Emmy Chatzigianni

Time-dependent mitochondrial-mediated programmed neuronal cell death prolongs survival in sepsis.

Objective:To investigate whether apoptosis is a possible mechanism of brain dysfunction occurring in septic syndrome. Design:Experimental prospective study. Setting:Laboratory of Surgical Research at the University of Athens. Subjects:Male pathogen-free Wistar rats. Interventions:Rats (n = 112) were subjected to sepsis by cecal ligation and puncture. Sham-operated animals (n = 40) underwent the same procedure but without ligation or puncture. Septic animals were either randomly divided (n = 62) in six groups and studied at 6, 12, 24, 36, 48, and 60 hrs after the operation or monitored (n = 50) for 48 hrs as a survival study group. Sham-operated animals were killed at 6, 12, 24, 36, 48, and 60 hrs after the procedure. Brain and cecum were then removed and postfixed in paraffin sections. Apoptosis was evaluated by light microscopy in hematoxylin and eosin-stained specimens and by transmission electron microscopy. In paraffin-embedded sections, immunostaining for bax, bcl-2, cytochrome c, and caspase-8 was done. Measurements and Main Results:In septic rats, increased apoptosis was detected in neurons of the CA1 region of the hippocampus, in choroid plexus, and in Purkinje cells of the cerebellum. Bax immunopositivity was found decreased after the septic insult (p = .03). Bax immunoreactivity was altered as the septic syndrome evolved; it was up-regulated in the early stages (6–12 hrs) and progressively decreased in the late phases (p = .001). Cytochrome c presented a similar regional pattern of expression and was found to be the sole gene marker carrying an independent prognostic role (p = .03). Both bcl-2 and caspase-8 expression remained at constant levels at all times evaluated. Conclusions:There is evidence that more neurons undergo apoptosis during sepsis than in normal brain tissue in certain sites where the blood-brain barrier is compromised. In this phenomenon, mitochondrial gene regulators such as bax and products such as cytochrome c seem to play important regulating and prognostic roles, respectively.

Administration of human protein C improves survival in an experimental model of sepsis

Objective:Study the effect of human protein C (PC) concentrate administration on organ damage and survival in septic rats. Design:Animal study. Setting:University laboratory. Subjects:Male Wistar rats. Interventions:Cecal ligation and puncture (CLP) was performed in 210 rats. Rats were randomly assigned to receive either human protein C (PC) IV 1, 7, and 13 hrs after CLP (CLP+PC) or placebo (CLP). Septic animals were again randomized in a survival group (CLP: n = 50 and CLP+PC: n = 40) that was monitored for 60 hrs and time groups (CLP: n = 60 and CLP+PC: n = 60) that were killed at 6, 12, 24, 36, 48, and 60 hrs after CLP. Brain, heart, lung, liver, kidney, gastric, and colon tissue were removed and postfixed in paraffin sections. Measurements and Main Results:PC infusion increased PC serum levels in early sepsis (median 7.25) compared with late sepsis (median 2, p = .001). Activated protein C/a1-antitrypsin complex levels in the CLP+PC group were significantly increased in late sepsis (60 hrs after CLP) compared with early sepsis (6, 12, and 24 hrs after CLP, p = .009, p = .004, and p = .008, respectively) and to late septic CLP and normal rats (p = .005 and p = .007, respectively). Their IL-6 and tumor necrosis factor a plasma levels were decreased (by 27% and 25%, respectively) at 6 hrs compared with placebo (p = .008 and p = .016). Their serum PC levels were also decreased in CLP+PC survivors compared with nonsurvivors of the same group (median = 1.5 vs. median = 7, p = .001). Apoptosis was reduced in brain (10% vs. 77.8%, p < .001), stomach (66.7% vs. 100%, p < .002) and intestine (33.3% vs. 85.2%, p < .001) compared with placebo. Finally, the survival of septic rats treated with human PC was significantly increased compared with placebo (75% vs. 54%, p = .033). Conclusions:Human Protein C administration increased survival in septic rats, decreased plasma inflammatory cytokines levels and tissue injury in vital organs.

Apoptotic death of renal tubular cells in experimental sepsis.

