting of these human cultures open the opportunity to new insights on culture and calcium signalling studies on the mechanism(s) of cell resistance to antiepileptic drugs, as well as to studies on plasticity, maturation and possible neurite emission for graft studies.

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors.

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca(2+) and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca(2+)], derived from intracellular stores, followed, only in myotubes, by a large Ca(2+) influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca(2+) permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca(2+) mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death.

Mitochondrial Ca2+ as a key regulator of cell life and death.

Mitochondrial Ca(2+) homeostasis is today at the center of wide interest in the scientific community because of its role both in the modulation of numerous physiological responses and because of its involvement in cell death. In this review, we briefly summarize a few basic features of mitochondrial Ca(2+) handling in vitro and within living cells, and its involvement in the modulation of Ca(2+)-dependent signaling. We then discuss the role of mitochondrial Ca(2+) in the control of apoptotic death, focusing in particular on the effects of pro- and anti-apoptotic proteins of the Bcl-2 family. Finally, the potential involvement of Ca(2+) and mitochondria in the development of two diseases, Ullrich muscular dystrophy and familial Alzheimer's disease, is briefly discussed.

Correlation between reduction in plasma HIV-1 RNA concentration 1 week after start of antiretroviral treatment and longer-term efficacy.

BACKGROUND: Early assessment of antiretroviral drug efficacy is important for prevention of the emergence of drug-resistant virus and unnecessary exposure to ineffective drug regimens. Current US guidelines for changing therapy are based on measurements of plasma HIV-1 RNA concentrations 4 or 8 weeks after the start of treatment with cut-off points of 0.75 or 1.00 log, respectively. We investigated the possibility of assessing drug efficacy from measurements of plasma HIV-1 concentrations made during the first week on therapy. METHODS: The kinetics of virus decay in plasma during the first 12 weeks of treatment was analysed for 124 HIV-1-infected patients being treated for the first time with a protease inhibitor. Patients with a continuous decline of HIV-1 concentrations and in whom HIV-1 was either undetectable or declined by more than 1.5 log at 12 weeks were defined as good responders; the rest were poor responders. FINDINGS: The individual virus decay rate constants (k) at day 6 correlated significantly (r>0.66, p<0.0001) with changes in HIV-1 concentrations at 4, 8, and 12 weeks, and correctly predicted 84% of the responses with a cut-off value of k=0.21 per day (in log scale). Reduction in plasma HIV-1 of less than 0.72 log by day 6 after initiation of therapy predicted poor long-term responses in more than 99% of patients. INTERPRETATION: These results suggest that changes in HIV-1 concentration at day 6 after treatment initiation are major correlates of longer-term virological responses. They offer a very early measure of individual long-term responses, suggesting that treatment could be optimised after only a few days of therapy.

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes.

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.

Long-term protease inhibitor-containing therapy results in limited improvement in T cell function but not restoration of interleukin-12 production in pediatric patients with AIDS.

This study investigated whether immune restoration occurred in 26 human immunodeficiency virus (HIV) type 1-infected children treated first with indinavir for 16 weeks and then with combination antiretroviral therapy for >2 years. Compared with baseline, a significant, although modest, decrease in virus loads (maximum median, -0.86 log(10)) and increase in the number of CD4(+) lymphocytes, especially naive cells, were observed at several time points after 2 years. A maximum of 7% of treated children achieved undetectable viremia. There was a marked increase in the proliferative response and skin reactivity to recall antigens. However, responses to an HIV antigen remained depressed, and the production of interleukin-12 remained unchanged and abnormally low. The magnitude of virus suppression did not correlate with these measures of functional immune reconstitution. These findings suggest that long-term nonsuppressive antiretroviral therapy can induce limited improvement in immune function in pediatric AIDS patients and that the effect of suppressive treatments should be investigated.

Long-term virologic and immunologic responses in human immunodeficiency virus type 1-infected children treated with indinavir, zidovudine, and lamivudine.

Virologic and immunologic responses were examined for 33 human immunodeficiency virus (HIV)-infected children who participated for > or = 96 weeks in a phase 1/2 protocol of 16 weeks of indinavir monotherapy, followed by the addition of zidovudine and lamivudine. At week 96, a median increase of 199 CD4+ T cells/microL and a median decrease of 0.74 log(10) HIV RNA copies/mL were observed. The relationship between control of viral replication and CD4) T cell count was examined. Patients were categorized into 3 response groups on the basis of duration and extent of control of viral replication. Of 21 children with a transient decrease in virus load of > or = 0.7 log(10) HIV RNA copies/mL from baseline, 7 experienced sustained increases in CD4+, CD4+ CD45RA+, and CD4+ CD45RO+ T cell counts. CD4+ CD45RA+ (naive) T cells were the major contributor to CD4+ T cell expansion. Continued long-term immunologic benefit may be experienced by a subset of children, despite only transient virologic suppression.

Work-family issues and perceptions of stress among pediatric faculty and house staff.

