Agapi Kataki

Lymphocyte apoptosis after major abdominal surgery is not influenced by anesthetic technique: a comparative study of general anesthesia versus combined general and epidural analgesia

STUDY OBJECTIVE To examine the influence of abdominal colectomy with combined general anesthesia and epidural analgesia versus general anesthesia on apoptosis of circulating lymphocytes. DESIGN Prospective, randomized, clinical comparison study. SETTING Tertiary-care general hospital. PATIENTS 40 ASA physical status I and II patients undergoing elective open colectomy for nonmetastatic colon carcinoma. INTERVENTIONS Patients were randomly allocated to two groups to receiver either general anesthesia alone (Group G) or general anesthesia combined with epidural analgesia (Group C). Group C comprised 21 patients while 19 patients constituted Group G. All patients underwent median longitudinal laparotomy. MEASUREMENTS Blood samples were collected preoperatively and 24 hours postoperatively for measurement of lymphocyte apoptosis, serum cortisol, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). MAIN RESULTS There were no differences between the two groups in age, weight, or duration of surgery. No significant alterations in total lymphocyte counts, as well as in lymphocyte subpopulations (early apoptotic, late apoptotic, viable, and necrotic), were observed between the general and combined anesthesia groups. Cortisol, ESR, and CRP were significantly increased postoperatively in both groups. Group C presented with lower serum cortisol levels postoperatively than Group G (b = -5.38, CI95%: -8.72 to -2.05, P = 0.002). CONCLUSIONS Epidural block could not suppress postoperative lymphocyte apoptosis, increases in cortisol, CRP, or ESR compared with general anesthesia.

Long-term primary cultures of human adult atrial cardiac myocytes: cell viability, structural properties and BNP secretion in vitro.

BACKGROUND Human adult cardiomyocytes (CM) have been used in short-term cultures for in vitro studies of the adult myocardium. However, little information is available regarding human adult CMs cultured for long term (>2 weeks). METHODS Human adult CMs were isolated from atrial specimens of 43 patients undergoing cardiopulmonary bypass surgery. Cell viability, cytoskeletal properties, intercellular junctional mediators and responsiveness to extracellular stimuli were monitored in CM cultures for 8 weeks. RESULTS Absolute numbers of CMs decreased through the first 2 weeks, with substantially lower rates of cell loss thereafter. Apoptosis predominated over necrosis as the principal mode of cell death, affecting 4.1+/-1.6% of freshly dissociated cells, that declined in culture (3.6+/-1.0% week 1, 1.3+/-0.5% week 2). CMs maintained rod-shaped morphology and cross-striated expression pattern of sarcomeric proteins desmin and beta-myosin heavy chain for the first 4 weeks. Levels of desmin remained stable on first 3 weeks, but declined thereafter. CMs expressed cardiac-specific adherence molecule N-cadherin throughout the culture duration, indicating conserved contractile potential. CMs remained functional early in culture, as indicated by BNP secretion, with maximal levels on 1st week that declined gradually by week 4. Cell responsiveness to metabolic stresses (serum deprivation) was detected, inducing an early (6 h) 1.8-fold increase in levels of BNP. CONCLUSION Long-term cultured human adult CMs maintain morphological integrity, adult-type cytoskeletal protein expression, cell-cell communication potential and functionality for 3-4 weeks in vitro.

Breast cancer nodal metastasis correlates with tumour and lymph node methylation profiles of Caveolin-1 and CXCR4

