Lung Allocation Score System: First Italian Experience.

BACKGROUND: Lung transplantation is an established therapeutic option for patients with end-stage pulmonary disease. In May 2005, the lung allocation score (LAS) was introduced in the United States to maximize the benefit to the recipient population and reduce waiting list mortality. The LAS has been applied in a region of Italy since March 2016 on a provisional basis. The aims of the study were describing waiting list characteristics and short-term outcomes after lung transplantation before and after LAS introduction. METHODS: All the patients who received transplants between January 1, 2011, and March 15, 2017, were included in our retrospective study. The study population was divided into 2 cohorts (historical cohort and post-LAS cohort) and a comparison among the main perioperative data was performed. RESULTS: The historical cohort consisted of 415 patients on the waiting list with 91 deaths and 199 lung transplants; the post-LAS cohort consisted of 134 patients with 10 deaths on the waiting list and 51 transplants. Median waiting time and mortality on the list decreased from 223 to 106 days (P = .03) and from 11.2% to 7.5% (P > .05), respectively. The transplantation rate increased from 25% to 38% (P = .001) and the probability to receive a transplant in the first year in the post-LAS era increased significantly (P = .004). CONCLUSIONS: The results of the introduction of the LAS system in our region are encouraging and have not shown any adverse short-term effects. The regional coordination decided to prolong the experimental application of LAS in order to accumulate more data and to evaluate medium-term outcomes.

Use of the Oto lung donor score to analyze the 2010 donor pool of the Nord Italia Transplant program.

INTRODUCTION: The feasibility and utility of a lung donor score that has been recently proposed was tested among a pool of lung donors referred to the Nord Italia Transplant program (NITp) organ procurement organization. MATERIAL AND METHODS: Each lung donor was assigned an Oto score including, age, smoking history, chest X-ray, secretions and ratio of arterial oxygen tension to inspired oxygen fraction (PaO(2)/FiO(2)). Based on clinical compromise, each variable received a score between 0 and 3, except for PaO(2)/FiO(2), which was scored between 0 and 6 given its overall relevance. RESULTS: Throughout 2010, 201 multiorgan donors were initially considered to be potential lung donors. Among these, 59 (29.4%) eventually yielded 67 lung transplantations (named "Used group"). Among the 142 (70.6%) refused lungs, 28 were not used due to logistic or medical problems ("general exclusion" group, GE) and 114, because of poor lung function ("lung exclusion" group, LE). Median lung donor scores were 1 (range, 0 to 3), 4 (range, 2.5 to 6.5), and 7 (range, 5 to 9) in the Used, GE, and LE groups, respectively (one-way analysis of variance, P < .001). Some donors with Oto scores ≤7 worsened over time so that the score had significantly increased by the time of organ retrieval. Overall, subjects who died after lung transplantation were characterized by higher lung donor scores, (2 [1-4] versus 0.5 [0-3], P = .003). CONCLUSION: Our analysis suggested that the use of a donor score as a dynamic tool over the donation process was of great utility to describe and analyze a pool of lung donors.

Lung procurement for transplantation: new criteria for lung donor selection.

In Italy, like everywhere in the world, the organ shortage for transplantation is a real problem. It is well known that lung donors (LD) are particularly difficult to procure and that management of the organ do not care during the diagnosis of cerebral death represents a difficult challenge. In this context, the salvage of the so-called "marginal donors" may increase the pool of donors, favoring organ retrieval. To increase lung procurement, the intensivist must recognize "marginal donors," optimizing organ selection and function. The aim of our study was to review LD procured in 2008, as identified by the unrestricted criteria, of the Nord Italian Transplant program Center (NITp). Particularly, the age and habits of donors and the presence of a parenchyma contusion were not sufficient per se to exclude donation. We revisited lung ventilation and monitoring modalities during cerebral death before retrieval. In 2008, the application of enlarged criteria for LD enabled us to collect 21 LD, namely 33% of all cerebral deaths, versus 13% in 2007. Seeking to maintain good gas exchange and lung function, we implemented a safe ventilation program avoided high peak pressures, and fluid therapy properly guided by the cardiac index and extravascular lung water index monitoring. Specific actions to improve LD procurement may help cope with the organ-donor shortage. Although our series was small, our results were encouraging; they underline the necessity to continuously review donor criteria and care, allowing good donor/recipient matching.

