Monoclonal mouse anti-I-Ak and anti-I-Ek antibodies cross-reacting with HLA-DR supertypic and subtypic determinants rather than classical DR allelic specificities

Thirty monoclonal alloantibodies (mAB) against mouse Iak antigens have been derived by fusion of mouse myeloma and spleen cells from A.TH (Ks Is Dd) mice immune to A.TL (Ks Ik Dd) lymphoid cells. Analysis of: (i) their reactivity (using 125I labelled protein A cell binding or cytotoxicity assays) on lymphoid cells from selected mouse strains with recombinant H-2 haplotypes; and (ii) the spatial arrangement of the specificities detected on the Iak molecules (studied by means of competitive inhibition of binding of radio-labelled monoclonal antibodies), permitted the identification of various epitopes present either on the I-Ak molecules (some of which were apparently identical to the conventional Ia.2, Ia.1 and Ia.19 specificities), or the I-Ek molecule (some being apparently analogous to the Ia.7 specificity) or on both I-Ak and I-Ek products. These mAB were tested in two different panels of human T and B lymphocytes. Panel (a) consisted of 28 Caucasian unrelated individuals, highly selected with regard to HLA-DR specificities, while panel (b) concerned 53 random HLA-A, B, C, DR typed individuals. The standard complement dependent lymphocytotoxicity microtechnique of histocompatibility workshop VIII was used throughout. All mAB were negative on resting T cells. Testing on B cells produced three patterns: 1) ten mAB did not react with any B cell tested; 2) four mAb reacted with all the panel cells; 3) sixteen mAb reacted with different sets of the panel indicating identification of polymorphic determinants. However, the strength of positivity obtained with a majority of single mAb varied considerably in the panel, suggesting identification of cross-reactive determinants. This necessitated the use of individual assignment criteria for each mAb. Following this procedure, 8 mAb were ascertained as reacting with HLA-DR supertypic determinants, 6 with associations to MT1, MT2, or both. Eight mAb reacted with HLA-DR subtypic determinants (more restricted than a classical DR allele). No mAb were ascertained, reacting exquisitely with acknowledged HLA-DR allelic specificities.     

Modification and benchmarking of MCNP for low-energy tungsten spectra

The MCNP Monte Carlo radiation transport code was modified for diagnostic medical physics applications. In particular, the modified code was thoroughly benchmarked for the production of polychromatic tungsten x-ray spectra in the 30-150 kV range. Validating the modified code for coupled electron-photon transport with benchmark spectra was supplemented with independent electron-only and photon-only transport benchmarks. Major revisions to the code included the proper treatment of characteristic K x-ray production and scoring, new impact ionization cross sections, and new bremsstrahlung cross sections. Minor revisions included updated photon cross sections, electron-electron bremsstrahlung production, and K x-ray yield. The modified MCNP code is benchmarked to electron backscatter factors, x-ray spectra production, and primary and scatter photon transport.     

Altered guanylyl-cyclase activity in vitro of pulmonary arteries from fetal lambs with congenital diaphragmatic hernia

Nitric oxide (NO) plays a major role in the modulation of perinatal pulmonary vascular tone. Congenital diaphragmatic hernia (CDH), a major cause of severe persistent pulmonary hypertension of the newborn (PPHN), is often refractory to inhaled NO. Alterations in NO/cyclic guanosine 3',5' monophosphate (cGMP)-mediated pulmonary vasodilatation may contribute to PPHN in CDH. We assessed NO/cGMP-mediated pulmonary vasorelaxation in vitro in 140-d gestational lamb fetuses with surgically created left CDH (term = 147 d) to age-matched controls. Relaxation of fourth generation intralobar pulmonary artery rings in response to the endothelium-dependent vasodilator, acetylcholine (ACh), and to the specific inhibitor of cGMP-phosphodiesterase (PDE), zaprinast, did not differ between the two groups. By contrast, relaxation in response to the calcium ionophore A23187 was impaired in CDH as compared with control animals. Relaxation in response to the NO donor sodium nitroprusside (SNP) (a direct activator of soluble guanylyl cyclase [sGC]) was also impaired in CDH animals as compared with controls. Repeating the challenge increased vasorelaxation in response to SNP in CDH as compared with control animals. Immunohistochemistry revealed the presence of endothelial NO-synthase in the endothelium of pulmonary arteries from both control and CDH animals. We conclude that endothelium-dependent vasodilatation in response to ACh and A23187 was differently affected in the fetal surgical CDH-lamb model. Furthermore, activity of sGC but not that of PDE was impaired in CDH animals. PPHN and decreased inhaled NO responsiveness in CDH may involve decreased sGC activity.     

