Donna I. Laturnus

Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages

Intracellular bacteria have been shown to cause autophagy, which impacts infectious outcomes, whereas extracellular bacteria have not been reported to activate autophagy. Here, we demonstrate that Pseudomonas aeruginosa, a Gram-negative extracellular bacterium, activates autophagy with considerably increased LC3 punctation in both an alveolar macrophage cell line (MH-S) and primary alveolar macrophages. Using the LC3 Gly120 mutant, we successfully demonstrated a hallmark of autophagy, conjugation of LC3 to phosphatidylethanolamine (PE). The accumulation of typical autophagosomes with double membranes was identified morphologically by transmission electron microscopy (TEM). Furthermore, the increase of PE-conjugated LC3 was indeed induced by infection rather than inhibition of lysosome degradation. P. aeruginosa induced autophagy through the classical beclin-1–Atg7–Atg5 pathway as determined by specific siRNA analysis. Rapamycin and IFN-γ (autophagy inducers) augmented bacterial clearance, whereas beclin-1 and Atg5 knockdown reduced intracellular bacteria. Thus, P. aeruginosa-induced autophagy represents a host protective mechanism, providing new insight into the pathogenesis of this infection.

Podocyte Loss in Aging OVE26 Diabetic Mice

Recent studies show that podocyte nuclear density (NV) and numbers of renal podocytes per glomerulus (N) are altered in experimental and spontaneous diabetes mellitus. NV and N are generally reduced, and it has been hypothesized that these morphological changes may relate to the loss of glomerular permselectivity in diabetic nephropathy (DN). In the current study, OVE26 transgenic diabetic mice and age‐matched (FVB) controls (60, 150, or 450 days) were fixed by vascular perfusion and renal cortical tissues were prepared for morphometric analyses. ImageJ software and point counting analyses were carried out on light and transmission electron micrographs to determine glomerular volume (VG), NV, and N. As expected, mean VG in OVE26 mice increased substantially (∼134%) over the course of the study and was significantly increased over FVB mice at all ages. At 60 days, NV and N were not statistically distinguishable in OVE26 and control mice, while at 150 days, NV was significantly reduced in diabetics but not N. In 450‐day‐old OVE26 animals, however, NV and N were both significantly decreased (∼231% and ∼99%, respectively) relative to age‐matched FVB mice. These data suggest that in the OVE26 model of diabetes, significant podocyte loss occurs relatively late in the course of the disease. Moreover, it seems possible that these podocytic changes could play a role in sustaining the increased permeability of the blood–urine barrier in the later stages of diabetic renal decompensation. Anat Rec, 291:114–121, 2007.

Podocyte‐specific overexpression of metallothionein mitigates diabetic complications in the glomerular filtration barrier and glomerular histoarchitecture: a transmission electron microscopy stereometric analysis

We previously demonstrated that cellular and extracellular components of the blood–urine barrier in renal glomeruli are susceptible to damage in OVE transgenic mice, a valuable model of human diabetic nephropathy that expresses profound albuminuria.

Diabetic basement membrane thickening does not occur in myocardial capillaries of transgenic mice when metallothionein is overexpressed in cardiac myocytes.

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension‐induced athero‐ or arteriosclerosis. This may be due either to generalized vascular disease, tissue‐based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia‐driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi‐transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ∼3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra‐ and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury. Anat Rec, 296:480–487, 2013.

Significant Retinal Capillary Basement Membrane Thickening in Hyperglycemic and Normoglycemic Diabetic-prone (DP) BB Wistar Rats

The diabetic-prone BioBreeding Wistar (BB/DP) rat is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 90% of the animals (BB/DPh) are hyperglycemic by 90–120 days of age, while the remaining ~10% (BB/DPn) and diabetes-resistant rats (BB/DR) are normoglycemic for life. The transmission electron microscope data from this study demonstrate expected significant age- and diabetes-related increases in retinal capillary basement membrane (RCBM) widths in (BB/DPh) rats relative to BB/DR animals. However, the data show, for the first time, an unexpected significant RCBM thickening in (BB/DPn) rats compared to BB/DR animals at 6 months and 1 year post-onset of hyperglycemia.

Host Genetic Variations and Sex Differences Potentiate Predisposition, Severity, and Outcomes of Group A Streptococcus-Mediated Necrotizing Soft Tissue Infections

ABSTRACT Host genetic variations play an important role in several pathogenic diseases, and we previously provided strong evidence that these genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive group A Streptococcus (GAS) patients, including sepsis and necrotizing soft tissue infections (NSTIs). The goal of the present study was to investigate how genetic variations and sex differences among four commonly used mouse strains contribute to variation in severity, manifestations, and outcomes of NSTIs. DBA/2J mice were more susceptible to NSTIs than C57BL/6J, BALB/c, and CD-1 mice, as exhibited by significantly greater bacteremia, excessive dissemination to the spleen, and significantly higher mortality. Differences in the sex of the mice also contributed to differences in disease severity and outcomes: DBA/2J female mice were relatively resistant compared to their male counterparts. However, DBA/2J mice exhibited minimal weight loss and developed smaller lesions than did the aforementioned strains. Moreover, at 48 h after infection, compared with C57BL/6J mice, DBA/2J mice had increased bacteremia, excessive dissemination to the spleen, and excessive concentrations of inflammatory cytokines and chemokines. These results indicate that variations in the host genetic context as well as sex play a dominant role in determining the severity of and susceptibility to GAS NSTIs.

Renoprotection from Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age‐matched OVE diabetic mice to produce bi‐transgenic OVE‐JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE‐JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day‐old OVE‐JTMT mice, blood glucose and HbA1c were indistinguishable from age‐matched OVE mice. However, endothelial‐specific MT overexpression in OVE‐JTMT mice mitigated several DN complications including significantly increased non‐fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE‐JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017.