The BCL6-associated transcriptional co-repressor, MTA3, is selectively expressed by germinal centre B cells and lymphomas of putative germinal centre derivation.

Metastasis-associated protein 3 (MTA3) is a recently described cell-type specific component of the Mi-2-NURD transcriptional co-repressor complex that is expressed in breast epithelia and germinal centre B cells. In model B cell lines, MTA3 physically interacts with BCL6 and appears to be instrumental in maintenance of the germinal centre B cell transcriptional programme that precludes premature plasmacytic differentiation. Here, we report selective, in situ cell-type specific expression of MTA3 among lymphoid cells largely confined to the germinal centre B cell compartment. Centroblasts display greater expression than smaller, less proliferative centrocytes, with undetectable expression in quiescent plasma cells. Among B cell neoplasms, germinal centre B cell-like lymphomas likewise exhibit selective expression that generally escalates with increasing proliferative capacity. MTA3 protein expression was, in accord, highly predictive of the germinal centre B cell-like gene expression profile for diffuse large B cell lymphomas. Lastly, relative repression of a subset of known BCL6 targets, including BLIMP1 and p27kip1, was highest in diffuse large B cell lymphomas that co-expressed both MTA3 and BCL6 protein. Together, these novel data suggest a role for MTA3 in BCL6-mediated lymphomagenesis in germinal centre B cell-like neoplasms.

Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide.

Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils. In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels. Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses. Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation. Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins. However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors. These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.

Histologic examination of bone marrow core biopsy specimens has limited value in the diagnosis of mycobacterial and fungal infections in patients with the acquired immunodeficiency syndrome.

Bone marrow cultures and biopsy specimens are commonly obtained to rule out disseminated infections, especially in persons with the acquired immunodeficiency syndrome (AIDS) and cytopenias. Using culture as the gold standard, we reviewed 130 consecutive bone marrow cores obtained from 114 AIDS patients along with results of concurrent blood and/or bone marrow aspirate cultures to determine the usefulness of histologic examination for diagnosis of mycobacterial and fungal infections. We also compared the ability of Ziehl-Neelsen, auramine-rhodamine (AR), polyclonal antibody to Mycobacterium bovis (Ab), and Gomori's methenamine silver staining to detect infections. Twenty-seven patients had mycobacterial infection (25 Mycobacterium avium-intracellulare complex cases and two Mycobacterium tuberculosis cases) detected by blood and/or bone marrow cultures. The maximum sensitivity of histology was 50% when the auramine-rhodamine stain and the polyclonal antibody to M bovis were used in combination. The single best stain was auramine-rhodamine, with a sensitivity of 44%, followed by the polyclonal antibody to M bovis (35%). Granulomas were observed in nine cases of mycobacterial infection and did not correlate with the presence of stainable organisms. Of seven patients with positive fungal cultures of bone marrow, four had granulomas and a positive Gomori's methenamine silver stain, one had only a positive stain, and two had neither granulomas nor a diagnostic stain. Overall, granulomas were not sensitive for the detection of infections when culture-proven mycobacterial and fungal cases were evaluated together. We conclude that bone marrow examination has a limited value in the routine evaluation of common opportunistic infections in AIDS patients and recommend that less-invasive tests, such as blood cultures, be obtained initially in most circumstances.

Neutrophil migration across intestinal epithelium.

Transmigration of neutrophils across epithelial surfaces is the hallmark of inflammatory mucosal diseases of diverse organs. In disorders such as Crohn's disease, ulcerative colitis, pyelonephritis, and bronchitis, for example, neutrophil transmigration correlates with clinical disease activity, is associated morphologically with injury to the epithelium, and is central to disease pathophysiology. The mechanisms by which neutrophils transmigrate across epithelia are, therefore, of considerable significance for numerous pathologic states. In this paper, we discuss current evidence that defines these mechanisms in intestinal epithelium, emphasizing the structural constituents determining adhesive interactions and a subset of the complex regulatory signals between neutrophils and epithelium.

An automated semiquantitative B and T cell clonality assay.

