D-line Approach for Safe Laparoscopic Cholecystectomy: Initial Experience.

Introduction The critical view of safety is an important concept for safe laparoscopic cholecystectomy. However, no standard step-by-step approach for achieving the critical view of safety has been established until now. Therefore, this study aims to evaluate a new approach for achieving the critical view of safety using the diagonal line of liver segment IV as an anatomical landmark. Patients and methods In this prospective non-randomized study, patients (n= 112) who underwent laparoscopic cholecystectomy for symptomatic cholelithiasis were included. The first 47 patients underwent laparoscopic cholecystectomy using the diagonal line approach (DLC group) whereas, the next 65 patients underwent laparoscopic cholecystectomy using the conventional method (CLC group).  Results No significant difference between both groups regarding the preoperative characteristics, operative time, and intraoperative blood loss. Laparoscopic subtotal cholecystectomy was performed more in the DLC group (6% vs 0%, p= 0.07). Whereas, in the CLC group, there was a tendency towards conversion to open cholecystectomy in difficult cases (6% vs 2%, p= 0.40). No intra- or postoperative complications occurred in either group.  Conclusion The diagonal line approach is a feasible and useful step-by-step technique to achieve the critical view of safety in laparoscopic cholecystectomy and enables surgeons to perform safe laparoscopic subtotal cholecystectomy in difficult cases.

Pancreatic Islet Transplantation into the Submandibular Gland: Our Experimental Experience and a Review of the Relevant Literature.

Pancreatic islet transplantation is a promising therapy for type 1 diabetes. Islet transplantation is clinically performed through intra-portal infusion, which is associated with several drawbacks, including poor engraftment. The histological resemblance between the submandibular gland and the pancreas renders it an attractive alternative site for islet transplantation. In this study, we refined the technique of islet transplantation into the submandibular gland to achieve good morphological features. Then, we transplanted 2600 islet equivalents into the submandibular glands of diabetic Lewis rats. Intra-portal islet transplantation was performed in diabetic rats as a control. Blood glucose levels were followed for 31 days, and an intravenous glucose tolerance test was performed. Immunohistochemistry was used to demonstrate the morphology of transplanted islets. Follow-up after transplantation showed that diabetes was cured in 2/12 rats in the submandibular group in comparison to 4/6 in the control group. The intravenous glucose tolerance test results of the submandibular and intra-portal groups were comparable. Immunohistochemistry showed large islet masses in the submandibular gland in all examined specimens with positive insulin staining. Our results show that submandibular gland tissue can support the islet function and engraftment but with considerable variability. Good morphological features were achieved using our refined technique. However, islet transplantation into rat submandibular glands did not demonstrate a clear advantage over conventional intra-portal transplantation.

Short-term inhalation of isoflurane improves the outcomes of intraportal hepatocyte transplantation.

Clinical hepatocyte transplantation (HTx) is only performed without general anesthesia, while inhalation anesthetics are usually used in animal experiments. We hypothesized that isoflurane may be a possible reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx. Syngeneic rat hepatocytes (1.0 × 107) were transplanted to analbuminemic rats with (ISO group) and without (AW group) isoflurane. The serum albumin, AST, ALT, LDH levels and several inflammatory mediators were analyzed. Immunohistochemical staining and ex vivo imaging were also performed. The serum albumin levels of the ISO group were significantly higher in comparison to the AW group (p < 0.05). The serum AST, ALT, LDH levels of the ISO group were significantly suppressed in comparison to the AW group (p < 0.0001, respectively). The serum IL-1ß, IL-10, IL-18, MCP-1, RNTES, Fractalkine and LIX levels were significantly suppressed in the ISO group. The ischemic regions of the recipient livers in the ISO group tended to be smaller than the AW group; however, the distribution of transplanted hepatocytes in the liver parenchyma was comparable between the two groups. Isoflurane may at least in part be a reason for the discrepancy between the results of animal experiments and the clinical outcomes of HTx.

KRP-203 Is a Desirable Immunomodulator for Islet Allotransplantation.

