[Clavien-Dindo classification and influencing factors analysis of complications after laparoscopic pancreaticoduodenectomy].

Objective: To semi-quantify the postoperative complications occurred after laparoscopic pancreaticoduodenectomy(LPD) using Clavien-Dindo score, thereafter exploring its impact factors. Methods: In this retrospective cohort study, the clinical data of 124 patients who had undergone LPD for periampullary tumor from June 2016 to June 2017 at Department of Biliary Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology were collected.Malignancy was confirmed based on postoperative pathological reports.Postoperative complications were semi-quantitated using Clavien-Dindo score.Multivariable logistic regression model was applied to explore the factors related to severe complications(Clavien-Dindo Ⅲb-Ⅴ). Results: Of the 124 patients, there were 64 males(51.6%) and 60 females(48.4%), with age of 57 years(range, 23-82 years). In total, postoperative complications occurred in 30 patients(24.2%). Among the 30 patients, 4 patients suffered Clavien-Dindo grade Ⅰ, 18 patients(14.5%) suffered Clavien-Dindo grade Ⅱ, 6 patients(4.8%) suffered Clavien-Dindo grade Ⅲa, 1 patient(0.1%) suffered Clavien-Dindo grade Ⅳb, and 1 patient(0.1%) suffered Clavien-Dindo grade Ⅴ.Intraabdominal hemorrhage occurred in 8 patients, pancreatic fistula was found in 10 patients(7 patients had biochemical leakage and 3 of them had grade B pancreatic fistula), both biliary fistula and gastrointestinal fistula were found in 1 patient.Abdominal infection occurred in 10 patients, both liver failure and renal failure occurred in one patient.Moreover, arrhythmia was found in two patients, and mortality occurred in one patient.Five patients suffered multiple complications.Univariable analysis showed that postoperative complications were associated with body mass index, American Society of Anesthesiologists(ASA) score, intraoperative blood transfusion, and pancreatic texture(P<0.05). In multivariable logistic regression, ASA grade Ⅲ, intraoperative blood transfusion, and pancreatic softness were independently related to postoperative complications after LPD(P<0.05). Conclusions: Clavien-Dindo score is feasible to be applied in management of patients with LPD.ASA score, texture of pancreas, and intraoperative blood transfusion were independently associated with postoperative complications.

Talin1 phosphorylation activates ß1 integrins: a novel mechanism to promote prostate cancer bone metastasis.

Talins are adaptor proteins that regulate focal adhesion signaling by conjugating integrins to the cytoskeleton. Talins directly bind integrins and are essential for integrin activation. We previously showed that ß1 integrins are activated in metastatic prostate cancer (PCa) cells, increasing PCa metastasis to lymph nodes and bone. However, how ß1 integrins are activated in PCa cells is unknown. In this study, we identified a novel mechanism of ß1 integrin activation. Using knockdown experiments, we first demonstrated that talin1, but not talin2, is important in ß1 integrin activation. We next showed that talin1 S425 phosphorylation, but not total talin1 expression, correlates with metastatic potential of PCa cells. Expressing a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 and C4-2B4 PCa cells, decreased activation of ß1 integrins, integrin-mediated adhesion, motility and increased the sensitivity of the cells to anoikis. In contrast, reexpression of the phosphorylation-mimicking mutant talin1(S425D) led to increased ß1 integrin activation and generated biologic effects opposite to talin1(S425A) expression. In the highly metastatic PC3-MM2 cells, expression of a non-phosphorylatable mutant, talin1(S425A), in talin1-silenced PC3-MM2 cells, abolished their ability to colonize in the bone following intracardiac injection, while reexpression of phosphorylation-mimicking mutant talin1(S425D) restored their ability to metastasize to bone. Immunohistochemical staining demonstrated that talin S425 phosphorylation is significantly increased in human bone metastases when compared with normal tissues, primary tumors or lymph node metastases. We further showed that p35 expression, an activator of Cdk5, and Cdk5 activity were increased in metastatic tumor cells, and that Cdk5 kinase activity is responsible for talin1 phosphorylation and subsequent ß1 integrin activation. Together, our study reveals Cdk5-mediated phosphorylation of talin1 leading to ß1 integrin activation is a novel mechanism that increases metastatic potential of PCa cells.

Localization of phosphorylated alphaB-crystallin to heart mitochondria during ischemia-reperfusion.

