Benefit of Primary Tumor Resection in Stage IV, Grade 1 and 2, Pancreatic Neuroendocrine Tumors: A Propensity-Score Matched Cohort Study.

Objective: To determine the association of primary tumor resection in stage IV pancreatic neuroendocrine tumors (Pan-NET) and survival in a propensity-score matched study. Background: Pan-NET are often diagnosed with stage IV disease. The oncologic benefit from primary tumor resection in this scenario is debated and previous studies show contradictory results. Methods: Patients from 3 tertiary referral centers from January 1, 1985, through December 31, 2019: Uppsala University Hospital (Uppsala, Sweden), Sahlgrenska University Hospital (Gothenburg, Sweden), and Brigham and Women's Hospital/Dana-Farber Cancer Institute (Boston, USA) were assessed for eligibility. Patients with sporadic, grade 1 and 2, stage IV pan-NET, with baseline 2000-2019 were divided between those undergoing primary tumor resection combined with oncologic treatment (surgery group [SG]), and those who received oncologic treatment without primary tumor resection (non-SG). A propensity-score matching was performed to account for the variability in the extent of metastatic disease and comorbidity. Primary outcome was overall survival. Results: Patients with stage IV Pan-NET (n = 733) were assessed for eligibility, 194 were included. Patients were divided into a SG (n = 65) and a non-SG (n = 129). Two isonumerical groups with 50 patients in each group remained after propensity-score matching. The 5-year survival was 65.4% (95% CI, 51.5-79.3) in the matched SG and 47.8% (95% CI, 30.6-65.0) in the matched non-SG (log-rank, P = 0.043). Conclusions: Resection of the primary tumor in patients with stage IV Pan-NET and G1/G2 grade was associated with prolonged overall survival compared to nonoperative management. A surgically aggressive regime should be considered where resection is not contraindicated.

Characterization of Human Medullary Thyroid Carcinoma Glycosphingolipids Identifies Potential Cancer Markers.

Medullary thyroid carcinoma (MTC) accounts for only 1-2% of thyroid cancers; however, metastatic MTC is a mortal disease with no cure. In this study, glycosphingolipids were isolated from human MTCs and characterized by mass spectrometry and binding of carbohydrate recognizing ligands. The tissue distribution of selected compounds was investigated by immunohistochemistry. The amount of acid glycosphingolipids in the MTCs was higher than in the normal thyroid glands. The major acid glycosphingolipid was the GD3 ganglioside. Sulfatide and the gangliosides GM3 and GD1a were also present. The majority of the complex non-acid glycosphingolipids had type 2 (Galß4GlcNAc) core chains, i.e., the neolactotetraosylceramide, the Lex, H type 2 and x2 pentaosylceramides, the Ley and A type 2 hexaosylceramides, and the A type 2 heptaosylceramide. There were also compounds with globo (GalαGalß4Glc) core, i.e., globotriaosylceramide, globotetraosylceramide, the Forssman pentaosylceramide, and the Globo H hexaosylceramide. Immunohistochemistry demonstrated an extensive expression av Ley in the MTC cells and also a variable intensity and prevalence of Globo H and Lex. One individual with multiple endocrine neoplasia type 2B expressed the Forssman determinant, which is rarely found in humans. This study of human MTC glycosphingolipids identifies glycans that could serve as potential tumor-specific markers.

Characterization of Glycosphingolipids in the Human Parathyroid and Thyroid Glands.

As part of a systematic investigation of the glycosphingolipids in human tissues, acid and non-acid glycosphingolipids from human thyroid and parathyroid glands were isolated and characterized with mass spectrometry and binding of carbohydrate-recognizing ligands, with a focus on complex compounds. The glycosphingolipid patterns of the human parathyroid and thyroid glands were very similar. The major acid glycosphingolipids were sulfatide and the gangliosides GM3, GD3, GD1a, GD1b, GT1b and Neu5Ac-neolactotetraosylceramide, and the major non-acid glycosphingolipids were globotriaosylceramide and globoside. We also found neolactotetra- and neolactohexaosylceramide, the x2 glycosphingolipid, and complex glycosphingolipids with terminal blood group O and A determinants in both tissues. A glycosphingolipid with blood group Leb determinant was identified in the thyroid gland, and the parathyroid sample had a glycosphingolipid with terminal blood group B determinant. Immunohistochemistry demonstrated the expression of blood group A antigens in both the thyroid and parathyroid glands. A weak cytoplasmatic expression of the GD1a ganglioside was present in the thyroid, while the parathyroid gland had a strong GD1a expression on the cell surface. Thus, the glycosylation of human thyroid and parathyroid glands is more complex than previously appreciated. Our findings provide a platform for further studies of alterations of cell surface glycosphingolipids in thyroid and parathyroid cancers.

