Cyclophosphamide affects the dynamics of granuloma formation in experimental visceral leishmaniasis

We observed histopathological and ultrastructural hepatic changes following the intracardiac inoculation ofLeishmania donovani amastigotes into inbred LHC hamsters (group I). Since granuloma formation is known to be T-cell-dependent, we also examined infected hamsters under cyclophosphamide immunosuppressive treatment (group ICy) and evaluated the production of interleukin-2 (IL-2) by their cells. Group I showed more intense hepatocyte and endothelial cell clasmatosis as well as hepatocyte degeneration and necrosis, deposits of connective tissue fibers, granulomas with multinucleated giant cells (MGCs) of foreign-body and Langhans' types and reduced production of IL-2 by spleen cells. In contrast, group ICy hamsters exhibited larger eosinophil and lymphocyte populations within sinusoids and peri-sinusoidal areas but showed no MGCs in granulomas. A striking decline in IL-2 production was noted. These results suggest that cyclophosphamide induces a delay in the natural evolution ofL. donovani-induced granulomatous hepatic inflammation.     

Cypress (Cupressus sempervirens) pollen allergens: identification by protein blotting and improved detection of specific IgE antibodies

On the basis of results of an investigation of the effects of different treatments employed, a dialysed and reduced extract of Cupressus sempervirens was separated electrophoretically on sodium dodecylsulphate-polyacrylamide gels before being transferred and then fixed with glutaraldehyde to nitrocellulose membrane. Probing with sera from 91 subjects allergic to C. sempervirens pollen followed by detection of bound IgE antibodies with [125I]-labelled anti-human IgE revealed 17 IgE-binding proteins in the molecular weight range 14-96 kilodaltons (kDa). One component, of molecular weight approximately 42 kDa, reacted with IgE antibodies in the sera of 81.3% of the allergic subjects and, for each of the subjects, this component bound the greatest quantity of IgE. Almost 50% of the sera recognized only the approximately 42 kDa component, reinforcing the conclusion that this component is the major allergen of C. sempervirens pollen. A comparative study employing C. sempervirens pollen allergen discs prepared commercially or in the laboratory showed that values of the uptakes of [125I]-anti-IgE indicating the presence of pollen-reactive IgE antibodies obtained with the latter discs were consistently higher (means 4.5 vs. 0.88), and that false-negative results were obtained when many sera were used with the commercial discs. The results of this study provide an essential basis for the production of standardized, safe and effective C. sempervirens pollen extract applicable to diagnosis and therapy of cypress pollen allergy.     

Cross-sensitization between cocaine and GBR 12909, a dopamine uptake inhibitor.

In the present experiment, rats which had received repeated injections of 20 mg/kg GBR 12909, a selective dopamine uptake inhibitor, were subjected to challenge doses of GBR 12909 and cocaine which were near or below the threshold for locomotor activation in drug-naive animals. The behavior measured was general motor activity, monitored in photocell testing cages. Results verify earlier findings which showed that rats treated chronically with GBR 12909 display a potentiated locomotor response to low doses of the drug (behavioral sensitization). In addition, it was found that GBR 12909-sensitized rats show a potentiated locomotor response to 5 mg/kg cocaine (cross-sensitization). These results are discussed with regard to various mechanisms which may underlie the sensitization phenomena observed, including possible neurochemical changes at the presynaptic level.     

Retrospective analysis of fetal vertebral defects: Associated anomalies,etiologies, and outcome

Our objectives were to describe fetal cases of vertebral defects (VD), assess the diagnostic yield of fetal chromosomal analysis for VD and determine which investigations should be performed when evaluating fetal VD. We performed a retrospective chart review for fetuses with VD seen between 2006 and 2015. Cases were identified from CHU Sainte‐Justine's prenatal clinic visits, postmortem fetal skeletal surveys, and medical records. Cases with neural tube defects were excluded. Sixty‐six fetuses with VD were identified at a mean gestational age of 20 weeks. Forty‐seven (71.2%) had associated antenatal anomalies, most commonly genitourinary, skeletal/limb, and cardiac anomalies. Thirteen mothers (19.7%) had pregestational diabetes (95% CI [10.1%–29.3%]). Fifty‐three cases had chromosomal analysis. Three had abnormal results (5.6%): trisomy 13, trisomy 22, and 9q33.1q34.11 deletion. Thirty‐four (51.5%) pregnancies were terminated, one led to intrauterine fetal demise and 31 (46.9%) continued to term. Of 27 children who survived the neonatal period, 21 had congenital scoliosis and 3 had spondylocostal dysostosis. Seven had developmental delay. In conclusion, prenatal evaluation of fetuses with VD should include detailed morphological assessment (including fetal echocardiogram), maternal diabetes screening, and chromosomal microarray if non‐isolated. Our findings provide guidance about management and counseling after a diagnosis of fetal VD.     