OBJECTIVES: To examine work-family balance issues and predictors of stress related to work-family balance among pediatric house staff and faculty. METHODS: Data were obtained through an anonymous mail survey. Univariate analyses assessed associations between work-family issues (work-related factors that affect work-family balance, perceived support, work-family--related stress, and proposed solutions) and the following variables: gender, parental status, working status of spouse, and academic rank. Multiple linear regression examined independent predictors of perceived stress. RESULTS: Fifty percent of the 327 respondents cared for dependent children, and 20% expected to care for an elderly person in the next 5 years. Only 5% strongly agreed that their division or department was concerned about supporting members' work-family balance, and 4% strongly agreed that existing programs supported their needs. Eighty-three percent reported feeling stressed as a result of efforts to balance work and family. Independent predictors of stress included perceived need to choose between career and family, increasing age, dependent children, less support from colleagues and supervisors, and female gender. CONCLUSIONS: Work-family balance issues are responsible for substantial perceived stress. Academic departments should consider a commitment to supporting faculty who are struggling with these issues, including creation of work-family policies and programs, development of mentoring systems, and reexamination of existing expectations for work practices.

Esophageal candidiasis in pediatric acquired immunodeficiency syndrome: clinical manifestations and risk factors.

BACKGROUND: Little is known about the epidemiology and clinical features of esophageal candidiasis (EC) in pediatric AIDS. We therefore investigated the clinical presentation and risk factors of EC in a large prospectively monitored population of HIV-infected children at the National Cancer Institute. PATIENTS AND METHODS: We reviewed the records of all HIV-infected children (N = 448) followed between 1987 and 1995 for a history of esophageal candidiasis to characterize the epidemiology, clinical features, therapeutic interventions and outcome of esophageal candidiasis. To understand further the risk factors for EC in pediatric AIDS, we then performed a matched case-control analysis of 25 patients for whom control cases were available. RESULTS: There were 51 episodes of EC documented in 36 patients with 23 male and 13 female patients (0.2 to 17 years; median CD4, count 11/microl), representing a frequency of EC of 8.0%. Concurrent oropharyngeal candidiasis (OPC) was the most common clinical presentation of EC (94%); other signs and symptoms included odynophagia (80%), retrosternal pain (57%), fever (29%), nausea/vomiting (24%), drooling (12%), dehydration (12%), hoarseness (6%) and upper gastrointestinal bleeding (6%). The causative organism documented in 36 episodes (18 from OPC, 17 from endoscopic biopsy and 1 from autopsy) was Candida albicans in all cases. Patients received treatment for EC with amphotericin B (63%), fluconazole (29%), ketoconazole (4%) or itraconazole (1%). A clinical response was documented in all 45 evaluable episodes. In 6 other cases, EC was a final event without contributing to the cause of death. By a conditional logistic regression model for matched data, the best predictor of EC was the presence of prior OPC (P<0.0001), followed by CD4 count and CD4 percentage (P = 0.0002) and use of antibacterial antibiotics (P = 0.0013). The risks associated with low CD4 count were independent of that of prior OPC. CONCLUSION: EC in pediatric AIDS is a debilitating infection, which develops in the setting of prior OPC, low CD4 counts and previous antibiotics.

Development and evaluation of an improved method for screening of amphetamines.

We developed a homogeneous immunoassay method to eliminate false-positive amphetamine results caused by cross-reactive substances, including over-the-counter allergy and cold medications. This method uses a neutralizing antibody that binds to amphetamines but does not bind to the labeled amphetamine conjugate used in the assay. The amount of neutralizing antibody is sufficient to reduce the assay signal resulting from authentic amphetamine and methamphetamine, but not the signal resulting from cross-reactants. This concept was implemented using the CEDIA DAU Amphetamines assay on Hitachi 747 and 717 clinical chemistry analyzers. Urine samples were tested using the standard, unmodified reagents in one channel and reagents containing the neutralizing antibody in a second channel. The difference in rate between the two tests was calculated by the analyzer; true-positive samples showed a significantly greater decrease in assay signal in response to neutralizing antibody as compared with false-positive samples. The neutralization method was evaluated in two studies using 448 samples that tested positive in the initial CEDIA DAU Amphetamines screening test. The samples were separated into categories of 154 true-positive samples and 294 false-positive samples based upon a secondary screen with the Abbott FPIA Amphetamines assay followed by gas chromatography-mass spectrometry (GC-MS) testing using the HHS (SAMHSA) cutoff criteria. The CEDIA neutralization test successfully identified all 154 of the GC-MS confirmed positive samples. The test successfully identified as false positive 251 out of the 294 (85.4%) samples that failed to confirm by GC-MS.

Preliminary evaluation of human immunodeficiency virus type 1 (HIV-1) immunogen in children with HIV-1 infection.

