Progressively developed myocardial apoptotic cell death during late phase of reperfusion.

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins and infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flow, measured by colored microspheres during ischemia. Necrotic cell death (TTC staining) was significantly increased during R, starting at 27 +/- 2% at 6 h R and increasing to 41 +/- 2% at 24 h R. There was no further change at 48 (37 +/- 3%) and 72 (36 +/- 6%) h R, respectively. TUNEL positive cells (% total normal nuclei) in the peri-necrotic zone progressively increased from 6 (26 +/- 2) to 24 (38 +/- 1), 48 (48 +/- 3) and 72 (59 +/- 4) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compared with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 +/- 0.8%) to 72 h R (78 +/- 2%), and increase in expression of pro-apoptotic proteins including Bax from 6 (30 +/- 3%) to 72 h R (66 +/- 3%), and p53 from 6 (12 +/- 1%) to 72 h R (91 +/- 2%), respectively. Immunohistochemical staining revealed that infiltrated neutrophils (mm(2) myocardium) were significantly correlated with development of necrotic and apoptotic cell death from 6 to 24 h R, respectively (P < 0.05), while large macrophage infiltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apoptosis; 3) inflammatory cells may play a role in triggering cell death during R. P < 0.05 vs. normal nuclei and tissue; P < 0.01 vs. 6 h R.

Dynamic progression of contractile and endothelial dysfunction and infarct extension in the late phase of reperfusion.

BACKGROUND: Myocardial injury during early reperfusion (R) has been well documented. However, the extent and time course of myocardial injury during late R are still unclear. The purpose of this study was to determine the extent of regional contractile and endothelial dysfunction and myocardial blood flow (MBF) defect as well as extension of infarction in association with neutrophil (PMN) actions during R. MATERIALS AND METHODS: A total of 29 dogs underwent a protocol of 1 h LAD ischemia followed by 6, 24, 48, and 72 h of R, respectively. Regional contractile function (sonomicrometry), MBF (colored microspheres), infarct size (triphenyltetrazolium chloride staining), and PMN localization (immunohistochemistry) were determined. RESULTS: Percentage segmental shortening at 6, 24, 48, and 72 h of R was significantly blunted (-1.8 +/- 1.2,* - 0.37 +/- 0. 6,* 0.04 +/- 0.2,* and 5.9 +/- 1.2* vs baseline 17.7 +/- 0.8). MBF (ml/min/g) was attenuated at 24 (0.27 +/- 0.03*), 48 (0.46 +/- 0. 07*), and 72 h of R (0.48 +/- 0.06*) vs 6 h of R (0.65 +/- 0.06). Infarct size increased from 6 (27 +/- 2%) to 24 h of R (41 +/- 2%*) with no further increase at 48 and 72 h of R, consistent with a peak of creatine kinase activity. PMN adherence (mm(2) endothelium) to left anterior descending coronary artery (LAD) segments was increased after 6 h of R (63 +/- 3*) vs nonischemic left circumflex coronary artery (LCX) segments (42 +/- 2) with a peak at 48 h of R (111 +/- 5*). Endothelium-dependent vascular relaxation in the LAD was also blunted at 6, 24, and 48 h of R. Immunostaining revealed CD18-positive PMNs were mainly accumulated in intravascular space during 6 h of R with an increase in migration of PMNs seen at 24 h of R, consistent with a peak of myeloperoxidase release. Myeloperoxidase activity in a given area at risk sample was significantly correlated with infarct extension during the first 24 h of R. CONCLUSIONS: These results provide pathologic evidence for myocardial injury during the extended R and a basis for exploration of interventions designed to limit myocardial injury after ischemia. (*P < 0.05 vs Baseline, 6 h of R and LCX segments.)

Ischemic preconditioning reduces neutrophil accumulation and myocardial apoptosis.

