Bioenergetics of fish spermatozoa with focus on some herring (Clupea harengus) enzymes.

Herring (Clupea harengus) shows the unique behavior of reproductive biology in which spermatozoa remains in the surrounding media for extended periods. It is an excellent model for studying the malic enzyme (ME) and creatine kinase (CK) biochemical properties because of their high activity and variability of molecular isoforms. The specific activity of NAD-preferring ME in herring spermatozoa is the highest among other fish spermatozoa and is localized in its large mitochondrion. Two different CK isoforms, dimer and octamer, were detected in herring spermatozoa. It has already been shown that CK isoforms play an important role in energy homeostasis by catalyzing a reversible transfer of the phosphate of ATP to creatine to yield ADP and creatine phosphate (CP) (creatine/CP circuit). Two lactate dehydrogenase (LDH) isoenzymes were also shown in herring spermatozoa, LDH-B4 and LDH-A2B2. In this mini-review, the role of ME and energy transport system with easily diffusible creatine and CP in herring spermatozoa is discussed.

Pancreas transplant rejection episodes are not revealed by biopsies of the donor duodenum in a prospective study with paired biopsies.

The surgical technique with duodeno-duodenal enteroanastomosis of pancreas transplants allows for representative endoscopic ultrasound-guided needle biopsies of the donor duodenum and the pancreas graft. We assessed whether histological findings in transplanted donor duodenal biopsies can indicate rejection in the transplanted pancreas. Since September 2012, a duodeno-duodenal enteroanastomosis has been the default technique for pancreas transplantations at our center. In 67 recipients we prospectively examined 113 endoscopic ultrasound-guided procedures with representative biopsies from the duodenum grafts and the pancreas grafts (97 per protocol and 16 on indication). All graft biopsies were evaluated according to established rejection criteria. A total of 22 biopsy-proven pancreas rejections were detected, with 2 matching duodenal biopsies showing rejection. This gives a sensitivity of 9% for detection of a pancreas rejection by duodenal biopsies. The other matching duodenal biopsies were either normal (n = 13) or indeterminate (n = 7). Rejection of the donor duodenum was found in only 6/113 biopsies, with 2 concurrent pancreas rejections. In conclusion, the donor duodenum is not a useful reporter organ for rejection in the pancreas graft.

Outcomes in Pancreas Transplantation With Exocrine Drainage Through a Duodenoduodenostomy Versus Duodenojejunostomy.

Until recently, pancreas transplantation has mostly been performed with exocrine drainage via duodenojejunostomy (DJ). Since 2012, DJ was substituted with duodenoduodenostomy (DD) in our hospital, allowing endoscopic access for biopsies. This study assessed safety profiles with DD versus DJ procedures and clinical outcomes with the DD technique in pancreas transplantation. DD patients (n = 117; 62 simultaneous pancreas-kidney [SPKDD ] and 55 pancreas transplantation alone [PTADD ] with median follow-up 2.2 years) were compared with DJ patients (n = 179; 167 SPKDJ and 12 PTADJ ) transplanted in the period 1998-2012 (pre-DD era). Postoperative bleeding and pancreas graft vein thrombosis requiring relaparotomy occurred in 17% and 9% of DD patients versus 10% (p = 0.077) and 6% (p = 0.21) in DJ patients, respectively. Pancreas graft rejection rates were still higher in PTADD patients versus SPKDD patients (p = 0.003). Hazard ratio (HR) for graft loss was 2.25 (95% CI 1.00, 5.05; p = 0.049) in PTADD versus SPKDD recipients. In conclusion, compared with the DJ procedure, the DD procedure did not reduce postoperative surgical complications requiring relaparatomy or improve clinical outcomes after pancreas transplantation despite serial pancreatic biopsies for rejection surveillance. It remains to be seen whether better rejection monitoring in DD patients translates into improved long-term pancreas graft survival.

Pancreas transplantation with enteroanastomosis to native duodenum poses technical challenges--but offers improved endoscopic access for scheduled biopsies and therapeutic interventions.