BACKGROUND: The current standard immunosuppressive regimens, calcineurin inhibitors, have diabetogenic and anti-vascularization effects on islet grafts. KRP-203, a sphingosine-1-phosphate functional antagonist, exerts its immunomodulatory function through lymphocyte sequestration. However, the effect of this antagonist on islets is unclear. We examined the effect of KRP-203 on the islet function and vascularization and sought a calcineurin-free regimen for islet allotransplantation. METHODS: KRP-203 was administered for 14 d to mice, then diabetogenic effect was evaluated by blood glucose levels and a glucose tolerance test. Static glucose stimulation, the breathing index, and insulin/DNA were examined using isolated islets. Islet neovascularization was evaluated using a multiphoton laser scanning microscope. After islet allotransplantation with either KRP-203 alone, sirolimus alone, or both in combination, the graft survival was evaluated by blood glucose levels and immunohistochemical analyses. A mixed lymphocyte reaction was also performed to investigate the immunologic characteristics of KRP-203 and sirolimus. RESULTS: No significant differences in the blood glucose levels or glucose tolerance were observed between the control and KRP-203 groups. Functional assays after islet isolation were also comparable. The multiphoton laser scanning microscope showed no inhibitory effect of KRP-203 on islet neovascularization. Although allogeneic rejection was effectively inhibited by KRP-203 monotherapy (44%), combination therapy prevented rejection in most transplanted mice (83%). CONCLUSIONS: KRP-203 is a desirable immunomodulator for islet transplantation because of the preservation of the endocrine function and lack of interference with islet neovascularization. The combination of KRP-203 with low-dose sirolimus may be promising as a calcineurin-free regimen for islet allotransplantation.

Decellularized Whole-Organ Pre-vascularization: A Novel Approach for Organogenesis.

Introduction: Whole-organ decellularization is an attractive approach for three-dimensional (3D) organ engineering. However, progress with this approach is hindered by intra-vascular blood coagulation that occurs after in vivo implantation of the re-cellularized scaffold, resulting in a short-term graft survival. In this study, we explored an alternative approach for 3D organ engineering through an axial pre-vascularization approach and examined its suitability for pancreatic islet transplantation. Methods: Whole livers from male Lewis rats were decellularized through sequential arterial perfusion of detergents. The decellularized liver scaffold was implanted into Lewis rats, and an arteriovenous bundle was passed through the scaffold. At the time of implantation, fresh bone marrow preparation (BM; n = 3), adipose-derived stem cells (ADSCs; n = 4), or HBSS (n = 4) was injected into the scaffold through the portal vein. After 5 weeks, around 2,600 islet equivalents (IEQs) were injected through the portal vein of the scaffold. The recipient rats were rendered diabetic by the injection of 65 mg/kg STZ intravenously 1 week before islet transplantation and were followed up after transplantation by measuring the blood glucose and body weight for 30 days. Intravenous glucose tolerance test was performed in the cured animals, and samples were collected for immunohistochemical (IHC) analyses. Micro-computed tomography (CT) images were obtained from one rat in each group for representation. Results: Two rats in the BM group and one in the ADSC group showed normalization of blood glucose levels, while one rat from each group showed partial correction of blood glucose levels. In contrast, no rats were cured in the HBSS group. Micro-CT showed evidence of sprouting from the arteriovenous bundle inside the scaffold. IHC analyses showed insulin-positive cells in all three groups. The number of von-Willebrand factor-positive cells in the islet region was higher in the BM and ADSC groups than in the HBSS group. The number of 5-bromo-2'-deoxyuridine-positive cells was significantly lower in the BM group than in the other two groups. Conclusions: Despite the limited numbers, the study showed the promising potential of the pre-vascularized whole-organ scaffold as a novel approach for islet transplantation. Both BM- and ADSCs-seeded scaffolds were superior to the acellular scaffold.

Preferable Transplant Site for Hepatocyte Transplantation in a Rat Model.

