New ABO intron 1 variant alleles.

Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29-5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor-binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A), and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies.Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29­5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor­binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A), and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies.

Using behavioural insights to increase HIV self-sampling kit returns: a randomized controlled text message trial to improve England's HIV self-sampling service.

OBJECTIVES: The aim of the study was to determine whether behaviourally informed short message service (SMS) primer and reminder messages could increase the return rate of HIV self-sampling kits ordered online. METHODS: The study was a 2 × 2 factorial design randomized control trial. A total of 9585 individuals who ordered a self-sampling kit from www.freetesting.hiv different SMS combinations: 1) standard reminders sent days 3 and 7 after dispatch (control); 2) primer sent 1 day after dispatch plus standard reminders; 3) behavioural insights (BI) reminders (no primer); or 4) primer plus BI reminders. The analysis was restricted to individuals who received all messages (n = 8999). We used logistic regression to investigate independent effects of the primer and BI reminders and their interaction. We explored the impact of sociodemographic characteristics on kit return as a secondary analysis. RESULTS: Those who received the primer and BI reminders had a return rate 4% higher than that of those who received the standard messages. We found strong evidence of a positive effect of the BI reminders (odds ratio 1.13; 95% confidence interval 1.04-1.23; P = 0.003) but no evidence for an effect of the primer, or for an interaction between the two interventions. Odds of kit return increased with age, with those aged ≥ 65 years being almost 2.5 times more likely to return the kit than those aged 25-34 years. Men who have sex with men were 1.5-4.5 times more likely to return the kit compared with other sexual behaviour and gender identity groups. Non-African black clients were 25% less likely to return the kit compared with other ethnicities. CONCLUSIONS: Adding BI to reminder messages was successful in improving return rates at no additional cost.

Monocytes-macrophages and cytokines/chemokines in fine-needle aspiration biopsy cultures: enhanced interleukin-1 receptor antagonist synthesis in rejection-free kidney transplant patients.

BACKGROUND: Monocytes-macrophages are found within kidney allografts during the first days after surgery, where they perform "housekeeping" tasks, participate in postreperfusion injury, and act as antigen-presenting cells, as well as become involved in the effector phase of acute rejection. They also seem to play a prominent role in chronic rejection. We quantified their presence in fine-needle aspiration biopsies and studied the growth factors that, we hypothesized, would mark the different implications of the presence of monocytes-macrophages. METHODS: Fine-needle aspiration biopsies were obtained from 56 adult renal transplants and analyzed for CD14+ using the alkaline phosphatase-anti-alkaline phosphatase procedure. Thirty-three patients were studied on the production of interleukin (IL)-1 receptor antagonist (IL-1ra), IL-6, IL-8, macrophage colony-stimulating factor, monocyte chemotactic protein 1, and macrophage inflammatory protein 1alpha by aspiration biopsies cultures using enzyme-linked immunosorbent assay techniques. RESULTS: CD14+ cells were present at significantly higher numbers in steroid-resistant acute rejections but also during the first days after surgery, especially if acute tubular necrosis was present. We found a significantly higher production of IL-1ra by rejection-free patients compared with acutely rejecting patients, and this difference was already established on day 7 after surgery (10+/-10.5 days before rejection). CONCLUSIONS: Monocytes-macrophages are present at higher numbers in aspiration biopsies of kidney transplant patients suffering either acute tubular necrosis or steroid-resistant rejections, but they are present during the first days after transplant in stable patients, too. The production of IL-1ra is significantly up-regulated in stable patients, which suggests that monocytes-macrophages may constitute an early key factor in the down-regulation of the anti-allograft immune response.

Analysis of fine-needle aspiration biopsies by flow cytometry in kidney transplant patients.