To facilitate endoscopic access for rejection surveillance and stenting of the pancreas, we have abandoned the duodenojejunostomy (DJ) in favor of duodenoduodenostomy (DD) in pancreas transplantation (PTx). From September 2012 to September 2013 we performed 40 PTx with DD; 20 solitary-PTx (S-PTx) and 20 simultaneous pancreas and kidney transplantation (SPK). We compared the outcomes with results from 40 PTx-DJ (10 S-PTx and 30 SPK) from the preceding era. The DD-enteroanastomoses were performed successfully. Endoscopic pancreas biopsies (endoscopic ultrasound examination [EUS]) yielded representative material in half of the cases. One exocrine fistula was treated by endoscopic stenting. PTxs-DD were associated with a higher rate of thrombosis compared to PTx-DJ (23% vs. 5%) and reoperations (48% vs. 30%), as well as inferior graft survival (80% vs. 88%). Time on waiting list, HLA A + B mismatches and reoperations were associated with graft loss. Only recipient age remained an independent predictor of patient death in multivariate analysis. PTx-DD showed a higher rate of thrombosis and inferior results, but facilitated a protocol biopsy program by EUS that was feasible and safe. Given that technical difficulties can be solved, the improved endoscopic access might confer long-term benefits, yet this remains to be proven.

IL1R1+ cancer-associated fibroblasts drive tumor development and immunosuppression in colorectal cancer.

Fibroblasts have a considerable functional and molecular heterogeneity and can play various roles in the tumor microenvironment. Here we identify a pro-tumorigenic IL1R1+, IL-1-high-signaling subtype of fibroblasts, using multiple colorectal cancer (CRC) patient single cell sequencing datasets. This subtype of fibroblasts is linked to T cell and macrophage suppression and leads to increased cancer cell growth in 3D co-culture assays. Furthermore, both a fibroblast-specific IL1R1 knockout and IL-1 receptor antagonist Anakinra administration reduce tumor growth in vivo. This is accompanied by reduced intratumoral Th17 cell infiltration. Accordingly, CRC patients who present with IL1R1-expressing cancer-associated-fibroblasts (CAFs), also display elevated levels of immune exhaustion markers, as well as an increased Th17 score and an overall worse survival. Altogether, this study underlines the therapeutic value of targeting IL1R1-expressing CAFs in the context of CRC.

Experimental evaluation of liver regeneration patterns and liver function following ALPPS.

BACKGROUND: The underlying mechanism of liver regeneration after Associating Liver Partition and Portal vein ligation (PVL) for Staged hepatectomy (ALPPS) is still unclear. The aim of this study was to evaluate the relationship between future liver remnant (FLR) volume, liver regeneration characteristics and restoration of function in an experimental model of ALPPS. METHODS: An ALPPS model in rats was developed with selective PVL, parenchymal transection and partial hepatectomy (step 1), followed by resection of the liver (step 2). Three different ALPPS groups with FLR sizes of 30, 20 and 10 per cent of total liver volume were compared with sham-operated controls and animals undergoing resection of left lateral lobe and 90 per cent PVL with respect to morbidity, mortality, liver regeneration and function. RESULTS: Three of 15 animals that had ALPPS with 10 per cent FLR (ALPPS10) died after step 1. Ascites developed in two of five rats that had ALPPS with 20 per cent FLR and in three of four animals in the ALPPS10 group after step 2. Although the relative increments in FLR size and growth rates were highest in the ALPPS groups, small FLR size was associated with a sustained increase in levels of serum aminotransferases and bilirubin, a lower albumin concentration, severe sinusoidal injury, increased expression of proliferation markers and increased activation of hepatic progenitor cells after step 2. CONCLUSION: There is discordance between FLR volume increase and functional restoration after the ALPPS procedure.

Monoclonal antibody identification and characterization of a Mr 43,000 membrane glycoprotein associated with human breast cancer.

