Factors affecting success of thymus transplantation for complete DiGeorge anomaly.

Thymus transplantation shows promise for the treatment of athymia in complete DiGeorge anomaly. This report reviews the effects of dose of thymus tissue, ABO compatibility, HLA matching, culture conditions, age of donor and immunosuppression of recipient on immune outcomes at 1 year after transplantation. Forty-nine athymic subjects have been treated with cultured postnatal allogeneic thymus tissue; 36 (73%) survive with only one subject on immunosuppression at 1.5 years. Of 31 surviving subjects more than 1 year after transplantation, 30 (97%) developed naive T cells, T-cell proliferative responses to mitogens and a diverse T-cell receptor beta variable (TCRBV) repertoire. The dose of thymus tissue, HLA matching and use of immunosuppression had nonsignificant effects on these outcome variables. Removal of deoxyguanosine from culture medium and length of culture did not adversely affect outcomes. Use of thymus tissue from donors over 1 month of age, versus under 1 month, resulted in higher total T-cell numbers (p = 0.03). However, this finding must be confirmed in a prospective trial. Although subtle immune effects may yet be associated with some of the factors tested, it is remarkable that consistently good immune outcomes result despite variation in dose, HLA matching and use of immunosuppression.

Comparison of PCR, culture, and histopathology for diagnosis of tuberculous pericarditis.

Nucleic acid amplification techniques for the diagnosis of tuberculosis (TB) are rapidly being developed. Scant work, however, has focused on pericardial TB. Using cryopreserved specimens from a prior study of pericarditis, we compared PCR to culture and histopathology for the diagnosis of tuberculous pericarditis in 36 specimens of pericardial fluid and 19 specimens of pericardial tissue from 20 patients. Fluid and tissue were cultured on Lowenstein-Jensen and Middlebrook solid media and in BACTEC radiometric broth. Tissue specimens were stained with hematoxylin-eosin, Ziehl-Neelsen, auramine O, and Kinyoun stains and were examined for granuloma formation and acid-fast bacilli. PCR was performed with both fluid and tissue with IS6110-based primers specific for the Mycobacterium tuberculosis complex by published methods. Sixteen of the 20 patients had tuberculous pericarditis and 4 patients had other diagnoses. TB was correctly diagnosed by culture in 15 (93%) patients, by PCR in 13 (81%) patients, and by histology in 13 of 15 (87%) patients. PCR gave one false-positive result for a patient with Staphylococcus aureus pericarditis. Considering the individual specimens as the unit of analysis, M. tuberculosis was identified by culture in 30 of 43 specimens (70%) from patients with tuberculous pericarditis and by PCR in 14 of 28 specimens (50%) from patients with tuberculous pericarditis (P > 0.15). The sensitivity of PCR was higher with tissue specimens (12 of 15; 80%) than with fluid specimens (2 of 13; 15%; P = 0.002). In conclusion, the overall accuracy of PCR approached the results of conventional methods, although PCR was much faster. Therefore, PCR merits further development in this regard. The sensitivity of PCR with pericardial fluid was poor, and false-positive results with PCR remain a concern.

Inclusion of a sensitivity control to add a quality-control parameter and improve reproducibility in BCR, immunoglobulin, and T-cell receptor gene-rearrangement studies.

Molecular analyses to determine clonality of T and B cells in malignant lymphoma and leukemia and to detect the (9;22) translocation in chronic myelogenous leukemia are commonly used in clinical molecular biology laboratories. We describe the inclusion of a sensitivity control in each of these assays derived from DNA of well-characterized cell lines. The inclusion of such a sample adds an important quality-control parameter to ensure assay-to-assay reproducibility and to satisfy accreditation and regulatory requirements.

Functional heterogeneity of mammalian TATA-box sequences revealed by interaction with a cell-specific enhancer.

A regulatory element upstream of the human myoglobin gene functions as a muscle-specific enhancer (MSE) in conjunction with core promoter elements of the myoglobin gene, but not in combination with the simian virus 40 (SV40) early promoter. These two promoters differ in the sequences of their 'TATA boxes': for the myoglobin gene, the sequence is TATAAAA, whereas for SV40, the sequence is TATTTAT. We have now tested the hypothesis that this sequence difference is responsible for the differential response of the promoters to the MSE. We found that when the TATA box sequence of the myoglobin promoter was changed to that of the SV40 promoter, responsiveness to the MSE was abolished; conversely, when the SV40 TATA box sequence was changed to that of the myoglobin promoter, the promoter became responsive to the MSE. We conclude that mammalian TATA-box elements are functionally heterogeneous, and suggest that this heterogeneity reflects differential interactions with distinctive TATA box-binding factors, only some of which can act cooperatively with MSE-binding proteins to generate an active transcriptional complex.

Identification of a muscle-specific enhancer within the 5'-flanking region of the human myoglobin gene.

A 2-kilobase fragment from the 5'-flanking region of the human myoglobin gene extending from -2038 to +7 relative to the cap site regulates expression of a heterologous reporter gene in a cell-specific and developmentally regulated manner. Functional analyses of 5' and internal deletions indicate that sequences located between -261 and -205 are essential for muscle-specific expression in cooperation with the myoglobin core promoter. A 167-base pair fragment containing these sequences (-371 to -205) enhances expression from myoglobin core promoter elements in a manner that is independent of its orientation and position relative to the cap site. When linked to the herpes simplex virus thymidine kinase promoter, this 167-base pair fragment of the myoglobin gene also enhances expression in differentiated myotubes but not in undifferentiated myoblasts or fibroblasts. Nucleotide sequences within the region that is essential for enhancing activity (-261 to -205) are conserved in other mammalian myoglobin genes (seal, mouse) and resemble sequences within control regions of other genes that are expressed selectively in striated myocytes. These data indicate that the 5'-flanking region of the human myoglobin gene contains an enhancer-like element that is important for transcriptional activation during myocyte differentiation.

Reentry into the cell cycle of differentiated skeletal myocytes.

The activation of muscle-specific myosin synthesis and its relationship to withdrawal from the cell cycle have been examined in cycle-synchronized myoblasts under growth-restrictive, fusion-impermissive (low Ca2+) culture conditions. Under these conditions, embryonic quail skeletal myoblasts, collected in mitosis by mechanical shake-off, complete one normal cycle and arrest in G1. The presence of skeletal muscle myosin is first detected, by indirect immunofluorescence, 8 hr into this protracted G1. Within the next 10-11 hr the percentage myosin positive (Myo+) cells increases with good synchrony, reaching approximately 95%. Refeeding with a proliferation--stimulating, low Ca2+ medium when approximately 50% of the cells are Myo+ induces reentry into S. Applying a 15-min pulse with [3H]TdR immediately preceding fixation at regular intervals following refeeding, cells can be detected which are Myo+ and whose nuclei have incorporated [3H]TdR. The numbers of such doubly labeled cells are small but consistent with the fraction of cells in S (by time-lapse analysis) at the postfeeding times sampled. These cinematographic studies also indicate that progression to mitosis following stimulation occurs slowly and asynchronously. The kinetics of progression of the stimulated cells suggest that they reenter S from a different compartment in G1 than do log-phase myoblasts. We conclude that in fusion-blocked quail myocytes irreversible withdrawal from the cell cycle is neither an obligate precondition for, nor an immediate consequence of the activation of the muscle-specific contractile gene set.