The cytosolic small heat shock protein alphaB-crystallin (alphaBC) is a molecular chaperone expressed in large quantities in the heart, where it protects from stresses such as ischemia-reperfusion (I/R). Upon I/R, p38 MAP kinase activation leads to phosphorylation of alphaBC on Ser(59) (P-alphaBC-S59), which increases its protective ability. alphaBC confers protection, in part, by interacting with and affecting the functions of key components in stressed cells. We investigated the hypothesis that protection from I/R damage in the heart by P-alphaBC-S59 can be mediated by localization to mitochondria. We found that P-alphaBC-S59 localized to mitochondria isolated from untreated mouse hearts and that this localization increased more than threefold when the hearts were subjected to ex vivo I/R. Mitochondrial P-alphaBC-S59 decreased when hearts were treated with the p38 inhibitor SB-202190. Moreover, SB-202190-treated hearts exhibited more tissue damage and less functional recovery upon reperfusion than controls. I/R activates mitochondrial permeability transition (MPT) pore opening, which increases cell damage. We found that mitochondria incubated with a recombinant mutant form of alphaBC that mimics P-alphaBC-S59 exhibited decreased calcium-induced MPT pore opening. These results indicate that mitochondria may be among the key components in stressed cells with which P-alphaBC-S59 interacts and that this localization may protect the myocardium, in part, by modulating MPT pore opening and, thus, reducing I/R injury.

Application of laser scanning cytometry for evaluation of DNA ploidy in routine cytologic specimens.

The laser scanning cytometer (LSC) is a relatively new instrument that combines the features of both flow and static image cytometry. The purpose of this study was to examine the application of the LSC for evaluation of DNA ploidy in routine cytologic specimens. The material for this study consisted of 60 routine cytologic specimens obtained from 33 males and 27 females ranging in age from 23-87 yr (mean, 58 yr). The specimens were simultaneously stained with propidium iodide and FITC-cytokeratin, either on Thin-Prep slide (35 cases) or in a concentrated cell suspension (25 cases). In each case a minimum of 500 cells was evaluated (range, 527-17,963; mean, 3,889). All abnormal cell populations were relocated for the presence of malignant cells. The results were defined as diploid and aneuploid/tetraploid. In 10 bladder washes, the results of LSC were compared to results of flow cytometry. Out of 60 specimens, 7 (11%: 6 bladder washes and 1 renal wash) were excluded due to low cellularity. Of the remaining 53 cases, 11 (20%) were aneuploid/tetraploid, and 42 (80%) were diploid. All but one cytologically diagnosed malignancy had abnormal DNA content. Additionally, two bladder washes diagnosed as suspicious and atypical were aneuploid. All abnormal LSC results were confirmed by relocation of the cells. The concordance between flow cytometry and LSC in the 10 control bladder washes was 100%. In conclusion, LSC proved to be a suitable instrument for the evaluation of DNA ploidy in routine cytologic specimens.

Oxidative stress and neurodegeneration in prion diseases.

Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are a group of fatal neurodegenerative diseases that affect humans and a number of other animal species. The etiology of these diseases is thought to be associated with the conversion of a normal protein, PrPC, into an infectious, pathogenic form, PrPSc. The PrPSc form shows greater protease resistance than PrPC and accumulates in affected individuals, often in the form of extracellular plaques. The pathogenesis and the molecular basis of neuronal cell death in these diseases are not well understood. Oxidative stress has been proposed to play an important role in the pathogenesis of several neurodegenerative disorders. In the present study, evidence of oxidative stress in scrapie, the archetype disease of the TSEs, is discussed. In addition, the mechanisms whereby oxidative stress could lead to neuronal degeneration are described.

Increased ferric iron content and iron-induced oxidative stress in the brains of scrapie-infected mice.

Scrapie is a transmissible neurodegenerative disease of sheep and goats. The neuropathological changes include vacuolation, astrocytosis, the development of amyloid plaques in some instances, and neuronal loss. The mechanisms involved in neuronal cell death in scrapie are not known. Recently, we reported the presence of oxidative stress in the brains of scrapie-infected animals and suggested that this is the main mechanism that induces neuronal cell loss. It is known that oxidative stress induced by free radicals is associated with iron accumulation; this association led to an examination of the levels of iron (total iron, Fe(2+) and Fe(3+)) in the brains of control and scrapie-infected mice by biochemical methods. In the scrapie-infected group, both the level of total iron and the Fe(3+) level were significantly increased in cerebral cortex, striatum, and brainstem as compared to the values in the control group. A shift in the ratio of Fe(2+)/Fe(3+) was observed in the same regions of infected mice. Additionally, in this scrapie model, we confirmed the presence of oxidative stress, as evidenced by the increase of free malondialdehyde. These results suggest that iron metabolism is changed and that iron-induced oxidative stress partly contributes to neurodegeneration in scrapie infection.

Induction of heme oxygenase-1 in the brains of scrapie-infected mice.

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in the degradation of heme to biliverdin, carbon monoxide and iron, and its expression can be used as a marker for oxidative stress. Oxidative stress has been reported to be associated with neurodegenerative diseases including Alzheimer's disease. It is possible that oxidative stress is also involved in the disease process seen in scrapie, the archetype transmissible spongiform encephalopathy. In this study, we report that HO-1 is significantly increased in the scrapie-infected group compared to an age-matched control group. Immunohistochemistry showed a pronounced increase of immunostaining of this protein in the infected group compared to the minimal amount of staining in the control group. These results support that oxidative stress is closely associated with the pathogenesis of scrapie and that it might contribute to neurodegeneration in this disease.