A monoclonal antibody (323/A3) with a high degree of selectivity for binding to breast cancer cells was produced by immunization of mice with MCF-7 human breast cancer cells. The antigen recognized by 323/A3 on MCF-7 appears to be surface localized, and by enzyme-linked immunosorbent assay, the antibody was found to bind strongly with four of six breast cancer cell lines examined while no binding was detectable with nonbreast cancer cell lines. In vivo distribution of the 323/A3 antigen was screened by immunoperoxidase staining of formalin-fixed paraffin sections of normal human tissues and tumors. Among breast tissues, positive staining was detected with 75% (6 of 8) of metastatic lymph nodes, 59% (76 of 128) of primary breast tumors, 20% (13 of 63) of benign breast lesions, and 0% (0 of 10) of normal breast. No immunostaining was detected with a large variety and number of other normal human tissues with the exception of staining observed with epithelium of normal colon. Antigen distribution appears not to be disease specific, since positive staining was also observed with adenocarcinomas other than breast. The antigen recognized by the 323/A3 antibody was identified by Western blot analysis as a Mr 43,000 protein. The glycoprotein nature of the antigen was demonstrated by its binding to concanavalin A, specific elution with sugar, and immunoprecipitation of a Mr 43,000 radiolabeled protein from extracts of MCF-7 cells after pulse labeling with [3H]glucosamine. The 323/A3 antigen appears to be the same Mr 43,000 protein in cell lines as in breast tumors in vivo. Based on a comparison with the molecular weights of other known tumor-associated antigens and with their immunocytochemical tissue distribution, the Mr 43,000 glycoprotein described here represents a tumor-associated antigen previously undescribed in breast cancer or in other tumors. Since the Mr 43,000 glycoprotein is present on the surface of most breast cancer cells and is either absent or expressed at very low levels in most normal tissues including normal breast, the monoclonal antibody described here may have potential applications in diagnosis and management of breast cancer.

Assessment of Mucosal Inflammation and Circulation in Response to Probiotics in Patients Operated with Ileal Pouch Anal Anastomosis for Ulcerative Colitis.

BACKGROUND: Pouchitis is a common and troublesome condition, and a disturbed microbiological flora and mucosal blood flow in the pouch have been suggested as possible causes. Laser Doppler flowmetry (LDF) has been used successfully to measure gastric and colonic mucosal perfusion in humans. The aim of this study was to evaluate the effect of intervention with probiotics on ileal pouch inflammation and perfusion in the pouch, assessed by endoscopy, histology, fecal calprotectin and LDF. METHODS: A fermented milk product (Cultura; 500 ml) containing live lactobacilli (La-5) and bifidobacteria (Bb-12) was given daily for 4 weeks to 10 patients operated with ileal-pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). Mucosal perfusion was measured with LDF and the degree of inflammation was examined at predefined levels of the distal bowel by endoscopy and histology. Stool samples were cultured for lactobacilli and bifidobacteria and calprotectin were measured before and after intervention. RESULTS: The LDF measurements were reproducible in the pelvic pouch at each of the predefined levels, but did not change after intervention. The mucosal perfusion was reduced in the distal compared to the proximal part of the pouch. Calprotectin levels did not change significantly after intervention. The median endoscopic score for inflammation was significantly reduced by 50% after intervention, whereas the histological score did not change significantly. CONCLUSION: The results suggest that probiotics primarily act superficially, with change of gross appearance of the mucosa at endoscopy, but without significant effect on histological picture, mucosal perfusion or faecal calprotectin, during a relatively short period of 4 weeks.

Colectomy for patients with ulcerative colitis and primary sclerosing cholangitis - what next?