BACKGROUND AND PURPOSE Renal dysfunction attributable to sepsis was long considered a result of hemodynamic instability and subsequent local ischemia. Recent data show that apoptosis may be implicated also. The purpose of this study was to evaluate the role of apoptosis and the expression of the bax, bcl-2, caspase-8, and cytochrome c proteins in the renal parenchymal cells of rats with sepsis. METHODS Sepsis was induced using cecal ligation and puncture (CLP) in 62 male Wistar rats, which were euthanized 6, 12, 24, 36, 48, or 60 h later. Ten sham-treated animals served as a control group. Another group of 50 animals were subjected to CLP and then supervised for 60 h. Renal apoptosis was evaluated using light and transmission electron microscopy, in situ nick-end labeling (TUNEL), and flow cytometry using 7-amino-actinomycin D (7-AAD). Caspase-mediated apoptosis was assessed using M30 antibody. The expression of the apoptosis-regulator proteins B-cell lymphoma 2 (bcl-2), bcl-2-associated x protein (bax), caspase-8, and cytochrome c was detected immunohistochemically. RESULTS Sepsis increased inflammatory infiltration (p < 0.001) and necrosis (p < 0.001) in renal parenchyma. Apoptosis was significantly more common than in the kidneys of control animals (p = 0.02). Nuclei stained by the TUNEL technique were predominant in the tubular cells of non-survivors (p = 0.05). The time distribution of all types of cell death was increased significantly 6 h after the induction of sepsis, and declined subsequently. Caspase-generated cytokeratin 18 (CK18) new epitope (M30) was significantly more abundant in the kidneys of animals with sepsis than in control rats, with peaks at 6 h and 60 h post-procedure (p < 0.001). In addition, cells initiating apoptosis were significantly more common at 6 h than at 48 h post-CLP (p = 0.014). Caspase-8 protein immunodetection followed the same time pattern as cell death, increasing as early as 6 h post-CLP and decreasing thereafter (p = 0.013). Bax protein expression was elevated significantly early in the course of sepsis (p = 0.037), whereas the other members of the mitochondrial-dependent pathway remained constant. Animals dying from sepsis had a significantly greater prevalence of bax- (p = 0.037) and caspase-8- (p = 0.031) immunoreactive renal cells. CONCLUSION Apoptosis in renal tissue was significantly more common in animals with sepsis than in controls. The time distribution of cell death markers showed a consistent pattern, making early sepsis the likely initiator of the apoptotic events.

Administration of Human Protein-C concentrate prevents apoptotic brain cell death after experimental sepsis

Activated Protein C renders anti-apoptotic properties in neurons and endothelial cells. The aim of the present study was to evaluate the in vivo cytoprotective role of Protein C zymogen (PC) administration in septic rat brain. Male Wistar rats (n=60) were subjected to sepsis via Cecal Ligation and Puncture (CLP). Animals were randomly divided either to receive 100 IU/kg human PC concentrate at 1, 7 and 13 h post CLP (CLP+PC group) or placebo treatment (CLP group). At pre-specified time points (6, 12, 24, 36, 48 and 60 h post CLP) five animals from either group were euthanized and the brain tissue was removed. Apoptosis in both neurons (Neu-N+) and astroglia (GFAP+) was assessed by flow cytometry using 7-aminoactinomycin D (7AAD). Immunohistochemical detection of cleaved caspase 3, bax, bcl-2, cytochrome c and caspase 8 was also performed. PC treated animals had significantly reduced apoptosis in neurons at 6 and 24 h post CLP (p=0.04 and p=0.016 respectively) and necrosis at 6, 12 and 60 h post CLP (p=0.008, p=0.012 and p=0.032 respectively). Astrocyte necrosis was also decreased in septic rats receiving PC (6, 12 and 60 h post CLP p=0.008, p=0.016 and p=0.008 respectively). In addition, active caspase 3, bax, cytochrome c and caspase 8 expression was significantly decreased during early sepsis (6-36 h) while bcl-2 expression was increased (24 h p=0.001 and 60 h p=0.001) in the PC treated animals compared to placebo. PC concentrate administration in experimental sepsis produced a time dependent inhibition of apoptosis in rat neurons and astrocytes. The inhibition of sepsis related apoptosis concerned both the mitochondrial and caspase 8 dependent pathways.

