Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children.

It still remains unclear whether patients with mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation (allo-BMT) have an increased risk of developing relapse or graft failure. To address this question, we monitored the individual dynamics of chimerism after allo-BMT in pediatric patients within a prospective case control study. The individual ratio of donor to recipient peripheral white cells was determined by quantification of genomic variable number of tandem repeats (VNTRs) with a polymerase chain reaction (PCR) approach. Within the study period from 1 January 1994 until 1 July 1996 we investigated 50 sequences of 46 pediatric patients after allo-BMT (32 with malignant, 18 with nonmalignant diseases). We found complete chimerism (CC) in 34/50 cases, MC in 12/50 follow-ups and 4/50 patients revealed autologous recovery (AC). Eight of 12 patients with MC showed increasing autologous patterns and subsequently relapsed or rejected their graft, 3/12 decreasing amounts of recipient DNA and turned to CC upon further follow-up. One patient of 12 who had severe combined immunodeficiency (SCID), attained engraftment with a stable MC pattern. Three patients of 34 with CC relapsed lacking a transitional MC interval. However, the time span between last CC confirmation and relapse in each of these three patients was 6 months or longer. We suggest that these patients also developed a stage of transitional MC but that the critical timepoint of molecular confirmation by PCR was missed as time intervals in the individual follow-up of these three patients were too long (> or = 6 months). In summary, the results demonstrate that the individual risk of developing relapse or graft failure is significantly enhanced in the MC situation (P < 0.0005). Therefore the quantitative analysis of MC at short time intervals might be of great value to identify high risk patients which will have a significantly/enhanced risk for relapse or graft rejection.

Quantitative assessment of mixed hematopoietic chimerism by polymerase chain reaction after allogeneic BMT.

Patients who develop mixed hematopoietic chimerism (MHC) after allogeneic bone marrow transplantation (allo-BMT) might have an increased risk of relapse or graft failure. In order to identify patients with MHC we established a quantitative polymerase chain reaction (PCR) approach to estimate the individual degree of autologous recovery and investigate the dynamics of MHC posttransplant. Therefore standardized mixed chimeric samples were generated in each individual case by mixing pretransplant recipient and donor DNA at a range of percentages. After amplification the samples were analyzed densitometrically and signal intensities were taken as the basis of individual standard curves. Subsequently posttransplant DNA samples were also analyzed, the intensity of the informative signals were compared to the standard curves and autologous recovery was expressed in percentage related to host DNA. We investigated 40 pediatric patients receiving allo-BMT by this method. 10/40 developed MHC during course of the observation period. Each patient with MHC revealed a certain dynamic. This approach might be helpful when deciding on early intervention and additional treatment to prevent graft failure or relapse.

[Antibody detection after antepartal rhesus prophylaxis: normal values or sensitization]. / Antikörper-Nachweis nach antepartaler Rhesus-Prophylaxe: Normalfall oder Sensibilisierung?

Antibody screening tests were performed in 29 unsensitized pregnant women after antepartum Rh immune prophylaxis, using the indirect Coombs test (ICT) and a more sensitive ID-microtyping-system (IDM). With the ICT, anti-D antibodies were detected in 85% for at least 4 weeks and at most 8 weeks after immunisation. The maximum titer was 1:8. With the IDM, 97% showed antibodies against 'D' for at least 4 weeks and at most 11 weeks with a maximum of 1:16. The IDM titer was always 1 to 3 steps more sensitive than the ICT. After postpartum Rh immune prophylaxis, anti-D titers were again positive in many of the patients (ICT: 42%; IDM: 60%). In conclusion, it is nearly always possible to measure antibodies against 'D' after antepartum Rh immune prophylaxis and IDM was superior in comparison to ICT. However, maternal isoimmunisation to the rhesus antigen cannot be excluded for sure and patients have then to be controlled. As isoimmunisation could not be confirmed in any of our patients, postpartum Rh immune prophylaxis has to be administered even after detection of an antibody titer against 'D' after antepartum Rh prophylaxis.

Pneumocystis carinii pneumonia following heart transplantation.

Pneumocystis carinii pneumonia represents a rare complication that is associated with a high mortality following heart transplantation. The cases of two heart transplant recipients who developed Pneumocystis pneumonia within the first 3 postoperative months are reported. Both patients had severe clinical symptoms of the disease; the diagnosis was confirmed by bronchoalveolar lavage, and the patients were treated with a combination of trimethoprim and sulfamethoxazole. Both patients recovered and are well at the time of this report.

Evaluation of elastase and alpha 1-proteinase inhibitor-elastase uptake by polymorphonuclear leukocytes and evidence of an elastase-specific receptor.

Neither resting nor stimulated isolated human polymorphonuclear leukocytes did bind or ingest preformed complexes of alpha 1-proteinase inhibitor and unlabeled/125I-labeled human leukocyte elastase. In contrast, granulocytes bound unlabeled/125I-labeled elastase and the extent of binding was reduced in the presence of respiratory burst stimulators, such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, E. coli endotoxin, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. In association/dissociation and competition inhibition experiments it was demonstrated that granulocyte-elastase binding was specific and saturable. From Scatchard and non-linear regression analysis there was evidence of a two-class receptor model with independent binding sites. Calculated by the non-linear regression method assuming a two-class receptor model the characteristics of the high affinity/low capacity binding site were K1 = 216 +/- 129 X 10(6) l X mol-1 (means +/- s; n = 3) and R1 = 1.38 +/- 0.95 nmol X l-1 corresponding to 0.083 X 10(6) receptors per cell, whereas the low affinity/high capacity binding site had the characteristics K2 = 0.50 +/- 0.09 X 10(6) l X mol-1 and R2 = 237 +/- 103 nmol X l-1 corresponding to 14.3 +/- 6.2 X 10(6) receptors per cell.