BACKGROUND: Peripheral blood lymphocyte (PBL) analysis by flow cytometry has been inconsistently reported as an adjunctive method for diagnosing acute kidney transplant rejection. However, there is good evidence that lymphocytes infiltrating renal grafts differ from those found at the peripheral level. We hypothesized that the study of aspiration biopsy samples in conjunction with PBL by flow cytometry would enable us to diagnose acute rejection crisis reliably. METHODS: Lymphocytes from PBL and aspiration biopsies of kidney transplant patients were analyzed. Fifty-one stable patients, rejection-free for the first 6 months, were studied on day 7 and day 30 after transplantation and were compared with 32 patients with 40 acute rejection episodes. RESULTS: Significant differences were observed for several lymphocyte subpopulations on aspiration biopsy samples comparing stable patients with rejection patients. In contrast, PBL analysis was not helpful in differentiating the two groups of patients. By combining the expression of several activation markers inside the graft with CD3DR and CD3CD25 aspiration biopsy to peripheral blood ratios, we obtained very good values for sensitivity and specificity-83.9% and 90.5%, respectively. The positive predictive value for rejection among dysfunctional grafts reached 85.8%. CONCLUSIONS: Flow cytometry study of aspiration biopsy samples of kidney transplant patients is a reliable and powerful method to diagnose acute rejection episodes, although it is needed to consider several lymphocyte phenotypes; cytofluorometric analysis of PBL is important because it provides graft-infiltrating cell to peripheral blood lymphocyte ratios. This safe and rapid test may significantly improve the management of kidney transplant patients.

Inconsistency in radiographic evaluation of progressive renal osteodystrophy.

Five radiologists graded 49 series of bone X-rays of 20 patients with chronic renal failure treated by hemodialysis. There was a high incidence of osteodystrophy which progressed identifiably over intervals exceeding 12 months. The severity grade of osteodystrophy was poorly reproducible among patients, among radiologists, and even between interpretations by the same radiologist after an interval of 10 months. Although the severity of osteodystrophy correlated with serum alkaline phosphatase values, the latter was not an accurate predictor of the severity of the bone lesions. Radiographic reassessment at intervals of one year or less in the asymptomatic patient has less reproducibility than the anticipated changes. More sensitive and reliable techniques are recommended.

Prediction of radiographic severity of renal osteodystrophy by serum values.

Renal osteodystrophy, a frequent complication of chronic renal failure, is usually assessed by periodic X-rays of bone which are both poorly reproducible and expensive. Seeking a better screening test for osteodystrophy, we evaluated the usefulness of serum alkaline phosphatase as a predictor of bone disease and of hyperparathyroidism. Alkaline phosphatase, despite nonspecificity, correlates with the severity of osteodystrophy and with the increase in serum parathyroid hormone concentration. Serial measurements of alkaline phosphatase can predict changes in these parameters.

Cultures of aspiration biopsy specimens in the immunological monitoring of renal transplants.

OBJECTIVE: Graft-infiltrating cells (GIC) have been studied in heart, lung, and liver transplants and have been shown to have greater proliferative ability when taken from rejecting allografts. Our aim was to study GIC harvested by fine-needle aspiration biopsy (FNAB) in renal transplant recipients. PATIENTS AND METHODS: 93 adult patients entered the study. The FNABs were done on the 7th, 14th, and 30th day after transplantation in stable cases and whenever a rejection crisis supervened. RESULTS: The proliferation responses of GIC were significantly higher in rejection than in stable cases during the 1st month after transplantation. The sensitivity for rejection was 96.4%, the specificity 91.3%, the negative predictive value 98.7%, and the positive predictive value was 93.3% among dysfunctioning grafts. CONCLUSIONS: The study of the proliferative capacity of graft-infiltrating cells in renal transplants is a safe and very useful immunologic monitoring tool, and it could improve the FNAB diagnostic accuracy.

[Acute renal insufficiency with diffuse intravascular coagulation in 2 fatal cases of septic abortion]. / Insuficiência renal aguda com coagulaçao intravascular disseminada em dois casos mortais de aborto inectado.

PIP: Acute renal insufficiency complicated by intravascular coagulation is an important cause of death in septic abortion. Whenever septic abortion is suspected, the patient should be promptly hospitalized. The report describes in detail the symptoms of the 2 patients, test findings, and the treatment used, and discusses the development of the infection, emphasizing the importance of secondary hemolysis in such cases.^ieng

Cytokine analysis of human renal allograft aspiration biopsy cultures supernatants predicts acute rejection.

