Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells.Antibodies to the a and ft chains of VLA4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA4 binding domain did not block adhesion, suggesting that VLA4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-l (VCAM-1), the other known counterreceptor for VLA4, was identified on bone marrowderived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment. (J. Clin. Invest. 1991. 88:995-1004

No abstract

Relatively little is known about the relationship of lymphoid-associated gene expression to the proliferation and differentiation potential of early human bone marrow lymphoid progenitors. Surface expression of interleukin-7 (IL-7) receptor-α (IL-7Rα), a component of the high-affinity receptor for the lymphoid precursor growth factor IL-7, defined a CD34+ progenitor subset lacking the CD19+ pro-B phenotype but demonstrating markedly enhanced lymphoid clonogenic capacity and the ability to differentiate into pro-B cells in short-term culture. These progenitors expressed mRNA for the lymphoid-associated genes Igβ, RAG-1, and PAX-5, and were uniformly TdT-positive (TdT+). In contrast, IL-7Rα−/CD19−/CD34+ progenitors had a 50-fold reduced lymphoid clonogenic capacity and did not differentiate into pro-B cells in short-term culture. Expression of TdT and the lymphoid-associated genes Igβ and RAG-1, but not PAX-5, was detected in this fraction, although at lower levels than in the IL-7Rα+ progenitors. In contrast to IL-7Rα, loss of the stem cell factor receptor c-kit was associated with enhanced lymphoid clonogenic potential and increased B-lineage differentiation potential. These results indicate that IL-7Rα expression defines entry into a developmental stage characterized by upregulation of multiple lymphoid-associated genes and enhanced fitness for B-lymphoid differentiation. The onset of IL-7Rα and PAX-5 expression immediately before acquisition of CD19 is consistent with evidence suggesting upregulation of CD19 through pathways involving PAX-5 and IL-7.

46 XY gonadal dysgenesis patients often develop gonadal tumors, including gonadoblastoma and other types of germ cell tumors. We report a case of a 16-year-old female adolescent with primary amenorrhea and a right adnexal mass. Subsequent study revealed that she is a 46 XY phenotypic female adolescent with complete gonadal dysgenesis and with no alterations of the sex-determining region Y gene. Microscopic examination of the gonads revealed bilateral gonadoblastoma mixed with dysgerminoma and mature teratoma. The tumor in the right gonad was also mixed with yolk sac tumor and immature teratoma with rhabdomyoblastic components, mimicking adult rhabdomyoma and rhabdomyosarcoma. No metastasis in the regional lymph nodes was identified and the patient is disease free 15 months postsurgery. The complexity of the tumorigenesis in this case indicates that the gonadal cells in gonadal dysgenesis are extremely unstable and highly tumorigenic. This tumorigenesis is not necessarily associated with sex-determining region Y gene alterations; therefore, it reinforces the recommendation of gonadectomy for gonadal dysgenesis patients, regardless of the sex-determining region Y gene status.

ᰔHepatitis C virus (HCV), a positive single-stranded RNA virus, is one of the major causes of end-stage liver disease worldwide (5). The nucleotide sequence of the HCV genome is an important predictor of response to antiviral therapy (6). Genotypic analysis is used together with measurement of viral load (VL) to help in the management of patients with HCV infection (1). The 5Ј untranslated region (UTR) is often used for monitoring VL because it is less variable than other regions of the genome and consequently less likely to suffer PCR failures due to sequence variation at the primer binding sites (3,6,8). Although this region is not ideal for determination of the genotype, it is often used for this purpose because of the availability of the template after a VL assay and also because the genotypic information needed for clinical use can be obtained from it (2, 4, 9, 10).Our initial attempt to sequence the HCV 5Ј UTR using the reverse transcription-PCR (RT-PCR) amplicon from the recently introduced Roche Diagnostics (Basel, Switzerland) Cobas AmpliPrep/Cobas TaqMan HCV test (HCV-T) (8) employed a set of sequencing primers originally described by Gargiulo et al. (2) for analysis of the RT-PCR amplicon from the Roche Cobas Amplicor HCV monitor test (HCV-M). This attempt failed because the sequence obtained was often inconsistent with the genome of HCV (data not shown). We suspected that this failure was due to the presence of an abundant amplicon from the internal quantitation standard (QS). The QS is a small nucleic acid that is added to the specimen in the VL assay as an internal control. It is amplified using the same primers used to amplify the HCV target but contains a different internal sequence so that it can be detected using a TaqMan probe with a different fluorophore than the one used to detect the HCV amplicon. To troubleshoot this problem, we sequenced the amplicon from a clinical specimen that was negative for HCV, using the sequencing primers KY78 and KY80 (2), to obtain the sequence of the QS amplicon that is generated by the Roche HCV-T. As shown in Fig. 1, the primers designed by Gargiulo et al. (2) to sequence the amplicon from the HCV-M assay (labeled MF and MR in Fig. 1) are no longer specific for HCV, but hybridize equally well to the QS. Presumably the QS in the HCV-T assay contains additional nucleotides from the HCV genome compared with the QS from the HCV-M assay. We designed new sequencing primers to regain specificity for HCV and avoid sequencing the QS to obtain an acceptable HCV sequence (data not shown). The upstream primer is 5Ј-AGCCATGGCGTTAGTATGAG-3Ј, and the downstream primer is 5Ј-GCAGTACCACAAGG CCTTTC-3Ј. As illustrated in Fig. 1, the upstream primer from Gargiulo et al. (MF) is moved downstream by 3 bp to produce the TF primer, and the downstream primer (MR) is moved upstream by 3 bp to produce the TR primer. Both of the present primers have three nucleotides at the 3Ј end to create specificity for the HCV 5Ј-UTR sequence and avoid amplification of the QS but still avoid the va...