Francesco Cacciabaudo

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.

Pancreatic islets from non-heart-beating donor pig: Two-layer preservation method in an in vitro porcine model

Purpose Pancreata from non-heart beating donors could represent an unlimited source of islets if their cell viability can be efficiently preserved during the time necessary to process the organs by the use of a better solution of preservation compared to the classic University of Wisconsin solution. The aim of this study was to determine whether it is possible to obtain functioning “alive islets” from non-heart-beating donors by comparing, on a porcine model, the classic “UW ice-store” method with a two-layer cold storage method (TLM) using oxygenated Perfluorocarbons (PFC) and UW. Methods Whole pancreata were harvested from 20 NHBDs female pigs with similar characteristics and preserved for 4 h in UW solution (n=10) or TLM (UW/PFC) solution (n=10). The isolated islets were then evaluated for number, viability, purity, and insulin secretion, also estimated after 8 weeks of cryopreservation. Results The total number of islets obtained from isolation, and their function assayed by the insulin stimulation index, before and after cryopreservation, showed a higher value in the TLM group. No significative differences in terms of purity and viability before and after cryopreservation were found when comparing the two groups. Conclusions TLM solution for NHBDs porcine pancreata with cold ischemia time lower than 4 h offers significant advantages over UW solution storage, thereby increasing the isolation yield and isolation success rate of the pancreatic porcine islets.

Towards an ideal source of mesenchymal stem cell isolation for possible therapeutic application in regenerative medicine

BACKGROUND The possibility of obtaining mesenchymal stem cells (MSCs) from fetal tissue such as amniotic fluid, chorionic villi and placenta is well-known and a comparison between MSCs originating in different sources such as fetal tissue and those from bone marrow in terms of yield and function is a topical issue. The mesenchymal stem cells isolated from bone marrow are well-characterized. Unfortunately the low quantitative yield during isolation is a major problem. For this reason, other tissue sources for MSCs are of paramount importance. CONCLUSION In this review, starting from a description of the molecular and cellular biology of MSCs, we describe alternative sources of isolation other than bone marrow. Finally, we describe the potential therapeutic application of these cells.

Abnormal espansion of segmented filamentous bacteria in the gut: a role in pathogenesis of chronic in fiammatory intestinal desease

Human intestinal microbiota create a complex polymicrobial ecology characterized by high population density, wide diversity, and complexity of interactions. Any imbalance of this complex intestinal microbiome, both qualitative and quantitative, might have serious health consequences including an increase in the number and/or alteration in the type of bacteria in the upper gastrointestinal tract, which is referred to as small intestinal bacterial overgrowth (SIBO) syndrome. SIBO is frequently found in persons fulfilling criteria for irritable bowel syndrome (IBS), and the large overlapping of symptoms of these two pathological conditions led some authors to believe that IBS is secondary to SIBO. Interestingly, SIBO is also found in about 25% of patients with Crohns disease. Emerging data show that specific components of gut microbiota, particularly segmented filamentous bacteria (SFB), activate intestinal immunocompetent cells, for example, Th17 cells which have a potential role in pathogenesis of inflammatory intestinal diseases. On the basis of the aforementioned data we postulate that a previously unidentified specific form of SIBO, involving in particular the aberrant expansion of SFB in the gut, could play a role in the onset of chronic intestinal inflammatory diseases through persistent activation of Th17 cells. From this point of view, a successful therapeutic approach to inflammatory intestinal disease patients could be the administration of specific antibiotics directed against SFB to restore the physiological levels of these bacteria in the gut. Furthermore, it could be very useful to identify appropriate laboratory methodologies to monitor the level of SFB in the gut with the aim of preventing their potentially dangerous increase in numbers.

Is secondary hyperparathyroidism-related myelofibrosis a negative prognostic factor for kidney transplant outcome?

