Large-volume leukapheresis in pediatric patients: processing more blood diminishes the apparent magnitude of intra-apheresis recruitment

Background: Recruitment of progenitors during a large-volume collection, as defined by increasing relative and absolute numbers of progenitors (colony-forming units-granulocyte-macrophage [CFU-GM] of CD34+ cells), has been reported previously. Study Design and Methods: To ascertain whether intra-apheresis recruitment occurs in pediatric patients who have undergone mobilization with chemotherapy and granulocyte-colony-stimulating factor (G-CSF), each hour's portion of a 4-hour leukapheresis was collected into separate bags, and assessed by complete blood count, CFU-GM, and CD34+ cell assays. Seven pediatric patients (median age, 7; range, 2–19) were studied in connection with 2 to 4 collections each, for a total of 21 collections (with hourly samples). The collections lasted for 4 hours, at an inlet rate of 1 to 3 mL per kg per minute, for daily processing totals of 5 to 12 blood volumes. (One blood volume [mL] is estimated by the patient's weight in kg × 70 mL/kg.) Smaller (younger) patients had inlet rates exceeding 2 mL per kg per minute, and larger (older) patients had rates of 1 to 1.5 mL per kg per minute. CFU-GM and CD34+ cell counts obtained each hour of the collection and divided by the first hour's value were compared by nonparametric repeated-measures ANOVA. Results: Second-, third- and fourth-hour CD34+ progenitor cell counts were arithmetically higher than first-hour counts, but the trend did not reach significance (p = 0.1561). Second-hour counts were higher than first-hour counts in the overall analysis (mean ± standard error [SE], 1.00 and 1.39 ± 0.1, respectively; p = 0.0525) and in children older than 5 years (1.00 vs. 1.70 ± 0.30, respectively; p = 0.0259), but not in children younger than 5 years (p = 0.8125). CFU-GM counts did not differ among the 4 hours of collection (p = 0.1717) or between the first and second hour (p = 0.9587). Conclusion: In larger (older) patients, from whom fewer blood volumes were collected, there is a trend toward intra-apheresis recruitment, although less than reported previously. In the smaller (younger) patients, from whom more blood volumes were collected, no trend was observed. Lack of (or submaximal) prior mobilization in previously reported studies may have facilitated intracollection recruitment. Alternatively, the larger number of blood volumes collected from the smaller (younger) patients may have masked intra-apheresis recruitment. The study documents the feasibility of large-volume, 4-hour leukapheresis in pediatric patients.     

TMD before and after correction of dentofacial deformities by orthodontic and orthognathic treatment

The aims of the study were to investigate the alteration of temporomandibular disorders (TMD) after correction of dentofacial deformities by orthodontic treatment in conjunction with orthognathic surgery; and to compare the frequency of TMD in patients with dentofacial deformities with an age and gender matched control group. TMD were evaluated in 121 consecutive patients (treatment group), referred for orthognathic surgery, by a questionnaire and a clinical examination. 18 months after treatment, 81% of the patients completed a follow-up examination. The control group comprised 56 age and gender matched subjects, of whom 68% presented for follow-up examination. TMD were diagnosed according to research diagnostic criteria for TMD. At baseline examination, the treatment group had a higher frequency of myofascial pain (P = .035) and arthralgia (P = .040) than the control group. At follow-up, the frequencies of myofascial pain, arthralgia and disc displacement had decreased in the treatment group (P = .050, P = .004, P = .041, respectively). The frequency of TMD was comparable in the two groups at follow-up. Patients with dentofacial deformities, corrected by orthodontic treatment in conjunction with orthognathic surgery, seem to have a positive treatment outcome in respect of TMD pain.     

