Acquisition of C3d-Binding Activity by De Novo Donor-Specific HLA Antibodies Correlates With Graft Loss in Nonsensitized Pediatric Kidney Recipients.

Alloantibody-mediated graft injury is a major cause of kidney dysfunction and loss. The complement-binding ability of de novo donor-specific antibodies (dnDSAs) has been suggested as a prognostic tool to stratify patients for clinical risk. In this study, we analyzed posttransplant kinetics of complement-fixing dnDSAs and their role in antibody-mediated rejection development and graft loss. A total of 114 pediatric nonsensitized recipients of first kidney allograft were periodically monitored for dnDSAs using flow bead assays, followed by C3d and C1q assay in case of positivity. Overall, 39 patients developed dnDSAs, which were C1q(+) and C3d(+) in 25 and nine patients, respectively. At follow-up, progressive acquisition over time of dnDSA C1q and C3d binding ability, within the same antigenic specificity, was observed, paralleled by an increase in mean fluorescence intensity that correlated with clinical outcome. C3d-fixing dnDSAs were better fit to stratify graft loss risk when the different dnDSA categories were evaluated in combined models because the 10-year graft survival probability was lower in patients with C3d-binding dnDSA than in those without dnDSAs or with C1q(+) /C3d(-) or non-complement-binding dnDSAs (40% vs. 94%, 100%, and 100%, respectively). Based on the kinetics profile, we favor dnDSA removal or modulation at first confirmed positivity, with treatment intensification guided by dnDSA biological characteristics.

Posttransplant de novo donor-specific hla antibodies identify pediatric kidney recipients at risk for late antibody-mediated rejection.

The emerging role of humoral immunity in the pathogenesis of chronic allograft damage has prompted research aimed at assessing the role of anti-HLA antibody (Ab) monitoring as a tool to predict allograft outcome. Data on the natural history of allografts in children developing de novo Ab after transplantation are limited. Utilizing sera collected pretransplant, and serially posttransplant, we retrospectively evaluated 82 consecutive primary pediatric kidney recipients, without pretransplant donor-specific antibodies (DSA), for de novo Ab occurrence, and compared results with clinical-pathologic data. At 4.3-year follow up, 19 patients (23%) developed de novo DSA whereas 24 had de novo non-DSA (NDSA, 29%). DSA appeared at a median time of 24 months after transplantation and were mostly directed to HLA-DQ antigens. Among the 82 patients, eight developed late/chronic active C4d+ antibody-mediated rejection (AMR), and four C4d-negative AMR. Late AMR correlated with DSA (p < 0.01), whose development preceded AMR by 1-year median time. Patients with DSA had a median serum creatinine of 1.44 mg/dL at follow up, significantly higher than NDSA and Ab-negative patients (p < 0.005). In our pediatric cohort, DSA identify patients at risk of renal dysfunction, AMR and graft loss; treatment started at Ab emergence might prevent AMR occurrence and/or progression to graft failure.

Allele frequencies of polymorphisms of TNFA, IL-6, IL-10 and IFNG in an Italian Caucasian population.

Polymorphisms in the regulatory and intronic regions of several cytokines have been associated with differential cytokine production. In this paper we genotyped, using the polymerase chain reaction-sequence-specific primers (PCR-SSP) method, a series of 363 healthy Italian Caucasians with the aim of obtaining a reference population for further studies on the role of cytokines in the inflammatory and immune responses. We also compared the results to those for other populations. The polymorphisms analysed were those of tumour necrosis factor alpha (TNFA), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNG). We found that the frequency of allele TNFA*1 at position -380 was 87.7% and that of TNFA*2 was 12.4%, significantly different from those of the UK and Japanese populations but not different from that of a population in Gambia. For IL-10 the frequencies of alleles -1082A and -1082G were 63.0% and 37.0% and those of alleles -819C, - 819T, -592C and -592A were 70.8, 29.2, 70.8 and 29.2%, respectively, significantly different from those observed in south-east England, in Manchester and in an Oriental population from southern China. The frequencies of IL-6 alleles - 174C and -174G were 29.0 and 71.0%, respectively; for IFNG polymorphisms at position -874, in the population under evaluation, the alleles -874T and -874A were present in 44.7 and 55.3% of the subjects, respectively. Genotype frequencies of IL-6 were significantly different from those observed in populations from Germany and from the UK. The analysis carried out by our group indicates that there is heterogeneity in the frequencies of the cytokine polymorphisms among the different Caucasian populations, and this underlines the importance of a 'local' reference population when evaluating the clinical relevance of cytokine gene polymorphisms.

[Lung base disease after orthotopic liver transplantation. Functional ultrasonography of the diaphragm and correlated alterations]. / Malattie della base polmonare dopo trapianto ortotopico del fegato. Ecografia funzionale del diaframma e alterazioni correlate.