BACKGROUND: Rearrangements of the antigen receptor genes in B and T cells generate products of unique length and sequence. Polymerase chain reaction (PCR) assays are routinely used to identify clonal lymphocyte populations by detecting clonal V-J rearrangements or chromosomal translocations within these antigen receptor loci. Multiple primer sets are, however, required to detect the majority of clonal B- and T-cell malignancies. Products from the individual reactions must be analyzed separately to avoid misinterpretation. Moreover, small clonal populations remain difficult to identify. To address these difficulties, we propose that an integrated fluorescence-based approach to clonal B- and T-cell detection would simultaneously identify both B- and T-cell neoplasia; increase amplicon resolution, analytic sensitivity, and assay throughput; produce more comprehensive and semiquantitative data useful for evaluation of hematologic malignancies; and eliminate labor intensive agarose and polyacrylamide gel electrophoresis. METHODS AND RESULTS: Samples were genomic DNA and cDNA. Differentially labeled primers were used to amplify regions diagnostic for B- and T-cell clonality in a single plate with a single thermocycler program. Combined amplicon products underwent capillary electrophoresis for high resolution fractionation and differential fluorescence detection and quantification. Data were automatically analyzed and archived. In a comparative analysis of a variety of clinical samples, this automated and integrated B- and T-cell assay showed >94% agreement (33 of 35 results) with individual B- and T-cell PCR assays. Furthermore, this assay had an overall monoclonality detection rate of 100%, and as little as 100 ng of sample DNA yielded complete B- and T-cell clonality test results. The limit of detection was approximately 10-2 cells, and amplicons were sized to within 0.1 basepair. Serial dilutions of clonal B- and T-cell lines comprising a coded proficiency panel were identified and correctly ranked. Specificity was 100% as determined by analysis of 18 control samples that were all negative for B- and T-cell clonality. CONCLUSIONS: Our data show that this automated and integrated B- and T-cell clonality assay system is a sensitive and specific tool useful for rapid identification of clonal lymphocyte populations and will likely have broad clinical applications.

Comparison of two rapid latex agglutination tests for detection of cryptococcal capsular polysaccharide.

The Murex Cryptococcus Test was compared with the Cryptococcal Antigen Latex Agglutination System (CALAS) for detecting cryptococcal polysaccharide in 173 cerebrospinal fluid (CSF) specimens and 117 serum samples with 99% and 97% concordance, respectively. Eighteen CSF samples and 17 serum samples were positive in both assays, and 249 were negative. The sensitivity and specificity of the Murex relative to the CALAS were 90% and 100%, respectively, for CSF, and 81% and 100%, respectively, for serum. Six discrepancies were arbitrated by retesting, using a third analytic method, review of other laboratory and clinical data, or both. The reaction in 1 CSF specimen was considered false positive by the CALAS, and the reactions in 2 serum samples were false negatives by the Murex. For 3 patients with previous cryptococcal meningitis but no active disease, only the CALAS detected antigen, suggesting that the Murex has less analytic sensitivity in this context. Titer differences dictate that direct comparisons between the 2 tests are not feasible. There were no false-positive reactions in limited testing with either method using specimens from patients with concurrent noncryptococcal infections or in rheumatoid factor-positive serum samples. Infections caused by Cryptococcus neoformans serotypes A or AD were detected equally by both assays. Based on our study, we have elected to continue to use the CALAS for routine testing for cryptococcal antigen.

The yield of bone marrow biopsy and culture compared with blood culture in the evaluation of HIV-infected patients for mycobacterial and fungal infections.

PURPOSE: To compare the clinical utility of bone marrow biopsy and culture specimens with blood cultures for mycobacterial and fungal infections among human immunodeficiency virus (HIV)-infected patients. PATIENTS AND METHODS: All bone marrow biopsies obtained from HIV-infected patients at the University of Alabama at Birmingham (UAB) Medical Center during 1993 to 1995 were blindly reviewed in a standardized format. Bone marrow culture results and blood culture results obtained within 6 weeks of each bone marrow study were compiled. Medical records were reviewed to determine indications for performing bone marrow biopsies, empiric or prophylactic antimicrobial therapies preceding the biopsy, and CD4 counts. RESULTS: Eighty-two bone marrow studies were obtained from 76 patients. Most were performed during the evaluation of fever, cytopenia, or weight loss. Of 55 bone marrow mycobacterial cultures, 13 yielded Mycobacterium avium complex (MAC) and 2 yielded M tuberculosis (MTB). Of 51 bone marrow fungal cultures performed, 2 yielded Cryptococcus neoformans and 1 Histoplasma capsulatum. All patients with a bone marrow culture positive for MAC had a CD4 count of 20 cells/mm3 or less. The mean CD4 count in this group (+/-95% confidence interval) (8+/-3 cells/mm3) was lower than that of culture-negative cases (41+/-25 cells/mm3); P <0.015). When bone marrow cultures and mycobacterial blood cultures were concurrently obtained, results were usually in agreement between the two sites. The mean time until the report of positive mycobacterial bone marrow cultures (22+/-5 days) was similar to that for blood cultures (24+/-3 days). Most (84%) patients with multiple mycobacterial cultures had completely concordant results (all positive or all negative). When blood or bone marrow culture yielded mycobacteria, only 29% of the corresponding bone marrow examinations revealed stainable acid-fast bacilli (AFB). In contrast, all 3 cases with positive fungal bone marrow cultures also had stainable organisms on histologic examination. CONCLUSIONS: The combined use of bone marrow biopsy and culture as well as blood cultures provide the maximum diagnostic yield when evaluating patients with AIDS for mycobacterial or fungal infections. However, when mycobacterial infections were diagnosed, bone marrow results seldom provided more immediate or specific information than lysis centrifugation blood cultures. A single lysis centrifugation blood culture should be the first step in the routine evaluation of HIV-infected patients when disseminated MAC infection is suspected.

