Hepatitis C Virus Positive Patients on the Waiting List for Liver Transplantation: Turnover and Characteristics of the Population on the Eve of the Therapeutic Revolution With Direct-Acting Antivirals.

INTRODUCTION: Antivirals direct acting (DAA) for hepatitis C virus (HCV) have brought a revolution in the field of transplantation. It is likely to think that in the future patients on the waiting list for liver transplantation (LT) will no longer be registered for HCV-related cirrhosis but for liver disease from other causes. On the eve of this change, we show a snapshot of the Italian waiting list for LT. METHODS: From October 1, 2012 to September 30, 2013, we estimated the total number of patients on the liver waiting list as intention to treat (ITT), the number of incident cases, and the delistings, particularly in the HCV positive (HCV+) population. Gender, median age, etiology and prognosis of liver disease, presence of hepatocellular carcinoma (HCC), reason for delisting, mean waiting time for LT, and rate of death on waiting list were evaluated. RESULTS: In the time period, there were 517 new patients who were HCV+ (median age, 53 years): 255 (49.3%) mono-infected with HCV, 236 (45.7%) co-infected with HCV and hepatitis B virus (HBV), 11 (2.1%) co-infected with HCV and human immunodeficiency virus (HIV), and 15 (2.9%) co-infected with HCV, HBV, and HIV. The median model for end-stage liver disease (MELD) score at listing was 17 and HCC was present in 206 (39.8%) cases. HCV+ patients delisted were 442 (61.9%), 355 (80.3%) for LT. The mean waiting time to transplantation was 1.9 months; the percentage of death was 7.6%. CONCLUSIONS: This snapshot of the waiting list for LT in the year before the advent of DAA drugs will allow us to assess whether and how they will change the waiting list for LT when we start to look at the impact of new therapies on the waiting list.

Optimizing the Use of Available Lungs.

BACKGROUND: Organ transplantation, the treatment of choice in organ failure, is penalized by the lack of organs. Because the increase in the number of donors is not proportional throughout the different age groups, there is no increase in lung transplantations. The aim of this work was to analyze the use of available lungs and evaluate strategies that may help increase transplantations. METHODS: We analyzed the activity of lung transplantation in 2015, divided into various allocation programs. We also examined the surplus organs, in particular, their origin, their destination, their offer's outcome, the characteristics of the donor and the proposed organ, and the reasons for rejection. RESULTS: In 2015, 112 lung transplantations were performed: 66 (68.9%) with regional organs, 46 (41.1%) with extraregional organs; 21 (45.6%) of these were allocated as emergencies/return, and 25 (54.4%) as surplus (19 in the North macroarea, 6 in the South macroarea). The number of surplus lungs was 148: 67 from the North macroarea, 71 from the South macroarea, and 10 from abroad. No organ procured in the North macroarea was transplanted in the South macroarea, whereas 6 lungs coming from the South macroarea were transplanted in the North. CONCLUSIONS: The acceptance criteria are not the same in different transplant centers and they include not only clinical parameters, but also ischemia time and composition of the waiting list at the time of the offer, quality and accessibility of the intensive care units where the donor is located, and organizational reasons. Offering organs which can not be transplanted within the region to other centers, without clinical foreclosures is a system that increases transplant activities by maximizing the available resources.

Liver Allocation in Urgent MELD Score ≥30: The Italian Experience.

INTRODUCTION: Patients with an urgent MELD score ≥30 are managed by the Italian Operative National Transplant Center on the basis of a division of Italy into 2 main areas, the northern macro area (NMA) and the southern macro area (SMA). The object of this study was to evaluate the possibility and the need to transform the MELD score ≥30 macro area-based program into a nationwide one. PATIENTS AND METHODS: When a region reports the presence of a patient with a MELD score ≥30, the same macro area-compatible donors, in the absence of urgent national and 1B status, are offered primarily to this recipient. RESULTS: From August 2014 to August 2015, 132 requests for patients with urgent MELD score ≥30, 98 from the NMA and 34 from the SMA, were handled. The average waiting list in the NMA was significantly different from that of the SMA (2.74 ± 2.29 vs 4.5 ± 3.98, P < .05). A total of 73.7% of the received requests (n = 97) were satisfied: the NMA met 80.4% of the requests (n = 77), whereas the SMA met 55.5% (n = 20). A total of 35 requests (26.5%), 21 from the NMA (60%) and 14 (40%) from the SMA, were not met. The average waiting time of these recipients for a liver was significantly different between the NMA and the SMA (3.14 ± 3.21 vs 5.78 ± 4.59; P < .05). CONCLUSIONS: The MELD score is a priority allocation, and the longer the waiting time to transplantation for these recipients, the more their mortality increases. Given the differences in waiting times between the NMA and SMA, we should start thinking about transforming the macro area program into a national one.