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) occurs in 2%-8% of patients who suffer from ulcerative colitis (UC). For patients who require colectomy, ileal pouch-anal anastomosis (IPAA) or ileorectal anastomosis (IRA) is employed to preserve continence.We evaluated the outcomes after IPAA and IRA for patients with UC-PSC, using patients with UC but without PSC as controls (UC-only group). PATIENTS: In a case-control study conducted at Sahlgrenska University Hospital, Sweden, patients with UC-PSC (N=48; 31 IPAA and 17 IRA) were compared to patients with UC only (N=113; 62 IPAA and 51 IRA). Functional outcomes (Öresland score), pouchitis, surgical complications, and failure were evaluated. RESULTS: For patients with IPAA, the median Öresland scores were similar for the two groups: 5 (range, 0-13) for the UC-PSC group and 5 for the UC-only group (range, 0-12; p>0.05). However, the IRA scores were significantly different at 7 (range, 2-11) and 3 (range, 0-11) for the respective groups (p=0.005). Pouchitis was more frequent in patients with UC-PSC. Complication rates did not differ. For patients with IPAA, the failure rate was 16% for those in the UC-PSC group versus 6% for those in the UC-only group (p>0.05); the corresponding results for IRA were 53% versus 22% (p=0.03). CONCLUSIONS: For cases of IPAA, pouchitis seems to be more common in patients with UC-PSC. However, the functional outcomes and failure rates are unaffected by concurrent PSC. For patients with UC-PSC, functional outcome is poor and the failure rate is high after IRA.

Reclassification of tumour origin in resected periampullary adenocarcinomas reveals underestimation of distal bile duct cancer.

BACKGROUND: Primary adenocarcinomas removed by pancreatoduodenectomy originate from the duodenum (DC), ampulla (AC), distal bile duct (DBC), or pancreas (PC). Pathobiology, staging, survival, and adjuvant chemotherapy vary among these cancers. The proximity of the structures of possible origin renders it difficult to obtain a correct diagnosis, which might lead to inconsistencies in reported data and inappropriate adjuvant treatment. METHODS: Records of 207 patients undergoing pancreatoduodenectomy (1998-2009) for periampullary adenocarcinoma were reviewed. Routine histopathology reports of tumour origin performed by multiple pathologists were independently re-evaluated based on predetermined criteria by two experienced pancreatic pathologists. RESULTS: Slide review changed the diagnosis in 55 (27%) patients. After reclassification, final distribution was 29 (14%) DC, 52 (25%) AC, 57 (28%) DBC, and 69 (33%) PC. The diagnosis was revised in 4 (14%) DC, 7 (17%) AC, 30 (53%) DBC and 14 (19%) PC. The underestimation of DBC during routine histopathology was caused by misinterpretation of DBC either PC or AC. Misclassification of PC was mainly due to erroneous diagnosis of AC. Reassignment of tumour origin caused no significant changes in survival within cancer type, but resulted in a significant difference in survival between DBC and PC (p = 0.004). CONCLUSION: Specialist slide review resulted in reassignment of tumour origin in 27% of periampullary adenocarcinomas. Distal bile duct cancer was found to be most frequently misdiagnosed (53%). Correct diagnosis of tumour origin is crucial for data quality, appropriate adjuvant therapy, and patient inclusion in clinical trials.

A possible difference in the heterogeneity of the heavy and light chains from a rabbit anti-p-azobenzoate antibody preparation.

CNBr cleavage of rabbit heavy (H) chains leads to the formation of a fragment, C-1, which consists of the N-terminal half of the H chain. Fragment C-1 is cleaved at methionyl residues but held together by intrachain S-S bonds so that smaller fragments can be liberated by total reduction and alkylation. In the case of the C-1 fragment from an anti-p-azobenzoate antibody preparation, which has a light (L) chain of markedly restricted heterogeneity, total reduction and alkylation liberated seven major fragments in good yield. The N-terminus of two of these fragments corresponds to position 35 of the H chain but their N-terminal sequences are clearly different. The H chain regions represented by the other fragments implied that they were derived from H chains having different distributions of methionyl residues. This hypothesis was supported by isolating six different antibody components from the antibody preparation by isoelectric focusing and then digesting them with CNBr. Comparison of the products showed that the six components all appeared to behave differently. These results are interpreted as suggesting that the process whereby H and L chains are paired in vivo may not be completely specific and may provide a simple means of generating a significant contribution to antibody diversity.

Assessment of mucosal inflammation and circulation in response to probiotics in patients operated with ileal pouch anal anastomosis for ulcerative colitis.