Intraportal injection is regarded as the current standard procedure of hepatocyte transplantation (HTx). In islet transplantation, which shares many aspects with HTx, recent studies have clarified that instant blood-mediated inflammatory reaction (IBMIR), characterized by strong innate immune responses, can cause poor engraftment, so other transplant sites to avoid such a reaction have been established. Although IBMIR was reported to occur in HTx, few reports have evaluated alternative transplant sites for HTx. In this study, we sought to determine the optimum transplant site for HTx. Rat hepatocytes (1.0 × 107) were transplanted at the 9 transplant sites (intraportal (IPO), intrasplenic (IS), liver parenchyma, subcutaneous, intraperitoneal, renal subcapsular, muscle, inguinal subcutaneous white adipose tissue, and omentum) of analbuminemic rats. The serum albumin levels, immunohistochemical staining (albumin, TUNEL, and BrdU), and in vivo imaging of the grafts were evaluated. The serum albumin levels of the IPO group were significantly higher than those of the other groups (p < .0001). The BrdU-positive hepatocyte ratio of liver in the IS group (0.9% ± 0.2%) was comparable to that of the IPO group (0.9% ± 0.3%) and tended to be higher than that of the spleen in the IS group (0.5% ± 0.1%, p = .16). Considering the in vivo imaging evaluation and the influence of splenectomy, the graft function in the IS group may be almost entirely achieved by hepatocytes that have migrated to the liver. The present study clearly showed that the intraportal injection procedure is more efficient than other procedures for performing HTx.

Improvement of hepatocyte engraftment by co-transplantation with pancreatic islets in hepatocyte transplantation.

Because of the fragility of isolated hepatocytes, extremely poor engraftment of transplanted hepatocytes remains a severe issue in hepatocyte transplantation. Therefore, improving hepatocyte engraftment is necessary to establish hepatocyte transplantation as a standard therapy. Since the pancreatic islets are known to have favorable autocrine effects, we hypothesized that the transplanted islets might influence not only the islets but also the nearby hepatocytes, subsequently promoting engraftment. We evaluated the effects of islet co-transplantation using an analbuminemic rat model (in vivo model). Furthermore, we established a mimicking in vitro model to investigate the underlying mechanisms. In an in vivo model, the hepatocyte engraftment was significantly improved only when the islets were co-transplanted to the nearby hepatocytes (p < 0.001). Moreover, the transplanted hepatocytes appeared to penetrate the renal parenchyma together with the co-transplanted islets. In an in vitro model, the viability of cultured hepatocytes was also improved by coculture with pancreatic islets. Of particular interest, the coculture supernatant alone could also exert beneficial effects comparable to islet coculture. Although insulin, VEGF, and GLP-1 were selected as candidate crucial factors using the Bio-Plex system, beneficial effects were partially counteracted by anti-insulin receptor antibodies. In conclusion, this study demonstrated that islet co-transplantation improves hepatocyte engraftment, most likely due to continuously secreted crucial factors, such as insulin, in combination with providing favorable circumstances for hepatocyte engraftment. Further refinements of this approach, especially regarding substitutes for islets, could be a promising strategy for improving the outcomes of hepatocyte transplantation.

Human Amniotic Epithelial Cells Secretome: Components, Bioactivity, and Challenges.

Human amniotic epithelial cells (hAECs) derived from placental tissue have received significant attention as a promising tool in regenerative medicine. Several studies demonstrated their anti-inflammatory, anti-fibrotic, and tissue repair potentials. These effects were further shown to be retained in the conditioned medium of hAECs, suggesting their paracrine nature. The concept of utilizing the hAEC-secretome has thus evolved as a therapeutic cell-free option. In this article, we review the different components and constituents of hAEC-secretome and their influence as demonstrated through experimental studies in the current literature. Studies examining the effects of conditioned medium, exosomes, and micro-RNA (miRNA) derived from hAECs are included in this review. The challenges facing the application of this cell-free approach will also be discussed based on the current evidence.

Strategy towards tailored donor tissue-specific pancreatic islet isolation.

