Surface immunoglobulin restriction in B-non Hodgkin's lymphomas in cell suspension and on frozen tissue sections

The diagnostic relevance of different tests for detection of surface immunoglobulin on tumour cells of B-type non-Hodgkin's lymphomas (B-NHL) was investigated by comparison of the direct antiglobulin rosetting reaction (DARR) in suspension with two-colour direct immunofluorescence (DIF) on frozen tissue sections. In benign lymph nodes (n = 27) the κ/λ ratio by DARR test ranged from 0.9 to 2.8. Tested by suspension and frozen tissue analysis, light chain restriction was found in 24 and 27 of 31 cases of B-NHL, respectively. Heavy chain restriction was found in half of the cases (14 of 26) studied in suspension and in almost all (28 of 31) tested on sections. In 9 cases DARR tests showed restriction of more than one Ig class on tumour cells, which was infrequent (2 of 28) in frozen section analysis. Although both tests appeared valuable for routine diagnostic purposes, we found the DIF analysis on tissue sections somewhat more discriminative, especially in detection of heavy chain restriction in B-NHL.     

Does exposure to swine leukocyte antigens after pig‐to‐nonhuman primate xenotransplantation provoke antibodies that cross‐react with human leukocyte antigens?

BACKGROUND: A potential concern of using pig kidney xenografts for human transplantation is that antibodies produced to swine leukocyte antigens (SLA) may cross-react with human leukocyte antigens (HLA) and thereby limit the scope for a subsequent human organ donor transplant. We therefore investigated whether exposure to SLA after pig-to-nonhuman primate kidney xenotransplantation gives rise to HLA cross-reactive antibodies. METHODS: Serum samples were obtained from 52 cynomolgus monkeys that received kidney transplants from human decay-accelerating factor (hDAF) transgenic pigs. Samples were collected pre-transplant and at time of autopsy (mean 20 days post-transplantation, range 1 to 53 days) and analyzed for IgG HLA class I and HLA class II specific antibodies by enzyme-linked immunosorbent assay (ELISA) against pooled purified HLA antigens. To ensure the ability of the HLA ELISA to detect cynomolgus monkey IgG binding, parallel experiments were performed to detect IgG Gal-alpha-1,3-Gal-specific antibodies known to be present in cynomolgus monkey serum. RESULTS: Analysis of both pre- and post-transplantation serum samples by ELISA demonstrated no detectable IgG antibody binding to HLA class I or class II antigens. Using the same ELISA antibody detection reagents, IgG Gal-alpha-1,3-Gal-specific antibodies were identified in 13 of 38 (34%) sera obtained before transplantation and 21 of 52 (40%) sera collected post-transplantation, confirming that the negativeHLA ELISA results were not due to a technical aspect of the assay. CONCLUSION: This study suggests that exposure to SLA following transplantation of porcine kidneys in nonhuman primates does not give rise to antibodies that cross-react with HLA.     

Influence of CYP3A4 inhibition on the steady-state pharmacokinetics of imatinib.

PURPOSE: To evaluate the effects of ritonavir, a potent inhibitor of CYP3A4, on the steady-state pharmacokinetics of imatinib. EXPERIMENTAL DESIGN: Imatinib pharmacokinetics were evaluated in cancer patients receiving the drug for at least 2 months, after which ritonavir (600 mg) was administered daily for 3 days. Samples were obtained on the day before ritonavir (day 1) and on the third day (day 4). The in vitro metabolism of imatinib with or without ritonavir and the effect of imatinib on 1-OH-midazolam formation rate, a probe for CYP3A4 activity, were evaluated with human CYP3A4 and pooled liver microsomes. RESULTS: In 11 evaluable patients, the geometric mean (95% confidence interval) area under the curve of imatinib on days 1 and 4 were 42.6 (33.0-54.9) microg.h/mL and 41.2 (32.1-53.1) microg.h/mL, respectively (P = 0.65). A population analysis done in NONMEM with a time-dependent covariate confirmed that ritonavir did not influence the clearance or bioavailability of imatinib. In vitro, imatinib was metabolized to the active metabolite CGP74588 by CYP3A4 and CYP3A5 and, to a lesser extent, by CYP2D6. Ritonavir (1 micromol/L) completely inhibited CYP3A4-mediated metabolism of imatinib to CGP74588 but inhibited metabolism in microsomes by only 50%. Imatinib significantly inhibited CYP3A4 activity in vitro. CONCLUSION: At steady state, imatinib is insensitive to potent CYP3A4 inhibition and relies on alternate elimination pathways. For agents with complex elimination pathways that involve autoinhibition, interaction studies that are done after a single dose may not be applicable when drugs are administered chronically.     