An immunogenetic map of Lombardy (Northern Italy).

For this study we consulted the Bone Marrow Donors' Registry of Lombardy (Italy) and analyzed 43937 HLA-A,B phenotypes and 13922 HLA-A,B,DR phenotypes. We estimated the HLA-A,B and HLA-A,B,DR haplotype frequencies via the maximum-likelihood method. We analyzed the genetic structure of the 11 provinces of Lombardy by means of Principal Component Analysis and Correspondence Analysis, and estimated the variety of the different haplotypes at provincial level and the percentage of unique phenotypes at village level. We found 11189 different HLA-A,B phenotypes, 661 different HLA-A,B haplotypes and more than 4000 different HLA-A,B,DR haplotypes. We identified 20 villages, in Western Lombardy, very rich in unique/rare phenotypes. Here we report a formula which allows the identification of a putative donor matched for two haplotypes with a recipient. This result may be of great importance for the genetic study of the population of Lombardy and, even more, for bone marrow transplantation programs.

Peripheral blood stem cell allograft rejection mediated by CD4(+) T lymphocytes recognizing a single mismatch at HLA-DP beta 1*0901.

Little is known about the molecular characteristics of alloantigens recognized by alloreactive T cells mediating hematologic stem cell graft rejection. In particular, it has never been shown that such alloantigens can be encoded by HLA-DP beta alleles. Indeed, matching for HLA-DP antigens is generally not considered to be of functional importance for the outcome of allogeneic bone marrow or peripheral blood stem cell transplantation. In this study, a case of peripheral blood stem cell allograft rejection was investigated in which the patient and donor differed for a single mismatch at HLA-DP in the rejection direction. Patient-derived T lymphocytes circulating at the time of rejection showed direct ex vivo cytotoxic activity against donor-derived B-lymphoblastoid cells as well as other HLA-DP beta 1*0901--expressing targets. The presence of HLA-DP beta 1*0901--specific effectors in vivo was further confirmed by in vitro stimulation experiments. CD4(+) T-cell lines and clones with specific cytotoxic activity against HLA-DP beta 1*0901--expressing targets including donor B-lymphoblastoid cells were generated both by nonspecific and by donor-specific in vitro stimulation. Taken together, these data demonstrate that HLA-DP can be the target antigen of cytotoxic CD4(+) T lymphocytes involved in peripheral blood stem cell allograft rejection. (Blood. 2001;98:1122-1126)

Molecular characterization of HLA class I in Colombians carrying HLA-A2: high allelic diversity and frequency of heterozygotes at the HLA-B locus.

Polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing was used to analyze HLA class I A, B and C alleles in three different Colombian populations. Fifty-nine samples were from Hispano-American Mestizos living in the urban areas of Cali (referred to here as Aso population). Forty-four and thirty samples were from the African Black populations of Zacarias (Zac) and Punta Soldado (PS), respectively. Samples were selected for expression of HLA-A2 by monoclonal antibody staining and allele-specific hybridization, and their HLA-A2 subtype distribution has been reported previously. Although only a limited number of samples was analyzed, the data suggest the existence of a remarkable degree of HLA class I polymorphism in the populations studied, with representatives of most serological classes. Despite their common African origin, the populations Zac and PS, both resident in malaria endemic regions, showed some striking differences in allelic distribution for all three class I loci. Furthermore, the samples from Aso and PS, but not Zac, showed a low percentage of blank alleles at the HLA-B locus (0 and 0.4%, respectively), suggesting the possibility of a heterozygote advantage for HLA-B alleles in Colombian populations.

HLA-A*02 subtype distribution in Caucasians from northern Italy: identification of A*0220.

