Altered muscarinic receptor expression in the cerebral cortex of epileptic rats: restorative role of Withania somnifera.

Temporal lobe epilepsy involves a sequence of events that can lead to neurotransmitter signalling alterations. There are many herbal extracts considered to be alternative therapeutic methods to manage epilepsy. In this study, we investigated the effect of Withania somnifera (WS) root extract and withanolide A (WA) in the management of temporal lobe epilepsy. Confocal imaging of TOPRO-3-stained cortical sections showed severe damage in the epileptic brain. We also observed a reduced antioxidant potential and increased peroxide levels in the epileptic test group of rats. Oxidative stress resulted in the down-regulation of CREB, NF-κB, and TNF-α, and with up-regulation of the apoptotic factors caspases 8 and 3 and Bax in the epileptic group. Epileptic condition also resulted in increased muscarinic receptor binding and mRNA expression in the cerebral cortex. Withania somnifera and withanolide A significantly reversed the altered muscarinic receptor expression and reversed the oxidative stress and resultant derailment in cell signalling. Thus our studies suggest that Withania somnifera and withanolide A play important roles in central muscarinic receptor functional balance and activation of the antioxidant system in the cerebral cortex in temporal lobe epilepsy. These findings can be of immense therapeutic significance for managing epilepsy.

The Role of the Medical Director in Ontario Long-Term Care Homes: Impact of COVID-19.

OBJECTIVES: The pandemic has uncovered a broad lack of understanding of the role of the Medical Director in Canadian Long-Term Care (LTC) Homes. Our objectives were to identify the current demographics and practices of LTC Medical Directors, discover how the pandemic affected their practice habits, and inform the content of the Ontario Long-Term Care Clinicians Medical Director Course, to ensure that Medical Directors have the requisite knowledge of the responsibilities of their role. DESIGN: Email survey. SETTING AND PARTICIPANTS: Medical directors in Ontario long-term care homes. METHODS: Responses to open-ended, close-ended, multiple-choice, and free-text questions. RESULTS: A total of 156 medical directors (approximately 24%) completed the survey. Ninety-four percent were family physicians. Approximately 40% of participants had been a medical director for fewer than 5 years, whereas more than 11% have been in the role for greater than 30 years. More than 60% spend fewer than 2 hours per week in their administrative role, with fewer than 23% completing formal evaluations of the attending clinicians. Greater than 75% are either satisfied or extremely satisfied in their medical director role, citing excellent engagement and collaboration with team members. Feelings of dissatisfaction were associated with pandemic stress, increased hours and responsibility, inadequate remuneration, lack of ability to make decisions and lack of acknowledgement that physicians add value to the interdisciplinary team. CONCLUSION AND IMPLICATIONS: It is clear that medical directors are in a unique position to impact the care of residents within LTC. It is imperative to engage medical directors as integral members of the LTC health care team. This can be achieved by acknowledging their medical expertise for improving outcomes, providing them with the authority for decision making, compensating them appropriately, and clearly defining the role. By making these changes, we can ensure that there is a higher likelihood to sustain effective medical leadership in LTC.

Long-term establishment of a human plasmacyte cell line derived from a patient with IgD multiple myeloma. I. Requirement of a plasmacyte-stimulating factor for the proliferation of myeloma cells in tissue culture.

Cell line LA-49, derived from pleural fluid cells of a patient with IgD multiple myeloma, was established in culture and maintained for more than 1 yr. The D-myeloma protein produced in culture was similar to the serum D-myeloma protein in electrophoretic mobility and in delta- and lambda-chain antigens. The plasma cell tumor culture, LA-49, differed from numerous immunoglobulin-producing B-lymphoblastoid cell lines established in this laboratory in: (a) Morphology (revealing various stages of maturation); (b) type of immunoglobulin produced (IgD vs. IgM, IgG, and/or, rarely, IgA); (c) growth characteristics (requirement of plasmacyte-stimulating factor); and (d) chromosomal features (polyploid vs. pseudodiploid). A growth factor was needed for cell division and maintenance of culture viability. This factor was supplied readily by irradiated feeder layers of normal human fibroblasts or conditional media from fibroblast cultures. Preliminary characterization of this factor revealed it to be a protein with a mol wt of approximately 150,000 daltons.

Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies.

Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.

Effect of pharmacological blockade of ACTH and TSH secretion on the acute stimulation of prolactin release by exposure to cold and ether stress.

Acute exposure of male rats to cold (5C)leads to a rapid increase of plasma levels of thyrotropin (TSH), prolactin (PRL), corticosterone, and L-thyroxine. Exposure to ether is similarly followed by a rapid increase of plasma levels of PRL and corticosterone, while TSH release is inhibited. Acute treatment with dexamethasone (500 mug) inhibits almost completely the PRL response to both exposure to cold and ether stress, while the plasma TSH response to cold is only delayed and the decrease of plasma TSH observed after ether stress is unchanged. Basal plasma levels of both TSH and PRL are lowered after treatment with the steroid. Thyroxine treatment lowers the plasma TSH concentration to undetectable levels without affecting the plasma PRL response to cold or ether exposure. The present data suggest that the rise of plasma PRL observed after cold exposure is not related to TRH and may suggest that common mechanisms control ACTH and PRL secretion during acute stress exposure.

Development of a reverse enzymoallergosorbent test (REAST) to detect timothy-specific IgE antibodies. Comparison with RAST.

Radioallergosorbent test (RAST) for the measurement of IgE antibodies has been introduced more than 15 years ago and a number of technical modifications have since improved its sensitivity and reproducibility. The test has been applied to the diagnosis of allergy and to determine changes in the levels of IgE antibodies following immunotherapy. However, specific IgG antibodies are raised during such a therapy and can interfere with the RAST. We have developed a reverse enzymoallergosorbent test (REAST) where microtiter plates are first coated with a purified polyclonal anti-IgE antibody, then with the serum to test and finally with peroxidase-labeled antigen. This assay is antigen specific as shown by the significant inhibition of binding of the labeled antigen in presence of unlabeled specific antigen (greater than 95%) and the absence of inhibition in presence of irrelevant antigens. The values found in atopic patients (85 subjects) were significantly higher than in the non-atopic donors (35 subjects) (1.14 U +/- 1.20 vs. 0.01 U +/- 0.02, P less than 0.0005) and there was a good correlation with the Pharmacia RAST (P less than 0.0005). The levels of specific IgE by both REAST and RAST correlated well with the clinical symptomatology.

Enhanced metabolic response to caffeine in exercise-trained human subjects.

The effect of caffeine on resting metabolic rate (RMR) was investigated in eight trained and eight nontrained young male subjects. The ingestion of 4 mg/kg caffeine produced a greater increase of RMR in trained subjects. This effect was associated with a greater increase in plasma free fatty acids and a larger fall in respiratory quotient, indicating an enhanced lipid oxidation following caffeine in exercise-trained subjects. An initial fall in plasma glucose was observed but only in trained subjects, and caffeine did not change plasma insulin in either group studied. Caffeine caused a significant fall in plasma norepinephrine and an increase in plasma epinephrine in both groups of subjects, but this action was significantly greater in trained subjects. It is suggested that the greater increase in RMR observed in trained subjects following caffeine ingestion is related to an enhanced lipid mobilization, possibly produced by a greater epinephrine secretion and by subsequent increased lipid oxidation.

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase.

Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.

Morphological difference between fibronectin sprayed on mica and on PMMA.

We have imaged with scanning force microscopy in air fibronectin (Fn) molecules sprayed on mica and on polymethylmetacrylate (PMMA), the latter being extensively used as biomaterial for implants. On mica we can observe small aggregates as well as individual molecules whose shape is influenced by the tip interaction during the scanning process, most of the isolated molecules showing a V-shape oriented in the scan direction. This indicates that the arms of the molecules are relatively free to move and the binding to the mica substrate is located near the disulfide bridge between the two subunits of the molecule. On the other side, when Fn molecules are sprayed on PMMA under the same conditions as for mica, we observe a thin network which we interpret as Fn molecules bound to each other. We relate our observation to the fact that mica is known to be strongly hydrophilic, which could reduce the Fn binding properties by interacting relatively strongly with molecules. On the other side, PMMA being hydrophobic, would interact less with molecules, leaving more binding sites for inter-molecular attachment.

