Occupational medicine programs for animal research facilities

Occupational medicine is a key component of a comprehensive occupational health and safety program in support of laboratory animal research and production facilities. The mission of the department is to maximize employee health and productivity utilizing a population health management approach, which includes measurement and analysis of health benefits utilization. The department works in close cooperation with other institutional health and safety professionals to identify potential risks from exposure to physical, chemical, and biological hazards in the workplace. As soon as exposures are identified, the department is responsible for formulating and providing appropriate medical surveillance programs. Occupational medicine is also responsible for targeted delivery of preventive and wellness services; management of injury, disease, and disability; maintenance of medical information; and other clinic services required by the institution. Recommendations are provided for the organization and content of occupational medicine programs for animal research facilities.     

p34cdc2-mediated phosphorylation mobilizes microtubule-organizing centers from the apical intermediate filament scaffold in CACO-2 epithelial cells

We have shown previously that centrosomes and other microtubule-organizing centers (MTOCs) attach to the apical intermediate filament (IF) network in CACO-2 cells. In this cell line, intermediate filaments do not disorganize during mitosis. Therefore, we speculated that the trigger of the G(2)-M boundary may also detach MTOCs from their IF anchor. If that was the case, at least one of the proteins involved in the attachment must be phosphorylated by p34(cdc2) (cdk1). Using confocal microscopy and standard biochemical analysis, we found that p34(cdc2)-mediated phosphorylation indeed released MTOCs from IFs in permeabilized cells. In isolated, immunoprecipitated multiprotein complexes containing both gamma-tubulin and cytokeratin 19, p34(cdc2) phosphorylated only one protein, and phosphorylation released cytokeratin 19 from the complexes. We conclude that this as yet unidentified protein is a part of the molecular mechanism that attaches MTOCs to IFs in interphase.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Cross-reactive and mono-reactive SARS-CoV-2 CD4+ T cells in prepandemic and COVID-19 convalescent individuals

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.     

Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense)

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.     

Occupational Exposure to Hydrazine and Subsequent Risk of Lung Cancer: 50-Year Follow-Up

Purpose

Hydrazine is carcinogenic in animals, but there is inadequate evidence to determine if it is carcinogenic in humans. This study aimed to evaluate the association between hydrazine exposure and the risk of lung cancer.

Methods

The cause specific mortality rates of a cohort of 427 men who were employed at an English factory that produced hydrazine between 1945 and 1971 were compared with national mortality rates.

Results

By the end of December 2012 205 deaths had occurred. For men in the highest exposure category with greater than two years exposure and after more than ten years since first exposure the relative risks compared with national rates were: 0.85 (95% CI: 0.18–2.48) for lung cancer, 0.61 (95% CI: 0.07–2.21) for cancers of the digestive system, and 0.44 (95% CI: 0.05–1.57) for other cancers.

Conclusions

After 50 years of follow up, the results provide no evidence of an increased risk of death from lung cancer or death from any other cause.     

Red Blood Cell Distribution Width as a Pragmatic Marker for Outcome in Pediatric Critical Illness

Background

Red cell distribution width (RDW) is a routine laboratory measure associated with poor outcomes in adult critical illness.

Objective

We determined the utility of RDW as an early pragmatic biomarker for outcome in pediatric critical illness.

Methods

We used multivariable logistic regression to test the association of RDW on the first day of pediatric intensive care unit (PICU) admission with prolonged PICU length of stay (LOS) >48 hours and mortality. The area under the receiver operating characteristic curve (AUROC) for RDW was compared to the Pediatric Index of Mortality (PIM)-2 score.

Results

Over a 13-month period, 596 unique patients had RDW measured on the first day of PICU admission. Sepsis was an effect modifier for LOS >48 hours but not mortality. In sepsis, RDW was not associated with LOS >48 hours. For patients without sepsis, each 1% increase in RDW was associated with 1.17 (95% CI 1.06, 1.30) increased odds of LOS >48 hours. In all patients, RDW was independently associated with PICU mortality (OR 1.25, 95% CI 1.09, 1.43). The AUROC for RDW to predict LOS >48 hours and mortality was 0.61 (95% CI 0.56, 0.66) and 0.65 (95% CI 0.55, 0.75), respectively. Although the AUROC for mortality was comparable to PIM-2 (0.75, 95% CI 0.66, 0.83; p = 0.18), RDW did not increase the discriminative utility when added to PIM-2. Despite the moderate AUROC, RDW <13.4% (upper limit of lower quartile) had 53% risk of LOS >48 hours and 3.3% risk of mortality compared to patients with an RDW >15.7% (lower limit of upper quartile) who had 78% risk of LOS >48 hours and 12.9% risk of mortality (p<0.001 for both outcomes).

Conclusions

Elevated RDW was associated with outcome in pediatric critical illness and provided similar prognostic information as the more complex PIM-2 severity of illness score. Distinct RDW thresholds best discriminate low- versus high-risk patients.