Effects of Salt Loading on the Regulation of Rat Hypothalamic Magnocellular Neurosecretory Cells by Ionotropic GABA and Glycine Receptors.

Synaptic and extrasynaptic transmission mediated by ionotropic GABA and glycine receptors plays a critical role in shaping the action potential firing (spiking) activity of hypothalamic magnocellular neurosecretory cells and therefore determines the rate at which vasopressin and oxytocin are released from the neurohypophysis. The inhibitory effect of these transmitters relies on the maintenance of a low concentration of intracellular chloride ions such that, when activated by GABA or glycine, a hyperpolarisation of the neuronal membrane potential results. In this review, we highlight the various ways by which the two types of inhibitory receptors contribute to homeostasis by fine-tuning the spiking rate of vasopressin-releasing magnocellular neurosecretory cells in a manner dependent on the hydration state of the animal. In addition, we review the currently available evidence on how the strength of these inhibitory pathways can be regulated during chronic hypernatraemia via a form of activity-dependent depolarisation of the chloride reversal potential, leading to an abolition of these inhibitory pathways potentially causing sodium-dependent elevations in blood pressure.

Circadian modulation of osmoregulated firing in rat supraoptic nucleus neurones.

The antidiuretic hormone vasopressin (VP) promotes water reabsorption from the kidney and levels of circulating VP are normally related linearly to plasma osmolality, aiming to maintain the latter close to a predetermined set point. Interestingly, VP levels rise also in the absence of an increase in osmolality during late sleep in various mammals, including rats and humans. This circadian rhythm is functionally important because the absence of a late night VP surge results in polyuria and disrupts sleep in humans. Previous work has indicated that the VP surge may be caused by facilitation of the central processes mediating the osmotic control of VP release, and the mechanism by which this occurs was recently studied in angled slices of rat hypothalamus that preserve intact network interactions between the suprachiasmatic nucleus (SCN; the biological clock), the organum vasculosum lamina terminalis (OVLT; the central osmosensory nucleus) and the supraoptic nucleus (SON; which contains VP-releasing neurohypophysial neurones). These studies confirmed that the electrical activity of SCN clock neurones is higher during the middle sleep period (MSP) than during the late sleep period (LSP). Moreover, they revealed that the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT was greater during the LSP than during the MSP. Activation of clock neurones by repetitive electrical stimulation, or by injection of glutamate into the SCN, caused a presynaptic inhibition of glutamatergic synapses made between the axon terminals of OVLT neurones and SON neurones. Consistent with this effect, activation of clock neurones with glutamate also reduced the excitation of SON neurones caused by hyperosmotic stimulation of the OVLT. These results suggest that clock neurones in the SCN can mediate an increase in VP release through a disinhibition of excitatory synapses between the OVLT and the SON during the LSP.

[Management of thyroid dysfunction by primary care physicians of the Province of Quebec: a transversal study]. / Etude transversale sur l'approche thérapeutique des patients avec troubles thyroïdiens par les omnipraticiens du Québec.

OBJECTIVES: This study aims to evaluate clinical practice of primary care physicians regarding common thyroid disorders. MATERIALS AND METHODS: A sample of 210 primary care physicians was randomly selected in three Quebec's administrative regions. Four clinical vignettes (V1 to V4) were presented by mail: two cases of subclinical hypothyroidism (women of 25 years - V1 - and 70 - V2 - years of age) for which physicians had to choose to either treat or not with thyroid replacement and two cases of hyperthyroidism (women of 30 - V3 - and 66 - V4- years of age) for which they had to choose a course of action (observation, treatment or referral to a specialist). V1 and V2 where followed by four sub-questions presenting supportive elements that could influence the decision to treat (presence of antithyroid antibodies, accumulation of symptoms, LDL cholesterol and thyreostimulin levels). RESULTS: The overall response rate was 22%. Forty-two percent of respondents would have treated V1 outright and 49% would have treated V2. The therapeutic approach in the face of these two vignettes, independently of the presence or absence of supportive clinical or biochemical elements, did not vary according to geographic practice area. However, one region was significantly more conservative for V4. The number of years in practice or assistance to continuous medical education activities did not affect management of vignettes. CONCLUSION: This study outlines the importance of clinical practice guidelines and tools to facilitate their application in clinical management of thyroid disorders.