BACKGROUND: Optimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. Collagen V (Col V) and collagen III (Col III) are crucial target matrices of ColG and ColH, respectively. We herein investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome. METHODS: Islet isolation was performed in Lewis and SD rats using different ColG:ColH ratios (5:1, 1:1 and 1:5; n = 7/group). The composition of Col III and Col V was examined using immunohistochemical staining, real-time polymerase chain reaction (PCR), Western blotting and mass spectrometry. Chain types in collagen I (Col I) were also assessed using mass spectrometry. RESULTS: No beneficial effects were observed by increasing the ColG amount, irrespective of the rat strain. In contrast, the islet yield in Lewis rats was considerably increased by high amounts of ColH but decreased in SD rats, suggesting that Lewis pancreas contains more Col III than SD pancreas. Neither immunohistochemical nor real-time PCR showed correlation with isolation outcome. However, Western blotting revealed that Lewis contained considerably higher amount of Col III than SD (p = 0.10). Likewise, Col-I(α1)/Col-III(α1) and Col-I(α2)/Col-III(α1) were significantly lower in Lewis than in SD rats (p = 0.007, respectively). Furthermore, the isolation outcome was considerably correlated with the composition of homotrimeric Col I. CONCLUSIONS: The Col III expression and the composition of homotrimeric Col I in pancreatic tissues determined using mass analyses appeared useful for optimizing the ColG:ColH ratio in islet isolation.

A Simple and Useful Predictive Assay for Evaluating the Quality of Isolated Hepatocytes for Hepatocyte Transplantation.

No optimal assay for assessing isolated hepatocytes before hepatocyte transplantation (HTx) has been established, therefore reliable and rapid assays are warranted. Isolated rat hepatocytes were dipped in a water bath (necrosis model), and were also cultured with Okadaic acid (apoptosis model) or vehicle, followed by cellular assessment including trypan blue exclusion (TBE) viability, ADP /ATP ratio, plating efficiency (PE), DNA quantity and ammonia elimination. Hepatocytes were transplanted into the liver of analbuminemic rats, subsequently engraftment was assessed by serum albumin and the histology of transplanted grafts. In the necrosis model, the ADP/ATP ratio was strongly and negatively correlated with the TBE (R2 = 0.559, P < 0.001). In the apoptosis model, the ADP/ATP ratio assay, PE, DNA quantification and an ammonia elimination test clearly distinguished the groups (P < 0.001, respectively). The ADP/ATP ratio, PE and DNA quantity were well-correlated and the ammonia elimination was slightly correlated with the transplant outcome. TBE could not distinguish the groups and was not correlated with the outcome. The ADP/ATP ratio assay predicted the transplant outcome. PE and DNA quantification may improve the accuracy of the retrospective (evaluations require several days) quality assessment of hepatocytes. The ADP/ATP ratio assay, alone or with a short-term metabolic assay could improve the efficiency of HTx.

Pattern of Bone Generation after Irradiation in Vascularized Tissue Engineered Constructs.

BACKGROUND: Regenerative medicine modalities provide promising alternatives to conventional reconstruction techniques but are still deficient after malignant tumor excision or irradiation due to defective vascularization. METHODS: We investigated the pattern of bone formation in axially vascularized tissue engineering constructs (AVTECs) after irradiation in a study that mimics the clinical scenario after head and neck cancer. Heterotopic bone generation was induced in a subcutaneously implanted AVTEC in the thigh of six male New Zealand rabbits. The tissue construct was made up of Nanobone (Artoss GmbH; Rostock, Germany) granules mixed with autogenous bone marrow and 80 µL of bone morphogenic protein-2 at a concentration of 1.5 µg/µL. An arteriovenous loop was created microsurgically between the saphenous vessels and implanted in the core of the construct to induce axial vascularization. The constructs were subjected to external beam irradiation on postoperative day 20 with a single dose of 15 Gy. The constructs were removed 20 days after irradiation and subjected to histological and immunohistochemical analysis for vascularization, bone formation, apoptosis, and cellular proliferation. RESULTS: The vascularized constructs showed homogenous vascularization and bone formation both in their central and peripheral regions. Although vascularity, proliferation, and apoptosis were similar between central and peripheral regions of the constructs, significantly more bone was formed in the central regions of the constructs. CONCLUSION: The study shows for the first time the pattern of bone formation in AVTECs after irradiation using doses comparable to those applied after head and neck cancer. Axial vascularization probably enhances the osteoinductive properties in the central regions of AVTECs after irradiation.