Characterization of glycosphingolipids from gastrointestinal stromal tumours.

Gastrointestinal stromal tumours (GISTs) are the major nonepithelial neoplasms of the human gastrointestinal tract with a worldwide incidence between 11 and 15 per million cases annually. In this study the acid and non-acid glycosphingolipids of three GISTs were characterized using a combination of thin-layer chromatography, chemical staining, binding of carbohydrate recognizing ligands, and mass spectrometry. In the non-acid glycosphingolipid fractions of the tumors globotetraosylceramide, neolactotetraosylceramide, and glycosphingolipids with terminal blood group A, B, H, Lex, Lea, Ley and Leb determinants were found. The relative amounts of these non-acid compounds were different in the three tumour samples. The acid glycosphingolipid fractions had sulfatide, and the gangliosides GM3, GD3, GM1, Neu5Acα3neolactotetraosylceramide, GD1a, GT1b and GQ1b. In summary, we have characterized the glycosphingolipids of GISTs and found that the pattern differs in tumours from different individuals. This detailed characterization of glycosphingolipid composition of GISTs could contribute to recognition of new molecular targets for GIST treatment and sub-classification.

Laparoscopic Lavage for Perforated Diverticulitis With Purulent Peritonitis: A Randomized Trial.

BACKGROUND: Perforated diverticulitis with purulent peritonitis has traditionally been treated with open colon resection and stoma formation with risk for reoperations, morbidity, and mortality. Laparoscopic lavage alone has been suggested as definitive treatment. OBJECTIVE: To compare laparoscopic lavage with open colon resection and colostomy (Hartmann procedure) for perforated diverticulitis with purulent peritonitis. DESIGN: Randomized, controlled, multicenter, open-label trial. (ISRCTN registry number: ISRCTN82208287). SETTING: 9 hospitals in Sweden and Denmark. PATIENTS: Patients who have confirmed Hinchey grade III perforated diverticulitis with purulent peritonitis at diagnostic laparoscopy. INTERVENTION: Randomization between laparoscopic lavage and the Hartmann procedure. MEASUREMENTS: Primary outcome was the percentage of patients having 1 or more reoperations within 12 months. Key secondary outcomes were number of reoperations, hospital readmissions, total length of hospital stay during 12 months, and adverse events. RESULTS: A total of 43 and 40 patients were randomly assigned to laparoscopic lavage and the Hartmann procedure with a median (first, third quartiles) follow-up of 372 days (336, 394) and 378 days (226, 396), respectively. Fewer patients in the laparoscopic group (12 of 43; 27.9%) than in the Hartmann group (25 of 40; 62.5%) had at least 1 reoperation within 12 months (relative risk reduction, 59%; relative risk, 0.41 [95% CI, 0.23 to 0.72]; P = 0.004). Mortality and severe adverse events did not differ between groups. Total length of hospital stay (days) within 12 months was shorter for the laparoscopic group than the Hartmann group, with a reduction of 35% (relative risk, 0.65 [CI, 0.45 to 0.94]; P = 0.047). After 12 months, 3 patients in the laparoscopic group and 11 in the Hartmann group had a stoma. LIMITATION: Not all patients presenting with suspected diverticulitis were enrolled. CONCLUSION: Laparoscopic lavage reduced the need for reoperations, had a similar safety profile to the Hartmann procedure, and may be an appropriate treatment of choice for acute perforated diverticulitis with purulent peritonitis. PRIMARY FUNDING SOURCE: ALF; Sahlgrenska University Hospital, Gothenburg.