The safety and preliminary activity of human immunodeficiency virus type 1 (HIV-1) immunogen were evaluated in 10 HIV-1-infected children with disease stage N1,2 or A1,2. Multiple inoculations of 2. 5 or 10 units (U) of HIV-1 immunogen were safe and well tolerated without an acceleration of disease progression. When antiretroviral agents were coadministered, the 10 U dose appeared to be associated with more sustained reduction in plasma HIV-1 RNA than the 2.5 U dose (median log10 HIV-1 RNA at month 18, 3.07 vs. 4.01 copies/mL in 10 U [n=4] vs. 2.5 U [n=3], respectively; P=.034). Levels of regulated-on-activation, normal T cell-expressed and -secreted chemokine produced from HIV-1 immunogen-stimulated lymphocytes in vitro were increased in the children who had HIV-1 immunogen-specific antibody responses (P<.02) and appeared to be inversely correlated with levels of plasma HIV-1 RNA (P<.01). These preliminary data warrant larger studies to determine the effectiveness of adjunctive therapy with HIV-1 immunogen in children with HIV-1 infection.

A double-blind comparison of empirical oral and intravenous antibiotic therapy for low-risk febrile patients with neutropenia during cancer chemotherapy.

BACKGROUND: Among patients with fever and neutropenia during chemotherapy for cancer who have a low risk of complications, oral administration of empirical broad-spectrum antibiotics may be an acceptable alternative to intravenous treatment. METHODS: We conducted a randomized, double-blind, placebo-controlled study of patients (age, 5 to 74 years) who had fever and neutropenia during chemotherapy for cancer. Neutropenia was expected to be present for no more than 10 days in these patients, and they had to have no other underlying conditions. Patients were assigned to receive either oral ciprofloxacin plus amoxicillin-clavulanate or intravenous ceftazidime. They were hospitalized until fever and neutropenia resolved. RESULTS: A total of 116 episodes were included in each group (84 patients in the oral-therapy group and 79 patients in the intravenous-therapy group). The mean neutrophil counts at admission were 81 per cubic millimeter and 84 per cubic millimeter, respectively; the mean duration of neutropenia was 3.4 and 3.8 days, respectively. Treatment was successful without the need for modifications in 71 percent of episodes in the oral-therapy group and 67 percent of episodes in the intravenous-therapy group (difference between groups, 3 percent; 95 percent confidence interval, -8 percent to 15 percent; P=0.48). Treatment was considered to have failed because of the need for modifications in the regimen in 13 percent and 32 percent of episodes, respectively (P<0.001) and because of the patient's inability to tolerate the regimen in 16 percent and 1 percent of episodes, respectively (P<0.001). There were no deaths. The incidence of intolerance of the oral antibiotics was 16 percent, as compared with 8 percent for placebo (P=0.07). CONCLUSIONS: In hospitalized low-risk patients who have fever and neutropenia during cancer chemotherapy, empirical therapy with oral ciprofloxacin and amoxicillin-clavulanate is safe and effective.

Adherence of Candida albicans to bladder mucosa: development and application of a tissue explant assay.

In order to study the interactions between Candida species and uroepithelial tissue, a tissue explant assay was developed using bladder mucosa harvested from New Zealand white rabbits. Blastoconidia of Candida albicans, Candida tropicalis and Candida glabrata attached to the uroepithelial tissue in similar quantities. However, there was significantly more adherence to the uroepithelium by pre-germinated C. albicans compared with C. albicans blastoconidia. Furthermore, the amount of uroepithelial tissue injury was directly related to the length of exposure of the tissue to Candida. Thus, this tissue explant assay may provide a useful method for investigating properties related to fungal adherence to transitional uroepithelium and organism-mediated tissue injury.

Immunoreconstitution after ritonavir therapy in children with human immunodeficiency virus infection involves multiple lymphocyte lineages.

OBJECTIVE: To evaluate lymphocyte reconstitution after protease inhibitor therapy in children with human immunodeficiency virus (HIV) infection. STUDY DESIGN: Forty-four HIV-infected children receiving ritonavir monotherapy followed by the addition of zidovudine and didanosine were evaluated during a phase I/II clinical trial. The cohort had a median age of 6.8 years and advanced disease (57% Centers for Disease Control and Prevention stage C, 73% immune stage 3) and was naive to protease inhibitor therapy. RESULTS: After 4 weeks of therapy, there was a significant increase in CD4(+) and CD8(+) T cells. CD4(+) T cells continued to increase, whereas CD8(+) T cells returned to baseline by 24 weeks. Unexpectedly, there was a significant increase in B cells. Changes in CD4(+) T-cell subsets revealed an initial increase in CD4(+) CD45RO T cells followed by a sustained increase in CD4(+) CD45RA T cells. Children <6 years of age had the highest increase in all lymphocyte populations. Significant improvement in CD4(+) T-cell counts was observed even in those children whose viral burden returned to pre-therapy levels. CONCLUSIONS: Early increases in lymphocytes after ritonavir therapy are a result of recirculation, as shown by increases in B cells and CD4(+) CD45RO and CD8(+) T cells. Children exhibited a high potential to reconstitute CD4(+) CD45RA T cells even with advanced disease and incomplete viral suppression.