Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

<div><p>Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)⁺PI (Propidium Iodide)⁺ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM⁺AnnV⁺PI⁺ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM^-AnnV⁺PI⁺ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM <i>in vivo</i> in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration.</p></div

Measurement of physical activity in the pre- and early post-operative period after total knee arthroplasty for osteoarthritis using a Fitbit Flex device

Total knee arthroplasty (TKA) is a standard treatment for patients with end stage knee Osteoarthritis (OA) to reduce pain and restore function. The aim of this study was to assess pre- and early post-operative physical activity (PA) with Fitbit Flex devices for patients with OA undergoing TKA and determine any benchmarks for expected post-operative activity. Significant correlations of pre-operative step count, post-operative step count, Body Mass Index (BMI) and Short Form 12 Physical Component Summaries (SF-12 PCS) were found. Mean step counts varied by 3,203 steps per day between obese and healthy weight patients, and 3,786 steps per day between those with higher and lower SF-12 PCS scores, suggesting the need for benchmarks for recovery that vary by patient pre-operative factors. A backwards stepwise regression model developed to provide patient specific step count predictions at 6 weeks had an R2 of 0.754, providing a robust patient specific benchmark for post-operative recovery, while population means from BMI and SF-12 subgroups provide a clinically practical alternative

Analysis of age-related changes in psychosine metabolism in the human brain

<div><p>α-Synuclein aggregation has been linked to Gaucher’s disease (GD) and Krabbe’s disease (KD), lysosomal conditions affecting glycosphingolipid metabolism. α-Synuclein pathology has been directly attributed to the dysregulation of glycosphingolipids in both conditions, specifically to increased galactosylsphingosine (psychosine) content in the context of KD. Furthermore, the gene (<i>GALC</i>) coding for the psychosine degrading enzyme galactosylceramidase (GALC), has recently been identified as a risk loci for Parkinson’s disease. However, it is unknown if changes in psychosine metabolism and GALC activity in the context of the aging human brain correlate with Parkinson’s disease. We investigated psychosine accumulation and GALC activity in the aging brain using fresh frozen post-mortem tissue from Parkinson’s (PD, n = 10), Alzheimer’s (AD, n = 10), and healthy control patients (n = 9), along with tissue from neuropsychiatric patients (schizophrenia, bipolar disorder and depression, n = 15 each). An expanded mutational analysis of PD (n = 20), AD (n = 10), and healthy controls (n = 30) examined if PD was correlated with carriers for severe <i>GALC</i> mutations. Psychosine content within the cerebral cortex of PD patients was elevated above control patients. Within all patients, psychosine displayed a significant (p<0.05) and robust regional distribution in the brain with higher levels in the white matter and substantia nigra. A mutational analysis revealed an increase in the incidence of severe <i>GALC</i> mutations within the PD patient population compared to the cohorts of Alzheimer’s patients and healthy controls tested. In addition to α-synuclein pathology identified in the KD brain, control patients identified as GALC mutational carriers or possessing a GALC pathogenic variant had evidence of α-synuclein pathology, indicating a possible correlation between α-synuclein pathology and dysregulation of psychosine metabolism in the adult brain. Carrier status for <i>GALC</i> mutations and prolonged exposure to increased psychosine could contribute to α-synuclein pathology, supporting psychosine metabolism by galactosylceramidase as a risk factor for Parkinson’s disease.</p></div

Immunohistological staining for α-synuclein.

<p>Immunohistological staining for α-synuclein was performed in brain tissue from 3 infantile KD patients (A-C), a late-onset KD patient (D), 2 patients found to be <i>GALC</i> mutation/variant carriers with no neurological disorders previously diagnosed (E,F), a Parkinson’s patient (G), and a healthy control with no <i>GALC</i> mutations (H). Accumulations of α-synuclein were observed in all Krabbe patients (A-D), the Parkinson’s brain (G), and also within the brains of <i>GALC</i> mutation/variant carriers (E,F). Accumulations in the infantile brains were abundant but smaller in size than the Lewy bodies observed in the Parkinson’s brain and the α-synuclein accumulations found in late-onset KD and <i>GALC</i> mutation carrier tissue. No significant α-synuclein accumulation was observed in the healthy diagnosed patient without GALC mutations (H). A secondary control experiment confirmed the specificity of the secondary antibody (I). Scale Bars: 100 μm.</p

GALC mutational effect on psychosine accumulation.

<p>Severe <i>GALC</i> mutations were identified in 2 patients diagnosed with Parkinson’s disease (PD). Psychosine levels in tissue from these two patients were elevated compared to that measured in tissue from PD patients without a severe <i>GALC</i> mutation.</p

GALC mutational analysis.

<p>GALC mutational analysis.</p

Psychosine content and GALC activity in neurodegenerative cohort.

<p>Psychosine concentration in white (WM) and gray (GM) cortical matter, substantia nigra, and caudate was examined in patients diagnosed with Parkinson’s, Alzheimer’s, Krabbe, or healthy controls. A-D) Psychosine was found to have a robust distribution, elevated in the white matter and substantia nigra of all patients. E-H) Psychosine in white and gray matter of Parkinson’s brains trended upwards compared to AD and healthy controls but did not reach significance. GALC activity in white (WM) and gray (GM) cortical matter, substantia nigra, and caudate was examined in patients diagnosed with Parkinsons’s, Alzheimer’s, Krabbe’s, or healthy controls. I-L) GALC activity was found to have a robust distribution, with lower levels found in the white matter and substantia nigra of all patients. M-P) GALC activity in white matter of Parkinson’s brains trended downwards compared to AD and healthy controls but did not reach significance. Q) Total cortical psychosine (summation of white and gray matter of cortex) found higher levels (p = 0.0554) of psychosine in Parkinson’s tissue compared to healthy controls. R) A total cortical GALC activity (summation of white and gray matter of cortex) did not find significantly different levels of GALC activity in Parkinson’s tissue compared to healthy controls. S) Correlation of psychosine to GALC activity as measured within the total cortical tissue of each group. (panel A-P: one way ANOVA with Tukey’s multiple comparison test, *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001, panel Q,R: t-test).</p

Psychosine content and GALC activity in neuropsychiatric cohort.

<p>Psychosine content and GALC activity was measured in white (WM) and gray (GM) matter of the cortex in patients diagnosed with schizophrenia, bipolar disorder, depression, or as age-matched healthy controls. A,D) The same robust distribution of psychosine and GALC activity was observed in this cohort as found in the older neurodegenerative cohort. B,C,E,F) No significant changes in psychosine content or GALC activity were observed between any disease state. (One way ANOVA with Tukey’s multiple comparison test, *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001).</p

Patient demographics.

<p>Patient demographics.</p

Psychosine content and GALC activity correlated with aging in mid-aged cohort.

<p>Psychosine content (A,B) and GALC activity (C,D) in cortical brain tissue from a combined cohort of mid-aged neuropsychiatric patients and healthy control patient tissue was compared to age at time of death. Linear regression revealed a significant positive correlation between psychosine content in the white matter (A) and age in this cohort, but a similar positive correlation did not reach significance in grey matter (B). However, a significant negative correlation between GALC activity and age was present in both white (C) and grey (D) matter. (Statistical test of slope significantly non-zero, *p<0.05).</p