Report on the Eleventh International Society of Blood Transfusion Platelet Genotyping and Serology Workshop.

BACKGROUND AND OBJECTIVES: The aims of the Eleventh International Workshop were to evaluate proficiency in platelet genotyping and antibody detection, to equip laboratories to perform Gov antigen system genotyping and antibody detection, and to evaluate the laboratory and clinical approach to cases of neonatal alloimmune thrombocytopenia (NAIT). MATERIALS AND METHODS: There were 34 participating laboratories from 22 countries on five continents. Participating laboratories were provided with 10 DNA samples, 15 unknown sera, and three monoclonal antibodies for titration, as well as primer pairs and a protocol for Gov genotyping and Gov antibody screening. They were also provided with a questionnaire on investigation and clinical management of patients with NAIT. RESULTS: Thirty-three participants reported human platelet antigen (HPA)-1, -2, -3 and -5 genotyping results, 25 reported HPA-4 typing results, 17 reported HPA-6 typing results and 24 reported Gov typing results. For HPA-1-6 genotyping, 23 laboratories were concordant with a majority vote for all allotypes tested, five laboratories reported one deviation, three laboratories reported two deviations and one laboratory reported three deviations. For Gov genotyping, six deviations occurred in three of the 24 laboratories reporting results. Antibody detection was 90% concordant for anti-HPA-1a, anti-HPA-5a and anti-HPA-5b detection. Anti-HPA-2b and anti-Gova were detected by 20 and 14 out of 33 laboratories, respectively. Approaches to the clinical management of NAIT vary widely, especially for mothers with a history of a previous infant with mild NAIT. CONCLUSIONS: The overall error rate for HPA-1-6 genotyping decreased from 2.7% in the tenth workshop to 0.8% in the eleventh workshop. The majority of laboratories were able to perform Gov genotyping, although the error rate was 7.5%. Detection of common clinically significant antibodies was good, although detection of the much rarer HPA-2b was problematic. There was considerable progress in the detection of anti-Gova. The lack of consensus over treatment of NAIT demonstrates uncertainty over optimal management of these patients.

Neonatal alloimmune thrombocytopenia due to anti-HPA-2b (anti-Koa).

Most severe cases of neonatal alloimmune thrombocytopenia (NAIT) are due to anti-HPA-1a (anti-PlA1) antibodies. We report a case of NAIT due to anti-HPA-2b that resulted in in utero intracranial hemorrhage.A 33-year-old G2P1A0 Caucasian woman had a routine ultrasound at 34 weeks. The fetus appeared to have a left hemispheric hematoma. IVIG, 1g/kg, was started immediately and administered weekly until delivery. One day after receiving the first dose of IVIG, fetal platelet count was 18 x 10(9)/L, and Hb was 116 g/L. Eleven mL of matched platelets compatible by monoclonal antibody immobilization of platelet antigens (MAIPA) assay were transfused in utero, raising the platelet count to 62 x 10(9)/L. Repeat transfusions were done later that week and 1 week later, with pretransfusion counts of 19 x 10(9)/L and 16 x 10(9)/L, respectively. Delivery by C section was done at 35.5 weeks, after the third platelet transfusion. Platelet count at birth was 77 x 10(9)/L. Drainage of the hematoma was performed after transfusion. Testing with a solid phase ELISA revealed reactivity against GP1b/IX. MAIPA testing after platelet treatment with the protease inhibitor leupeptin demonstrated the presence of anti-HPA-2b. On PCR-SSP the mother was HPA-2a homozygous, the father was HPA-2a/2b. Antibodies against the HPA-2b antigen located on the GP1b/IX complex have been reported in rare cases of NAIT. Testing is complicated by proteolytic degradation of the antigen-bearing fragment. Compatible platelets are easily found since approximately 85 percent of donors are HPA-2a/2a.