Secondary hyperparathyroidism (HP) presenting with hypocalcemia and subsequent increased parathormone (PTH), is mainly identified in patients with chronic renal failure, which has been associated with variable degrees of bone marrow fibrosis. For suitable patients with end-stage renal disease (ESRD), kidney transplantation is recognized as the therapy of choice, being superior to dialysis in terms of quality of life and long-term mortality risk; in this regard interesting data show that increased time on dialysis prior to kidney transplantation is associated with decreased graft and patient survival. In our opinion an important and until now underestimated determinant of graft survival is the proper activity of bone marrow because of the emerging role of hematopoietic stem cells (HSC) in repair of ischemia/reperfusion (IR) damage. We postulate that in ESRD patients, who usually undergo long dialytic treatment, a myelofibrosis caused by an overt secondary HP could drastically decrease the HSC potential for IR damage repair after kidney transplant; this could irremediably lead to a delay in graft function with all related complicances. If the curative role of bone marrow-derived stem cells was confirmed by more data obtained in experimental animal models, it could be possible to try a cellular-based therapeutic approach in the management of ESRD patients which are in waiting list for a kidney transplant.

Sistemic calciphylaxis and thrombotic microangiopathy in a kidney transplant patient: Two mixing fatal syndromes?

Abnormalities in calcium and phosphorus metabolism are common and metabolic bone diseases develop often in patients with chronic renal failure (CRF). Effective clinical management includes measures to control phosphorus retention and prevent hyperphosphataemia, to maintain serum calcium concentrations within the normal range and to prevent excess parathyroid hormone (PTH) secretion by the judicious use of vitamin D sterols. Certain of these interventions, however, appear to increase the risk of soft tissue and vascular calcification in patients with End Stage Renal Disease (ESRD), so current therapeutic approaches are thus being re-evaluated in an effort to limit these risks. Patients with calciphylaxis have an extremely high mortality rate, diagnosis requires a high degree of clinical suspicion and the role and extent of parathyroidectomy in the management of this condition remain controversial. In some cases renal transplant patients could suffer from a comorbidity in which vascular function is compromised not only by secondary hyperparathyroidism-related calcification but also by other pathological condition as haemolytic uremic syndrome (HUS), leading to a fatal clinical outcome. We postulate that in these cases a secondary hyperparathyroidism not properly diagnosed in an early phase of the renal disease (probably before the kidney transplant) could cause a vascular calcification which, adding to the pre-existing HUS-related vascular compromission, gave rise to catastrophic clinical consequences. In the management of ESRD patients, in particular in the cases of pre-existing angiopathies, could be therefore crucial the early and proper diagnosis of an alteration of calcium-posphorus metabolism and effort of medicine could be oriented in these cases also towards identification of screening methodologies to undoubtedly assess such a diagnosis.

A complex case of fatal calciphylaxis in a female patient with hyperparathyroidism secondary to end stage renal disease of graft and coexistence of haemolytic uremic syndrome

BACKGROUND Calciphylaxis is a potentially fatal complication of persistent secondary hyperparathyroidism; its cause is still not clear. Unfortunately there is no close relation in severity of clinical picture, serological and pathological alteration. For this reason the prognosis is difficult to establish. Administration of sodium thiosulphate may reduce the precipitation of calcium crystals and improve the general clinical conditions before surgical parathyroidectomy, which seems the only therapeutic approach able to reduce the mortality risk in these patients. METHODS AND RESULTS A 60 year old female patient suffering from End Renal Stage Disease, on haemodialysis from 2001 due to the onset of haemolytic uremic syndrome, underwent a kidney transplant in April 2008. After transplantation there was a recurrence of the haemolytic uremic syndrome, with temporary worsening of the graft. Six months later there was a definite loss of graft and return to dialysis treatment. On April 2010 a severe systemic calciphylaxis related to secondary hyperparathyroidism was diagnosed. The patient underwent parathyroidectomy but, because of the unimproved clinical picture, treatment with sodium thiosulphate was initiated. There was only improvement in cutaneous lesions. The worsening general clinical condition of the patient caused death due to general septic complications. CONCLUSIONS The coexistence of haemolytic uremic syndrome and secondary hyperpathyroidism makes the prognosis poor and, in this case, therapy, which counteracts calcium crystals precipitation, has no effect. Preventive parathyroidectomy can be considered as the only possible treatment.