Abstract DNA methylation is the best characterised epigenetic change so far. However, its role in breast cancer metastasis has not as yet been elucidated. The aim of this study was to investigate the differences between the methylation profiles characterising primary tumours and their corresponding positive or negative for metastasis lymph nodes (LN) and correlate these with tumour metastatic potential. Methylation signatures of Caveolin-1, CXCR4, RAR-β, Cyclin D2 and Twist gene promoters were studied in 30 breast cancer primary lesions and their corresponding metastasis-free and tumour-infiltrated LN with Methylation-Specific PCR. CXCR4 and Caveolin-1 expression was further studied by immunohistochemistry. Tumours were typified by methylation of RAR-β and hypermethylation of Cyclin-D2 and Twist gene promoters. Tumour patterns were highly conserved in tumour-infiltrated LN. CXCR4 and Caveolin-1 promoter methylation patterns differentiated between node-negative and metastatic tumours. Nodal metastasis was associated with tumour and lymph node profiles of extended methylation of Caveolin-1 and lack of CXCR4 hypermethylation. Immunodetection studies verified CXCR4 and Caveolin-1 hypermethylation as gene silencing mechanism. Absence of Caveolin-1 expression in stromal cells associated with tumour aggressiveness while strong Caveolin-1 expression in tumour cells correlated with decreased 7-year disease-free survival. Methylation-mediated activation of CXCR4 and inactivation of Caveolin-1 was linked with nodal metastasis while intratumoral Caveolin-1 expression heterogeneity correlated with disease progression. This evidence contributes to the better understanding and, thereby, therapeutic management of breast cancer metastasis process.

Alternations of 14‐3‐3 θ and β protein levels in brain during experimental sepsis

The 14‐3‐3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14‐3‐3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time‐dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14‐3‐3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis‐induced, time‐dependent alteration of 14‐3‐3 θ and β isoforms in both Neun+ and GFAP+ cells. 14‐3‐3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down‐regulation of θ isoform in alive neurons and astrocytes. The phospho‐P38 (pP38) MAP kinase levels were altered in a time‐dependent manner during sepsis, presenting a peak at 6 hr post‐CLP. A significant correlation between the two isoforms of 14‐3‐3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia‐like cells showed intense immunohistochemical reactivity for 14‐3‐3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14‐3‐3 θ and β isoforms in a time‐, cell‐, and topography‐dependent manner.

An in vitro model for investigating human autologous neuronal–astrocyte and immune cell interactions underlying neurodegenerative and immunosuppressive processes in neuropathy

Primary mixed neuronal-astrocytic cultures were established from human brain tissues from elective surgical procedures and maintained in vitro for over 21 days. The majority of cells (a) expressed morphological and cytoskeletal markers of differentiated neurons (MAP2a&b; Tau) or astrocytes (GFAP) in anticipated proportion (1:2), and (b) regenerated synaptic connections and neural-astrocytic associations. Co-cultures with autologous blood leukocytes established that alterations in the viability (by Annexin V/PI) of brain and immune cells over 3 days were indicative of neurodegenerative or immunosuppressive processes. During co-culture, B-cells (CD19+) remained largely unaffected while T-lymphocytes (CD3+) and monocytes (CD14+) declined, consistent with immunosuppressive process. Indications of immunosuppression were not observed when immune cells were maintained in free of neural cells medium collected from neuro-cultures. Decline in brain cell viability in neuro-immune co-cultures may be associated with density of activated monocytes (HLA-DR+/CD14+), consistent with neurodegenerative process. Our findings, though preliminary and associated with significant variability between individuals, establish an approach to investigate neuroimmune pathology in humans.

The spectrum of myocardial homeostasis mechanisms in the settings of cardiac surgery procedures (Review)

Classic cardiac surgery, determined through the function of cardiopulmonary bypass machine and myocardial cardioplegic arrest, represents the most controlled scenario for cardiomyocyte homeostatic disturbances due to systemic inflammatory response and myocardial reperfusion injury. An increasing number of studies have demonstrated that myocardial cell homeostasis in cardiac surgery procedures is a sequence of molecularly interrelated and overlapping mechanisms in the form of apoptosis, autophagy and necrosis, which are activated by a plethora of induced inflammatory mediators and gene-related signaling pathways. In this study, we outline the molecular mechanisms of the cardiomyocyte adaptive homeostatic process and the associated clinical implications, in the settings of classic cardiac surgery procedures.