Lectin histochemistry as a predictor of dysplasia grade in colorectal adenomas

Lectins are sugar-binding proteins that bind to specific cellular carbohydrates, commonly affecting cellular physiology. Phaseolus vulgaris leucoagglutinin (PHA), ulex europaeus isoagglutinin-I (UEA-I), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) are among the most well studied lectins in various tissues. The purpose of this study was to detect the above lectins’ binding sites and so examine alterations in glycoconjugate expression in neoplastic cells of 52 colorectal adenomas with various clinicopathologic characteristics and proliferation rates. Lectin histochemistry was performed in paraffin sections with and without neuraminidase treatment. Proliferative fraction was determined by immunolabelling for Proliferating Cell Nuclear Antigen. PHA was the more frequently positive lectin in the examined specimens; however, it was simultaneously detected in normal colonic mucosa and so was WGA. The frequency of high grade dysplasia was significantly greater in older patients and in samples with UEA-I positivity without neuraminidase pretreatment. UEA-I-reactive adenomas were generally characterized by high cell proliferation rates. A statistical model based on patients’ age and UEA-I binding without neuraminidase treatment can generally predict grade of dysplasia in 83% of adenomas and particularly high grade dysplasia in up to 93% of adenomas; so, such a model may be potentially useful for the early detection of neoplasia, for instance in exfoliative cells from the large intestine.

Sepsis: Prognostic Role of Apoptosis Regulators in Gastrointestinal Cells

BackgroundIntestinal epithelial cell apoptosis has been reported in sepsis as a mechanism of organ failure. The aim of this study was to clarify the role of apoptosis-regulating proteins (bcl-2, bax, cytochrome-c, and caspase-8) in septic rats by studying their expression in gastric and intestinal epithelial cells.MethodsAdult Wistar rats were subjected to the cecal ligation and puncture (CLP) model of sepsis and randomly divided into two study groups. Sixty-two animals were sacrificed 6, 12, 24, 36, 48, and 60 h post-procedure, and 50 animals served as the survival study group. Sham-operated animals (n = 40) were used as controls. Gastric and intestinal tissue was excised, and immunohistochemical detection of bcl-2, bax, cytochrome-c, and caspase-8 protein expression was performed.ResultsIn gastric mucosa, sepsis induced upregulation of bax and downregulation of caspase-8 expression (p = 0.053 and p = 0.05, respectively). Both bax and caspase-8 were upregulated as early as 6 h post CLP and progressively decreased (p = 0.001, p = 0.004 respectively). In contrast, the expression of the anti-apoptotic bcl-2 was upregulated progressively during the sepsis syndrome (p = 0.03). In intestine, sepsis induced a fourfold upregulation of the cytoprotective bcl-2 (p = 0.0001), accompanied by a remarkable increase in bax (p = 0.002) and caspase-8 (p = 0.0001) expression and a decrease in cytochrome-c expression (p = 0.02). The time distribution of the apoptosis regulators followed the same pattern as in gastric tissue, showing an upregulation of the proapoptotic bax and cytochrome c (p = 0.04) during the early phases and a progressively increased expression of bcl-2 during the late phases (p = 0.0001). Bax expression in gastric epithelium of subjects with septic syndrome was detrimental to survival (p = 0.0001), whereas the expression of the cytoprotective bcl-2 in intestinal epithelium appeared to favor a good prognosis (p = 0.0001).ConclusionsSepsis results in alterations of apoptosis regulators in gastrointestinal cells. Alterations of bax and bcl-2 expression in gastric and intestinal epithelial cells may predict the outcome in septic rats.

Increased apoptosis in the alveolar microenvironment of the healthy human lung.

BACKGROUND Apoptosis represents a physiological clearance mechanism in human tissues. The role of apoptosis has not been examined in normal lung cell populations, such as alveolar macrophages and polymorphonuclear cells. What is the percentage, as well as the role, of apoptosis in the alveolar microenvironment of the healthy human lung? PATIENTS AND METHODS Bronchoalveolar lavage was obtained from 21 volunteers without lung disease. The specimens were analyzed using: Annexin V binding, DNA laddering, light microscopy and immunohistochemistry for bcl-2 expression. RESULTS Apoptosis of the total bronchoalveolar lavage cell population was 51.2%. Both alveolar macrophages and polymorphonuclear cells had a high apoptotic rate (62.1% and 48.3%, respectively) as determined by Annexin V binding. These findings were further confirmed using morphological criteria for apoptosis and gel electrophoresis for DNA fragmentation. In the majority of the individuals examined, (8 out of 21), the bcl-2 gene was expressed in the lymphocyte population mainly. CONCLUSIONS The percentage of apoptosis in lung cells of healthy humans is high. Apoptosis plays a key role in normal lung cell death. It appears to be the mechanism that opposes cell proliferation by eliminating, aged or damaged cells thus facilitating the process of lung remodeling.