Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation.

Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

Remote Ischemic Conditioning on Recipients of Deceased Renal Transplants Does Not Improve Early Graft Function: A Multicenter Randomized, Controlled Clinical Trial.

Delayed graft function is a frequent complication following deceased donor renal transplantation, and is closely related to ischemia-reperfusion injury. Experimental and clinical studies have shown protection by remote ischemic conditioning (RIC). We hypothesized that recipient RIC before kidney graft reperfusion reduces the time to graft recovery. This multicenter, blinded, randomized, controlled clinical trial included 225 adult recipients of renal transplants from deceased donors at four transplantation centers in Denmark, Sweden, and the Netherlands. Participants were randomized 1:1 to RIC or sham-RIC. RIC consisted of 4 × 5-min thigh occlusion by an inflatable tourniquet each followed by 5-min deflation, performed during surgery prior to graft reperfusion. The tourniquet remained deflated for sham-RIC. The primary endpoint was the estimated time to a 50% decrease in baseline plasma creatinine (tCr50) calculated from plasma creatinine measurements 30 days posttransplant or 30 days after the last, posttransplant dialysis. No significant differences were observed between RIC and sham-RIC-treated patients in the primary outcome median tCr50 (122 h [95% confidence interval [CI] 98-151] vs. 112 h [95% CI 91-139], p = 0.58), or the number of patients receiving dialysis in the first posttransplant week (33% vs. 35%, p = 0.71). Recipient RIC does not reduce the time to graft recovery in kidney transplantation from deceased donors. ClinicalTrials.gov: NCT01395719.

A Grading Score for Colon Preservation Injury in the Rat.

BACKGROUND: Colon transplantation is rarely performed because of the fear for an advanced ischemic injury that may favor septic complications. Systematic studies on colon preservation are missing. The score used to evaluate the preservation injury of the colon is adapted from that used for the small intestine, despite histological and biological differences between the two organs. We studied sequentially the tissue changes in the rat colon during prolonged cold storage (CS) in histidine-tryptophan-ketoglutarate (HTK) solution and designed a grading score specific for the colon. METHODS: Large bowels of Sprague-Dawley rats (n = 9) were perfused in situ with HTK and stored at 4°C for 6 hours, 12 hours, 18 hours, and 24 hours. Samples from the proximal colon were stained with hematoxylin-eosin and alcian blue. Tight junction protein zonulla occludens (ZO)-1 was also studied. RESULTS: Minimal subepithelial edema (hallmark of small intestinal preservation injury) was observed throughout the 24 hours of CS. The two major changes observed during the colonic CS were progressive submucosal edema and the depletion of Goblet cells (GC). The submucosal edema was absent at 6 hours, started after 12 hours, and become significant (over 50% of the circumference) after 18 hours of CS. Depletion of GC started in the luminal half of the crypts between 12 and 18 hours of CS, and all samples revealed significant GC depletion only after 24 hours. The overall appearance of the mucosa was little affected under the CS, and ZO-1 expression was frequently maintained throughout the first 18 hours. CONCLUSIONS: The colon is more resilient to cold ischemia than the small bowel and maintains its histological epithelial features longer than the small intestine. On the basis of these serial observations, we suggest the following grading score: grade 0: normal mucosa, repleted GC, mucosa adhering to the muscular layer; grade 1: limited submucosal edema, repleted GC; grade 2: limited submucosal edema, GC depletion in the luminal half of the crypts; grade 3: advanced (>50% of circumference) submucosal edema, GC depletion in the luminal half of the crypts; grade 4: advanced mucosal injury (edema, GC depletion, epithelial breakdown).

Optimal Solution Volume for Luminal Preservation: A Preclinical Study in Porcine Intestinal Preservation.

