1. The aim of this study was to test whether CRF enhanced nuclear factor kappa B (NF-kappaB)-directed gene transcription in leukocytes and the receptor specificity of the effect. Initially, we examined the ability of CRF to modulate an antigen-specific, in vitro antibody response. Since that could be mediated by NF-kappaB transcription factor activity, we tested CRF in a NF-kappaB driven luciferase gene expression reporter assay. 2. CRF enhanced the antigen-specific antibody production in a dose- and time-dependent manner. RAW 264.7 macrophage cells and splenocytes stained by immunohistochemistry were positive for CRF receptors and CRF. Expression of both was up-regulated by mitogen treatment of the splenocytes. CRF also enhanced the NF-kappaB-regulated reporter assay and this could be blocked by a CRF-R1 receptor antagonist. 3. In light of these findings, it seems likely that CRF enhanced the antigen-specific antibody response through the CRF-R1 receptor by elevation of NF-kappaB activity. This study provides further support for the concept that CRF can act as an immunomodulator mediating neuro-immune interactions.
Univ. 734 S s . ,'iepe. P z i : : : i a s , Baltimore. We have identified 14 mutations of the factor VIII (FB) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects by restriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22 and exon 24, and a single CG to TG transition was identified in exon 23. In addition. a Taq I site alteration in intron 4 of the F8 gene was identified in a patient with mild hemophilia. Cloning and sequencing of this region suggests that a change in this Taq I site may create a new splice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. We estimate that the mutation rate of CG to TG in the FB gene is at least 100 times greater than the average mutation rate per nucleotide. Six different deletions were also characterized. In one family. the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.Of the 14 mtations. 6 occurred de novo within 2 generations; 4 in males and 2 in females. In these de novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man. In the presence of 20.001 IU/ml of ASNase, the amount of 14C ASN recovered was <20% of that without ASNase, within 5 minutes. Ueilizing this assay we have studied the effect of several known inhibitors of ASNase in an attempt to improve ASN recovery. In the presence of ASA, ASN levels in the presence of 0.001 to 1.0 IUIml of ASNase remain at '90% of control, Blood samples drawn from patients at 1 and 7 days following ASNase injection were collected directly into tubes containing 1 4~ ASN +/-ASA. Recovery with ASA, was 60-110% and 100-110% of control (i.e., no ASNase) on days 1 and 7 respectively. We conclude that: 1) continued hydrolysis of ASN by ASNase can result in falsely low serum ASN measurements; 2) ASA is a potent inhibitor of E. Coli ASNase; 3) ASA present in collection tubes obviates the problem of continued ASNase activity. Thus, for accurate measurements of the rate and degree of ASN depletion by ASNase. ASA should be present in the blood collection system. To determine the manner i n which erythrocyte changes occur during ontogeny, several red cell parameters were analyzed i n 19 fetuses, 14 newborn infants, 19 children am3 45 adults. Although M C V and Hb F levels decreased a s expected during i n utero development, the coefficient af variation of red cell size (%CV = SD/mean), or red c e l l distribution width (RDW), increased from fetuses t o newborn infants, and then decreased i n children to a d u l t l e v e l s . I n normal c h i l d r e n and a ...
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