This study describes a comprehensive easy to perform PCR-SSOP typing approach suitable for complete genomic subtyping of HLA-A*02. A single 1.6 kb PCR-amplificate spanning exons 2, 3 and 4 of the HLA-A*02 gene was used for hybridization with a panel of twenty-four SSOPs. This allowed unequivocal assignment of all so far known HLA-A2 subtypes, including A*0209 and A*0215N which differ for nucleotide substitutions in exon 4, without the need for two separate amplifications. Using this approach, HLA-A*02 subtype distribution was analyzed in 218 samples from unrelated, healthy individuals from northern Italy enrolled in the Italian Bone Marrow Registry and typed as HLA-A2 by serology or generic molecular analysis. As expected, A*0201 was found in the majority (92.6%) of samples. However, a significant number (6.8%) of individuals carried A*0205. Furthermore, A*0202, A*0208, A*0209 and A*0217, so far not described in Caucasians, were detected in a low number of samples (frequency ranging from 0.45% to 1.8%). Finally, a novel HLA-A*02 subtype, A*0220, was detected in 0.9% of the samples. As confirmed by DNA sequencing of exons 2 and 3, this allele is identical to A*0201 except for a single nucleotide substitution in codon 66 which changes the predicted amino acid sequence form Lys to Asn. The findings of this study have implications for the selection of HLA-A*02+ donors in unrelated bone marrow transplantation and of patients for specific immuno-therapy with HLA-A*02 restricted peptide vaccines.

Association of IA-2 autoantibodies with HLA DR4 phenotypes in IDDM.

Insulin, glutamate decarboxylase (GAD) and the protein tyrosine phosphatase-like molecule IA-2 are major targets of humoral autoimmunity in insulin-dependent diabetes mellitus (IDDM). These autoantibodies are heterogeneous with respect to age and patient human leukocyte antigen (HLA) phenotype. We have previously demonstrated that GAD and IA-2 antibodies potentially identify different subsets of IDDM patients. The aim of this study was to determine whether GAD and IA-2 autoantibodies were associated with different HLA DR phenotypes. We studied 160 patients with IDDM onset before age 16 years. At disease onset serum was tested for GAD and IA-2 antibodies by immunoprecipitation by in vitro-translated 35S-methionine labelled recombinant proteins. IA-2 antibodies were significantly associated with HLA DR4: 67 (86%) of 78 patients with HLA DR4 vs 31 (38%) of 82 non-DR4 patients had IA-2 antibodies (Pc < 0.0001) and IA-2 antibody levels were higher in patients with HLA DR4 (Pc < 0.0001). In contrast, GAD antibodies were more prevalent (Pc < 0.05) and antibody levels highest (Pc < 0.01) in patients with HLA DR3 phenotypes. These data provide further evidence that, in IDDM, production and titre of major autoantibody specificities are associated with HLA class II alleles.

Complete generic and extensive fine-specificity typing of the HLA-B locus by the PCR-SSOP method.

This study describes sequence specific oligonucleotide probe (SSOP) typing of hypervariable regions in exons 2 and 3 of HLA-B locus genes. A single HLA-B specific PCR-product spanning from bp 84 in exon 2 to bp 241 in exon 3 was used for dot blot hybridization to forty-seven chemiluminescent labeled oligonucleotide probes. Thirty-one of these probes were derived from four hypervariability zones in exon 3 of HLA-B genes and covered most known sequence polymorphisms within these regions. In addition, sixteen probes derived from polymorphic regions in exon 2 were used to discriminate alleles not unequivocally characterized by the exon 3 based probes. This SSOP panel gave rise to eighty-six distinct hybridization patterns that could be used to unequivocally define all WHO-designated serological HLA-B specificities except for HLA-B54 in all homo- and heterozygous combinations. Furthermore, sixty-six out of ninety-seven molecularly defined HLA-B subtypes were characterized by unique hybridization patterns in all homozygous and most (possibly all) heterozygous combinations. The reproducibility of these results was confirmed by analysis of forty-four Workshop reference cell lines and of seventy-eight randomly chosen samples (one-hundred forty-seven alleles) from unrelated individuals serologically typed in the laboratory. For sixty-five samples (one-hundred-thirty-three alleles), molecular typing confirmed the results obtained by serology and allowed molecular subtype assignment for ninety-one alleles tested. A serologically blank allele could be defined by molecular analysis in three cases. The method presented here for molecular typing of the HLA-B locus can be used as an alternative to biochemical methods such as one-dimensional isoelectric focusing for assignment of serologically cross-reacting HLA-B molecules as well as for subtype characterization of a large variety of HLA-B alleles.

Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation.

The infusion of donor lymphocytes after allogeneic bone marrow transplantation is a promising therapeutic tool for achieving a graft versus leukemia (GvL) effect in case of leukemic relapse (1-7), and for the treatment of other complications related to the severe immunosuppressive status of transplanted patients, such as Epstein Barr virus-induced lymphoproliferative disorders (EBV-BLPD) (8) or reactivation of CMV infection (9). Although the delay in the administration of T lymphocytes is expected to reduce the risk of severe GvHD, this risk is still present at higher doses of donor T-cells. The transfer of a suicide gene into donor lymphocytes could allow the in vivo selective elimination of cells responsible for severe GvHD. Additionally, under appropriate conditions, it may allow in vivo modulation of donor anti-tumor responses, and to separate GvL from GvHD. Finally, crucial questions concerning survival and function of donor lymphocytes could be answered by their gene marking. Previous studies documented that T lymphocytes are suitable targets for gene transfer through retroviral vectors (10, 11). This protocol has been designed to evaluate in the contest of allogeneic BMT: 1--the safety of increasing doses of donor lymphocytes transduced with a suicide retroviral vector; 2--the efficacy in terms of survival and immunologic potential of donor lymphocytes after in vitro activation, gene transduction, and immunoselection; 3--the possibility of in vivo down regulation of GvHD by the administration of ganciclovir to patients treated by tk-transduced donor lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic T cells specific for glutamic acid decarboxylase in autoimmune diabetes.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that results in the destruction of the pancreatic islet beta cells. Glutamic acid decarboxylase (GAD) has been recently indicated as a key autoantigen in the induction of IDDM in nonobese diabetic mice. In human diabetes, the mechanism by which the beta cells are destroyed is still unknown. Here we report the first evidence for the presence of GAD-specific cytotoxic T cells in asymptomatic and recent diabetic patients. GAD65 peptides displaying the human histocompatibility leukocyte antigen (HLA)-A*0201 binding motif have been synthesized. One of these peptides, GAD114-123, binds to HLA-A*0201 molecules in an HLA assembly assay. Peripheral blood mononuclear cells from individuals with preclinical IDDM, recent-onset IDDM, and from healthy controls were stimulated in vitro with the selected peptide in the presence of autologous antigen-presenting cells. In three cases (one preclinical IDDM and two recent-onset IDDM), we detected specific killing of autologous antigen-presenting cells when incubated with GAD114-123 peptide or when infected with a recombinant vaccinia virus expressing GAD65. These patients were the only three carrying the HLA-A*0201 allele among the subjects studied. Our finding suggests that GAD-specific cytotoxic T lymphocytes may play a critical role in the initial events of IDDM.

Use of 19F nuclear magnetic resonance spectroscopy for probing substitution of Al for Ga in cloverite.

The synthesis of gallophosphate cloverite in the presence of aluminium leads to a partial substitution of Ga by Al. 27Al and 19F magic-angle spinning nuclear magnetic resonance (MAS NMR) studies on this material confirm this substitution. 27Al MAS NMR shows a broad line at 30 ppm which can be attributed to tetrahedrally coordinated Al in the solid. The 19F MAS NMR results are more complex. The spectrum presents five lines between -68 ppm and -95 ppm which can be assigned to fluorine in D4Rs with variable composition in Ga and Al. Two other lines, at -145.6 ppm and -178.2 ppm, may correspond to impurities and to a fluorine atom in a pseudo-sodalite cage environment, respectively.