Scanning force microscopy and cryo-electron microscopy of tobacco mosaic virus as a test specimen.

In this study, tobacco mosaic virus (TMV) provides a resolution criterion for specimen preparation methods as well as for imaging parameters of the scanning force microscope (SFM). We present scanning force microscopic images of the virus embedded in 0.5% buffered phosphotungstic acid solution adsorbed on a freshly cleaved mica surface, and imaged under atmospheric conditions. Individual TMV particles were clearly identified with a characteristic shape of long rods of about 300 nm long and 60-70 nm in apparent width due to the geometric parameters of the tip. The structure of the virus was compared with cryo-electron microscopic data of vitrified suspensions observed to a resolution of 1.15 nm. Uncoated TMV particles were also deposited on evaporated titanium thin films and imaged by SFM.

Protein quality and microbiological changes in aerobically- or vacuum-packaged, irradiated fresh pork loins.

The effect of gamma-irradiation on the physicochemical, organoleptic and microbiological properties of pork was studied, during 43 days of storage at 4±1°C. Irradiation treatments were carried out under air or vacuum packaging on fresh pork loins at a dose of 6 kGy, at two different dose rates: 2 kGy/h and 20 kGy/h. The loins were evaluated for protein sulphydryl content and emulsifying capacity, surface hydrophobicity of proteins and sensorial evaluation. Regardless of the type of packaging and dose rate of irradiation, all irradiated pork samples were effectively prevented from bacterial spoilage for at least 43 days. Meat redness and texture of irradiated loins were relatively well preserved during the storage period, especially when samples were stored under vacuum. Overall, the physicochemical and organoleptic changes in pork loins appeared to be relatively little affected by the 6 kGy dose. No marked changes in emulsifying capacity and protein sulphydryl content of proteins were noted throughout the storage period. However, the hydrophobicity was reduced (P⩽0.05) by the faster dose rate of irradiation and by longer storage.

Stearic acid content of abdominal adipose tissues in obese women.

OBJECTIVE: Subcutaneous (SC) adipose tissue stearic acid (18:0) content and stearoyl-CoA desaturase-1 (SCD1)-mediated production of oleic acid (18:1) have been suggested to be altered in obesity. The objective of our study was to examine abdominal adipose tissue fatty acid content and SCD1 mRNA/protein level in women. SUBJECTS AND METHODS: Fatty acid content was determined by capillary gas chromatography in SC and omental (OM) fat tissues from two subgroups of 10 women with either small or large OM adipocytes. Samples from 10 additional women were used to measure SCD1 mRNA and protein expression, total extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 protein as well as insulin receptor (IR) expression levels. RESULTS: OM fat 18:0 content was significantly lower in women with large OM adipocytes compared with women who had similar adiposity, but small OM adipocytes (2.37±0.45 vs 2.75±0.30 mg per 100 g adipose tissue, respectively, P0.05). OM fat 18:0 content was negatively related to the visceral adipose tissue area (r=-0.44, P=0.05) and serum triglyceride levels (r=-0.56, P<0.05), while SC fat 18:0 content was negatively correlated with total body fat mass (BFM) (r=-0.48, P<0.05) and fasting insulin concentration (r=-0.73, P<0.005). SC adipose tissue desaturation index (18:1/18:0), SCD1 expression and protein levels were positively correlated with BFM. Moreover, obese women were characterized by a reduced OM/SC ratio of SCD1 mRNA and protein levels. A similar pattern was observed for ERK1/2 and IR expression. CONCLUSION: The presence of large adipocytes and increased adipose mass in a given fat compartment is related to reduced 18:0 content and increased desaturation index in women, independently of dietary fat intake. The depot-specific difference in ERK1/2 expression and activation, as well as in SCD1 and IR expression in obese women is consistent with the hypothesis that they may predominantly develop SC fat, which could in turn help protect from metabolic disorders.