Laparoscopic Lavage Is Feasible and Safe for the Treatment of Perforated Diverticulitis With Purulent Peritonitis: The First Results From the Randomized Controlled Trial DILALA.

OBJECTIVE: To evaluate short-term outcomes of a new treatment for perforated diverticulitis with purulent peritonitis in a randomized controlled trial. BACKGROUND: Perforated diverticulitis with purulent peritonitis (Hinchey III) has traditionally been treated with surgery including colon resection and stoma (Hartmann procedure) with considerable postoperative morbidity and mortality. Laparoscopic lavage has been suggested as a less invasive surgical treatment. METHODS: Laparoscopic lavage was compared with colon resection and stoma in a randomized controlled multicenter trial, DILALA (ISRCTN82208287). Initial diagnostic laparoscopy showing Hinchey III was followed by randomization. Clinical data was collected up to 12 weeks postoperatively. RESULTS: Eighty-three patients were randomized, out of whom 39 patients in laparoscopic lavage and 36 patients in the Hartmann procedure groups were available for analysis. Morbidity and mortality after laparoscopic lavage did not differ when compared with the Hartmann procedure. Laparoscopic lavage resulted in shorter operating time, shorter time in the recovery unit, and shorter hospital stay. CONCLUSIONS: In this trial, laparoscopic lavage as treatment for patients with perforated diverticulitis Hinchey III was feasible and safe in the short-term.

Treatment of acute diverticulitis laparoscopic lavage vs. resection (DILALA): study protocol for a randomised controlled trial.

BACKGROUND: Perforated diverticulitis is a condition associated with substantial morbidity. Recently published reports suggest that laparoscopic lavage has fewer complications and shorter hospital stay. So far no randomised study has published any results. METHODS: DILALA is a Scandinavian, randomised trial, comparing laparoscopic lavage (LL) to the traditional Hartmann's Procedure (HP). Primary endpoint is the number of re-operations within 12 months. Secondary endpoints consist of mortality, quality of life (QoL), re-admission, health economy assessment and permanent stoma. Patients are included when surgery is required. A laparoscopy is performed and if Hinchey grade III is diagnosed the patient is included and randomised 1:1, to either LL or HP. Patients undergoing LL receive > 3L of saline intraperitoneally, placement of pelvic drain and continued antibiotics. Follow-up is scheduled 6-12 weeks, 6 months and 12 months. A QoL-form is filled out on discharge, 6- and 12 months. Inclusion is set to 80 patients (40+40). DISCUSSION: HP is associated with a high rate of complication. Not only does the primary operation entail complications, but also subsequent surgery is associated with a high morbidity. Thus the combined risk of treatment for the patient is high. The aim of the DILALA trial is to evaluate if laparoscopic lavage is a safe, minimally invasive method for patients with perforated diverticulitis Hinchey grade III, resulting in fewer re-operations, decreased morbidity, mortality, costs and increased quality of life. TRIAL REGISTRATION: British registry (ISRCTN) for clinical trials ISRCTN82208287http://www.controlled-trials.com/ISRCTN82208287.

Perforated diverticulitis operated at Sahlgrenska University Hospital 2003-2008.

All patients with perforated diverticulitis admitted as emergency cases and having undergone colon resection during their initial hospitalisation in the period from 1 January 2003 to 30 June 2008 at one institution were analysed with regard to morbidity and mortality. The group consisted of 106 patients (mean age 65 years, range 32-98 years), 60% of whom had comorbidity. Hartmann's procedure was the initial procedure in 77% and primary resection and anastomosis in 23%. Of these patients 18% underwent reoperation, leading to a mean number of surgical procedures during the initial hospitalisation of 1.3 (range 1-10). The mean length of stay was 17 days, the median stay 12 days (range 1-111 days). A total of 43% of the patients underwent surgery during readmissions. Among the 82 patients operated with Hartmann's procedure, permanent stoma was the end result for 35 patients (43%). Six patients died. This retrospective study confirmed that perforated diverticulitis requiring colon resection was associated with a high risk of reoperation, long hospital stay, readmittance with renewed surgery and permanent stoma. Furthermore, the procedure caused suffering and a considerable drain on resources. The results will be used as the basis for a randomised trial on laparoscopic lavage versus Hartmann's procedure.

