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.

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.

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.

Improved acid tolerance of a recombinant strain of Escherichia coli expressing genes from the acidophilic bacterium Oenococcus oeni.

AIMS: Oenococcus oeni is a lactic acid bacterium used in wine fermentation. Two open reading frames (orfB and orfC) were identified in the upstream region of the hsp18 gene, encoding the small heat-shock protein Lo18. Expression of these genes in conditions of acid stress was studied in Escherichia coli. METHODS AND RESULTS: Sequence analysis showed that orfB encodes a putative transcriptional regulator of the LysR family. The protein encoded by orfC shares homologies with multi-drug resistance systems. Heterologous expression of orfB, orfC and hsp18 genes in Escherichia coli significantly enhanced the viability of the host strain under acidic conditions. CONCLUSION: It was demonstrated that the three genes were needed for acquisition of this acid tolerance phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: Heterologous expression of Oenococcus genes could be used to confer acidophilic behaviour on strains of biotechnological interest.

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.

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.

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.

Influence of surface treatments developed for oral implants on the physical and biological properties of titanium. (I) Surface characterization.

We present an investigation of the physico-chemical surface properties of commercially pure titanium coverslips which were submitted to various treatments designed to optimize their topography in view of application in oral implantology. The surface microroughness, chemical composition and water wettability were analyzed on titanium coverslips prepared by mechanical polishing, acid attack in HCl/H2SO4, after mechanical polishing or sandblasting, and titanium plasma-spray. The chemical composition has been measured by Auger electron spectroscopy. The treatments have no major influence on the surface chemical composition and all the samples display a composition approaching that of TiO2 with minor amounts of carbon, sulfur, silicon and calcium as impurities. The roughness has been measured by scanning force microscopy on an area of 20 microns x 20 microns on each sample. Polished titanium is smooth (peak-to-valley roughness 81 nm), whereas the acid-attacked surfaces exhibit a micro-roughness in the microns range (2100 nm for polished and acid attacked; 3600 nm for sandblasted and acid attacked) which is quite reproducible over large areas of the sample. The acid attacked samples present a subsurface layer which contains hydrogen below the native passivating oxide layer. Water wettability measurement shows that all surfaces are hydrophobic with a slightly higher contact angle for the acid attacked surfaces. The different treatments analyzed in this study essentially influence the surface roughness by preserving the chemical composition and the wettability properties of titanium native oxide surface layer.

A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase.

In Leuconostoc oenos, a malolactic bacterium, the synthesis of a stress protein called LO18 with an apparent molecular mass of 18 kDa was greatly induced after heat (42 degrees C), acid (pH 3) or ethanolic (12% (v/v)) shocks. Moreover, the LO18 protein synthesis was induced in stationary growth phase and was detected for a long time (30 h) during this growth phase. Significant identity was found between the N-terminal parts of the LO18 protein and the Hsp18 from Clostridium acetobutylicum suggesting that LO18 protein belongs to the family of small heat shock proteins conserved in prokaryotic and eukaryotic cells.

Molecular characterization of the gene encoding an 18-kilodalton small heat shock protein associated with the membrane of Leuconostoc oenos.

In Leuconostoc oenos, different stresses such as heat, ethanol, and acid shocks dramatically induce the expression of an 18-kDa small heat shock protein called Lo 18. The corresponding gene (hsp18) was cloned from a genomic library of L. oenos constructed in Escherichia coli. A 2.3-kb DNA fragment carrying the hsp18 gene was sequenced. The hsp18 gene encodes a polypeptide of 148 amino acids with a calculated molecular mass of 16,938 Da. The Lo18 protein has a significant identity with small heat shock proteins of the alpha-crystallin family. The transcriptional start site was determined by primer extension. This experiment allowed us to identify the promoter region exhibiting high similarity to consensus promoter sequences of gram-positive bacteria, as well as E. coli. Northern blot analysis showed that hsp18 consists of a unique transcription unit of 0.6 kb. Moreover, hsp18 expression seemed to be controlled at the transcriptional level. This small heat shock protein was found to be peripherally associated with the membrane of L. oenos.

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.

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk.

The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and beta-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 10 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.

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.

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.

Imaging of DNA by scanning force microscopy.

The scanning probe microscopies applied to the sequencing of DNA is a challenging goal attempted by several groups. But one limitant parameter has been the sample preparation of DNA molecules. Here we report how to hold DNA molecules fixed on mica substrate and we show the three-dimensional configuration of double-stranded DNA obtained with our scanning force microscope. We can image DNA under negative supercoiling, a feature of general importance controlling the activities of DNA. We compared the electron micrographs of a carbon replica of the same DNA specimen with scanning force images which demonstrates well the feasibility and accuracy of our scanning probe measurements.

Allergic rhinitis to ragweed pollen. I. Reassessment of the effects of immunotherapy on cellular and humoral responses.

This work presents a double-blind, placebo-controlled study of 27 patients with allergic rhinitis to ragweed who received preseasonal desensitization immunotherapy [IT] with alum-precipitated aqueous ragweed extracts. We reassessed the following parameters in relation to clinical responses: clinical scores, nasal reactivity to a provocative dose of ragweed causing a 75% fall in airflow rate (PD75), ragweed IgE and IgG, and ragweed-induced basophil histamine release (BHR). First, the nasal PD75 correlated with the severity of nasal symptoms (p less than 0.05). Second, we confirmed a significant symptomatic improvement in the IT-treated group either by clinical scores (p less than 0.05) or the prevention of the seasonal fall of the PD75 (p less than 0.005). Also, IT reduced the seasonal rise of IgE (p less than 0.02) and induced an increase in IgG (p less than 0.01) and a decrease in BHR (p less than 0.03). There was a significant correlation between IgE and BHR (r = 0.80; p less than 0.01). After selecting out the effects of IgE, the BHR was still higher in the placebo-treated group than in the IT-treated group (p less than 0.02), suggesting the involvement of other modulating factors. Symptomatic improvement after IT correlated only with the summation of both IgE and BHR (PD75; r = 0.64; p less than 0.005). This observation suggests that the severity of clinical symptoms is determined by several interacting factors and not by the antibody response alone.

Allergic rhinitis to ragweed pollen. II. Modulation of histamine-releasing factor production by specific immunotherapy.

A number of cytokines, including histamine-releasing factors (HRFs), have a role to play in IgE-mediated asthma. However, the influence of HRF in allergic rhinitis without asthma remains to be revealed. This article presents a double-blind, placebo-controlled study on the role of HRF in ragweed-allergic rhinitis and its modulation by natural pollen exposure and specific immunotherapy (IT). Twenty-seven patients allergic to ragweed were randomly assigned to receive either preseasonal alum-precipitated aqueous extracts of ragweed or placebo. Before the onset of therapy and during the ragweed-pollen season, subjects were evaluated for each of the following: clinical scores, ragweed IgE and IgG antibody levels, and spontaneous and allergen-driven HRF production. Thirteen nonatopic volunteers were also studied in the same protocol. First, before the initiation of therapy, more HRF was produced by both unstimulated and ragweed-stimulated mononuclear cells (MNCs) of atopic subjects as compared to MNCs of nonatopic subjects. Second, MNCs of the placebo-treated group produced significantly more spontaneous and ragweed-specific HRF during the pollen season compared to the preseasonal values. Finally, specific IT not only improved the clinical manifestation of allergy but also prevented the seasonal rise of spontaneous and ragweed-driven HRF production, along with a well-known change in other immunologic parameters associated with successful IT.

Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory.

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.

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.