Colony-forming cell proliferation: a rapid and sensitive assay system for murine granulocyte and macrophage colony-stimulating factors

A microculture assay for murine granulocyte-macrophage colony- stimulating factor (GM-CSF) has been developed using fetal liver GM colony-forming cells (CFC) isolated by fluorescence-activated cell sorting. These GM-CFC are free of mature hemopoietic cells, such as granulocytes and macrophages, which may interfere with direct assays for GM-CSF. The assay procedure allows the quantitation of GM-CSF within 48 hr by measuring the number of cells produced from 50 GM-CFC in microcultures (15 microliter). The assay is particularly simple to set up and score and yet, because of the reduced volumes, this assay is still capable of detecting 0.2 pg (i.e., 0.2 U) of GM-CSF within 48 hr, i.e., 100 times less GM-CSF than the conventional soft agar assay. By allowing the microcultures to develop for 7 days, the extra proliferation allows a further tenfold increase in the sensitivity of CSF detection. The time and cost of setting up hundreds of GM-CSF assays for fractions from chromatographic columns, e.g., reverse phase high performance liquid chromatography, is reduced by at least five- fold. Enough GM-CFC can be isolated and stored frozen in one afternoon to provide sufficient cells for the daily assay of 200 samples of GM- CSF for several months. Microassay results for several sources of GM- CSF at different stages of purification are compared to the results obtained from the soft agar assay.     

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

Characterization of non-human primate antisera to acute lymphoblastic leukemia (ALL): evidence for unique antigen(s) on childhood ALL of "T" phenotype

A non-human primate antiserum was prepared to acute lymphoblastic leukemia of T-cell phenotype (T-ALL) and, after absorptions with normal blood elements, reacted by immunofluorescence and microcytotoxicity to all the T-ALL tested. In addition, the antiserum reacted with cells from about 70% of the common ALL studied and immunoprecipitated the common ALL antigen of 100,000 daltons. However, when the anti-T-ALL serum was absorbed with with lymphoblasts from common ALL, it failed to react with common ALL lymphoblasts, yet reacted significantly with cells from patients with T-ALL phenotype and defined a 100,000-dalton membrane component not found on common ALL lymphoblasts. In addition, sequential immunoprecipitation of 125I-labeled T-ALL membranes by anti- common-ALL serum followed by anti-T-ALL serum detected the T-ALL membrane component of 100,000 daltons that was not found on common ALL. Thus, our results demonstrate the presence of of a unique human T-ALL antigen present on all T-ALL distinct from the common ALL antigen.     

A 3-base pair deletion,c.9711_9713del,in DMD results in intellectual disability without muscular dystrophy

We have identified a deletion of 3 base pairs in the dystrophin gene (DMD), c.9711_9713del, in a family with nonspecific X-linked intellectual disability (ID) by sequencing of the exons of 86 known X-linked ID genes. This in-frame deletion results in the deletion of a single-amino-acid residue, Leu3238, in the brain-specific isoform Dp71 of dystrophin. Linkage analysis supported causality as the mutation was present in the 7.6 cM linkage interval on Xp22.11–Xp21.1 with a maximum positive LOD score of 2.41 (MRX85 locus). Molecular modeling predicts that the p.(Leu3238del) deletion results in the destabilization of the C-terminal domain of dystrophin and hence reduces the ability to interact with β-dystroglycan. Correspondingly, Dp71 protein levels in lymphoblastoid cells from the index patient are 6.7-fold lower than those in control cell lines (P=0.08). Subsequent determination of the creatine kinase levels in blood of the index patient showed a mild but significant elevation in serum creatine kinase, which is in line with impaired dystrophin function. In conclusion, we have identified the first DMD mutation in Dp71 that results in ID without muscular dystrophy.     

Cytogenetic studies in non-African Burkitt lymphoma

A particular translocation between chromosomes 8 and 14 has been found repeatedly in cytogenetic studies of Burkitt lymphoma, both of African and non-African origin. We report here our findings in cytogenetic studies of direct tumor preparations from 18 non-African Burkitt lymphoma patients, 9 of whom also had cell lines available for study. A t(8;14) was found in direct tumor material in 10 of the 18 patients. Seven of the 9 cell lines had a t(8;14). A total of 15 patients had either a t(8;14) or a 14q+ present in tumor material and/or cell lines. In addition, 8 patients had a peculiar marker chromosome 1. The t(8;14) was not found in every malignant cell and, where present, it was rarely the sole karyotypic abnormality. The relationship of the t(8;14) to the evolution of the tumor is discussed.     