Interactions between the pituitary, thyroid and adrenal cortex during acute exposure to cold or to electric shocks in the rat.

In intact rats acclimated to 25 +/- 1 degrees C, acute exposure to cold resulted in simultaneous stimulation of TSH and ACTH secretion. The plasma TSH response to cold was identical at temperatures varying from +14 to -10 degrees C, whereas the adrenocortical response increased proportionally to the severity of cold. Acute stimulation of ACTH secretion by exposure to a stressful situation (electrical shocks) did not alter the TSH response to cold. Conversely, acute blockade of the pituitary-adrenocortical response by dexamethasone treatment did not enhance the TSH response to cold. Chronic stimulation of ACTH secretion resulting from adrenalectomy did not interfere with the TSH response during subsequent exposure to cold. However, a reduced adrenocortical response to cold was observed during chronic hypersecretion of TSH resulting from previous thyroidectomy. These findings do not support the hypothesis of an inverse relationship between TSH and ACTH secretions during acute cold exposure, but rather suggest that these secretions are independent.

The effect of face fanning during recovery from exercise hyperthermia.

Hyperthermia was induced in nine subjects on two separate occasions by a progressive treadmill run, which resulted in an average esophageal temperature (Tes) of 39.77 +/- 0.07 degree C after 30-57 min. Fanning the face during exercise to simulate conditions during running (wind at 3.75 m X s-1) maintained a tympanic temperature (Tty) that was lower than Tes; the difference was 1.5 degrees C at the end of exercise. In one session, face fanning was interrupted at the end of running, whereas in the other it was maintained for 15 min after exercise stopped. Face fanning had no significant influence on the fall of Tes during recovery, but it markedly influenced the course of Tty during this period. When face fanning was stopped at the end of the run, Tty rose by nearly 0.5 degree C, peaked after 4.5 min, and thereafter decreased slowly to a value close to Tes. In contrast, when face fanning was maintained throughout the recovery period, Tty rose only slightly (0.1 degree C) and remained significantly lower than Tes at all times. The results suggest that following hyperthermic exercise, face fanning could be helpful in preventing acute cerebral hyperthermia.

Biological activity of monoclonal anti-idiotypic antibody representing the internal image of the major allergenic component of Lolium perenne pollen.

Upon immunization with an anti-Lol p I (major allergenic component of Lolium perenne pollen) monoclonal antibody, we have previously produced anti-idiotypic monoclonal antibody (A7H2) displaying some internal image properties. The present study was designated to evaluate the capacity of this anti-idiotypic monoclonal antibody to mimic functionally the antigen by triggering histamine release from basophils of patients allergic to Lol p I. Anti-idiotypic monoclonal antibody, as the antigen, could induce histamine release in a dose-response fashion in all of the atopic patients (6/6). The inhibition of this histamine release by the addition of the idiotype (290A-167) confirmed the specificity of the reaction. Binding inhibition of human IgE to Lol p I demonstrated that the anti-idiotypic antibody recognized an idiotope expressed in the antigen-combining site of IgE molecules. Altogether, these data confirmed the internal properties of our anti-idiotypic antibody and it can mimic the original antigen in its capacity to trigger histamine release.

Effect of exposure to cold on hypothalamic TRH activity and plasma levels of TSH and prolactin in the rat.

No significant change in hypothalamic TRH content was found in rats during acute (5-240 min) exposure to cold (5 degrees C), in spite of rapid and sustained elevations in plasma TSH and thyroxine. Plasma PRL rose markedly in the first 15 min, but returned to normal thereafter. Chronic exposure to cold (32 days) was characterized by elevated plasma and pituitary levels of both TSH and PRL in the presence of an unaltered hypothalamic TRH content. If increased TRH release from the hypothalamus occurs during exposure to cold, as suggested by the pituitary-thyroid stimulation, either it is compensated for by an equal rise in synthesis, or the extra amount released is negligible in comparison with the hypothalamic content of TRH. The acute PRL response to exposure to cold may be related to an acute TRH release but could also result from the accompanying stress response acting by a mechanism independent of TRH.