BACKGROUND: Rodent studies suggest that luminal solutions alleviate the mucosal injury and prolong intestinal preservation but concerns exist that excessive volumes of luminal fluid may promote tissue edema. Differences in size, structure, and metabolism between rats and humans require studies in large animals before clinical use. METHODS: Intestinal procurement was performed in 7 pigs. After perfusion with histidine-tryptophan-ketoglutarate (HTK), 40-cm-long segments were cut and filled with 13.5% polyethylene glycol (PEG) 3350 solution as follows: V0 (controls, none), V1 (0.5 mL/cm), V2 (1 mL/cm), V3 (1.5 mL/cm), and V4 (2 mL/cm). Tissue and luminal solutions were sampled after 8, 14, and 24 hours of cold storage (CS). Preservation injury (Chiu score), the apical membrane (ZO-1, brush-border maltase activity), and the electrolyte content in the luminal solution were studied. RESULTS: In control intestines, 8-hour CS in HTK solution resulted in minimal mucosal changes (grade 1) that progressed to significant subepithelial edema (grade 3) by 24 hours. During this time, a gradual loss in ZO-1 was recorded, whereas maltase activity remained unaltered. Moreover, variable degrees of submucosal edema were observed. Luminal introduction of high volumes (2 mL/mL) of PEG solution accelerated the development of the subepithelial edema and submucosal edema, leading to worse histology. However, ZO-1 was preserved better over time than in control intestines (no luminal solution). Maltase activity was reduced in intestines receiving luminal preservation. Luminal sodium content decreased in time and did not differ between groups. CONCLUSIONS: This PEG solution protects the apical membrane and the tight-junction proteins but may favor water absorption and tissue (submucosal) edema, and luminal volumes >2 mL/cm may result in worse intestinal morphology.

Screening of early antigen genes of adult-stage Trichinella spiralis using pig serum from different stages of early infection.

The goal of this work was to identify novel, early antigens present in Trichinella spiralis. To this end, a cDNA library generated from 3-day old adult worms (Ad3) was immunologically screened using serum from a pig infected with 20,000 muscle larvae. The serum was obtained from multiple, time course bleeds coinciding with early worm development. Seventeen positive clones were isolated using serum obtained at 20 days post infection (dpi). All clones corresponded to one gene that exhibited high sequence identity with the T. spiralis ATP-dependent RNA helicase DDX19B which is involved in parasite growth and development. In addition, nine additional positive clones representing 5 unique genes were identified when the library was screened with 30 dpi serum; four of these five genes displayed high similarity with members of a putative T. spiralis serine protease family known to be involved in host invasion and host-parasite interactions. The remaining gene aligned with the T. spiralis hypothetical ORF 11.30. The identification of these antigens provides potential candidates for the early diagnosis of trichinellosis and for the development of a vaccine against this parasite.

First detection of Echinococcus granulosus G1 and G7 in wild boars (Sus scrofa) and red deer (Cervus elaphus) in Romania using PCR and PCR-RFLP techniques.

The epidemiological status of CE in our country places Romania into the top of the European countries and among the first countries worldwide. Two hundred ninety wild animals (267 wild boars, 21 red deer and 2 mouflons) hunted in a private ground from Bihor county from western Romania were the subject of the necropsy in January 2012.Out of 290 wild animals, 35 were positive during necropsy for hydatic cysts and from these, 33 wild boars and 2 red deer had hydatic cystsonly in the liver parenchyma. This paper presents the first identification of Echinococcus granulosus G1 in cervids (100%). In wild boars it were identified G1 (45.5%) and G7 genotypes (39.4%). The mouflons were free of hydatic cysts. Our resultsconfirmthat the sheep strain (G1) is the predominant Echinococcus genotype, which occurs, followed closely by the pig strain (G7) and that wildlife reservoirs should be taken in consideration for the management of the disease.

Intraluminal polyethylene glycol stabilizes tight junctions and improves intestinal preservation in the rat.