BACKGROUND: A Th1 response is said to be associated with transplant rejection and Th2 with tolerance, although this is not agreed by all. Cytokines evaluation in peripheral blood and urine in kidney transplants produces variable results. We hypothesized that measurement of major cytokines involved in Th1/Th2 paradigm on transplant renal-infiltrating cells could bring valuable scientific and clinical information. METHODS: Fifty-six adult cadaver kidney transplants were subdivided into 21 stable patients (group A), 22 suffering acute rejection (group B), 10 with chronic rejection (group C) and three with CMV disease (group D). Fine-needle aspiration biopsies were cultured and their supernatants analysed for IL-2, IL-4, IL-10 and IFN-gamma. RESULTS: Group A produced small amounts of both IL-2 and IL-10 while group B synthetized significantly higher IL-2 and significantly lower IL-10 amounts than group A. Group B produced significantly more IL-2 than A on day 7 post-transplantation, several days before rejection supervened. Group C produced IL-10 and very low amount of IL-2. Group D produced both IL-2 and IL-10. We did not find any IL-4, and IFN-gamma was present in a few samples. For IL-2, sensitivity, specificity, negative and positive predictive values for acute rejection were 100, 87.2, 94.7 and 83.3%, respectively. CONCLUSIONS: Cytokine analysis in fine-needle aspiration biopsy cultures supernatants is a very useful immunological screening method for kidney transplants. IL-2 synthesis on day 7 post-transplantation reliably predicted the risk of impending acute rejection during the first weeks. The cytokine pattern suggests that acute rejection is associated with Th1, stable patients with Th0/Th2, and chronic rejection with Th2 patterns.

Assessment of renal dopaminergic system activity during the recovery of renal function in human kidney transplant recipients.

BACKGROUND: The urinary excretion of free dopamine has been used as an index of the renal synthesis of amine. However, it is now well recognized that in the kidney, newly-formed dopamine is significantly inactivated through deamination to 3,4-dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase (MAO). The aim of the present study was to assess the renal dopaminergic system activity during the recovery of renal function in kidney transplant recipients and to assess which parameters are appropriate for the evaluation of renal amine synthesis under these conditions. METHODS: Twenty-four-hour urinary excretion of L-DOPA, dopamine and its metabolites (DOPAC; 3-MT; HVA) were continuously monitored in 19 renal transplant recipients from the first day of surgery until the twelfth day post-transplantation. RESULTS: In 11 patients (Group 1), renal function consistently recovered throughout the study (plasma creatinine levels decreased from 6.2 +/- 0.4 to 2.1 +/- 0.1 mg/dl). Eight patients presented with acute tubular necrosis (Group 2) and minimal renal function was maintained until the twelfth post-operative day. The urinary excretion of L-DOPA did not differ throughout the study between the two groups of patients. In contrast, the 24-h urinary levels of dopamine, DOPAC and HVA were significantly higher throughout the study in patients of Group 1: dopamine (Group 1, 179 +/- 26 to 422 +/- 51 nmol/24 h; Group 2, 25 +/- 3 to 57 +/- 13 nmol/ 24 h), DOPAC (Group 1, 698 +/- 57 to 3487 +/- 414 nmol/ 24 h; Group 2, 158 +/- 22 to 1014 +/- 193 nmol/24 h) and HVA (Group 1, 13,058 +/- 1199 to 20,387 +/- 1559 nmol/ 24 h; Group 2, 4140 +/- 848 to 15,219 +/- 1037 nmol/24 h). CONCLUSIONS: The recovery of renal function in renal transplant recipients is accompanied by an enhanced ability to synthesize dopamine and inactivate it to DOPAC and HVA. It is suggested that the urinary levels of DOPAC may be a useful parameter for the assessment of dopamine formation in renal tissues.

T lymphocyte subsets and cytokine production by graft-infiltrating cells in FSGS recurrence post-transplantation.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) aetiology remains undefined although a derangement of lymphocytes and monocytes macrophages, at least, has been strongly suspected. We report the graft-infiltrating phenotypes and their cytokine production in a case of FSGS recurrence post-transplantation. METHODS: The kidney transplant recipient suffered immediate FSGS recurrence. Aspiration biopsies were done at the first and second week post-surgery and were analysed by flow cytometry. The cytokine analysis was done on aspiration sample culture supernatants and serum by enzyme-linked immunosorbent assay. RESULTS: High expression of CD3CD69, CD3CD71 and CD4CD29 was found on infiltrating lymphocytes. Biopsy cultures pointed to a Th0/Th1 pattern of cytokine production as well as significant synthesis of transforming growth factor-beta1. Interestingly, monocyte chemokines were absent. CONCLUSION: We report evidence of intragraft lymphocyte activation in the early days of FSGS recurrence. Aspiration biopsy cultures showed failure of cyclosporin A to inhibit interleukin-2 (IL-2) production by infiltrating lymphocytes. If our findings are confirmed in similar patients, a trial with anti-IL-2-receptor antibody could be warranted.