SummaryIntracellular 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-c (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.

Capillary basement membrane (CBM) thickening is an ultrastructural hallmark in diabetic patients and in animal models of diabetes. However, the wide variety of tissues sampled and diverse methods employed have made the interpretation of thickness data difficult. We showed previously that acellular glomerular BMs in OVE26 transgenic diabetic mice were thickened beyond normal age-related thickening, and in the current study we hypothesized that other microvascular BMs likewise would show increased widths relative to age-matched controls. Accordingly, a series of tissues, including skeletal and cardiac muscle, ocular retina and choriod, peripheral nerve, lung, pancreas, and renal glomerulus was collected from 300 -350-day-old normal and transgenic mice. Transmission electron micrographs of cross sections through capillary walls were prepared, and CBM thickness (CBMT) was determined by the "orthogonal intercept" method. Morphometric analyses showed highly variable transgene-related BMT increases in the sampled tissues, with glomerular BM showing by far the greatest increase (ϩ87%). Significant thickness increases were also seen in the retina, pulmonary alveolus, and thoracoabdominal diaphragm. BMT increases were not universal; however, most were modestly widened, and those that were thickest in controls generally showed the greatest increase. Although the pathogenesis of diabetes-related increases in CBM is poorly understood, data in the current study showed that in OVE26 transgenic mice increased BMT was a frequent concomitant of hyperglycemia. Accordingly, it seems likely that hyperglycemia-induced microvascular damage may be a contributing factor in diabetic BM disease, and that microvessel cellular and extracellular heterogeneity may limit the extent of CBM thickening in diverse tissues. Anat Rec Part A 271A: 332-341, 2003. © 2003 Key words: diabetes; basement membrane thickness; electron microscopy; transgenic diabetic mice OVE26 transgenic mice are diabetic as a result of the specific overexpression of calmodulin in pancreatic beta cells (Epstein et al., 1989(Epstein et al., , 1992. Histological studies show that this results in beta cell destruction early in life, and by 30 -55 days of age glucose values exceed 450 mg/dl. At the same time, insulin levels are reduced to 42% of control levels. OVE26 mice are a particularly valuable model of diabetes, because beta cell toxins need not be administered to induce the disease, and the animals can survive well over a year without exogenous insulin administration (probably because of a small amount of residual insulin secretion). Moreover, because both males and females are fertile for several months without insulin therapy, the transgenic line has been maintained continuously for more than 14 years.Several studies have shown that the OVE26 diabetic model is useful for examining chronic complications of diabetes. This was particularly evident in a previous study of the kidney (Carlson et al., 1997) in which transmission electron microscopy (TEM) morphometry sh...

Recent studies show that podocyte nuclear density (N V ) and numbers of renal podocytes per glomerulus (N) are altered in experimental and spontaneous diabetes mellitus. N V 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 (V G ), N V , and N. As expected, mean V G 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, N V and N were not statistically distinguishable in OVE26 and control mice, while at 150 days, N V was significantly reduced in diabetics but not N. In 450-day-old OVE26 animals, however, N V 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. 2007 Wiley-Liss, Inc.

These results provide evidence of oxidative damage to podocytes induces primary diabetic nephropathic features including severe and sustained albuminuria, specific glomerular filtration barrier damage and alterations in glomerular endothelial and mesangial cell number. Importantly, these diabetic complications are significantly mitigated by podocyte targeted metallothionein overexpression.

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 (128% and 139.5%, 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 intraand perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury. Anat Rec,