High-dose therapy and peripheral blood progenitor cell transplantation: effects of recombinant human granulocyte-macrophage colony-stimulating factor on the autograft

Between June 1989 and June 1992, 144 patients participated in sequential clinical trials using peripheral blood progenitor cells (PBC) as their sole source of hematopoietic rescue following high-dose chemotherapy. All patients had received prior extensive combination chemotherapy and had marrow defects that precluded autologous bone marrow transplantation (ABMT). PBC were collected according to a single apheresis protocol. The initial 86 patients (group 1) had PBC collected without mobilization. Beginning in April 1991, PBC were mobilized solely with recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF). Thirty-four patients (group 2) received rHuGM-CSF at a dose of 125 micrograms/m2/d by continuous intravenous infusion, and 24 patients (group 3) received rHuGM-CSF at a dose of 250 micrograms/m2/d by continuous intravenous infusion. Patients underwent at least six aphereses and had a minimum of 6.5 x 10(8) mononuclear cells (MNC)/kg collected. Cytokines were not routinely administered immediately after transplantation. A median of nine aphereses were required to collect PBC in group 1 and seven aphereses for groups 2 and 3 (P = .03). The time required to recover 0.5 x 10(9)/L granulocytes after transplant was significantly shorter (P = .0004) for the mobilized groups; the median time to recovery was 26 days for group 1, 23 days for group 2, and 18 days for group 3. Transplantation of PBC mobilized with rHuGM-CSF resulted in a shorter time to platelet (P = .04) and red blood cell (P = .01) transfusion independence. Mobilization with rHuGM-CSF alone resulted in efficient collection of PBC, that provided rapid and sustained restoration of hematopoietic function following high-dose chemotherapy. Mobilization of PBC with rHuGM-CSF alone is an effective method for patients who have received prior chemotherapy and have bone marrow abnormalities.     

Correlation between serum lipoprotein (a) and angiographic coronary artery disease in non-insulin-dependent diabetes mellitus

Abstract. Comlekqi A, Biberoglu S, Kozan 0, Bahqeci 0, Ergene 0, Nazli C, Kinay 0, Guner G (Dokuz Eylul University, Medical School, Inciralti, Izmir, Turkey). Correlation between serum lipoprotein(a) and angio-graphic coronary artery disease in non-insulin-dependent diabetes mellitus. J Intern Med 1997; 242:449-54.
Objectives: To examine the impact of diabetic state on the concentrations of lipoprotein(a) [Lp(a)] in patients with non-insulin-dependent diabetes mellitus (NIDDM) and the correlation between angiographic coronary artery disease (CAD) and serum Lp(a) concentrations in NIDDM.
Design: In this cross-sectional study of 26 patients with NIDDM and 19 nondiabetic sex- and agematched patients who underwent coronary angiography, CAD was assessed visually using coronary artery score (CAS), and plasma Lp(a) was measured by an enzyme-linked immunosorbent assay.
Setting: The study was performed in an internal medicine clinic at a university hospital.
Subjects: Twenty-six age- and sex-matched patients with NIDDM and 19 control patients without diabetes.
Results: There was no significant difference between the Lp(a) concentrations of patientswith NIDDM and nondiabetic subjects (P > 0.05). When patients with NIDDM were stratified by absence or presence of CAD, patients with CAD had higher levels of Lp(a) (P < 0.05). However, there was no significant correlation between the concentrations of Lp(a) and CAS (P > 0.05).
Conclusions: Diabetic state does not have any impact on Lp(a) concentrations. Lp(a) excess seems to be atherogenic in patients with NIDDM as shown in nondiabetic patients in previous studies. Although diabetic patients with CAD have higher Lp(a) concentrations than the diabetic patients without CAD, Lp(a) levels were not correlated with CAS.