Rapidly progressing mucosal breakdown limits the intestinal preservation time below 10 h. Recent studies indicate that intraluminal solutions containing polyethylene glycol (PEG) alleviate preservation injury of intestines stored in UW-Viaspan. We investigated whether a low-sodium PEG solution is beneficial for intestines stored in histidine-tryptophane-ketoglutarate (HTK) preservation solution. Rat intestines used as control tissue (group 1) were perfused with HTK, groups 2 and 3 received either a customized PEG-3350 (group 2) or an electrolyte solution (group 3) intraluminally before cold storage. Tissue injury, brush-border maltase activity, zonula occludens-1 (ZO-1) and claudin-3 expression in the tight junctions (TJ) were analyzed after 8, 14 and 20 h. We measured epithelial resistance and permeability (Ussing chamber) after 8 and 14 h. Group 2 had superior morphology while maltase activity was similar in all groups. TJ proteins rapidly decreased and decolocalized in groups 1 3; these negative events were delayed in group 2, where colocalization persisted for about 14 h. Intestines in group 2 had higher epithelial resistance and lower permeability than the other groups. These results suggest that a customized PEG solution intraluminally reduces the intestinal preservation injury by improving several major epithelial characteristics without negatively affecting the brush-border enzymes or promoting edema.

Ixodes ricinus is the dominant questing tick in forest habitats in Romania: the results from a countrywide dragging campaign.

In 2010 and 2011, questing ticks were collected from 188 forested locations in all the 41 counties of Romania using the dragging method. The total of 13,771 ticks collected belonged to eleven species: Ixodes ricinus (86.9 %), Dermacentor marginatus (9.5 %), Haemaphysalis punctata (2.6 %), H. concinna (0.6 %), H. sulcata (0.3 %), H. parva (0.1 %), Hyalomma marginatum (0.02 %), D. reticulatus (0.02 %), I. crenulatus (0.007 %), I. hexagonus (0.007 %) and I. laguri (0.007 %). Ixodes ricinus was present in 97.7 % (n = 180) of locations, occurring exclusively in 41.7 % of the locations, whereas it was the dominant species in 38.8 % of the other locations, accounting for over 70 % of the total tick community. The following most common questing ticks were D. marginatus, H. punctata and H. concinna. Ixodes ricinus co-occurred with one, two or three sympatric species. The occurrence of D. reticulatus in forested habitats from Romania was found to be accidental.

Natural killer group 2 member D cell recruitment driven by major histocompatibility complex class I chain-related antigens A and B: a possible mechanism during acute intestinal allograft rejection in the mouse.

Intestinal allograft rejection occurs frequently despite potent T-cell depletion protocols. We investigated the interaction of major histocompatibility complex class I chain-related antigens A and B (MICA/B; a ligand for natural killer [NK] cells) and NK group 2 member D (NKG2D) cells as an alternative mechanism for acute rejection (AR) of the intestinal graft. Heterotopic intestinal allotransplantation was performed from BalbC to C57Bl mice. Samples of grafted and native intestine were obtained at days 1, 3, 6, and 8 after transplantation (n = 4-6). We performed immunostaining for MICA/B and NKG2D. Moderate AR with increased crypt apoptosis was observed at day 6 and advanced AR with crypt destruction and mucosal sloughing was present by day 8. Low MICA/B levels were observed in grafted and native intestines on day 1. MICA/B expression gradually increased in the grafts during AR but not in the native intestines. The up-regulation was found mostly in the crypts. NKG2D+ cell counts that increased in the graft colocalized with MICA/B. The increase was most prominent in the crypt and villus. Together, these results suggest that MICA/B up-regulation and its subsequent interaction with the NK cells may represent an important link between innate and adaptive immune responses early after intestinal transplantation.

Infectious complications after multivisceral transplantation in adults.

