BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

Prolonged exposure to arsenic in UK private water supplies: toenail, hair and drinking water concentrations.

Chronic exposure to arsenic (As) in drinking water is an established cause of cancer and other adverse health effects. Arsenic concentrations >10 µg L(-1) were previously measured in 5% of private water supplies (PWS) in Cornwall, UK. The present study investigated prolongued exposure to As by measuring biomarkers in hair and toenail samples from 212 volunteers and repeated measurements of As in drinking water from 127 households served by PWS. Strong positive Pearson correlations (rp = 0.95) indicated stability of water As concentrations over the time period investigated (up to 31 months). Drinking water As concentrations were positively correlated with toenail (rp = 0.53) and hair (rp = 0.38) As concentrations - indicative of prolonged exposure. Analysis of washing procedure solutions provided strong evidence of the effective removal of exogenous As from toenail samples. Significantly higher As concentrations were measured in hair samples from males and smokers and As concentrations in toenails were negatively associated with age. A positive association between seafood consumption and toenail As and a negative association between home-grown vegetable consumption and hair As was observed for volunteers exposed to <1 As µg L(-1) in drinking water. These findings have important implications regarding the interpretation of toenail and hair biomarkers. Substantial variation in biomarker As concentrations remained unaccounted for, with soil and dust exposure as possible explanations.

Determination of HLA-A, -C, -B, -DRB1 allele and haplotype frequency in Japanese population based on family study.

The present study investigates the human leucocyte antigen (HLA) allele and haplotype frequencies in Japanese population. We carried out the frequency analysis in 5824 families living across Japanese archipelago. The studied population has mainly been typed for the purpose of transplant, especially the hematopoietic stem cell transplantation (HSCT). We determined HLA class I (A, B, and C) and HLA class II (DRB1) using Luminex technology. The haplotypes were directly counted by segregation. A total of 44 HLA-A, 29 HLA-C, 75 HLA-B, and 42 HLA-DRB1 alleles were identified. In the HLA haplotypes of A-C-B-DRB1 and C-B, the pattern of linkage disequilibrium peculiar to Japanese population has been confirmed. Moreover, the haplotype frequencies based on family study was compared with the frequencies estimated by maximum likelihood estimation (MLE), and the equivalent results were obtained. The allele and haplotype frequencies obtained in this study could be useful for anthropology, transplantation therapy, and disease association studies.

Lack of association between KIR and HLA-C type and susceptibility to idiopathic bronchiectasis.

INTRODUCTION: Idiopathic bronchiectasis is a poorly defined disease characterised by persistent inflammation, infection and progressive lung damage. Natural killer (NK) cells provide a major defense against infection, through the interaction of their surface receptors, including the activating and inhibitory killer immunoglobulin-like receptors (KIR), and human leukocyte antigens (HLA) class I molecules. Homozygosity for HLA-C has been shown in a single study to confer increased genetic susceptibility to idiopathic bronchiectasis. We aimed to assess whether the KIR and HLA repertoire, alone or in combination, may influence the risk of developing idiopathic bronchiectasis, in an independent replication study. METHODS: In this prospective, observational, case-control association study, 79 idiopathic bronchiectasis patients diagnosed following extensive aetiological investigation were compared with 98 anonymous, healthy, age, sex and ethnically-matched controls attending blood donor sessions in the same geographical location. DNA extraction was performed according to standardised techniques. Determination of presence or absence of KIR genes was performed by a sequence specific oligonucleotide probe method. Allele frequencies for the proposed KIR, HLA-B and HLA-C risk alleles both individually and in combinations were compared. RESULTS: We found no significant differences in allele frequency between the idiopathic bronchiectasis and control samples, whether considering HLA-C group homozygosity alone or in combination with the KIR type. DISCUSSION: Our results do not show an association between HLA-C and KIR and therefore do not confirm previous positive findings. This may be explained by the lower frequency of HLA-C1 group homozygosity in the control population of the previous study (27.2%), compared to 42.3% in our study, which is consistent with the genetic profiling of control groups across the UK. The previous positive association study may therefore have been driven by an anomalous control group. Further larger prospective multicentre replication studies are needed to determine if an association exists.

Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway-involving AKT, CREB, Bcl-xL, survivin, and Bcl-2-downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

'Immunogenetics of Aging': report on the activities of the 15th International HLA and Immunogenetics Working Group and 15th International HLA and Immunogenetics Workshop.