Transportation of Organs by Air: Safety, Quality, and Sustainability Criteria.

INTRODUCTION: The outcomes of organ transplantation activities are greatly affected by the ability to haul organs and medical teams quickly and safely. Organ allocation and usage criteria have greatly improved over time, whereas the same result has not been achieved so far from the transport point of view. Safety and the highest level of service and efficiency must be reached to grant transplant recipients the healthiest outcome. OBJECTIVES: The Italian National Transplant Centre (CNT), in partnership with the regions and the University of Bologna, has promoted a thorough analysis of all stages of organ transportation logistics chains to produce homogeneous and shared guidelines throughout the national territory, capable of ensuring safety, reliability, and sustainability at the highest levels. METHODS: The mapping of all 44 transplant centers and the pertaining airport network has been implemented. An analysis of technical requirements among organ shipping agents at both national and international level has been promoted. A national campaign of real-time monitoring of organ transport activities at all stages of the supply chain has been implemented. Parameters investigated have been hospital and region of both origin and destination, number and type of organs involved, transport type (with or without medical team), stations of arrival and departure, and shipping agents, as well as actual times of activities involved. RESULTS: National guidelines have been issued to select organ storage units and shipping agents on the basis of evaluation of efficiency, reliability, and equipment with reference to organ type and ischemia time. Guidelines provide EU-level standards on technical equipment of aircrafts, professional requirements of shipping agencies and cabin crew, and requirements on service provision, including pricing criteria. CONCLUSIONS: The introduction in the Italian legislation of guidelines issuing minimum requirements on topics such as the medical team, packaging, labeling, safety and integrity, identification, real-time monitoring of temperature, and traceability of the organ during the logistics chain is deemed a valid response to the necessity of improving safety, reliability, and sustainability of organ transplantation activities in Italy.

Peptide-based radioimmunoassay for the two isoforms of the human insulin receptor.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms employing antibodies raised against two peptides, one (Pep-12) corresponding to residues encoded by exon 11, and the other (Pep-13) corresponding to a COOH-terminal domain of the alpha-subunit which is common to both HIR-A and HIR-B isoforms. These peptides were iodinated and used as both ligands and standards. The assay is specific, highly reproducible, and sensitive with a detection limit of 10 fmol of receptor. One mole of purified insulin receptor, measured by Scatchard analysis, is read as one mole of receptor in the radioimmunoassay with either Pep-12 or Pep-13 as standards. The radioimmunoassay is applicable to the measurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to measure the relative abundance of the two isoforms in fat and muscle from normal, obese non-diabetic and non-insulin-dependent diabetic (NIDDM) subjects. Results demonstrate that expression of the low-affinity HIR-B form is significantly increased in obese and NIDDM subjects compared with control subjects. In addition, the increased expression of the HIR-B isoform was significantly correlated with both body mass index (r = 0.52; p = 0.006) and fasting glucose levels (r = 0.59; p = 0.001).

Role of the exon 11 of the insulin receptor gene on insulin binding identified by anti-peptide antibodies.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the C-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. It was shown that the two isoforms exhibit different binding affinities for insulin, thus suggesting that the sequence encoded by exon 11 may be important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the two HIR alpha-subunit variants. Herein, we characterized two antibodies, PA-11 and PA-12, directed against the C-terminus or the N-terminus of the sequence encoded by exon 11, respectively, and one (PA-13) directed against a sequence in the carboxy-terminal region of the alpha-subunit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [125I]insulin binding to both isoforms. We found that PA-13 immunoprecipitates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusively the HIR-B, and PA-11 fails to precipitate both isoforms. Interestingly, PA-12 inhibits specifically insulin to the HIR-B, whereas other PAs fail to affect insulin binding to either isoforms. Furthermore, PA-12 linearises the Scatchard plot of binding data, and retards the dissociation rate of insulin, thus suggesting that antibody affects cooperative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insulin to the receptor and negative cooperativity.

Androgens increase insulin receptor mRNA levels, insulin binding, and insulin responsiveness in HEp-2 larynx carcinoma cells.