UNLABELLED: It is thought that multivisceral transplantation requires high levels of immunosuppression and therefore, patients run an increased risk of infection. We retrospectively reviewed our center's experience with clinically relevant infectious complications. PATIENTS: Between 2000 and 2005, 10 adult patients underwent multivisceral transplantation. Two immunosuppression protocols were used: between 2000 and 2003, a high immunosupression protocol (six patients; daclizumab induction, tacrolimus trough levels >20 ng/mL and steroids) and an immunomodulatory, low imunosuppression scheme from 2003 onward (four patients; ATG induction, tacrolimus levels 5 to 10 ng/mL, no steroids). Standard antimicrobial prophylaxis consisted of vancomycin, meropenem, and amphotericin B. Cytomegalovirus (CMV) prophylaxis was used in all but first two cases. Donor and recipient CMV status were D+/R+ (n = 7), D+/R- (n = 2), D-/R+ (n = 1). RESULTS: The median follow-up period was 627 days (range, 19 to 2207 days). A total of 47 infectious episodes were recorded in all patients (range 1 to 14 per patient). The etiology was bacterial in 32 (69%), viral in 8 (17%), and fungal in 7 (14%) cases. The most frequent were catheter related (n = 13) followed by respiratory (n = 7), intraabdominal (n = 6), and wound infections (n = 5). Symptomatic viral infection of the graft (CMV gastritis or enteritis, adenoviral enteritis) was also encountered. Epstein-Barr virus was transiently detected in the serum of nine patients, one of whom later developed posttransplant lymphoproliferative disorder (PTLD). Three deaths all among patients receiving high immunosuppression were owing to infectious complications: pulmonary PTLD at 4 months posttransplantation, ruptured mycotic aneurysm after 8 weeks, and sepsis after 3 weeks. CONCLUSIONS: Infections accounted for a high morbidity after multivisceral transplantation, representing the leading cause of mortality. Exhaustive monitoring, early antimicrobial intervention, and lower immunosuppression may improve the outcome.

Laser-Doppler flowmetry in the monitoring of the human intestinal allograft: a preliminary report.

During acute rejection, graft endothelium becomes a prime target for recipient immune cells. Animal studies have shown reduced microvascular perfusion, probably due to increased endothelial-leukocyte interaction and endothelial impairment, leading to graft damage. Using laser-Doppler flowmetry (LDF), we correlated the microvascular blood flow in the intestinal mucosa of five patients receiving multivisceral grafts with clinical events and pathology results. Measurements (n = 75) were performed during the first 4 weeks posttransplantation by inserting the LDF flexible probe through the ileostomy for 25 to 30 cm. Forty-six of the 75 measurements were performed within 24 hours of endoscopy and biopsy. In uncomplicated cases, we recorded a gradual increase in mucosal perfusion during the first week posttransplantation that presumably reflected regeneration after reperfusion injury. Increased mucosal perfusion did not seem to correlate with rejection or other adverse clinical events. Sudden and sustained decreases in mucosal perfusion by 30% or more compared to the previous measurements were associated with septic episodes, rejection, or both. LDF revealed a good sensitivity in monitoring the intestinal microcirculation. It was able to indicate perfusion changes associated with acute rejection. The relatively low specificity of LDF may be compensated by the low invasivity, allowing frequent investigation. LDF may be an additional tool for routine monitoring of intestinal allografts.

Transplantation of preconditioned intestinal grafts is associated with lower inflammatory activation and remote organ injury in rats.

Reperfused grafts--particularly the intestine--release free radicals and cytokines into the systemic circulation. The type of discharge, which is greatly dependent on the local injury, may also induce inflammatory activation in distant organs and leading to multiple system and organ failure. It has been suggested that intestinal grafts from tacrolimus (TRL)-pretreated donors show improved morphology and microcirculation. We studied whether transplantation of intestines from TRL-pretreated donors influenced inflammatory response and remote organ injury posttransplantation. Donor Sprague Dawley rats received TRL or saline (controls) intravenously at 6 hours prior to graft harvest. The intestinal grafts were preserved in saline for 3 hours before transplantation. At 6 and 12 hours postreperfusion hepatic and renal cortical microcirculation were assessed using laser-Doppler flowmetry (n = 8-12 per group). Blood pressure was measured; liver, kidney, and serum samples were obtained. We analyzed hepatic and renal ICAM-1 expression and caspase-3-like activity as well as plasma content of tumor necrosis factor-alpha and interleukin-6. Pretreated graft recipients had higher mean arterial pressure (82 +/- 10 vs 51 +/- 17 mm Hg, P < .05) and renal perfusion at 6 hours whereas liver perfusion was similar at both 6 and 12 hours. Liver and renal functions were also superior among recipients of pretreated grafts. Both caspase-3-like activity and ICAM-1 expression in liver and kidney were lower in pretreated graft recipients. Plasma IL-6 levels were lower in animals receiving pretreated grafts. Transplantation of intestines from TRL-pretreated donors was followed by a lower systemic inflammatory response, improved organ function and decreased remote injury early posttransplantation compared with animals receiving grafts from untreated donors.