Double Endocrine Neoplasia in a Renal Transplant Recipient: Case Report and Review of the Literature

INTRODUCTION The incidence of cancer compared for age groups is 3-4 times higher in transplant recipients than the general population. The increased risk is related to immunosuppressive therapy as well as the use of increasingly older donors and recipients. Although cardiovascular disease with a functioning transplant is the leading cause of death (47%), cancer mortality is significant especially among older patients. However, the most frequent posttransplantation cancers relate to hemolymphopoietic organs and skin, whereas the occurrence of solid tumors elsewhere is rare. Herein we have described a rare case of synchronous double malignancy of endocrine organs (thyroid-adrenal) in a young woman who underwent renal transplantation. CASE REPORT A 37-year-old woman with end-stage renal disease for 18 years underwent transplantation when she was 30 years old with a 17-year-old standard cadaveric donor receiving immunosuppressive therapy with mycophenolate mofetil, cyclosporine, and steroids. Follow-up demonstrated good indices of renal function with negative tumor pathology at 79 months when, at an annual ultrasound monitoring, we found a lesion in the right lobe of the thyroid and left adrenal neoplasm of dubious interpretation. The cytology for the thyroid was highly suspicious of papillary carcinoma, whereas the histological examination after surgery diagnosed a thyroid multifocal papillary microcarcinoma (mpT1NxMx) and an oxyphil cell adrenocortical carcinoma (pT2, N0). RESULTS Six months after total thyroidectomy with central lymphadenectomy and left kidney and adrenal gland removal the patient showed no evidence of recurrent lesions and stable graft function. CONCLUSIONS The rare occurrence of solid tumors after transplantation has no known etiopathogenetic relation. Despite the young age of the patient and the double neoplasm that could have produced an unfavorable outcome for the patient and the graft, careful follow-up for tumor pathologies and multidisciplinary management achieved an early diagnosis of both tumors with a surgical eradication without adjuvant therapy, preserving the life of the patient and the function of the graft.

A Good Breath of Oxygen for Beta-Like Cells Obtained From Porcine Exocrine Pancreatic Tissue

Ischemia is the most important factor that affects organ survival during harvesting. The two-layer method (TLM) is one of several cold storage solutions that seeks to preserve organs and cells avoiding in vivo and in vitro ischemia. We compared the retrieval of beta-like elements from exocrine pancreatic cells using TLM versus University of Wisconsin (UW) solutions. For this purpose pancreata laparoscopically harvested from 20 female pigs were preserved in UW solution or TLM before digestion. The resulting exocrine cells were divided into 2 groups: the first was cultured in a designed medium to allow differentiation into beta-like cells and the second was cryopreserved before the differentiation process at -196 °C for 8 weeks before culture in the same medium. The results revealed that TLM was better than UW as a preservation solution in terms of beta-cell viability and insulin secretion. We suggest that the use of TLM solution allows one to obtain less damaged cells for research purposes.

Histologic Effects of University of Wisconsin Two-Layer Method Preservation of Rat Pancreas

Marginal donors represent a poorly utilized source of organs for transplantation despite their availability. The key is to reduce the ischemic damage in the effort to improve organ quality. This study investigated the histologic effects after in situ perfusion of preservation with a two-layer method compared with the classic University of Wisconsin preservation in term of tissue integrity and number of viable exocrine cells in the rat pancreas both after exsanguination and at 8 weeks of cryopreservation. Pancreata harvested from 60 rats were collected using 3 methods: two-layer method following University of Wisconsin perfusion; exsanguination; and classic University of Wisconsin perfusion/storage. In addition to histologic analysis of collected pancreata, we analyzed the number of CK19(+) cells and their viability using chi-square tests with values P < .05 considered to be significant. Rat pancreas histology showed as University of Wisconsin in situ perfusion and preservation by the two-layer method to be more effective to maintain the morphologic integrity of both exocrine and endocrine tissues. There were a larger number of CK19(+) cells with good viability. Moreover, the effects of oxygenation were visible in pancreas biopsies preserved after exsanguination. In situ University of Wisconsin perfusion and preservation for 240 minutes with the two-layer method yielded greater numbers and viability of CK19(+) cells even after 8 weeks of cryopreservation.