Parent and health professional perspectives in the management of adolescents with diabetes: Development of assessment instruments for international studies

Objective: Assessment of quality of life (QOL) in adolescents with diabetes requires patient, parent and health professional input. Psychometrically robust instruments to assess parent and professional perspectives are required. Research design and methods: Questionnaires concerning adolescent QOL were developed for completion by parents and health professionals. In an international study assessing QOL in 2,101 adolescents with diabetes (median age 14 years, range 10–18; from 17 countries including Europe, Japan and North America), parents and health professionals completed their respective questionnaires between March and August 1998. Results: Feasibility and acceptability of the new questionnaires were indicated by high questionnaire completion rates (adolescents 92%; parents 89%; health professionals 94%). Internal consistency was confirmed (Cronbach’s α coefficients 0.80 parent; 0.86 health professional). Correlations of Diabetes Quality of Life Questionnaire for Youths (DQOLY) scores with parent and health professional global QOL ratings were generally low (r ranging from 0.12 to 0.36). Parent-rated burden decreased incrementally across adolescence, particularly for girls. Professional-rated burden followed a similar profile but only after age 15 years. Until then, burden was rated as uniformly high. Clinically relevant discrepancies in parent and professional burden scores were noted for one-parent families and families where adolescents had been referred for psychological help. In both cases, health professionals but not one-parent families perceived these as high burden situations. The clinical significance of this relates to the significantly poorer metabolic control recorded for adolescents in both situations. Conclusions: Parent and health professional questionnaires were found to have adequate internal consistency, and convergent and discriminant validity in relation to key clinical and QOL outcomes. The questionnaires are brief, easy to administer and score. They may also enable comparisons across countries and languages to facilitate development of international health outcome parameters. The inclusion of the parent and health professional perspectives completes a comprehensive assessment of adolescent QOL relevant to diabetes.     

Elicited antibodies in baboons exposed to tissues from alpha1,3-galactosyltransferase gene-knockout pigs

BACKGROUND: This study investigates anti-nonGal antibodies (Abs) in baboons after alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pig heart transplantation (Tx). METHODS: Four baboons underwent pig heart Tx under chronic immunosuppression, which was discontinued after graftectomy. During follow-up, one baboon also received a pig splenocyte infusion. Hearts and splenocytes were from GalT-KO pigs (n = 3) or pigs with low Gal expression (Gal-low, n = 2), all of swine leukocyte antigen (SLA) dd haplotype. Several weeks after graftectomy, sera were tested by flow cytometry and cytotoxicity assay on porcine peripheral blood mononuclear cells (PBMC) for elicited anti-nonGal Abs. Sera were adsorbed on a Gal immunoaffinity matrix, and tested for SLA haplotype specificity using PBMC from SLA aa, cc, and dd haplotypes. RESULTS: Before heart Tx, no baboon had anti-nonGal Abs demonstrable by binding or cytotoxicity to GalT-KO PBMC. All four baboons developed anti-nonGal Abs after Tx, demonstrable by flow cytometry, and three sera from baboons showed cytotoxicity to GalT-KO PBMC of SLA(dd) haplotype. After adsorption of anti-Gal Abs, the elicited anti-nonGal Abs showed similar binding to PBMCs from pigs of all three haplotypes (SLA(dd), SLA(aa), SLA(cc)). CONCLUSIONS: Anti-nonGal Abs developed after GalT-KO pig heart Tx into baboons. The most potent of these antibodies appeared to detect antigens shared by the three pig haplotypes tested. It remains unclear whether these antibodies are directed towards shared SLA determinants or other pig antigens, and whether antibodies with specificity for allelic SLA determinants are also present, but at lower titer.     

Arrestin-related proteins mediate pH signaling in fungi

Metazoan arrestins bind to seven-transmembrane (7TM) receptors to regulate function. Aspergillus nidulans PalF, a protein involved in the fungal ambient pH signaling pathway, contains arrestin N-terminal and C-terminal domains and binds strongly to two different regions within the C-terminal cytoplasmic tail of the 7TM, putative pH sensor PalH. Upon exposure to alkaline ambient pH, PalF is phosphorylated and, like mammalian beta-arrestins, ubiquitinated in a signal-dependent and 7TM protein-dependent manner. Substitution in PalF of a highly conserved arrestin N-terminal domain Ser residue prevents PalF-PalH interaction and pH signaling in vivo. Thus, PalF is the first experimentally documented fungal arrestin-related protein, dispelling the notion that arrestins are restricted to animal proteomes. Epistasis analyses demonstrate that PalF posttranslational modification is partially dependent on the 4TM protein PalI but independent of the remaining pH signal transduction pathway proteins PalA, PalB, and PalC, yielding experimental evidence bearing on the order of participation of the six components of the pH signal transduction pathway. Our data strongly implicate PalH as an ambient pH sensor, possibly with the cooperation of PalI.     