LARP7 variants and further delineation of the Alazami syndrome phenotypic spectrum among primordial dwarfisms: 2 sisters

Alazami syndrome (AS) (MIM# 615071) is an autosomal recessive microcephalic primordial dwarfism (PD) with recognizable facial features and severe intellectual disability due to depletion or loss of function variants in LARP7. To date, 15 patients with AS have been reported. Here we describe two consanguineous Algerian sisters with Alazami PD due to LARP7 homozygous pathogenic variants detected by whole exome sequencing. By comparing these two additional cases with those previously reported, we strengthen the key features of AS: severe growth restriction, severe intellectual disability and some distinguishing facial features such as broad nose, malar hypoplasia, wide mouth, full lips and abnormally set teeth. We also report significant new findings enabling further delineation of this syndrome: disproportionately mild microcephaly, stereotypic hand wringing and severe anxiety, thickened skin over the hands and feet, and skeletal, eye and heart malformations. From previous reviews, we summarize the main etiologies of PD according to the involved mechanisms and cellular pathways, highlighting their clinical core features.     

Bcl-x(L)-mediated changes in metabolic pathways of breast cancer cells: from survival in the blood stream to organ-specific metastasis

Bcl-x(L) protein plays a role in breast cancer dormancy, promoting survival of cells in metastatic foci by counteracting the proapoptotic signals in the microenvironment. The aim of this study was to identify phenotypes mediated by Bcl-x(L) in breast cancer cells that enhance in vivo survival of clinical metastases. 435/Bcl-x(L) or 435/Neo human breast cancer cells were injected into the inguinal mammary gland of nude mice, and tumors, metastases in lymph node, lung, and bone, and bloodstream surviving cells were examined. Proteomic analysis identified 17 proteins that were overexpressed (more than twofold) or underexpressed (less than twofold) in metastases. A protein interaction program allowed us to functionally associate peroxiredoxin 3, peroxiredoxin 2, carbonyl reductase 3, and enolase 1, suggesting a role for cellular responses to oxidative stress in metastasis organ selection. The prediction included proteins involved in redox systems, kinase pathways, and the ATP synthase complex. Furthermore, the interaction of redox proteins with enolase 1 suggests a connection between glycolysis and antioxidant pathways, enabling achievement of a high metastatic activity. In conclusion, Bcl-x(L) mediates a phenotype in which redox pathways and glycolysis are coupled to protect breast cancer metastatic cells during transit from the primary tumor to the metastatic state.     

Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10 s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh burst of quanta was also observed at unclamped junctions. At –100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 M TTX to the Ringer's solution, or total replacement of sodium with quanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca²⁺-free, EGTA solutions, or in Ca²⁺-free solutions containing 2 mM Mn²⁺. Sr²⁺ effectively substituted for Ca²⁺. Addition of 2 mM Co²⁺ or Mn²⁺ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl--methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to –100 mV and disappearing at –60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.     

Identification of orchard grass (Dactylis glomerata) pollen allergens following electrophoretic transfer to nitrocellulose

Orchard grass (cocksfoot) pollen extracts, fractionated by polyacrylamide gradient electrophoresis or SDS gel electrophoresis were electroblotted onto nitrocellulose membranes and probed with sera from orchard grass pollen-allergic patients and 125I-anti-human IgE. The IgE-binding components of the pollen were detected by autoradiography. Elution studies showed that allergens could be extracted immediately and continuously over a 3-hour period. Two fractions of MWs 28,000 and 30,000 could be detected only after 20 min extraction. SDS-PAGE separations gave the better resolution revealing 19 electrophoretically-separate components, 13 of which bound human IgE. All of the IgE-binding components had MWs in the range 14,000 - 70,000. Three of the bands bound IgE from more than 85% of the serum samples. Following gradient gel electrophoresis, IgE binding was exhibited by 10 bands in the range MW 5,000 to greater than 669,000. The technique used allows one to quantitatively examine patients' sera for allergen-specific IgE antibodies and to identify the clinically important allergens. Results revealed numerous allergenic components over a wide MW range while patterns of IgE binding with different patients' sera demonstrated a great diversity of IgE antibody responses. This study demonstrates the suitability of the electroblotting technique combined with autoradiography for the investigation of allergenic components of grass pollen extracts and hence has application to extract standardization and immunotherapy. Such studies can be carried out rapidly, economically and with a high degree of sensitivity.     

A re-examination of ryegrass (Lolium perenne) pollen allergens

Electroblotting of crude ryegrass pollen extracts and purified group I, II and III allergens identified 14 IgE-binding components, 8 of which were previously unrecognized. In addition to allergen groups I, II and III, which are already regarded as clinically important, and on the basis of the frequencies and intensities of IgE binding with sera from 42 ryegrass pollen-allergic patients, proteins with molecular weights (MWs) of 60, 32, 30 and 28 kD were identified as allergens of possible major clinical importance. Six other pollen components with MWs ranging from 23 to 80 kD and which reacted with IgE antibodies in the sera of 33-50% of patients, should also be viewed as proteins with potential clinical relevance for at least a proportion of the patients. The electrophoretic separation patterns of ryegrass pollen extracts in both alkaline and acid gels and IgE-probed membrane transfers produced in this study should serve as useful reference patterns for standardization purposes. In addition, the identification of the complete allergen recognition pattern by individual patients will permit safer and more effective diagnosis and therapy of ryegrass pollen sensitivities.     

Detection of house dust mite allergens and frequency of IgE binding following electroblotting and enzyme immunoassay

Mite allergens, fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 45 mite atopic sera and an enzyme immunostaining assay for IgE. More than 20 different mite components bound IgE and almost every serum showed a different pattern of binding. 7 of the 20 components were bound by half of the sera and 70% were bound by at least 20% of the sera. These results demonstrate a greater number of house dust mite allergens and a far greater diversity of the IgE antibody response to mite allergens than has previously been described.