PURPOSE: To demonstrate the greater accuracy of B- and M-probe ultrasound (US) compared to traditional examinations in the evaluation of diseases of the lung-base, a frequent localisation of pathology in patients undergoing orthotopic liver transplantation (OLT). MATERIAL AND METHODS: Twenty-one patients, 13 males and 8 females aged 24 to 63, awaiting OLT were examined using the three modalities. B-mode US was performed as a preliminary study to identify the districts of interest and the profile of the diaphragm wall, searching for any related alterations. This was followed by M-mode US, with an approach along the left and right posterior axillary lines during spontaneous and forced maximal expiration, to calculate and document the curve representing diaphragm mobility. All patients were also studied pre- and postoperatively by standard chest X-ray double projections. The parameters evaluated by US were diaphragmatic inspiratory slant and diaphragm range while the standard chest X-ray was used to assess hypoventilation, diaphragm range and pleural effusion. RESULTS: For each parameter considered we obtained the following results: presence or absence of pleural effusion (sensitivity: 100% with US vs 64% with chest X-ray) and diaphragmatic hypomobility with related hypoventilatory phenomena (sensitivity: 85% with US, with 15% false negatives). In 15 cases the chest X-ray revealed a clear elevation of the diaphragm, a finding supported by US in 11 cases. In 7 cases US showed a reduction in the diaphragm range curve without, however, any radiological evidence of any ventilatory dysfunction of the lung base and/or elevation of the corresponding hemidiaphragm. CONCLUSION: Our results suggest that radiology, B-mode US and color Doppler US, which are widely used for monitoring OLT patients, can be usefully integrated by M-mode US to evaluate diaphragmatic mobility both pre- and post-operatively. This method is fast, easy to use and widely available.

Comparison of serological and molecular typing for HLA-A and -B on cord blood lymphocytes.

HLA class I typing by standard microcytotoxicity testing has been unsatisfactory for 14.5% of 1644 cord blood samples. In this study, we evaluated the capacity of PCR-SSP in solving problems in HLA-A,B typing with serological methods. With this aim we have compared serology with PCR-SSP in 100 cord blood samples with doubtful or unreliable HLA-A,B typing. PCR-SSP was successful in amplifying HLA-A,B alleles in all 100 cord blood samples. Forty-six typings gave discrepant results with the 2 methods (serology and PCR-SSP). Typings were considered discrepant also in the case of inability to define a split. For 19 specimens, no serological conclusion was drawn due to high mortality of the cell suspension, while PCR-SSP allowed the definition of a clear typing. In 6 cases it was necessary to infer information from serology to define the current typing. Finally, in 3 other cases it was impossible to exclude or attribute the antigen/allele B67 or B4802. PCR-SSP for HLA-A,B can improve the overall reliability of HLA-A,B typing requiring a small amount of blood although, with the set of sequence specific primers adopted, a number of alleles are still poorly defined.

Genomic HLA-DR compatibility in long-term surviving recipients of cadaver kidney transplants.

The finding that HLA-DR compatibility assessed by DNA typing correlates with short-term graft outcome better than serology prompted us to study the degree of genomic HLA-DR compatibility on 55 patients with a graft functioning for more than 10 years (group A), compared with 82 patients with more recent transplants regardless of survival (group B). Because adequate blood donor samples were not available for group A long-term survivors, we used donor renal cells obtained by fine needle aspiration biopsy as a source of DNA. We found that in long-term survivors, the distribution of HLA-DR mismatches was significantly different from that observed in group B patients. In particular, whereas a similar proportion of patients with 1 mismatch was seen in both groups, 27.3% of group A patients vs. 6.1% of group B patients had no mismatch, and 23.6% of group A vs. 41.5% of group B patients received transplants with no HLA-DR compatibility (P = 0.001). We also investigated a possible correlation between number of incompatibilities and graft function. Well-matched patients received less steroid pulses than less well-matched recipients, and steroid-resistant rejection episodes were more common among less well-matched recipients. These results suggest a prognostic role of genomic HLA-DR compatibility on long-term success of cadaver kidney transplantation.

Prenatal paternity testing by DNA analysis.

Prenatal paternity testing was evaluated by DNA analysis in chorionic villus biopsies obtained during the 7th-22nd weeks of gestation. Using highly polymorphic variable number of tandem repeats (VNTR) probes, we analysed four cases consisting of mother/child/alleged father trios. In all cases, we were able to detect maternal and paternal alleles and could establish or exclude paternity. The application of DNA analysis represents a new important diagnostic aid for all cases that require a prenatal identification of paternity.

[Detection of varicella virus with a DNA probe in fetal blood and amniotic fluid]. / Nachweis des Varizellenvirus mit der DNA-Sonde im fetalen Blut und im Fruchtwasser.

In our Centre for Prenatal Diagnosis, we undertook a punctuation of the umbilical cord on a pregnant woman infected with varicella, complicated by viral encephalitis, to diagnose a foetal viraemia. In cooperation with the Toma Laboratory and Clonit Ltd., who have long been working on the development of specific viral genome probes, we succeeded in proving, that the foetus had contracted varicella.