BACKGROUND: Pouchitis is a common and troublesome condition, and a disturbed microbiological flora and mucosal blood flow in the pouch have been suggested as possible causes. Laser Doppler flowmetry (LDF) has been used successfully to measure gastric and colonic mucosal perfusion in humans. The aim of this study was to evaluate the effect of intervention with probiotics on ileal pouch inflammation and perfusion in the pouch, assessed by endoscopy, histology, fecal calprotectin and LDF. METHODS: A fermented milk product (Cultura; 500 ml) containing live lactobacilli (La-5) and bifidobacteria (Bb-12) was given daily for 4 weeks to 10 patients operated with ileal-pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). Mucosal perfusion was measured with LDF and the degree of inflammation was examined at predefined levels of the distal bowel by endoscopy and histology. Stool samples were cultured for lactobacilli and bifidobacteria and calprotectin were measured before and after intervention. RESULTS: The LDF measurements were reproducible in the pelvic pouch at each of the predefined levels, but did not change after intervention. The mucosal perfusion was reduced in the distal compared to the proximal part of the pouch. Calprotectin levels did not change significantly after intervention. The median endoscopic score for inflammation was significantly reduced by 50% after intervention, whereas the histological score did not change significantly. CONCLUSION: The results suggest that probiotics primarily act superficially, with change of gross appearance of the mucosa at endoscopy, but without significant effect on histological picture, mucosal perfusion or faecal calprotectin, during a relatively short period of 4 weeks.

Assessment of mucosal inflammation and blood flow in response to four weeks' intervention with probiotics in patients operated with a J-configurated ileal-pouch-anal-anastomosis (IPAA).

BACKGROUND: Pouchitis is a common and troublesome condition in patients operated on with ileal-pouch-anal-anastomosis (IPAA). A disturbed mucosal perfusion in the pouch has been suggested as a possible cause. Laser Doppler flowmetry (LDF) has been used successfully to measure gastric and colonic mucosal perfusion in humans. In a previous study, we demonstrated a reduced mucosal perfusion in the distal part of the pouch, during probiotic intervention, examined by LDF measurement. The aim of the present study was to confirm our previous results in a much larger material, and to compare the results of LDF measurements and inflammatory activity in ulcerative colitis (UC) patients with those in familial adenomatous polyposis (FAP) patients. METHODS: Five hundred millilitres of a fermented milk product (Cultura), containing live lactobacilli (La-5) and bifidobacteria (Bb-12), was given daily for 4 weeks to 41 UC and 10 patients with FAP, operated on with IPAA. Mucosal perfusion was measured with LDF and the degree of inflammation was examined at predefined levels of the distal bowel by histology and faecal calprotectin measurements both before and after intervention. We also evaluated the applicability of a Pouchitis Disease Activity Index (PDAI). RESULTS: The LDF measurements were reproducible in the pelvic pouch at each of the predefined levels, but did not change during intervention. Mucosal perfusion was significantly reduced in the distal compared to the proximal part of the pouch in the UC group (P < 0.05). The perfusion levels were higher in the FAP patients compared to the UC patientsat all predefined levels (P < 0.05). Calprotectin levels and histological score did not change significantlyafter intervention in any of the groups. The calprotectin level was significantly lower in the FAP compared to the UC group both before and after intervention. The PDAI decreased in both groups from alevel considered diagnostic for pouchitis to a level considered as not active pouchitis. The decreasewas significant for the UC patients. CONCLUSIONS: The results did not demonstrate an effect of probiotics on histology, although a significant effect on the PDAI was achieved, which concurs with the previously reported effect on symptoms and endoscopic score. The significantly reduced blood flow in the UC group compared to the FAP group, operated on with the same procedure, and the significantly increased calprotectin levels in the UC group, are original findings. Both findings may be related to an increased risk for pouchitis among UC patients. The lack of effect of intervention on mucosal perfusion does not exclude a role for reduced circulation as a cause of pouchitis based on the reduced LDF measurements in the distal part of the pouch.