'Immunogenetics of Aging' is a component that was first included in the 14th International HLA and Immunogenetics Workshop (IHIWS) and developed further within the 15th Workshop. The aim of this component was to assess the impact of human leukocyte antigen (HLA) genes, cytokine genes, and some innate immunity genes such as killer-cell immunoglobulin-like receptors (KIRs) and mannose-binding lectin 2 (MBL2) in successful aging and their contribution to the better understanding of immune dysfunction in old age. Within the 15th IHIWS new populations were included in the analysis. Additional cytokine gene polymorphisms were assessed and innate immunity genes were analyzed for possible relevance in longevity. The results showed that longevity might be associated with anti-inflammatory cytokine gene profiles, decreased frequency of interleukin-10 (IL-10) and transforming growth factor-B1 haplotypes associated with a low level of gene expression, and increased frequency of haplotypes determining a high level of expression. Extended tumor necrosis factor-A and IL-12B genotypes were also likely relevant to longevity. Data also showed that innate immunity genes are associated with susceptibility to infections in the elderly and showed that these genes might be an important genetic marker in aging. Decreased frequencies of KIR2DS5 and A1B10 haplotypes, and an increased proportion of MBL2-deficient haplotypes were found in the group with higher cytomegalovirus-specific IgG antibody levels. Together, these studies emphasize the relevance of genes regulating immune functions in maintaining human longevity and stress the importance of further clarifying their impact on successful aging.

Analysis of the HLA population data (AHPD) submitted to the 15th International Histocompatibility/Immunogenetics Workshop by using the Gene[rate] computer tools accommodating ambiguous data (AHPD project report).

During the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), 14 human leukocyte antigen (HLA) laboratories participated in the Analysis of HLA Population Data (AHPD) project where 18 new population samples were analyzed statistically and compared with data available from previous workshops. To that aim, an original methodology was developed and used (i) to estimate frequencies by taking into account ambiguous genotypic data, (ii) to test for Hardy-Weinberg equilibrium (HWE) by using a nested likelihood ratio test involving a parameter accounting for HWE deviations, (iii) to test for selective neutrality by using a resampling algorithm, and (iv) to provide explicit graphical representations including allele frequencies and basic statistics for each series of data. A total of 66 data series (1-7 loci per population) were analyzed with this standard approach. Frequency estimates were compliant with HWE in all but one population of mixed stem cell donors. Neutrality testing confirmed the observation of heterozygote excess at all HLA loci, although a significant deviation was established in only a few cases. Population comparisons showed that HLA genetic patterns were mostly shaped by geographic and/or linguistic differentiations in Africa and Europe, but not in America where both genetic drift in isolated populations and gene flow in admixed populations led to a more complex genetic structure. Overall, a fruitful collaboration between HLA typing laboratories and population geneticists allowed finding useful solutions to the problem of estimating gene frequencies and testing basic population diversity statistics on highly complex HLA data (high numbers of alleles and ambiguities), with promising applications in either anthropological, epidemiological, or transplantation studies.

Killer immunoglobulin-like receptors (KIR2DL2 and/or KIR2DS2) in presence of their ligand (HLA-C1 group) protect against chronic myeloid leukaemia.

We have analysed the frequency of killer immunoglobulin-like receptors (KIR) in cohorts of patients from Turkey with acute lymphocyte leukaemia (n = 52), acute myeloid leukaemia (n = 54) and chronic myeloid leukaemia (CML) (n = 52) and compared the results with 154 controls. We also examined the frequencies of human leucocyte antigen (HLA)-C groups, -Bw4, -Bw6 and where appropriate the combination of the KIR gene and its ligand. We found several statistically significant results between the patients and the controls. We proposed a model in CML of protection via KIR2DL2 and/or KIR2DS2 with the presence of the ligand HLA-C1 group and susceptibility via HLA-Bw4 homozygosity (i.e. absence of HLA-Bw6).

Optimizing BeLPT criteria for beryllium sensitization.

BACKGROUND: The beryllium lymphocyte proliferation test (BeLPT) is used to identify persons sensitized to beryllium. ATSDR convened an expert panel of physicians and scientists in April 2006 to discuss this test and to consider what BeLPT test results actually establish beryllium sensitization. The three criteria proposed by panel members were (1)one abnormal result, (2)one abnormal and one borderline result, and (3)two abnormal results. METHODS: Complete algorithms were developed for each of the three proposed criteria. Using single-test outcome probabilities developed by Stange et al. [2004. Am J Ind Med 46:453-462], we calculated and compared the sensitivity, specificity, and positive predictive values (PPVs) for each set of criteria. RESULTS: The overall sensitivity and specificity of the three criteria were similar. When the criteria required confirmation of an abnormal result the PPV was higher--whether the requirement was satisfied by a borderline result, or only by another abnormal result. Confirmation also reduced the likelihood of false positives. The differences between the three criteria decreased as the prevalence of sensitization increased. CONCLUSIONS: A single unconfirmed abnormal is usually insufficient to establish sensitization for an apparently healthy person. When the prevalence of beryllium sensitization in a group is high, however, even a single abnormal BeLPT can be a strong predictor.