Changes in T-Lymphocytes' Viability After Laparoscopic Versus Open Cholecystectomy

Laparoscopic surgery results in decreased immune and metabolic stress response compared to open surgery. Our aim was to evaluate the suspension of host immune defense in terms of apoptosis, necrosis, and survival of peripheral T-lymphocytes in patients undergoing laparoscopic versus open cholecystectomy. Apoptosis, necrosis and viability of peripheral T-lymphocytes were measured preoperatively and postoperatively by means of flow cytometry in 27 patients undergoing laparoscopic cholecystectomy and 25 undergoing open cholecystectomy. White cell count, CRP, and serum glucose levels were also measured. Viable peripheral T-lymphocytes were significantly decreased in open cholecystectomy (P = 0.02), while their late apoptotic as well as the overall necrotic rate were significantly increased (P = 0.01 and P < 0.01, respectively). Open cholecystectomy was also associated with lower levels of surviving circulating T-lymphocytes (P = 0.01) and higher percentage of necrotic T lymphocytes (P = 0.03) 24 hours postoperatively compared to laparoscopic cholecystectomy. Serum CRP was increased 24 hours after open cholecystectomy (P = 0.04). All differences failed to sustain more than 48 hours postoperatively. Increased viability and decreased necrosis of circulating T-lymphocytes were observed in laparoscopic cholecystectomy. Necrosis (and not apoptosis) seems to be the predominant pathway of T-lymphocyte death in open cholecystectomy, in a process reaching its peak at 24 hours and further attenuating 48 hours postoperatively.

14-3-3 gene expression exerts isoform-dependent functions in sinonasal pathophysiology

The expression profiles of 14-3-3β and θ isoforms, known to exert both oncogenic and antiapoptotic effects, were assessed in different entities of nasal pathophysiology. Flow cytometry and immunohistochemistry were used on paraffin-embedded sections of 51 inverted papillomas (IP), 26 nasal polyps (NP), 9 polyps with IP (NPIP) and 10 specimens of normal epithelium (NE). 14-3-3β expression was significantly upregulated in IP as compared with both NP (p=0.015) and NE (p=0.002). 14-3-3β was also increased in NPIP as compared with NE (p=0.008). 14-3-3β cytoplasmic staining was more pronounced in basal cells of the respiratory epithelium although serous glands and the vascular system were often positive as well. High 14-3-3β immunopositivity in IP patients concurred with increased proliferative activity shown by PCNA immunostaining (p=0.04). Expression of 14-3-3θ was also found increased in IP and NPIP patients, compared to NP (p=0.005, p=0.002 respectively) and NE (p=0.004 and p=0.001 respectively). 14-3-3θ cytoplasmic immunopositivity was detected in columnar epithelium, particularly in basal and subluminal cells, whereas no immunoreactivity was observed in NP and NE. Our results demonstrate differential expression of 14-3-3β and θ isoforms in sinonasal pathophysiology, supporting their implication, respectively, in the proliferative and inflammatory process engaged in the formation of IP.

ENDOTHELIAL FUNCTION AND CIRCULATING CD4 T CELLS IN PATIENTS WITH UNSTABLE ANGINA

Previous studies support the crucial role of immune responses in the development and progression of atherosclerosis. CD4+CD28null T cells and CD4+CD31- T cells represent two specific subsets of circulating CD4+ T cells that affect endothelium. However, their accurate role on endothelial function

A misquoted mutation in exon16 of the BRCA2 gene

AbstractA pathogenic mutation in the BRCA2 gene, nt7602del16, has been misquoted as a mutation, possibly due to the incorrect inclusion of the last 16 nucleotides of exon15 of the BRCA2 gene as part of the intron15-exon16 BRCA2 gene sequence in publicly available databases. This was concluded following mutational screening by sequencing and enzymatic mapping of the BRCA2 gene exon15-exon16 region in DNA from peripheral blood samples from a total of 74 breast cancer and non-breast cancer patients as well as healthy individuals and the cell line MCF7. Careful interpretation of genetic variants and direct feedback to the corresponding sequence databases prevent systemic errors by integrating updated data into broadly referenced sources.