Human immunodeficiency virus type 1 Tat protein induces an intracellular calcium increase in human monocytes that requires DHP receptors: involvement in TNF-alpha production

HIV-1 Tat protein, acting at the cell membrane, stimulates the production by human monocytes of TNF-α, a cytokine implicated in both HIV-1 replication and pathogenesis. Here, we analyze, in primary human monocytes, the mechanisms involved in Tat-stimulated calcium mobilization and its relationship with TNF-α production. We show that the Tat protein induces a calcium signal by mobilizing calcium from extracellular stores. This calcium signal is totally blocked when cells are stimulated in the presence of DHP receptor inhibitors such as nimodipine or calcicludine, thus suggesting the implication of this L-type calcium channel. By using RT-PCR amplification, Western blot with antibodies directed against the α1D subunit, binding assays with specific agonists or antagonists, and inhibition with specific antisense oligonucleotides, we show that DHP receptors are expressed and functional in primary human monocytes. Interestingly, we demonstrate that Tat-induced calcium mobilization is tightly linked to TNF-α production, thus indicating that Tat-induced mobilization and TNF-α production are entirely mediated by DHP receptors, as shown by their total inhibition by nimodipine, calcicludine, or anti-α1D antisense oligonucleotides.     

Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis

Background

The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children.

Objective

We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model.

Methods

The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified.

Results

Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis.

Conclusion

We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

     

Molecular cytogenetic characterization of nasopharyngeal carcinoma cell lines and xenografts by comparative genomic hybridization and spectral karyotyping

Nasopharyngeal carcinoma (NPC) cell lines and xenografts represent valuable models for functional and therapeutic studies on this common malignancy in Southeast Asia. The karyotypic information in most NPC cell lines and xenografts, however, remains largely unclear to date. We have characterized the chromosomal aberrations in six commonly used human NPC cell lines and xenografts using the molecular cytogenetic technique of comparative genomic hybridization (CGH). Genomic imbalances identified in cell lines were further correlated with structural abnormalities indicated from spectral karyotyping (SKY) analysis. CGH revealed consistent overrepresentations of 8q (six out of six cases) with a smallest overlapping region identified on 8q21.1q22. Other common gains included 7p (4/6 cases), 7q (4/6 cases), 12q (4/6), and 20q (4/6 cases), where minimal overlapping regions were suggested on 7p15p14, 7q11.2q21, and 12q22q24.1. Common losses were detected on 3p12p21 (4/6 cases) and 11q14qter (4/6 cases). Although SKY analysis on cell lines revealed predominantly unbalanced rearrangements, reciprocal translocations that involved chromosome 2 [i.e., t(1;2), t(2;3), and t(2;4)] were suggested. Furthermore, SKY examination illustrated additional breakpoints on a number of apparently balanced chromosomes. These breakpoints included 3p21, 3q26, 5q31, 6p21.1p25, 7p14p22, and 8q22. Our finding of regional gains and losses and breakpoints represents information that may contribute to NPC studies in vitro.     

Familial Similarities of Changes in Cognitive, Behavioral, and Physiological Variables in a Cardiovascular Health Promotion Program

A number of studies have demonstrated that physiological andbehavioral cardiovascular disease (CVD) risk factors aggregatewithin families. This fact, and the potential mediating rolethat the family plays in behavior change, have led to the developmentof family-based CVD risk reduction programs, including the SanDiego Family Health Project. The aggregation of behavioral,physiological, and cognitive changes within families was assessedduring a 1-year intervention. We found evidence of modest butsignificant aggregation of change. There was more aggregationof change in behavioral variables than in physiological or cognitivevariables. More significant correlations were found among 3-dayfood record measures than among 24-hour recall dietary measures,suggesting an influence of assessment method. Aggregation ofchange within families was stronger within generations thanacross generations. These data point to the importance of involvingall age groups in health promotion programs.     