Intestinal preservation injury: a comparison between different rat strains.

AIMS: Comparison between the extensive information on intestinal preservation injury in the rat is challenging since various preservation solutions, technical details, and grading systems have been used. This study investigates if strain represents another relevant variable for preservation injury outcome. METHODS: Grafts from Piebald-Viral-Glaxo (PVG), Lewis, Brown Norway (BN), Wistar, and Sprague-Dawley (SD) male rats (n = 8/strain) were used. Grafts were perfused and stored at 4 degrees C in University of Wisconsin (UW) solution (with or without luminal preservation). Intestinal histology was evaluated at 8, 16, and 24 hours of preservation using the Park score. Lactate, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and glucose were measured in the preservation solution. RESULTS: Grafts from PVG and Lewis showed significantly lower injury scores compared to BN, Wistar, and SD at 8 and 16 hours, a difference that disappeared at 24 hours of preservation. Luminal preservation significantly reduced the injury score for all strains, except SD, at 8 and at 16 hours, a difference that disappeared at 24 hours. Biochemical analyses of LDH and lactate levels show a similar pattern both between different strains and the effect of luminal preservation, although not statistically significant. CONCLUSION: Different rat strains seem to have different susceptibility to intestinal preservation injury under identical conditions. These results indicate that PVG and Lewis are more resistant to preservation injury than Wistar, BN, or SD. The beneficial effect of luminal preservation with UW solution is limited after 8 to 16 hours.

Monitoring of the intestinal mucosal perfusion using laser Doppler flowmetry after multivisceral transplantation.

BACKGROUND: Graft endothelium constitutes a prime target during acute rejection. Infiltration of T cells, monocytes, and enhanced endothelial-leukocyte interactions result in microvascular impairment and altered perfusion. MATERIALS AND METHODS: We measured mucosal blood flow using a laser Doppler flowmeter in three patients undergoing multivisceral transplantation. Thirty-seven measurements were performed through the ileostomy over the first 4 weeks posttransplantation. Most measurements were performed within a 24-hour interval from endoscopy and biopsy. RESULTS: Mucosal perfusion increased throughout the first postoperative week and eventually stabilized around levels specific for each patient. Mucosal perfusion remained stable during graft pancreatitis, but decreased 35% to 55% from baseline (the average value of the previous measurements) during acute rejection and sepsis. During the first week posttransplantation there was a gradual increase in mucosal perfusion, which might reflect regeneration after reperfusion injury. Increased mucosal perfusion did not seem to correlate with rejection or other adverse clinical events. A sudden decrease in mucosal perfusion of 30% or more compared to the previous measurements was associated with septic episodes and/or rejection.

FK506 donor pretreatment improves intestinal graft microcirculation and morphology by concurrent inhibition of early NF-kappaB activation and augmented HSP72 synthesis.

