CD40 Expression by B cells is Required for Optimal Immunity to Murine Pneumocystis Infection.

CD40-CD40L interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well-defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout (KO) mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell depleted splenocytes and unstimulated bone-marrow derived dendritic cells (BMDCs) were unable to control infection in CD40 KO mice. Pneumocystis antigen-pulsed BMDCs showed early, but limited, control of infection. Consistent with recent studies that have suggested a role for antigen presentation by B cells, using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis.

CD40 Expression by B cells is Required for Optimal Immunity to Murine Pneumocystis Infection.

CD40-CD40L interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well-defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout (KO) mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell depleted splenocytes and unstimulated bone-marrow derived dendritic cells (BMDCs) were unable to control infection in CD40 KO mice. Pneumocystis antigen-pulsed BMDCs showed early, but limited, control of infection. Consistent with recent studies that have suggested a role for antigen presentation by B cells, using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Swine influenza A virus isolates containing the pandemic H1N1 origin matrix gene elicit greater disease in the murine model.

Since the 1990s, endemic North American swine influenza A viruses (swFLUAVs) contained an internal gene segment constellation, the triple reassortment internal gene (TRIG) cassette. In 2009, the H1N1 pandemic (pdmH1N1) virus spilled back into swine but did not become endemic. However, the pdmH1N1 contributed the matrix gene (pdmM) to the swFLUAVs circulating in the pig population, which replaced the classical swine matrix gene (swM) found in the TRIG cassette, suggesting the pdmM has a fitness benefit. Others have shown that swFLUAVs containing the pdmM have greater transmission efficiency compared to viruses containing the swM gene segment. We hypothesized that the matrix (M) gene could also affect disease and utilized two infection models, resistant BALB/c and susceptible DBA/2 mice, to assess pathogenicity. We infected BALB/c and DBA/2 mice with H1 and H3 swFLUAVs containing the swM or pdmM and measured lung virus titers, morbidity, mortality, and lung histopathology. H1 influenza strains containing the pdmM gene caused greater morbidity and mortality in resistant and susceptible murine strains, while H3 swFLUAVs caused no clinical disease. However, both H1 and H3 swFLUAVs containing the pdmM replicated to higher viral titers in the lungs and pdmM containing H1 viruses induced greater histological changes compared to swM H1 viruses. While the surface glycoproteins and other gene segments may contribute to swFLUAV pathogenicity in mice, these data suggest that the origin of the matrix gene also contributes to pathogenicity of swFLUAV in mice, although we must be cautious in translating these conclusions to their natural host, swine. IMPORTANCE: The 2009 pandemic H1N1 virus rapidly spilled back into North American swine, reassorting with the already genetically diverse swFLUAVs. Notably, the M gene segment quickly replaced the classical M gene segment, suggesting a fitness benefit. Here, using two murine models of infection, we demonstrate that swFLUAV isolates containing the pandemic H1N1 origin M gene caused increased disease compared to isolates containing the classical swine M gene. These results suggest that, in addition to other influenza virus gene segments, the swFLUAV M gene segment contributes to pathogenesis in mammals.

The Host Adapted Fungal Pathogens of Pneumocystis Genus Utilize Genic Regional Centromeres.

Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of Schizosaccharomyces pombe. Using organisms from a short-term in vitro culture or infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.

Mucosal-Associated Invariant T Cells Accumulate in the Lungs during Murine Pneumocystis Infection but Are Not Required for Clearance.

Pneumocystis is a fungal pathogen that can cause pneumonia in immunosuppressed hosts and subclinical infection in immunocompetent hosts. Mucosal-associated invariant T (MAIT) cells are unconventional lymphocytes with a semi-invariant T-cell receptor that are activated by riboflavin metabolites that are presented by the MHC-1b molecule MR1. Although Pneumocystis can presumably synthesize riboflavin metabolites based on whole-genome studies, the role of MAIT cells in controlling Pneumocystis infection is unknown. We used a co-housing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to characterize the response of MAIT cells to infection in C57BL/6 mice, and, using MR1-/- mice, which lack MAIT cells, to examine their role in clearing the infection. MAIT cells accumulated in the lungs of C57BL/6 mice during Pneumocystis infection and remained at increased levels for many weeks after clearance of infection. In MR1-/- mice, Pneumocystis infection was cleared with kinetics similar to C57BL/6 mice. Thus, MAIT cells are not necessary for control of Pneumocystis infection, but the prolonged retention of these cells in the lungs following clearance of infection may allow a more rapid future response to other pathogens.

COVID-19: The Effects on the Course, Outcomes, and Discharge Destination From Acute Rehabilitation.

DESIGN: A descriptive cohort study was performed. METHODS: Retrospective data were collected for 63 patients aged >18 years with COVID-19- or non-COVID19-related debility between January 2015 and September 2020. RESULTS: Patients admitted with COVID-19-related debility had a mean length of stay of 20.35 days. 15.4% were transferred back to acute service, 92.3% required respiratory interventions, and 88.5% were discharged to the same residence.Patients with COVID-19-related debility were significantly older, required more respiratory interventions, and had a longer length of stay than the non-COVID-19 group. No significant differences were found in functional outcomes. CONCLUSIONS: Functional outcomes improved, allowing most patients to return to their same residence. CLINICAL RELEVANCE: Patients with COVID-19-related debility responded to rehabilitation as well as the non-COVID-19 group.

Genetic and Epidemiologic Analyses of an Outbreak of Pneumocystis jirovecii Pneumonia Among Kidney Transplant Recipients in the United States.