Design of functionalized lipids and evidence for their binding to photosystem II core complex by oxygen evolution measurements, atomic force microscopy, and scanning near-field optical microscopy.

Photosystem II core complex (PSII CC) absorbs light energy and triggers a series of electron transfer reactions by oxidizing water while producing molecular oxygen. Synthetic lipids with different alkyl chains and spacer lengths bearing functionalized headgroups were specifically designed to bind the Q(B) site and to anchor this large photosynthetic complex (240 kDa) in order to attempt two-dimensional crystallization. Among the series of different compounds that have been tested, oxygen evolution measurements have shown that dichlorophenyl urea (DCPU) binds very efficiently to the Q(B) site of PSII CC, and therefore, that moiety has been linked covalently to the headgroup of synthetic lipids. The analysis of the monolayer behavior of these DCPU-lipids has allowed us to select ones bearing long spacers for the anchoring of PSII CC. Oxygen evolution measurements demonstrated that these long-spacer DCPU-lipids specifically bind to PSII CC and inhibit electron transfer. With the use of atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), it was possible to visualize domains of PSII CC bound to DCPU-lipid monolayers. SNOM imaging has enabled us to confirm that domains observed by AFM were composed of PSII CC. Indeed, the SNOM topography images presented similar domains as those observed by AFM, but in addition, it allowed us to determine that these domains are fluorescent. Electron microscopy of these domains, however, has shown that the bound PSII CC was not crystalline.

The interaction between lipid derivatives of colchicine and tubulin: consequences of the interaction of the alkaloid with lipid membranes.

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin-water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.

Prevention of cytokine accumulation in platelets obtained with the COBE spectra apheresis system.

BACKGROUND AND OBJECTIVES: Febrile nonhemolytic transfusion reactions frequently accompany platelet transfusions and may be due to accumulation of cytokines mediating inflammation during storage of platelet concentrates (PCs). We wished to determine whether PCs collected using the COBE(R) SpectraTM Apheresis System (Version 4) were sufficiently leukocyte reduced (LR) to limit cytokine accumulation during storage. MATERIALS AND METHODS: Cytokine accumulation - interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha) - and release of platelet alpha-granule - P-selectin, transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor AB (PDGF-AB), von Willebrand factor (vWf) - or dense granule (serotonin) markers were investigated during a 7-day storage period comparing apheresis-collected, LR PCs (LR PCs) and random donor platelets prepared from whole blood (WB). RESULTS: Leukocyte counts were reduced 99.95% comparing LR PCs (5.7 x 10(5)/l) and WB PCs (1.09 x 10(9)/l). Little or no accumulation of leukocyte-derived cytokines was observed in LR PCs during storage in contrast to WB PCs. A reduction in the release of platelet alpha-granule proteins, such as P-selectin, TGF-beta1 and PDGF-AB, was observed on day 0 for LR PCs compared to WB PCs with little or no difference observed from day 3 to 7. Plasma vWf levels were higher in LR PCs compared to WB PCs on days 0-7. CONCLUSION: Leukocyte levels in PCs collected with the COBE Spectra Apheresis System are sufficiently low to limit cytokine production during 7 days of storage.

Expression of stem cell factor and c-kit mRNA in cultured endothelial cells, monocytes and cloned human bone marrow stromal cells (CFU-RF).

