Immunophenotyping and cellular immune responses of cadaveric donor bone marrow cells.
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Biomedical subjects
Publications and source records attributed to A Alamo.
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A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.
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Between February and April 1990 the first five cases of human paragonimiasis, tentatively due to Paragonimus africanus, have been detected in Equatorial Guinea, thanks to the normal activities of the National Schistosomiasis Project and its coordination with the National Tuberculosis Project.
Peripheral blood lymphocytes were obtained from 65 individuals: 34 nondiabetic patients with islet cell autoantibodies (ICA) (prediabetic phase), 9 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM), 6 ICA-negative siblings or offsprings of IDDM patients, and 16 ICA-negative controls. The presence of lymphocyte abnormalities and/or activation was examined with dual fluorescence flow cytometry. The percentages of B cells, total T-lymphocyte, and helper T-lymphocyte (Th) and cytotoxic/suppressor T-lymphocyte (Tc/s) subsets and their ratio were not significantly different among the patient groups. No increased expression of interleukin 2 receptor on T-lymphocyte was found in newly diagnosed IDDM or prediabetic individuals. Sixteen of 49 patients had significantly increased number of T-lymphocyte expressing HLA-DR. A significant increase in the number of both Th and Tc/s subsets expressing HLA-DR was found in only 3 of 16 patients. This increase was unrelated to the patients's relative ICA titer or HLA-DR phenotype. On the other hand, the relative density of the DR antigen (RAD-DR) was significantly increased on both Th (886 +/- 120) and Tc/s (1250 +/- 273) in 13 of 38 patients compared with control patients (Th 484 +/- 129 and Tc/s 460 +/- 166). The RAD-DR on Tc/s correlated with the relative ICA titer and was greatest on DR3/4-phenotyped T-lymphocytes. In addition, significantly increased RAD-DR was found in noncontrol patients with impaired insulin release responses at 1 and 3 min to intravenous glucose.(ABSTRACT TRUNCATED AT 250 WORDS)
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A newborn baby boy was diagnosed with the mixed form of congenital mesoblastic nephroma (CMN) representing both classic and cellular histology features in the renal tumor. Additionally, the patient had skin and bone lesions consistent with multifocal involvement of a generalized infantile fibromatosis (IFS). Both skin and bone lesions were distinctly different from CMN and did not represent metastasis. The primary tumor cell line (MCH-MN-1), established from the resected right kidney tumor, had a diploid DNA content. Cytogenetic studies revealed deletion on the long arm of chromosome 3 (q21q24) and duplication on the short arm of chromosome 11 (p15). MCH-MN-1 cells expressed ETV6-NTRK3 gene fusion transcripts, characteristic of cellular and mixed forms of CMNs. The cells had high p21 and low Bax mRNA expression in the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The high level of proliferative marker (Ki67) mRNA expression correlated well with the pluripotent nature of MCH-MN-1 in tissue culture (cell doubling time = 12.4 h). Our results showed that MCH-MN-1 might be a good model cell line for investigations on mesoblastic nephroma.
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The expression of genes associated with apoptosis, cell proliferation and drug resistance in tumor cells was investigated in two pediatric Wilms' tumor patients (MCH-WT-1 and MCH-WT-3) for their association with cell cycle, daunorubicin accumulation and clinical data. DNA content, cell cycle and drug accumulation were analyzed immediately after surgery by flow cytometry and mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Primary cell cultures were established from tumor specimens and tumor cells in both cases showed epithelial morphology. Although cell proliferation markers (Ki67 and PCNA) were expressed in both cases, MCH-WT-3 showed higher levels of mRNA expression, which corresponded, with metastatic behavior of the tumor in the patient. While p53 and p21 mRNAs were expressed at low levels in MCH-WT1, MCH-WT-3 showed high levels of p21 mRNA only. The increased expression of cyclin kinase inhibitor (p21) in MCH-WT-3 compared to MCH-WT-1 correlated with a higher percentage of G0/G1 cell population in the tumor specimen. Despite the expression of multidrug resistance markers (MDR1 and LRP) in MCH-WT-1, flow cytometric analysis showed tumor cell populations with very low and high daunorubicin accumulation and with the absence of any effect for verapamil and dipyridamole on daunorubicin accumulation of tumor cells.