Peptide amidating activity in human bronchoalveolar lavage fluid

Monitoring respiratory epithelial biology may reveal individuals with incipient lung cancer. The expression of neuroendocrine (NE) markers in pulmonary epithelium is thought to be central to lung development, repair of injury and may contribute to carcinogenesis. In this study, we evaluate several candidate NE markers to determine the feasibility of prospective analysis of clinical specimens. The potential NE markers include the enzyme L-DOPA decarboxylase (DDC), the neuropeptide gastrin releasing peptide (GRP), and peptidyl-glycine alpha-amidating monooxygenase (PAM), the bifunctional enzyme responsible for the final bioactivation step of many neuropeptides. A comparison of PAM activity and DDC levels in 30 lung cancer cell lines indicated that peptide amidating activity may be an indicator of NE status. Bronchoalveolar lavage (BAL) fluid from subjects at risk of developing second primary lung cancer and from volunteers was obtained. The activity of the first PAM enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), ranged from not detectable to 507 pmol/h/mg protein in 57 specimens. The second PAM enzyme, peptidylamidoglycolate lyase (PAL), ranged from not detectable to 414 pmol/h/mg protein in 56 specimens. Using cluster analysis by the average linkage method, a group of enzyme values with PHM greater than 230 pmol/h/mg protein was determined. Long-term follow-up of these patients for new second primary lung cancers may help to determine the potential predictive value of PAM detected in the BAL fluid.     

Direct evidence for the role of haem doming as the primary event in the cooperative transition of haemoglobin

The study of cooperative ligand binding among the four subunits of haemoglobin has played a central role in the understanding of allosteric transitions in a large number of enzymes. Haem iron out-of-plane motion has been suggested to be the trigger for the cooperative transition of haemoglobin. To function as a trigger in a dynamic sense, haem-iron doming must be the first conformational change to occur following ligand dissociation. Here we present the first direct demonstration that haem-iron doming occurs on the same time scale as the breaking of the iron-ligand bond, thus establishing haem-iron doming as the primary event which lead to the R-->T transition in haemoglobin.     

Enhanced cytotoxicity with interleukin-1 alpha and 5-fluorouracil in HCT116 colon cancer cells

Recombinant human interleukin-1 alpha (rIL-1 alpha), at concentrations that were not growth-inhibitory when given alone (100-10,000 U/ml), enhanced the growth inhibition resulting from a 72-h fluorouracil (FUra) exposure in HCT116 colon cancer cells. Median-effect analysis of clonogenic assays indicated that rIL-1 alpha, given 24 h prior to and following a 24-h exposure to FUra, increased lethality in a more than additive fashion. rIL-1 alpha did not appear to significantly affect [3H]-FUra metabolism, total [3H]-FUra-RNA incorporation or RNA retention after drug removal, inhibition of thymidylate synthase, or thymidine triphosphate pool depletion. During continuous exposure to rIL-1 alpha, transient stimulation of RNA and DNA synthesis was observed at 72 h, with a return to normal by 96 h. A 24-h exposure to 10 microM FUra altered the elution profile of newly synthesized DNA as monitored by pH step alkaline elution. An accumulation of lower-MW single-stranded DNA species was noted with FUra compared to control, accompanied by a significantly decreased proportion of DNA retained on the polycarbonate filter: 10% retained vs. 32% for control (P = 0.01). A 48-h exposure to rIL-1 alpha alone did not affect the elution profile of nascent DNA species, nor did it enhance the effects of FUra. Although FUra did not appreciably affect pulse [3H]-uridine incorporation into RNA for the initial 8-24 h of FUra exposure, progressive inhibition of net RNA synthesis was observed thereafter. FUra prevented the stimulatory effect of rIL-1 alpha on RNA synthesis, and net RNA synthesis was significantly inhibited (by 64-79% after 72 and 96 h) with the combination compared to rIL-1 alpha alone. Continuous exposure to 10 microM thymidine did not rescue cells from the lethality of FUra alone or the combination of FUra plus rIL-1 alpha, suggesting that depletion of deoxythymidine triphosphate as a consequence of thymidylate synthase inhibition was not the most important component of FUra toxicity. In contrast, 1 mM uridine provided partial protection against the toxicity of FUra alone or with rIL-1 alpha. Although uridine did not affect FUra metabolism, it decreased FUra-RNA incorporation by 42-60%, presumably as a consequence of the 2-fold expansion of UTP pools. [125I]-rIL-1 alpha binding was nonspecific; with a 24-h exposure, however, internalized [125I]-rIL-1 alpha exceeded cell surface-bound material by 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.     