Analysis of KIR gene frequencies in HLA class I characterised bladder, colorectal and laryngeal tumours.

Three cohorts of patients with laryngeal, bladder or colorectal tumours were investigated for frequency of killer immunoglobulin-like receptor (KIR) genes compared with a normal control population. The frequency of KIR3DL1 and KIR2DS4 was significantly increased (but not after correction for number of comparisons made) in patients with bladder tumour compared with controls. No other significant differences were found in gene frequencies or in the frequencies of those KIR genes with and without their human leucocyte antigen (HLA) ligands. Furthermore, no significant differences were found in KIR gene frequencies, taking into consideration the type of loss of HLA expression in the individual tumours. Finally, in the group of colorectal carcinomas, there was an overall significant difference in the frequencies of C group heterozygosity and homozygosity with HLA alterations on the tumour.

14th International HLA and Immunogenetics Workshop: report on KIR receptors and their applications.

During the last few years many laboratories have developed a keen interest in detecting killer immunoglobulin-like receptor (KIR) receptor genes in various populations, diseases and in stem cell transplantation. At the 14th International Histocompatibility Workshop held in Melbourne in December 2006, many of these laboratories presented their findings at a special session. To introduce this work, we provide an introduction to KIR receptors and an outline of previous applications of KIR receptor typing prior to the Workshop.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

KIR allele and gene polymorphism group (KAG).

Recent research presented at this meeting verifies the extent of the polymorphism of KIR genes and their alleles and shows their association with various diseases. Methods were described for the determination of alleles of several of the KIR genes in different populations including a novel allele formed by an unequal recombination event. Associations of KIR genes were found with several diseases: type I diabetes; psoriatic arthritis; and hepatitis C infection. Discussion took place on nomenclature issues of the KIR genes and their alleles, and a website has been developed to collect frequencies of KIR genes and alleles in various diverse populations.

A squamous cell carcinoma and an adenocarcinoma in Australian treefrogs.

We describe the pathology in a captive adult White lipped treefrog (Litoria infrafrenata) with a squamous cell carcinoma of the skin and a free-living adult Common green treefrog (L. caerulea) with an adenocarcinoma of the skin. Although many amphibian neoplasms have been reported world wide, none had been described in Australian species.

Are human platelet alloantigens (HPA) minor transplantation antigens in clinical bone marrow transplantation?

The role of human platelet alloantigens (HPA) in clinical bone marrow allotransplantation was investigated. The leading hypothesis was that HPA alloepitopes act as minor histocompatibility antigens and aggravate graft-versus-host disease (GVHD). To exclude the effect of MHC disparity, only HLA identical donor-recipient pairs were entered into the study. The influence of HPA compatibility on overall survival, occurrence of relapses and haematopoietic recovery was also investigated. A total of 223 patients who received a graft from an HLA-identical sibling, genotyped for HPA -1, -2, -3, -4 and -5, were observed over a post-transplant period of 24 months following the protocol recommended by EBMT. The data from patients having received grafts from HPA compatible donors were compared to data from patients having received grafts that were mismatched in HPA allotypes in the GVH direction. Analysis of the incidence of acute and chronic (GVHD), overall survival, relapse incidence, haematopoietic recovery and some other clinical parameters did not reveal any significant difference between the HPA-matched and -mismatched groups of patients, regardless of their age. Our results give no evidence that HPA-1, -2, -3 and -5 alloantigens should be considered minor transplantation antigens in clinical bone marrow transplantation.

Plastic particle migration during intravenous infusion assisted by a peristaltic finger pump in an animal model.

The contamination of intravenously administered fluid with foreign material has always been of major concern, but the in-vivo impact of silicone embolisation from administration of fluid via a peristaltic finger pump (PFP) has not previously been assessed. To determine whether silicone particles enter the lungs and to review the histological response, 10 rabbits received an IV infusion of 0.9% saline at 10 ml/kg per hour over a 72-h period, via an IVAC 591 PFP. The lungs were analysed for silicone particles with scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDXA). These results were compared with a control group of non-infused animals. Silicone particles were found in 8 of 10 animals in the experimental group and in 2 of 9 control animals, indicating that silicone particles are dislodged during pump-assisted IV infusions. The difference between the control and infused animals was statistically significant using Fisher's exact test (P = 0.023). However, silicone plastic particles in control animals suggest that there is also environmental exposure to silicone in addition to those particles that come from a therapeutic source. The additional finding of elemental silicon (which is one of the constituents of silicone plastic) in both infused and control animals in which silicone plastic was not found indicates that not all elemental silicon in animals reflects the presence of silicone plastic. The clinical significance of each of these two findings is yet to be determined.