The Optimization of the Prevascularization Procedures for Improving Subcutaneous Islet Engraftment.

BACKGROUND: Subcutaneous islet transplantation is associated with minimal invasiveness, but poor vascularization. Thus, the optimization of the prevascularization procedures is crucial for improving the outcomes. Although the effectiveness of basic fibroblast growth factor (bFGF) was reported, the optimal procedures remain unclear. We sought to optimize the prevascularization procedures including the use of a novel scaffold, recombinant peptide (RCP). METHODS: Devices containing various amount of bFGF with/without heparin or RCP were implanted into the subcutaneous space of diabetic C57BL/6 mice. Syngeneic islets were transplanted into the prevascularized space. Blood glucose, intraperitoneal glucose tolerance, and immunohistochemistry were evaluated. RESULTS: The cure rates in all the device groups irrespective of bFGF doses were considerably higher than in the nondevice group. The cure rate in the bFGF0 group was unexpectedly higher than that in the subcutaneous islet transplant alone group (the None group) (57.1% vs 28.6%). Glucose tolerance was ameliorated in the bFGF10(-), 10(+) and 15(-) groups. The number of von Willebrand factor-positive vessels in the bFGF10(+) group was significantly higher than that in the None and bFGF0 groups (P < 0.01). Taken together, the bFGF10(+) group was considered to have received optimized procedures. In a marginal graft model, the efficiency in the RCP group was better than that in the bFGF10(+) group, furthermore, comparable to that in the intraportal transplantation group. Unlike bFGF, no bleeding and effusion were observed in the RCP group. CONCLUSIONS: These results suggest that optimizing biomaterials to induce efficient prevascularization could be a novel strategy for improving subcutaneous islet transplantation.

Heterotopic thyroid autotransplantation: A preliminary clinical study.

BACKGROUND: Thyroid autotransplantation offers an attractive alternative to subtotal thyroidectomy and replacement therapy. However, it has not been sufficiently clinically investigated. METHODS: Total thyroidectomy was performed for 20 patients with benign thyroid disorders, and at least 10 g of thyroid tissue were implanted intramuscularly. Graft monitoring was achieved through 99m Tc scan at 2 months, free triiodothyronine (FT3), FT4, and thyroid-stimulating hormone (TSH) measurements at 1, 2, 4, 6, 8, 10, and 12 months postoperatively, and clinical examination. RESULTS: Grafts survived and gradually functioned in all patients to a variable extent after a latent period (mean 99m Tc uptake = 1.28 ± 0.37%). Mean values for FT3, FT4, TSH at 12 months were 1.75 ± 0.48 pg/mL, 1.06 ± 0.26 ng/dL, and 28.08 ± 34.01 µIU/mL respectively. CONCLUSION: Thyroid autotransplantation restored euthyroid status in 33.3% of patients after 12 months. A role of age, operative time, and 99m Tc-uptake in determining the final graft outcome is possible.

Knowledge, Attitudes, and Beliefs Regarding Antimicrobial Therapy and Resistance Among Physicians in Alexandria University Teaching Hospitals and the Associated Prescription Habits.