Androgen receptors have been found in human larynx and androgens have been supposed to play an important role in promoting the growth of laryngeal carcinomas. The molecular mechanism underlaying this phenomenon is not at all understood. Aim of this work was to investigate the effects of two androgens (testosterone and dihydrotestosterone) on insulin receptor mRNA levels and insulin binding activity as well as on either metabolic or growth-promoting actions of insulin in a human larynx carcinoma cell line (HEp-2). We found that HEp-2 cells express a high affinity insulin receptor. Both androgens significantly increase insulin receptor mRNA levels and insulin receptor number in HEp-2 cells. Insulin action, evaluated either as total glucose utilization or as [3H]thymidine incorporation into DNA, significantly increased in HEp-2 treated with androgens in comparison to control cultures. Altogether, our data allow us to speculate that the increased insulin effectiveness we observed in the larynx carcinoma cell line HEp-2 after androgen treatment might be involved in the regulation of larynx cancer cells growth.

Evidence that two naturally occurring human insulin receptor alpha-subunit variants are immunologically distinct.

The IgG from a patient (Italy 2 [I2]) with hypoglycemia, due to autoantibodies to the insulin receptor, was purified on protein A Sepharose into two fractions that were tested in various human tissues and cells. The IgG fraction that bound protein A (absorbed IgG [IgGa]) nearly completely inhibited the binding of 125I-labeled insulin to various cells or tissues (placenta, IM-9, adipocytes, HEp-2-larynx cells, Epstein-Barr virus lymphocytes) but not greater than 50% of 125I-labeled insulin binding to human liver membranes. Conversely, both the IgG fraction from this patient, which did not bind protein A (flow-through IgG [IgGb]), and the IgGa fraction from a second similar patient (Italy 1 [I-1]) almost completely inhibited the binding of 125I-labeled insulin to liver membranes. The IgGa fraction from patient I-2 did not change receptor affinity because 50% inhibition of 125I-labeled insulin binding was not affected by either the presence or absence of these IgG fractions. Furthermore, liver binding data were not due to cross-reaction of 125I-labeled insulin to the insulinlike growth factor I receptor, and treatment of liver membranes with neuraminidase did not alter the inhibitory effect of the IgGa fraction from patient I-2 on 125I-labeled insulin binding to liver. Binding inhibition experiments performed with cells transfected with and overexpressing the -12 (human insulin receptor [HIR]-A) or the +12 (HIR-B) variant of HIR revealed that the IgGa fraction from patient I-2 inhibited 125I-labeled insulin binding to the HIR-A receptor but not to the HIR-B receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered expression of the two naturally occurring human insulin receptor variants in isolated adipocytes of non-insulin-dependent diabetes mellitus patients.

The human insulin receptor gene is expressed in two variant isoforms which differ by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the extracellular alpha-subunit as a consequence of alternative splicing of exon 11. Expression of the two variant isoforms is regulated in a tissue-specific manner. In this study, we have measured the levels of the two receptor variants in isolated adipocytes from 10 non-insulin-dependent diabetes mellitus (NIDDM) and 11 normal subjects using an immunological assay, based on the ability of a human anti-receptor autoantibody to discriminate between HIR-A and HIR-B. Results indicate that levels of HIR-B variant are increased in NIDDM patients.

Evidence that human and porcine insulin differently affect the human insulin receptor: studies with monoclonal anti-insulin receptor antibodies.

Binding studies have been carried out with radioiodinated monoclonal antibodies directed to various epitopes of the insulin receptor in order to detect differences between human and porcine insulin in the interaction with the human insulin receptor. Human insulin was more effective that porcine insulin at inhibiting the binding of 125I-MA-5 to IM-9 cells, Hep-2 human larynx cells and human placenta membranes. On the contrary, human and porcine insulin showed similar inhibitory effect on the binding of two other labeled anti-insulin receptor monoclonal antibodies, thus ruling out the possibility that results were due to experimental artifacts. Although several interpretations are possible, data reported suggest that human insulin and porcine insulin might differently affect the insulin receptor, even if, the biological significance of these findings remains unknown.

[Primary lymphoma of the thyroid gland. Review of the literature and presentation of a clinical case]. / I linfomi primitivi della tiroide. Revisione della letteratura e presentazione di un caso clinico.

Primary malignant lymphoma of the thyroid is a very uncommon disease and represents approximately 5% of all thyroid malignant neoplasms. The Authors report a case whose clinical and histopathological features are discussed and compared with those of previously published series. Primary malignant lymphomas raise a number of issues: abrupt onset, age and sex patterns, histological typing (virtually 100% of cases are non-Hodgkin lymphomas), differential diagnosis with either Hashimoto's chronic thyroiditis (often associated with thyroid primary lymphomas) or genuine anaplastic carcinoma of the thyroid. Routine histological study can be complemented by immunohistochemistry. The treatment of choose is total thyroidectomy plus radiotherapy in cases where radical surgery is possible, or radiotherapy alone which usually carries good results.