Insulin binding to erythrocytes before and after changing from porcine to biosynthetic human insulin in children with type-I diabetes.

The binding of [125I]insulin to isolated erythrocytes from diabetic children (n = 27) before (group a) as well as one and five months after changing from porcine to biosynthetic human insulin (groups b and c) was investigated. An analysis of variance of the binding parameters, determined by a nonlinear regression procedure, yielded statistically significant differences between the receptor affinities Ka as well as between the receptor concentrations Xo of the groups a and b, a and c and b and c. (formula: see text). The results suggest that the change from porcine insulin to biosynthetic human insulin induces a short-term as well as long-term increase in the affinity, and a decrease in the concentration of the erythrocyte insulin receptors.

Insulin binding to erythrocytes in children with type-I diabetes mellitus.

Specific binding of [125I]insulin to isolated erythrocytes was investigated in four groups of children (A) Healthy children under 10 years of age (n = 20) (B) Healthy children over 10 years of age (n = 13) (C) Diabetic children under 10 years of age (n = 12) (D) Diabetic children over 10 years of age (n = 63). In addition, all diabetic children (n = 75) were subdivided into four groups according to the duration of diabetes. (E) less than 2 years, (F) 2--4 years, (G) 4--6 years, and (H) greater than 6 years. By means of a nonlinear regression analysis for the extraction of binding parameters (assuming a single receptor class model with independent receptor sites) and methods of variance analysis, statistically significant differences were observed for the receptor affinities Ka (10(8) l/mol) and the receptor concentrations X0 (nmol/l) between groups A and C, B and D, C and D, but not between A and B. The affinities of groups C and D were found to be higher than the corresponding values of groups A and B, whereas the receptor concentrations exhibited an inverse behaviour. A significant increase of the receptor concentration and decrease of the receptor affinity depending on the duration of diabetes could only be proved to exist during the first 2 years of the disease.

[Effect of total hormone concentration on the determination of amount of binding in the hormone/receptor interaction between erythrocytes and insulin]. / Zum Einfluss der totalen Hormonkonzentration auf die Ermittlung von Bindungskenngrössen bei der Hormon/Receptor-Wechselwirkung zwischen Erythrocyten und Insulin.

The interaction of erythrocytes and [125I]insulin/insulin were studied up to a total insulin concentration of 409 mumol/l. Assuming a single class receptor model the evaluation of receptor affinity Ka and concentration R0 may be performed either by non-linear regression analyses with iteration procedures of R0, Ka and U (nonspecific binding), or by a linear regression analysis of the initial part of the Scatchard plot. Nonlinear fitting of data to a two class receptor model gives results that are reliable only for the high affinity receptor site. The non-definable step of R0 determination leads to uncertainties in results determined by the negative cooperativity model. From the results of this investigation and from considerations of signal modulation by receptor occupancy, some recommendations have been formulated for the evaluation of binding parameters; these should contribute to an improvement in the comparability of studies on the interactions of erythrocytes and insulin.

Insulin binding to erythrocytes after acute 16-methyleneprednisolone ingestion.

The binding of [125I]insulin to erythrocytes, glucose and insulin were determined before and 1, 7 and 35 days after ingestion of 2 X 60-methyleneprednisolone. None of two groups of volunteers (7 males, 4 females showed clear alterations of the insulin binding parameters (Ka and R0), or of the fasting cortisol, glucose and insulin concentrations. These results exclude the possibility that the diabetogenic effect of glucocorticoides is accompanied by an alteration of the insulin receptor characteristics of erythrocytes.

Insulin binding to erythrocytes from pregnant, postpartum, follicular and luteal states.

Specific binding of [125I] insulin to isolate erythrocytes from four groups of women was investigated: (A) pregnant subjects between weeks 38 and 40 of pregnancy (n = 18), (B) postpartum subjects within 6 days after delivery (n = 20), (C) normal women during the follicular phase of the menstrual cycle (n = 12) and (D) normal women during the luteal phase of the menstrual cycle (N = 11). Specific [125I] insulin binding (fraction), fasting plasma glucose concentrations (mmol/l) and the corresponding insulin concentrations (mU/l) were 0.074 +/- 0.012 / 4.00 +/- 0.58 / 29.4 +/- 21.4 for group A, 0.065 +/- 0.016 / 4.40 +/- 0.75 / 41.5 +/- 26.2 for group B, 0.052 +/- 0.008 / 4.58 +/- 0.62 / 6.7 +/- 4.0 for group C and 0.054 +/- 0.011 / 4.49 +/- 0.63 / 8.3 +/- 5.9 for group D. By using a modified Scatchard analysis, statistically significant differences were observed between the receptor affinities of the groups A and D, B and D, A and C. The receptor affinities and concentrations were not significantly different between the follicular and the luteal phases. From the data, no inverse correlation between the plasma insulin concentration and receptor binding was seen, i.e. the phenomenon of downregulation of insulin receptor concentration with hyperinsulinaemia seemed not to apply to erythrocytes.