High-flux acetate haemodialysis: a single-centre experience.

One hundred haemodialysed patients have been treated for 4 h thrice weekly by acetate haemodialysis with high-flux dialysers (HAHD) 1.4-1.8 m2 and automated ultrafiltration control for 18.9 +/- 8.3 months. The aim of the study was to evaluate the efficacy of treatment as regards urea and beta 2-microglobulin removal, cardiovascular stability, acid-base balance and plasma Il-1 variations. Moreover medium-term observation of both lipid profile and basal beta 2-microglobulin concentrations were performed. With our current adequacy criteria (urea clearance greater than or equal to 120 l/week; KT/V greater than or equal to 1.2; beta 2-microglobulin removal greater than or equal to 150 mg/treatment), the occurrence of hypotension was 6% during 24,500 treatments. Cardiovascular stability was preserved by the increase of total peripheral resistances in response to cardiac output decrease. No disturbances of acid-base parameters were observed in spite of plasma acetate concentrations greater than 6 mmol/l. No variations of plasma Il-1 occurred during the session or 2 h later.

Effects of acute administration of acetazolamide and frusemide on lithium clearance in humans.

To investigate the mechanism(s) of acute frusemide-induced increases in FELi, the effects of frusemide and acetazolamide, the carbonic anhydrase inhibiting agent, were evaluated in 19 healthy subjects either before or after pretreatment with acetazolamide or frusemide (11 subjects and eight subjects respectively). Acetazolamide pretreatment did not modify frusemide-induced increases in FELi (delta FLi 12.8 +/- 4.1% compared to 14.6 +/- 7.3%, P = NS); similarly, frusemide pretreatment did not modify acetazolamide-induced increases in FELi (delta FLi: 11.3 +/- 5.9% compared to 9.8 +/- 3.8%, P = NS). Acetazolamide-induced changes of FELi were correlated significantly with acetazolamide-induced increases of FEHCO3 (r = 0.61, P less than 0.05), FENa (r = 0.46, P less than 0.05) but not of FECl (r = 0.25, P = NS). On the other hand, frusemide-induced changes of FELi were correlated significantly with frusemide-induced increases of FENa (r = 0.62, P less than 0.005), FEC1 (r = 0.52, P less than 0.025) but not of FEHCO3 (r = 0.3, P = NS). Thus, frusemide effects on tubular lithium reabsorption are not related to carbonic anhydrase inhibition; furthermore, it appears that frusemide and acetazolamide affect lithium reabsorption by different and independent mechanism(s), possibly acting at different nephron site(s).

Radiosensitivity of NK lytic activities and NK-mediated hematopoietic colony inhibition: effect of activation with IL-2 and blocking of the T-200 molecule.

NK cells are capable of a variety of effector functions including the unprimed lysis of certain tumor cell targets and the nonspecific suppression of hematopoietic colony formation. In this study we have evaluated in parallel the radiosensitivity of lytic and colony inhibitory activity (CIA) by peripheral blood lymphocytes and large granular lymphocytes before and after activation by 72-96 hr of culture in medium containing interleukin 2 (IL-2). Our results show that (1) cells exhibiting CIA and lytic activities have almost identical patterns of radioresistance both before and following activation by IL-2; (2) the amount of preculture irradiation which completely blocks IL-2-stimulated cell proliferation only minimally affects CIA and lytic activities; (3) lysis of K562 and inhibition of CFU-GM are more resistant to preculture irradiation than is the killing of NK insensitive targets; (4) both the CIA and the lytic activities of PBL, but not of LGL, can be increased by 500-1000 rad of gamma-irradiation; and (5) NK lytic activity and CIA both before and following activation by IL-2 are primarily mediated by cells bearing the NKH1A antigen. Evidence that suggests NK cells mediate both the lysis of K562 and the inhibition of myeloid cells by a common mechanism came from experiments which showed that a monoclonal antibody to the T-200 molecule (13.3) could block both activities.