FK506 protects against ischemia-reperfusion injury but the mechanisms remain unclear. We investigated the impact of donor pretreatment using FK506 on graft microcirculation and morphology after intestinal transplantation. FK506 was given intravenously to SD rats (0.3 mg/kg) 6 hours before graft harvesting while controls received saline (n = 7/group). Grafts were stored for 3 hours in saline, then transplanted. Preservation induced similar lesions in both groups, but pretreated grafts showed better morphology than controls at 20 minutes after reperfusion. Six hours post-reperfusion, preconditioned grafts revealed near-normal morphology, whereas controls showed short villi, denuded areas, and intense inflammation. Pretreated grafts displayed a lower apoptotic rate and reduced caspase-3 activity. Hsp72 expression was enhanced in preconditioned grafts at harvesting, after preservation, and 20 minutes post-reperfusion compared to controls. Control grafts showed intranuclear p65 (activation of NFkappaB) at 20 minutes post-reperfusion; whereas pretreated grafts displayed no intranuclear p65. However, at 6 hours, comparable intranuclear p65 levels were found in both groups. ICAM-1 was low in both groups after preservation and early post-reperfusion, but greatly increased in controls at 6 hours post-reperfusion. In contrast, pretreated grafts continued to lack ICAM-1. Microvascular perfusion was comparable at 20 minutes. Six hours later, pretreated grafts had 30% increased perfusion, while in controls it was slightly decreased. FK506 alleviated reperfusion injury by blocking NF-kappaB activation and ICAM-1 transcription, thus decreasing endothelial activation and improving the microcirculation. It also induces Hsp72, therefore inhibiting apoptosis and accelerating morphologic restoration.

Accelerated acute rejection of the intestinal graft in CD28-deficient mice.

OBJECTIVE: Multiple in vivo studies have shown that the pace and severity of graft rejection is little or not at all changed by deleting CD28 molecules in the recipient. These findings contrast with the effects of monoclonal antibody therapy aimed the same costimulatory target. The objective of the present study was to evaluate how the acute rejection process is affected in CD28-deficient mice using a fully allogeneic, highly immunologically reactive transplant model. METHODS: Heterotopic vascularized small bowel transplants were performed in 24 recipient mice divided into 4 groups: 2 wild-type and 2 knockout groups. Each group consisted of 5 to 7 animals in which BalbC mice were used as intestinal donors to either wild-type C57BL6 or C57BL6 background CD28-deficient recipient mice. Selected endpoints were 3 and 6 postoperative days (POD). Intestinal rejection was evaluated by mucosal laser Doppler flowmetry (expressed in perfusion units) and histology (expressed in rejection grades). RESULTS: Acute rejection occurred in both wild-type and CD28-deficient groups. At POD 3, no significant difference was noted between groups in terms of mucosal perfusion and histology. At POD 6, significant differences in graft mucosal perfusion and histology revealed a more aggressive rejection in the CD28-deficient group compared to the wild-type group. CONCLUSIONS: The present study showed that the severity of intestinal graft rejection responses was amplified by deleting CD28 molecules. Together with data from other studies, these results suggest a different pattern of distribution and/or activation of CD28/B7 receptors in various organs.

Intragraft heat shock protein-60 expression after small bowel transplantation in the mouse.

The time course of heat shock protein 60 (hsp 60) expression after intestinal transplantation in syngeneic and allogeneic combination was correlated with the degree of rejection. Hsp 60 expression was assessed by immunostaining; rejection degree was established by histologic examination on posttransplantation days 1, 3, 6, and 8. No signs of rejection occurred in syngeneic grafts at any time. In the allogeneic setting, rejection was absent in all but 1 case on postoperative day 3. Three days later moderate rejection was evident based on focal crypt destruction and focal mucosal ulceration, whereas at postoperative day 8 extensive mucosal sloughing was the dominant feature, consistent with advanced rejection. Hsp 60 remained undetectable in the syngeneic setting at all times. In allografts, hsp 60 was initially expressed on posttransplant day 3, increasing synchronously with the progression of rejection at days 6 and 8. Hsp 60 expression was localized almost exclusively to the crypt area and the lower third of the villi. In conclusion, the rejection of murine allogeneic intestinal grafts is characterized by a progressive expression of hsp 60 in the epithelium.