BACKGROUND: Pneumocystis jirovecii is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised hosts. Over an 11-month period, we observed a rise in cases of PCP among kidney-transplant recipients (KTR), prompting an outbreak investigation. METHODS: Clinical and epidemiologic data were collected for KTR diagnosed with PCP between July 2019 and May 2020. Pneumocystis strain typing was performed using restriction fragment length polymorphism analyses and multilocus sequence typing in combination with next-generation sequencing. A transmission map was drawn, and a case-control analysis was performed to determine risk factors associated with PCP. RESULTS: Nineteen cases of PCP in KTR were diagnosed at a median of 79 months post-transplantation; 8 received monthly belatacept infusions. Baseline characteristics were similar for KTR on belatacept versus other regimens; the number of clinic visits was numerically higher for the belatacept group during the study period (median 7.5 vs 3). Molecular typing of respiratory specimens from 9 patients revealed coinfection with up to 7 P. jirovecii strains per patient. A transmission map suggested multiple clusters of interhuman transmission. In a case-control univariate analysis, belatacept, lower absolute lymphocyte count, non-White race, and more transplant clinic visits were associated with an increased risk of PCP. In multivariate and prediction power estimate analyses, frequent clinic visits was the strongest risk factor for PCP. CONCLUSIONS: Increased clinic exposure appeared to facilitate multiple clusters of nosocomial PCP transmission among KTR. Belatacept was a risk factor for PCP, possibly by increasing clinic exposure through the need for frequent visits for monthly infusions.

Use of a structural equation model for prediction of pain symptoms in patients with orofacial pain and temporomandibular disorders.

AIMS: To develop and test a biopsychosocial model using structural equation modeling for predicting orofacial pain symptoms in a sample of patients with masticatory muscle pain (MMP). METHODS: Data were collected from clinic records of 251 adult patients who presented for initial evaluation to the Orofacial Pain Center at the University of Kentucky College of Dentistry and were subsequently diagnosed with MMP. Data were used to fit a model relating stressors, psychological distress, arousal, sleep problems, oral parafunction, and pain symptoms. Items from the Multidimensional Pain Inventory (MPI) and the IMPATH:TMJ, a comprehensive biopsychosocial assessment of patients with temporomandibular disorders (TMD), were used to construct a measurement model of five latent variables. RESULTS: Estimation of the model indicated a good fit to the data and significant associations between stressors, psychological distress, arousal, sleep problems, and pain symptoms. Sleep problems partially mediated the relation between arousal and pain symptoms. Contrary to hypotheses, no association occurred between oral parafunction and pain symptoms, possibly indicating that any relationship between oral parafunction and pain symptoms may not exist. CONCLUSION: Results from the model tested in the present study are an additional step toward developing a more comprehensive biopsychosocial model explaining the nature and etiology of MMP in orofacial pain and TMD. With additional development and testing, it may also serve as an aid to planning interventions, especially psychosocial interventions targeting stress management, psychophysiological regulation, psychological distress, and sleep problems.

Ecological momentary assessment of fatigue following breast cancer treatment.

Fatigue is a common and debilitating symptom often experienced during and following cancer treatment. An Ecological Momentary Assessment (EMA) approach was used to examine the diurnal pattern of off-treatment fatigue in breast cancer survivors. Twenty-five breast cancer (BC) survivors 6-26 months posttreatment and age-matched groups of healthy women (HC; n = 25) and women with benign breast problems (BBP; n = 24) completed four daily diary measures of fatigue, pain, and mood for 5 consecutive days. Type of activity engaged in at the time of the diary assessments, as well as daily pedometer activity level, and nightly sleep duration were also assessed. While BC survivors reported greater levels of fatigue relative to BBP anid HC groups, no group differences in mood, activity type or level, sleep duration, or diurnal pattern of fatigue were evident. The results confirm that fatigue mav continue to be experienced long after conclusion of cancer treatment while questioning its clinical significance, provide insight into potential etiological mechanisms underlying off-treatment fatigue in, and demonstrate the value of EMA approaches to the study of cancer-related fatigue.

Psychological and physiological parameters of masticatory muscle pain.

The objective of this research was to identify the psychological and physiological variables that differentiate persons reporting masticatory muscle pain (MMP) from normal controls (NC). This study examined the characteristics of 35 MMP patients in comparison to 35 age-, sex-, and weight-matched NCs. All subjects completed a series of standardized questionnaires prior to undergoing a laboratory evaluation consisting of a psychosocial stressor and pressure pain stimulation at multiple body sites. During the evaluation, subjects' emotional and physiological responses (heart rate, blood pressure, respiration, skin temperature, and muscle activity) were monitored. Results indicated that persons with MMP reported greater fatigue, disturbed sleep, depression, anxiety, menstrual symptoms, and less self-deception (P's < 0.05) than matched controls. At rest, MMPs had lower end tidal carbon dioxide levels (P < 0.04) and lower diastolic blood pressures than the NCs (P < 0.02). During laboratory challenge, both groups responded to the standard stressor with significant physiological activity and emotional responding consistent with an acute stress response (P < 0.01), but there were no differences between the MMPs and NCs. Muscle pain patients reported lower pressure pain thresholds than did NCs at the right/left masseter and right temporalis sites (P's < 0.05); there were no differences in pressure pain thresholds between MMPs and NCs for the left temporalis (P < 0.07) and right/left middle finger sites (P's > 0.93). These results are discussed in terms of the psychological and physiological processes that may account for the development of muscle pain in the masticatory system.