The Oenococcus oeni clpX homologue is a heat shock gene preferentially expressed in exponential growth phase.

Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococcus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed clpX gene products from other organisms. An open reading frame located upstream of the clpX gene was identified as the tig gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an increase in clpX mRNA amount after a temperature shift from 30 to 42 degrees C. The clpX transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike hsp18, the gene encoding one of the major small heat shock proteins of Oenococcus oeni, clpX expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5' end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as Escherichia coli.

Plasma catecholamines and cardiovascular responses to cold and mental activity.

Plasma epinephrine (E) and norepinephrine (NE) as well as blood pressure and heart rate variations were measured in 12 male subjects before, during, and after a cold hand test (5 degrees C for 2 min), a mental arithmetic test, and a combination of both these tests. Although the cold and mental tests had comparable effects on blood pressure, the heart rate response was greater than the mental test. The mental test produced a greater increase of E than the cold test, but the effect of the cold test was greater on NE than on E. Changes in heart rate were significantly correlated with E variations but not with NE, whereas changes in blood pressure were correlated with NE. Resting NE was correlated with resting blood pressure and resting E with resting heart rate. Finally the levels of basal E were positively correlated with the increase in E during the test. Cardiovascular changes were shown to be differently modified by a mental and a cold test. Evidence was given indicating that these changes are related to differences in E and NE responses in the presence of these tests.

Effects of H1 and H2 receptor agonists on nonspecific proliferative response of human peripheral blood lymphocytes.

Histamine is known to modulate immune responses through the induction of suppressor cell subsets. The inhibition studies with antagonists suggest that the H2 agonist accounts for most of the suppression. This work studies the effects of various concentrations of 3-methyl histamine (as negative control), histamine, and pure H1 (2-methyl histamine, 2-pyridyl ethylamine) and H2 (4-methyl histamine, dimaprit) receptor agonists on the mitogenic (phytohemagglutinin A) proliferative response of normal human lymphocytes. At high concentrations of agonists (10(-3), 10(-4) M) the suppression induced by the two types of agonists is comparable to that of histamine. At lower concentrations (10(-6) M) the suppression is seen only in the presence of the H2 agonist. The suppression induced by the two agonists is generally reversed in the presence of an H2 receptor antagonist. The H1 receptor antagonist did not abolish and even increased the suppression induced by histamine and the two agonists.

Possible dual role of anti-idiotypic antibodies in combined passive and active immunotherapy in honeybee sting allergy.

BACKGROUND: Passive infusion of beekeepers' plasma was shown to protect patients against systemic reactions occurring during active immunotherapy by mechanisms still to be clarified. It is tempting to speculate that anti-idiotypic antibodies could play a role because they are found in beekeepers' plasma and are involved in the regulation of IgE synthesis. METHODS: In this report we studied the effects of passive infusion of a beekeeper's plasma rich in anti-idiotypic antibodies to a patient who experienced systemic reactions to honeybee venom. RESULTS: We reported, during the days after the infusion, a decrease of clinical sensitivity to the honeybee venom. Indeed, the patient tolerated a cumulative dose of 280 micrograms of venom without adverse reactions. We also observed decreases in skin mast cell and in basophil sensitivity. After the plasma infusion, a modified rush immunotherapy with honeybee venom was initiated in our patient. In the following 76 weeks, increased levels of anti-idiotypic antibodies in the serum of the patient were associated with a diminution of specific antibodies (IgG and IgE) to honeybee venom. CONCLUSION: These results suggest a dual role of anti-id in our combined protocol of passive and active immunotherapy: an immediate action on clinical sensitivity along with a decrease of skin mast cell and basophil sensitivity and an immunoregulatory role on specific antibody production.