Previously, we have shown that conditioned medium from a subpopulation of human marrow stromal cells (CFU-RF) contain an activity able to stimulate the growth of macroscopic epo-dependent erythroid colonies. The ligand for the product of the c-kit proto-oncogene (also known as stem cell factor or SCF), among other activities, has been reported to have similar effects on erythroid colony growth. We have also presented data showing that SCF together with phytohemagglutinin-stimulated leukocyte conditioned medium can stimulate erythroid colony growth in the presence of antibodies to erythropoietin. Using the human SCF cDNA probe (K. Zsebo, Amgen Inc.) we now show that cells derived from CFU-RF colonies express SCF but not c-kit. Human umbilical vein endothelial cells were also found to express SCF and this expression was increased by addition of monocyte supernatant, IL-1 beta or thrombin. Cells of the human erythroleukemia cell line HEL were found to express c-kit but not SCF. Neither c-kit nor SCF mRNA were detected in phytohemagglutinin-stimulated lymphocytes. Together, these data support the view that the behaviour of proliferating erythroid stem cells in the marrow, which may express c-kit, could be regulated by membrane-bound SCF present on surrounding stromal cells.

Characterization of novel platelet and endothelial cell target antigens in a family with genetic susceptibility to autoimmunity.

This report describes a French Canadian family whose members exhibit a high incidence of allo- and autoantibodies to antigens present on both platelets and endothelial cells. This is correlated with various HLA specificities known to be associated with autoimmunity, such as A1, B8, DR3, and, in some cases, with clinical disorders, including nephritis, hypertension, and thrombocytopenia. Immunoblot analysis using platelet and endothelial cell lysates showed serum antibodies to a 75 kDa endothelial cell surface polypeptide and to polypeptides with apparent mass of 115 kDa and 26 kDa found on both platelets and endothelial cells. This 115 kDa internal platelet protein was also found in a variety of other cell types, such as mononuclear cells, and increased following cell activation. Monoclonal antibody immunobilization assays were used to characterize the 26 kDa polypeptide; in three of the four patients tested, an antibody to leukocyte differentiation antigen CD9 was identified. The asymptomatic child of the propositus also exhibited an autoantibody against an 80 kDa platelet protein which was sensitive to thrombin digestion, suggesting that this polypeptide may be platelet glycoprotein V. In addition, P1A1 alloantibody was identified in one sister who had given birth to a severely thrombocytopenic boy and who herself had a severe vascular rejection to a cadaver kidney 2 years prior to this study. The propositus also developed hypertensive renal disease following a pregnancy and became dialysis dependent. Thus, members of this family have developed a variety of antibodies, particularly to platelet and endothelial cell antigens. Some subjects have remained asymptomatic in spite of having autoantibodies. However, others have been seriously ill, and their immune response to these antigens is believed to have played a role in the pathogenesis of their neonatal alloimmune thrombocytopenic purpura, hypertensive renal disease, renal graft rejection, and thrombocytopenia.

Alloimmunization to the PlA1 platelet antigen: results of a prospective study.

The natural history of alloimmunization to the PlA1 platelet antigen is uncertain. We followed 50 PlA1-negative pregnant women during pregnancy and for 6 months post-partum in order to determine this natural history. The cohort of PlA1-negative women was obtained by PlA1 typing 5000 women. Three PlA1-negative women formed anti-PlA1 antibodies during this prospective study, two in pregnancy and one in the immediate post-partum period. All three PlA1 antibody producers were HLA-DR3 positive, a histocompatibility phenotype that is strongly associated with alloimmunization to the PlA1 antigen. One of the three infants delivered to these mothers was thrombocytopenic (platelet count 9 x 10(9)/l). The remaining two infants had normal platelet counts at birth (160 and 174 x 10(9)/l). The HLA-A1, -B8, -DR3 and -DRw52 phenotype frequencies in the group of PlA1-negative women who did not form PlA1 antibodies (n = 47) was similar to that found in their husbands, and that expected in a normal Caucasian population. From our data we estimate that alloimmunization to the PlA1 antigen occurs in approximately one out of every 1000 pregnancies in a Caucasian population. It is important to recognize that not all pregnancies in which a mother has formed PlA1 alloantibodies will result in the delivery of a thrombocytopenic infant. These findings are relevant to programs designed to either prevent alloimmunization to the PlA1 antigen (through passive administration of anti-PlA1 immunoglobulin to at-risk PlA1-negative mothers), or to identify women at risk of delivery of thrombocytopenic infants (by antenatal screening to detect women alloimmunized to the PlA1 antigen).