Pathology of xenograft rejection: a commentary

Abstract: Trends in solid organ xenograft pathology are presented, with the focus on pig-to-nonhuman primate models. A simplified classification of rejection is followed, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and acute cellular xenograft rejection (ACXR). The main components in HAR are natural xenoreactive antibodies in combination with complement activation. This is evident from the prevention of HAR in recipients in whom either antibodies or complement activation is depleted or inhibited. However, these strategies generally fail to prevent AHXR, which occurs later. AHXR is a multifactorial process in which natural and elicited antibodies may play roles, possibly in conjunction with complement, coagulation factors, and white blood cells. A main target appears to be the microvasculature which, in kidney grafts, is associated with a glomerular thrombotic microangiopathy. It is not clear to what extent species-specific physiologic disparities in complement and coagulation processes may play a role, separate from antibody-initiated processes. As rejection of solid organ xenografts is currently from AHXR, ACXR has not yet received close attention. In addition to intragraft rejection events, systemic complications following host–graft interactions have emerged, including (often fatal) consumptive coagulopathy and immune complex disease. It is anticipated that rejection processes will change when pigs with new genetic modifications become available. For instance, the precise role of natural antibodies to Galα1,3Gal will be able to be distinguished from other factors when pigs that lack the target antigen are available, and their organs can be evaluated in large animal xenotransplantation models.     

Anti-pig antibody levels in naïve baboons and cynomolgus monkeys

Anti‐pig antibodies (APA) were analysed in serum from 28 naïve wild‐caught baboons (originating from Kenya) and 31 naïve captive‐bred cynomolgus monkeys (13 from the Philippines and 18 from Mauritius), using a haemolytic assay with pig erythrocytes (APA), flow cytometry on the porcine lymphoma T‐cell cell line L35, and enzyme linked immunosorbent assay (ELISA) using α‐Gal type II and type VI antigen. This was extended in baboon samples by the evaluation in two laboratories (Imutran, Cambridge, UK and Immerge, Boston, USA), and by antibody absorption using either immobilized α‐Gal type II or α‐Gal type VI. Anti‐porcine antibodies were demonstrated in all assays with substantial variability within and between the three non‐human primate groups. Immunoglobulin (Ig)M antibody levels tended to be similar to or higher than those in a pooled normal human standard serum while IgG levels tended to be lower. Highest antibody levels were recorded in Mauritius cynomolgus monkeys. There were statistically significant correlations between assays for IgM or IgG class anti‐Gal antibodies using either α‐Gal type II or α‐Gal type VI as antigen, both for different assays and two laboratories involved. Also, significant correlations were observed between the anti‐Gal and L35 binding assays. Baboon sera before and after absorption to immobilized α‐Gal type II or type VI were analysed for anti‐Gal type VI or type II antibody: levels were almost undetectable indicating that most anti‐Gal antibodies react to epitopes shared between α‐Gal type II and type VI oligosaccharides. Finally, the relation between APA and outcome of porcine heart xenotransplantation in cynomolgus monkeys and baboons showed no apparent relation between pre‐transplant APA levels and the occurrence of hyperacute rejection (HAR) when compared with non‐immunological cause of organ/recipient dysfunction or acute humoral xenograft rejection during the first 4 days post‐transplantation or survival exceeding 4 days post‐transplantation.     

Cost-Benefit Analysis of Vaccination Against Influenza of Employees from an Academic Medical Centre

Objective: To determine whether vaccination against influenza in the Academic Medical Centre (AMC) of the University of Amsterdam, The Netherlands, may lead to an economic benefit by avoiding the loss in productivity associated with an outbreak of influenza illness among its employees. Design: A newly developed cost-benefit model from an employer’s perspective was applied and several scenario and sensitivity analyses were performed. Methods: The model inputs were vaccination-, personnel- and influenza-related elements, which were obtained from data specific to the AMC where possible, otherwise these were based on published literature. The output (net benefit) was defined as the difference between the benefits of vaccination due to reducing workplace absenteeism (productivity) of employees and the costs of vaccination, excluding campaign and administration costs. The net benefits of baseline, vaccination promotion and influenza-persistent scenarios were assessed and sensitivity analysis was performed. Results: The net benefits for all the scenarios was positive, being 120 000 Euros (EUR), EUR460 000 and EUR180 000 (2000 values) for the baseline, vaccination promotion and influenza-persistent scenarios, respectively. The vaccination compliance rate appeared to be the element with greatest impact on the net benefits. Conclusion: An influenza vaccination program in an institution with the characteristics of the AMC can be performed with an economic benefit for the organization. Our results suggest that vaccination of medical residents provides greater economic benefits than vaccination of other personnel.     

Phase I Study of Dalteparin in Combination With Sunitinib in Patients With Metastatic Clear Cell Renal Carcinoma

Background

Based on the tumor-driven concomitant activation of angiogenesis and coagulation we conducted a phase I combination study of sunitinib with the low molecular weight heparin dalteparin in patients with metastatic clear cell renal cell carcinoma (ccRCC).