BACKGROUND: This study tested the hypothesis that ischemic preconditioning (IP) inhibits myocardial apoptosis after a short period of ischemia and reperfusion. METHODS: In 9 anesthetized dogs, the left anterior descending (LAD) coronary artery was occluded for 30 min and reperfused for 3 h (control), while in 9 others, LAD occlusion was preceded by 5 min of occlusion and 5 min of reperfusion (IP). DNA from frozen myocardial tissue samples was extracted, and apoptosis were identified as "ladders" by agarose gel electrophoresis or confirmed histologically using the terminal transferase UTP nick end-labeling (TUNEL) assay. Neutrophil accumulation was detected by measuring cardiac myeloperoxidase activity. RESULTS: Thirty minutes of LAD occlusion caused a significant decrease in blood flow (colored microspheres), which was comparable between groups. In the control group, DNA ladders occurred in the area at risk (AAR) in six out nine experiments. In contrast, DNA laddering in the AAR was not observed in any of the IP group. AAR in the control group showed a greater percentage of apoptotic cells than IP (6.7 +/- 0.9% vs 1.2 +/- 0.2%; p < 0.01). Cardiac myeloperoxidase activity (U/g tissue) was significantly reduced from 0.07 +/- 0.004 in control to 0.04 +/- 0.01 in IP group (p < 0.05). CONCLUSIONS: We conclude that ischemic preconditioning attenuates apoptosis and neutrophil accumulation in the AAR in a model of nonlethal acute ischemia and reperfusion.

An unusual cervical carcinoma showing exception to epitheliotropism of human papillomavirus.

Human papillomaviruses (HPV) infect epithelial tissues but have not been previously detected within mesenchymal cells. During a systematic investigation of FIGO stage Ib cervical cancers with colorimetric in situ hybridization, we detected HPV 16 DNA within the stromal compartment of an unusual undifferentiated carcinoma. The mesenchymal nature of the HPV-containing cells was confirmed by immunohistochemistry and electron microscopy. No viral particles were identified. Sequencing the majority of the HPV 16 genome identified few changes from the revised reference clone; all previously reported in other HPV 16 variants. These viral changes are unlikely to explain the exceptional mesenchymal localization of the HPV 16 DNA in this case.

Skin necrosis indicating antiphospholipid syndrome in patient with AIDS.

Although antiphospholipid antibodies have been detected in patients with human immunodeficiency virus (HIV) disease, the clinical manifestations of the antiphospholipid syndrome are extremely rare. We describe a woman with acquired immunodeficiency syndrome (AIDS) and elevated antiphospholipid antibodies who developed necrotic skin lesions and was subsequently shown to have antiphospholipid syndrome. This finding contradicts the general belief that such antibodies are not clinically significant in patients with HIV disease. We follow the report with a brief description of the antiphospholipid syndrome and its relation to AIDS.

Disseminated microsporidiosis (Encephalitozoon hellem) and acquired immunodeficiency syndrome. Autopsy evidence for respiratory acquisition.

Microsporidia are obligate intracellular protozoal parasites that infect a variety of cell types in a broad range of invertebrates and vertebrates. They have recently come to medical attention due to the increased frequency with which members of two microsporidian genera, Enterocytozoon and Encephalitozoon, are being diagnosed in patients with the acquired immunodeficiency syndrome (AIDS). The majority of published reports of human microsporidiosis describe Enterocytozoon infection of small intestinal enterocytes. In addition, a growing number of AIDS patients have been identified with infection due to the two species of Encephalitozoon-Encephalitozoon cuniculi and Encephalitozoon hellem, observed in conjunctival, corneal, and, recently, sinonasal tissues. However, there are scant data regarding the systemic pathology and epidemiology of these infections. This article describes a patient with AIDS who died with systemic Encephalitozoon infection. The etiologic microsporidian was found to be E hellem by using antemortem biochemical and antigenic analyses. A complete autopsy, the first to be reported in a patient with this infection, revealed organisms in the eyes, urinary tract, and respiratory tract. A surprising observation was the occurrence of numerous organisms within the lining epithelium of almost the entire length of the tracheobronchial tree, suggestive of respiratory acquisition. Detailed light and electron microscopic findings and the biological and diagnostic features of microsporidiosis are discussed.

The Ehlers-Danlos syndrome: recognition, characterization, and importance of a milder variant of the classic form. A preliminary study.

The diagnosis of Ehlers-Danlos syndrome is based on the clinical criteria of joint hypermobility, increased skin extensibility, abnormal scarring, and easy bruisibility. The literature reports this syndrome as rare, yet our experience dictated to the contrary. The present study of the prevalence of Ehlers-Danlos syndrome in a general dermatology population revealed (1) a milder variant of the classic mitis form of Ehlers-Danlos syndrome was common and present in 9% of the population studied, (2) these patients could be easily identified by the use of a defined clinical scoring system, and (3) a statistically significant association existed between clinical findings in Ehlers-Danlos syndrome and electron microscopic collagen abnormalities. The identification of this syndrome may be important prognostically in patients with diseases or conditions in which collagen plays a major role, such as joint disease (dislocations), bruising disease (pigmented purpura), and potentially scarring diseases (acne, patients undergoing cutaneous surgery).