Clinical applications of C-reactive protein in pediatrics.

The body of literature concerning studies of the applications of CRP measurement in the pediatric population continues to grow. Based on current data serial CRP measurements appear to be most useful for monitoring patient response to therapy after the primary diagnosis of invasive infectious or inflammatory diseases, for monitoring patients after major surgical procedures and those with serious burns. Monitoring CRP over time may be used to assess for recrudescent disease, a secondary process or ineffective therapy. In addition CRP appears to be suited to most applications for which the ESR is used but offers many advantages. At present there are no objective outcome-based clinical trial data to justify using CRP values alone, whether elevated or normal, as a basis for management decisions regarding instituting or withholding antimicrobial therapy, or its early discontinuance for patients suspected of having neonatal sepsis, meningitis, bacteremia or pneumonia, regardless of immune status. In addition, because of significant inconsistencies among studies for which CRP has been applied to differential diagnosis of bacterial vs. viral diseases, including meningitis, acute otitis media and lower respiratory tract infection, we cannot recommend it for this purpose. Data do not support a role for CRP in differential diagnosis of acute appendicitis or for localizing urinary tract infections.

Gene transfer to avian embryos with a recombinant adenovirus.

Replication-defective adenoviral vectors are widely used for gene transfer into mammalian cells. We show here that these vectors are also useful for delivering genes to chick embryos. Cells of many tissues expressed Escherichia coli beta-galactosidase following infection in vitro or in ovo with a recombinant adenoviral vector carrying this reporter gene. Labeled cells were morphologically normal and integrated normally into host tissues. Moreover, cells labeled in neural crest and somites subsequently migrated along normal paths. However, restricted subsets of cells were infected in some tissues at some stages, suggesting complex control of adenoviral infectivity and/or expression.

The properdin-like type I repeats of human thrombospondin contain a cell attachment site.

Thrombospondin (TS) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to TS. These include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an RGDA sequence of TS. We have characterized a mAb against human TS, designated A4.1, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for A4.1 lies within the amino terminal half of the central stalklike region of TS which is distinct from the three known cell attachment sites. This region of TS is recovered in a 50-kD peptide after chymotryptic digestion of TS in EDTA. It contains the procollagen-like domain of TS as well as three type I repeats of a 60-residue segment homologous to two malarial proteins and the complement proteins properdin, and factors C6 through C9. The purified chymotryptic fragment is an effective attachment factor for G361 cells. A4.1 blocks adhesion to the 50-kD domain, as do some sulfated glycoconjugates. RGD (and RGE) peptides and mAbs against other domains of TS are not inhibitory. Peptides (19 mers) based on the core homology sequence of the three type I repeats of TS are potent attachment factors for these cells, and this adhesion is also inhibited by sulfated glycoconjugates. A polyclonal antibody raised against one of these peptides inhibits adhesion of G361 cells to the peptides, to the 50-kD fragment and to intact TS. Thus a new cell-adhesion site has been identified in TS whose sequence is very similar to the site identified in region II of the circumsporozoite protein of malaria parasites (Rich, K. A., F. W. George IV, J. L. Law, and W. J. Martin. 1990. Science (Wash. DC) 249:1574-1577. Thus there may be a common receptor which binds TS, malarial proteins, and properdin.

Purification and characterization of an abundant cytosolic protein from human neutrophils that promotes Ca2(+)-dependent aggregation of isolated specific granules.

Intracellular ionized calcium has been strongly implicated in mediating several responses of human neutrophils to stimulation. However, proteins that serve as effectors of these responses have not been well characterized. To identify proteins that might serve as mediators of the effects of Ca2+ in human neutrophils, we isolated proteins that bind to membrane phospholipids in a Ca2(+)-dependent manner. The most abundant of these, a protein of 33 kD, was readily purified to homogeneity, and was found to bind to phosphatidylserine vesicles in the presence of 2 microM ionized Ca2+. In addition, this purified protein promoted Ca2(+)-dependent aggregation of isolated specific granules from human neutrophils, indicating that it might mediate membrane-membrane contact during processes such as phagosome-lysosome fusion or degranulation. This protein was localized to the cytoplasm of unstimulated neutrophils and found to account for approximately 1% of the cytosol protein. Amino acid sequence of several peptides derived from the purified protein revealed that it is identical to lipocortin III, a recently described member of the annexin family that is scarce in other cells and tissues. The abundance of this protein, together with its Ca2(+)-dependent membrane effects, suggest that it mediates membrane-localized events in stimulated neutrophils, such as phagosome-lysosome fusion or degranulation.