Irresponsible prescription of antimicrobials (AMs) is the driving factor for the growing antimicrobial resistance (AMR) crisis. In this study, we assessed the knowledge, attitudes, perceptions, and beliefs regarding AMs and AMR together with the prescription habits of physicians in three University hospitals in Alexandria, Egypt. A 40-question survey was used. Physicians were stratified into residents and practicing staff members, and further into various departments. Clinical pharmacists at the University main hospital were included for comparative purposes. A total of 319 questionnaires were completed (response rate = 91.4%). Participants demonstrated fair average knowledge about AMs (4.71 ± 1.29 out of 7), with no significant difference between residents and staff members, whereas clinical pharmacists scored significantly higher on knowledge questions (p < 0.005). Participants showed poor awareness regarding local AMR patterns of Klebsiella pneumoniae and Pseudomonas aeruginosa (13% and 23%, respectively). AMR was perceived as a global (95%), national (97%), and local (85%) problem. High confidence regarding use of AMs was noticed with significantly higher levels among staff members (70.3% vs. 86.7%, p < 0.05). Most participants agreed that the patients' demands (78.5%) and socioeconomic statuses (76.3%) do influence their choices. The most significant knowledge deficit was regarding dosage adjustment in renal patients, and the survey highlighted poor engagement in educational activities, limited awareness of local resistance patterns, and neglect in explaining the side-effects to patients. Patients' demands and socioeconomic statuses were also shown to influence the physicians' decisions.

Rapid hepatic perfusion decellularization: technique and critique.

BACKGROUND: Organ shortage facing the increasing success of liver transplantation has provoked research into the utilization of animal organs for clinical transplantation. The technique of whole-organ decellularization aims at the removal of the antigenic cellular content, thus evading the immune rejection cascade and the production of complex three-dimensional extracellular matrices of the entire organs with preservation of their intrinsic vascular networks rendering them transplantable. The aim of this study was the production of decellularized rabbit liver matrices by applying a simple, rapid perfusion decellularization technique and their characterization (both qualitatively and quantitatively). MATERIALS AND METHODS: Decellularization of the caudate hepatic lobes of New Zealand white rabbits (n = 22) was achieved through sequential perfusion of the portal venous system with deionized water, 0.8% Triton X-100 and 0.8% sodium dodecyl sulphate (SDS). Decellularized specimens were characterized both qualitatively (histology, fluoroscopy, corrosion casting and scanning electron microscopy) and quantitatively (total collagen assay [colorimetric] and total DNA assay [Hoechst 33258]). A Student's t-test was used to compare quantitative laboratory results before and after decellularization. A probability (P) value of <0.05 was considered significant. RESULTS: Effective decellularization was achieved as proven by histology and quantitative assessment (DNA remnants <1.5%, P = 0.0009), while preserving 68% of the total collagen content (P = 0.003). Portal vascular network integrity was confirmed by fluoroscopy and corrosion casting. Scanning electron microscopy also confirmed the preservation of the three-dimensional architecture. CONCLUSIONS: Liver perfusion decellularization technique using both 0.8% Triton X-100 and 0.8% SDS is a simple and rapid technique, yielding efficiently decellularized liver matrices preserving their vascular integrity, 3D architecture and 68% of total collagen content.

Review of tumoral calcinosis: A rare clinico-pathological entity.

Tumoral calcinosis (TC) has long been a controversial clinico-pathological entity. Its pathogenesis and genetic background have been gradually unravelled since its first description in 1943. According to the presence or absence of an underlying calcifying disease process, TC has been divided into primary and secondary varieties. Two subtypes of the primary variety exist; a hyper-phosphatemic type with familial basis represented by mutations in GalNAc transferase 3 gene (GALNT3), KLOTHO or Fibroblast growth factor 23 (FGF23) genes, and a normo-phosphatemic type with growing evidence of underlying familial base represented by mutation in SAMD9 gene. The secondary variety is mainly associated with chronic renal failure and the resulting secondary or tertiary hyperparathyroidism. Diagnosis of TC relies on typical radiographic features (on plain radiographs and computed tomography) and the biochemical profile. Magnetic resonance imaging can be done in difficult cases, and scintigraphy reflects the disease activity. Treatment is mainly surgical for the primary variety; however, a stage-oriented conservative approach using phosphate binders, phosphate restricted diets and acetazolamide should be considered before the surgical approach is pursued due to the high rate of recurrences and complications after surgical intervention. Medical treatment is the mainstay for treatment of the secondary variety, with failure warranting subtotal or total parathyroidectomy. Surgical intervention in these patients should be kept as a last resort.