The long-term efficacy of ibopamine in treating patients with severe heart failure: a multicenter investigation.

This randomized, double-blind, placebo-controlled, multicenter trial compared the effects of treatment with ibopamine with those of placebo in patients with severe heart failure who still showed symptoms although they were receiving standard therapy with digitalis and diuretics. The results showed a significant and sustained improvement in exercise tolerance (+70% about in average), clinical condition, and NYHA functional class and a decrease in cardiothoracic ratio and left ventricular end-systolic wall stress in patients treated with ibopamine, digitalis, and diuretics (group 1) compared with patients treated with digitalis, diuretics, and placebo (group 2).

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies.

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.

[Preparation of 125I-labelled monoclonal antibodies of the insulin receptor]. / Préparation d'anticorps monoclonaux du récepteur de l'insuline marqués par 125I.

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.

Species specificity of insulin binding and insulin receptor protein tyrosine kinase activity.

The effect of monoclonal anti-insulin receptor antibody MA 10 on [125I]insulin binding and on insulin receptor protein tyrosine kinase activity was investigated in human and rat tissues. It was observed that MA 10 inhibits insulin binding to human, but not rat, tissues while inhibiting insulin-stimulated receptor autophosphorylation and protein tyrosine kinase activity in both human and rat tissues. These data suggest that MA 10 is directed against a region of the insulin receptor that is in between the insulin-binding domain and the beta-subunit and that in human, but not rat, tissues, this region is involved in insulin binding.

Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation.

Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.

Human autoantibodies directed against the human, but not the rat, insulin receptor.

Prior studies with monoclonal antibodies produced against the human insulin receptor in mice revealed that these antibodies may be species specific. Whether species-specific antibodies to the insulin receptor occur spontaneously in patients, however, has not been previously investigated. Recently, we found that the serum immunoglobulin G from a patient with lupus nephritis, insulin resistance, and hypoglycemia contained multiple subpopulations of antibodies directed at the human insulin receptor. We report herein that one such subpopulation has a high affinity for the human insulin receptor. This antibody subpopulation at 10 nM half-maximally inhibited [125I]insulin binding to human IM-9 lymphocytes, circulating erythrocytes and monocytes, isolated adipocytes, and placenta membranes. In contrast, this antibody subpopulation did not inhibit [125I]insulin binding to isolated rat adipocytes and hepatocytes, even at concentrations as high as 100 nM. These studies indicate that species-specific antibodies can occur spontaneously in patients with antiinsulin receptor antibodies.

Corpus luteum function during the early puerperium.

Progesterone and 17 beta-estradiol peripheral plasma levels have been determined during labor and after 1, 3, 6, 12, 24, 48, 72, 96, 120 and 144 h from delivery in a group of 7 women, whose corpus luteum had been removed at delivery and in a corresponding control group with intact corpus luteum. In both groups the results indicate a progressive fall of progesterone and 17 beta-estradiol, that is evident until 144 h for progesterone and 72 h from 17 beta-estradiol. The analysis of the two groups with the Student's t test has shown significantly lower levels in the group of women, whose corpus luteum had been removed, at the 6th h (p less than 0.01), 12th h (p less than 0.02) and 24th h (p less than 0.05) for progesterone and at 3rd h (p less than 0.01) and 6th (p less than 0.01) for 17 beta-estradiol. The significant difference for progesterone appearing later than for 17 beta-estradiol could be due to the fact that progesterone is also dismissed by the adipose tissue, where it is stored. A further statistical elaboration of the results, carried out by calculating the regression line and the transfer function, confirmed the different pattern in time of progesterone and 17 beta-estradiol plasma levels after delivery in the two groups of patients considered.

Androstenedione, 17 beta-estradiol and progesterone plasma levels in gonadotropins induction of ovulation.

A comparison of peripheral patterns of androstenedione (A), 17 beta-estradiol (E2) and progesterone (P) is reported in ten infertile women during HMG-HCG induction of ovulation, in order to assess the site of ovarian secretion of plasma A and the possible influence on conception. Evidence for both the follicular and luteal secretion of plasma A is suggested, in addition to the stromal and adrenal contributions, since a highly significant (p less than 0.001) correlation between A and E2 plasma levels was shown during the treatment. In three cycles of conception, plasma A showed a periovulatory peak and drop, followed by a luteal increase, all of which are characteristic of E2.