Differential expression of Bax and Bcl-2 in the brains of hamsters infected with 263K scrapie agent.

To study the mechanism(s) of neuronal cell death during scrapie infection, we investigated the expression of Bax and Bcl-2 in brains of hamsters infected with 263K scrapie agent. The expression of Bcl-2 mRNA was significantly decreased in the brains of 263K scrapie-infected hamsters compared with controls, whereas the expression levels of Bax mRNA were significantly increased in scrapie-infected brain. The levels of Bax and Bcl-2 proteins in brains of scrapie and control animals reflected the difference in mRNA levels. Immunoreactivity for Bax and Bcl-2 were found predominantly within neurons. In scrapie-infected brains, the number of neuronal cells positive for Bcl-2 was significantly lower in the hippocampal CA3 region and was decreased in the cerebral cortex, whereas the number of neuronal cells positive for Bax was significantly increased in both regions. The possibility that differential regulation of Bax and Bcl-2 expression may play an important role in neuronal cell death induced by scrapie infection is discussed.

Increased expression of CaM kinase II alpha in the brains of scrapie-infected mice.

We investigated the distribution of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the brains of mice infected with ME7 scrapie strain. CaM kinase II is an enzyme that plays a major role in the regulation of long-term potentiation, a form of synaptic plasticity associated with learning and memory. Immunoreactivity of CaM kinase II alpha, measured by Western blot, increased markedly in scrapie-infected brains compared with control brains. Immunohistochemically, CaM kinase II alpha immunoreactivity was upregulated in the cerebral cortex and hippocampal CA1 area of scrapie-positive mice infected with ME7 scrapie strain. This result implies that this enzyme is associated with aberrant function of synaptic transmission and LTP of the pyramidal neurons in the hippocampal CA1 area of mice infected with ME7 scrapie strain.

Telomerase activity, telomere length, and DNA ploidy in prostatic intraepithelial neoplasia (PIN).

PURPOSE: To investigate the relationship of telomerase activity, telomere length, and DNA ploidy in high grade prostatic intraepithelial neoplasia (PIN). MATERIALS AND METHODS: Tissue samples were carefully microdissected to obtain adenocarcinoma or PIN-containing tissue free of cancer. Telomerase activity was measured using the PCR-based telomeric repeat amplification protocol (TRAP). Telomere length was estimated from Southern blots of telomere restriction fragments (TRFs). DNA ploidy of PIN and carcinoma was determined by image analysis of adjacent Feulgen stained tissue sections. RESULTS: Telomerase activity was found in 4 of 25 samples (16%) of high grade PIN. All telomerase positive PIN foci had a diploid DNA content. Although 5 of 25 samples (25%) of high grade PIN foci analyzed were DNA aneuploid, none of these demonstrated telomerase activity. Telomerase positive foci of prostate carcinoma (69% of all cancer foci analyzed) displayed heterogeneity in TRF length, with a mean TRF length two kilobase pairs shorter than that of telomerase negative specimens. CONCLUSIONS: Telomerase activity is present in a low percentage of high-grade PIN foci, which are diploid by DNA content measurements.

DNA ploidy and proliferation heterogeneity in human prostate cancers.

DNA ploidy determinations have been shown to have clinical application in predicting disease progression, survival, or response to anti-androgen therapies in prostate carcinomas. Since intra-tumor heterogeneity may have a profound effect on DNA measurements, we determined the frequency of DNA ploidy and proliferation (here S-phase fraction) heterogeneity in early prostatic carcinomas, and estimated the potential impact of heterogeneity on predicting disease course, survival, or response to therapy. Using image and flow cytometric analysis of archival, paraffin-embedded prostate tumors, we measured DNA ploidy in individual foci of prostatic carcinoma in stage T1a, T1b and T1c disease. Image analysis studies included the use of Feulgen stained tissue sections, and a comparison of these results with flow cytometric DNA ploidy determinations on nuclei isolated from the same tumor foci. Flow cytometry was also used to measure DNA Index and tumor S-phase fraction, in some cases using multiparameter analysis of isolated nuclei to determine DNA content and the level of the proliferation-associated antigen, p105. Our results indicate that DNA aneuploid foci of prostate carcinoma are infrequently seen in stage T1a disease (13% of the individuals studied), and that the presence of both DNA diploid and aneuploid foci in the same sample is seen in less than 10% of these individuals. Stage T1b and T1c tumors containing only DNA diploid nuclei are seen, though these are likely most common in low volume, low Gleason grade tumors. By using flow cytometry to compare these results with those using image analysis of the same tumor foci, we demonstrated that the majority (> 75%) of these aneuploid tumors are DNA tetraploid. Our data on prostate tumor S-phase fractions indicate that DNA diploid tumors generally have a lower S-phase than DNA aneuploid foci (including comparisons of DNA diploid and aneuploid foci in the same prostate tumor). These results support the model that early prostate tumors are DNA diploid and have a low S-phase, and that these tumors likely evolve to DNA tetraploid tumors with a similar low S-phase fraction.