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.     

Intravascular ultrasound predictors of restenosis after balloon angioplasty of the femoropopliteal artery

The skepticism surrounding the potential benefits of resistance exercise training prevalent just decades ago has evolved over the years to an understanding of the integral nature muscular overload plays in the training programs for athletes. The science of training elite athletes is progressing rapidly, as insights into the physiological adaptations resulting from varying program configurations become available. Resistance training impacts several body systems, including muscular, endocrine, skeletal, metabolic, immune, neural, and respiratory. An understanding and appreciation of basic scientific principles related to resistance training is necessary in order to optimize training responses. Careful selection of the acute program variables in a workout to simulate sports-specific movements is required for optimal transfer of gains made in training to competition. Thus, whether athletes require predominantly eccentric, isometric, slow-velocity, or high-velocity strength or power in their athletic event will dictate the time commitment to each component and form the basis for designing individual workouts. Program variation over a training period is essential to maximize gains and prevent overtraining.     

Interleukin 8 (IL-8) induces the expression of kinin B1 receptor in human lung fibroblasts

Near-haploidy is a rare cytogenetic finding in childhood acute lymphoblastic leukaemia (ALL) and is associated with a poor prognosis. A second hyperdiploid line, occurring presumably by endoreduplication of the near-haploid stemline, is often observed. We present a case of common ALL in relapse characterized morphologically by a dual population of small and large lymphoblasts. Cytogenetic analysis supplemented with fluorescence in-situ hybridization (FISH) studies localized near-haploidy and hyperdiploidy to the small and large blast population respectively. DNA ploidy determination confirmed two abnormal clones with near-haploidy as the predominant one. A novel t(9;12)(q11;q13) was present in the near-haploid clone and was duplicated in the hyperdiploid clone. This finding identified cells bearing near-haploidy to be the clonogenic population following malignant transformation and confirmed endoreduplication as the mechanism for the presence of associated hyperdiploidy.     

Food allergy to peanuts in France--evaluation of 142 observations

BACKGROUND: The increase in frequency of peanut allergy and fatal cases have been reported. OBJECTIVES: The objective of this study is to document the severity of food allergy to peanuts by evaluating the reactive dose of peanuts and to search for the role of peanut oil. METHODS: This study is carried out on the basis of 142 observations collected according to the same diagnostic methodology in two allergy centres in France. Skin-prick-tests were performed with peanut powder, peanut oil and peanut oil proteinic extract. Labial provocation tests were performed on 121 patients. The reactive dose of peanuts and the role of peanut oil were determined by standardized oral provocation tests in 50 and 62 patients respectively. The data are computerized and the data bank includes 509 food allergic patients. RESULTS: Allergy to peanuts represents 28% of food allergies and occurs under 1 year of age in 46% of cases, under 15 years of age in 93%. The clinical features were atopic dermatitis (40%), angioedema (37%), asthma (14%), anaphylactic shock (6%) and digestive symptoms (1.4%). The specific IgE were class 3 or higher in 80% of cases. The total reactive dose was less than 100 mg in 25% of cases, from 100 mg to 1 g in 62.5%. All patients reacted to a dose of less than 7.1 g. The threshold of peanut reactivity was lower than the threshold of egg reactivity. An allergy to peanut oil was demonstrated in 14 patients. CONCLUSION: The severity of peanut allergy and the early onset of the occurrence of this allergy is documented. The role of residual allergenic proteins in peanut oil is established by positive skin-prick tests to proteic extracts from peanut oil and by double-blind placebo-controlled challenges to peanut oil. The increased consumption of allergens in the form of peanut oil and fats can contribute to the occurrence or persistence of symptoms and may be suspected to increase the risk of sensitisation.