Absence of mutations in the interspecies conserved regions of the CFTR promoter region in cystic fibrosis (CF) and CF related patients.

This study was aimed at testing if a 5.2 kb untranslated region on both sides of the first CFTR exon, shown to contain regulatory elements, could carry mutations responsible for cystic fibrosis (CF) or CF related phenotypes. Selection of the DNA segments studied within this region was based upon the identification of conserved sequences throughout evolution (phylogenetic footprints, PFs). Comparison of the CFTR sequences in eight species representing four orders of mammals (man, gibbon, rhesus monkey, squirrel, monkey, rabbit, cow, rat, and mouse) identified four clusters of PFs within the 3.9 kb of DNA sequence upstream from the initiation codon, as well as two nearby PFs at +1 kb within intron 1. Six DNA segments containing PFs were scanned for mutations by denaturing gradient gel electrophoresis (DGGE) in patients with CF (n = 29), congenital bilateral absence of the vas deferens (n = 143), or disseminated bronchiectasis (n = 33), for whom only one or no mutations had been identified despite extensive DGGE analysis of the 27 CFTR exons and exon/intron boundaries. Only one polymorphism (-966 T-->G) was identified with a frequency of 2.2% and no other sequence variations were found. This study reinforces the idea that the promoter region in the CFTR is not frequently mutated.     

Effects of the order of running and cycling of similar intensity and duration on pulmonary diffusing capacity in triathletes

To study the pathophysiological mechanisms involved in the decrease of post-triathlon diffusing capacity (DLco), blood rheologic properties (blood viscosity: _b; changes in plasma volume: PV) and atrial natriuretic factor (ANF) were assessed in ten triathletes during cycle-run (CR) and run-cycle (RC) trials at a metabolic intensity of 75% of maximal oxygen consumption (O_2max). The DLco was measured before and 10 min after trials. ANF and PV were measured at rest, after the cycle and run of CR and RC trials, and at the end of and 10 min after exercise. RC led to a greater DLco decrease, a lower ANF concentration and a lower PV than did CR, whereas for both CR and RC _b was increased throughout exercise and 10 min after. In addition, after CR the DLco decrease was inversely correlated (r=–0.764; P<0.01) with PV. The association of decreased plasma volume, increased _b, and lower ANF concentrations after RC suggested that lower blood pulmonary volume may have caused the greater decrease in Dlco as compared with CR. The inverse correlation between PV and DLco reinforces the hypothesis that fluid shifts limit the post-exercise DLco decrease after the CR succession in triathletes. Lastly, cycling in the crouched position might increase intra-thoracic pressure, decrease thorax volume due to the forearm position on the handlebars, and weaken peripheral muscular pump efficacy, all of which would limit venous return to the heart, and thus result in low pulmonary blood volume. Compared with cycling, running appeared to induce the opposite effects.     

Role of ATP-dependent potassium channels in pulmonary vascular tone of fetal lambs with congenital diaphragmatic hernia

High mortality in newborn babies with congenital diaphragmatic hernia (CDH) is principally due to persistent pulmonary hypertension. ATP-dependent potassium (K(ATP)) channels might modulate pulmonary vascular tone. We have assessed the effects of Pinacidil, a K(ATP) channel opener, and glibenclamide (GLI), a K(ATP) channel blocker, in near full-term lambs with and without CDH. In vivo, pulmonary hemodynamics were assessed by means of pressure and blood flow catheters. In vitro, we used isolated pulmonary vessels and immunohistochemistry to detect the presence of K(ATP) channels in pulmonary tissue. In vivo, pinacidil (2 mg) significantly reduced pulmonary vascular resistance (PVR) in both controls and CDH animals. GLI (30 mg) significantly increased pulmonary arterial pressure (PAP) and PVR in control animals only. In vitro, pinacidil (10 microM) relaxed, precontracted arteries from lambs with and without CDH. GLI (10(-5) microM) did not raise the basal tone of vessels. We conclude that activation of K(ATP) channels could be of interest to reduce pulmonary vascular tone in fetal lambs with CDH, a condition often associated with persistent pulmonary hypertension of the newborn.