Mechanisms of peptide YY release induced by an intraduodenal meal in rats: neural regulation by proximal gut

 Peptide YY (PYY) release in anaesthetized rats was studied during the 2 h following the intraduodenal administration of a semi-liquid meal of 21 kJ. Surgical and pharmacological manipulations were performed in order to analyse the mechanisms of PYY release. Postprandial PYY release was suppressed or strongly decreased by caecocolonectomy, truncal vagotomy, tetrodotoxin, hexamethonium, sensory denervation by perivagal capsaicin, and by the NO-synthase inhibitor L-N-arginine methyl ester, while atropine, adrenergic blockers, antagonists of type-A or type-B cholecystokinin (CCK) receptors or bombesin receptors had no effect. Comparing the digestive transit of the semi-liquid meal with the amount of PYY contained in the small bowel wall showed that nutrients had not reached the area rich in cells containing PYY by 30 min, the time at which there was a large PYY release in plasma. By 120 min, the meal front had travelled 72% of the small intestine length, just beginning to reach the PYY-rich part of the ileum. We conclude that the main postprandial PYY release studied in this model comes from ileal and colonic L-cells indirectly stimulated through a neural mechanism originating in the proximal gut and involving sensory vagal fibres, nicotinic synapses and NO release, while CCK and bombesin do not seem to be physiologically involved. Received: 17 July 1996 / Received after revision: 11 October 1996 / Accepted: 18 October 1996     

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

BACKGROUND: DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. PATIENTS AND METHODS: Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 x 1014 molecules) or peptides (10 versus 100 mug/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. RESULTS: The GMP process allowed to harvest about 5 x 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. CONCLUSION: The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.     

Effect of sensory stimulus on striatal dopamine release in humans and cats: a [(11)C]raclopride PET study

BACKGROUND: Sensory stimulation of the forelimb extremities constitutes a well-established experimental model that has consistently shown to activate dopamine (DA) neurotransmission in the mammals' forebrain. OBJECTIVES: To visualize in vivo this modification of striatal DA release in healthy human volunteers using Positron Emission Tomography (PET) and [(11)C]raclopride. Experiments in humans were paralleled by experiments in anesthetized cats. Changes in endogenous DA release were assessed through its competition with [(11)C]raclopride binding (BP(raclo)), a radioligand probing DA D2-receptors. RESULTS: In humans no significant difference of BP(raclo) in caudate (with sensory stimulation: 2.0 +/- 0.3 versus without sensory stimulation: 2.2 +/- 0.3; P = 0.3) or putamen (2.6 +/- 0.3 versus 2.6 +/- 0.2; P = 0.9) ipsilateral to the stimulus was disclosed as a result of sensory stimulation. Similarly, no change of BP(raclo) was observed contralaterally to the stimulation in the caudate nucleus (with sensory stimulation: 2.0 +/- 0.4 versus without sensory stimulation: 2.1 +/- 0.2; P = 0.5) and the putamen (2.5 +/- 0.4 versus 2.6 +/- 0.2; P = 0.4). In cats the same results were obtained in the ipsilateral to stimulation striatum (with sensory stimulation: 2.5 +/- 0.03 versus without sensory stimulation: 2.4 +/- 0.05; P = 0.7). No change was also observed contralaterally to the stimulation (2.4 +/- 0.04 versus 2.5 +/- 0.06; P = 0.6). The [(11)C]raclopride binding remained unchanged by sensory stimuli in both humans and cats. CONCLUSION: This suggests that the DA release induced by sensory stimulus is mostly extrasynaptic whereas the synaptic DA release is probably small, which fits well with the absence of [(11)C]raclopride displacement. The mechanism of this extrasynaptic DA release could be related to a local action of glutamate on dopaminergic terminals via a thalamo-cortico-striatal loop. Present results also underline homology between cat and human responses to sensory stimuli and validate the use of cat brain to find physiological concepts in humans.