A model of reversible inhibitors in the gastric H+/K+-ATPase binding site determined by rotational echo double resonance NMR.

Several close analogues of the noncovalent H(+)/K(+)-ATPase inhibitor SCH28080 (2-methyl-3-cyanomethyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine) have been screened for activity and examined in the pharmacological site of action by solid-state NMR spectroscopy. TMPIP, the 1,2,3-trimethyl analogue of SCH28080, and variants of TMPIP containing fluorine in the phenylmethoxy ring exhibited IC(50) values for porcine H(+)/K(+)-ATPase inhibition falling in the sub-10 microm range. Deuterium NMR spectra of a (2)H-labeled inhibitor titrated into H(+)/K(+)-ATPase membranes revealed that 80-100% of inhibitor was bound to the protein, and K(+)-competition (2)H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from (13)C-(19)F dipolar coupling measurements using the cross-polarization magic angle spinning NMR method, REDOR. It was found that the inhibitor adopts an energetically favorable extended conformation falling between fully planar and partially bowed extremes. These findings allowed a model to be proposed for the binding of this inhibitor to H(+)/K(+)-ATPase based on the results of independent site-directed mutagenesis studies. In the model, the partially bowed inhibitor interacts with Phe(126) close to the N-terminal membrane spanning helix M1 and residues in the extracellular loop bridging membrane helices M5 and M6 and is flanked by residues in M4.

An IgH enhancer that drives transcription through basic helix-loop-helix and Oct transcription factor binding motifs. Functional analysis of the E(mu)3' enhancer of the catfish.

The transcriptional enhancer (E(mu)3') of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian E(mu) enhancer in both location and structure. It occurs between the mu and delta genes and contains numerous transcription factor binding sites, predominantly octamer and muE5 motifs of consensus and variant sequences. It lacks the classical muA-muE3(CBF)-muB core array of binding motifs seen within mammalian IgH E(mu) enhancers. To determine the functionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of consensus and variant octamer motifs (individually or in multiples) decreased enhancer function, mutation of a single consensus muE5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus muE5 site, combined with mutations of certain octamer sites, destroyed function in catfish B cells. Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal E(mu)3' enhancer (a) contains a consensus muE5 site and two octamer sites, (b) is B cell-specific, and (c) is active across species. The dependence of an Ig enhancer on sites that bind basic helix-loop-helix and Oct transcription factors has not previously been observed and confirms large differences in structure and function between fish and mammalian IgH enhancers.

Association between polymorphism in regulatory region of gene encoding tumour necrosis factor alpha and risk of Alzheimer's disease and vascular dementia: a case-control study.

BACKGROUND: Deposition of beta-amyloid in the brains of patients with Alzheimer's disease is thought to precede a chain of events that leads to an inflammatory response by the brain. We postulated that genetic variation in the regulatory region of the gene for the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) leads to increased risk of Alzheimer's disease and vascular dementia. METHODS: A polymorphism in the regulatory region of the TNF-alpha gene was analysed in a case-control study. The polymorphism (C-850T) was typed in 242 patients with sporadic Alzheimer's disease, 81 patients with vascular dementia, 61 stroke patients without dementia, and 235 normal controls. These groups of individuals were also genotyped for the apolipoprotein E polymorphism, and the vascular dementia and stroke groups were typed at the HLA-DR locus. FINDINGS: The distribution of TNF-alpha genotypes in the vascular dementia group differed significantly from that in the stroke and normal control groups, giving an odds ratio of 2.51 (95% CI 1.49-4.21) for the development of vascular dementia for individuals with a CT or TT genotype. Logistic regression analysis indicated that the possession of the T allele significantly increased the risk of Alzheimer's disease associated with carriage of the apolipoprotein E epsilon4 allele (odds ratio 2.73 [1.68-4.44] for those with apolipoprotein E epsilon4 but no TNF-alpha T, vs 4.62 [2.38-8.96] for those with apolipoprotein E epsilon4 and TNF-alpha T; p=0.03). INTERPRETATION: Possession of the TNF-alpha T allele significantly increases the risk of vascular dementia, and increases the risk of Alzheimer's disease associated with apolipoprotein E. Although further research is needed, these findings suggest a potential role for anti-inflammatory therapy in vascular dementia and Alzheimer's disease, and perhaps especially in patients who have had a stroke.