Regulation of ER alpha signaling pathway in neuronal HN10 cells: role of protein acetylation and Hsp90.

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ER alpha signaling have recently been found to be acetylated, including ER alpha itself and Hsp90, a key chaperone in the functional regulation of ER alpha. ER alpha complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ER alpha signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17beta-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pER alpha-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ER alpha signaling. Our results reveal that ER alpha signaling is dependent on protein acetylation and epigenetic regulation.

Coaccumulation of calcium and beta-amyloid in the thalamus after transient middle cerebral artery occlusion in rats.

Transient occlusion of the middle cerebral artery (MCAO) in rats leads to abnormal accumulation of beta-amyloid (Abeta) peptides in the thalamus. This study investigated the chemical composition of these deposits. Adult male human beta-amyloid precursor protein (APP) overexpressing (hAPP695) rats and their wild-type littermates were subjected to transient MCAO for 2 h or sham operation. After 26-week survival time, histological examination revealed an overlapping distribution pattern for rodent and human Abeta in the thalamus of hAPP695 rats subjected to MCAO. X-ray microanalysis showed that the deposits did not contain significant amount of iron, zinc, or copper typical to senile plaques. In contrast, the deposit both in hAPP695 and non-transgenic rats contained calcium and phosphorus in a ratio (1.28+/-0.15) characteristic to hydroxyapatites. Alizarin red staining confirmed that calcium coaccumulated in these Abeta deposits. It is suggested that APP expression is induced by ischemic insult in cortical neurons adjacent to infarct, which in turn is reflected as increased release of Abeta peptides by their corticothalamic axon endings. This together with insufficient clearance or atypical degradation of Abeta peptides lead to dysregulation of calcium homeostatis and coaccumulation in the thalamus.

Overexpression of APP provides neuroprotection in the absence of functional benefit following middle cerebral artery occlusion in rats.

Cerebral ischaemia leads to a transient accumulation of beta-amyloid precursor protein (APP) and beta-amyloid (Abeta) peptides adjacent to the ischaemic lesion. There is conflicting evidence that APP/Abeta fragments may either enhance neuronal plasticity or be neurotoxic. The aim of the current study was to assess the effect of overexpression of human APP in rats on functional recovery following cerebral ischaemia. Adult APP-overexpressing (hAPP695 Tg) rats subjected to transient middle cerebral artery occlusion (MCAO) had significantly smaller infarct volumes than non-transgenic littermates, yet did not perform better on a series of sensorimotor or learning tests during a 6-month follow-up period. In fact, transgenic animals were found to be significantly more impaired in both the beam-walking and Morris water maze tests following MCAO. Immunohistochemistry showed human Abeta-positive staining in the cortex and hippocampus of APP transgenic rats. The present data suggest that while overexpression of APP in rats may provide some histological neuroprotection in the event of cerebral ischaemia, this does not translate into significant functional recovery.

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins.

The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high-throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production. Here, we present a generic and integrated parallel HTP strategy for cloning and expression screening of membrane proteins in their detergent solubilized form. Based on this strategy, we provide overall success rates for membrane protein production in Escherichia coli, as well as initial benchmarking statistics of parameters such as expression vectors, strains, and solubilizing detergents. The technologies were applied to 49 E. coli integral membrane proteins with human homologs and revealed that 71% of these proteins could be produced at sufficient levels to allow milligram amounts of protein to be relatively easily purified, which is a significantly higher success rate than anticipated. We attribute the high success rate to the quality and robustness of the methodology used, and to introducing multiple parameters such as different vectors, strains, and detergents. The presented strategy demonstrates the usefulness of HTP technologies for membrane protein production, and the feasibility of large-scale programs for elucidation of structure and function of bacterial integral membrane proteins.

The ETS transcription factor GABPalpha is essential for early embryogenesis.

The ETS transcription factor complex GABP consists of the GABPalpha protein, containing an ETS DNA binding domain, and an unrelated GABPbeta protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpalpha gene. Embryos homozygous for the null Gabpalpha allele die prior to implantation, consistent with the broad expression of Gabpalpha throughout embryogenesis and in embryonic stem cells. Gabpalpha(+/-) mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpalpha protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpalpha function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.