Early induction of cyclin D2 expression in phorbol ester-responsive B-1 lymphocytes.

B-1 lymphocytes represent a distinct B cell subset with characteristic features that include self-renewing capacity and unusual mitogenic responses. B-1 cells differ from conventional B cells in terms of the consequences of phorbol ester treatment: B-1 cells rapidly enter S phase in response to phorbol ester alone, whereas B-2 cells require a calcium ionophore in addition to phorbol ester to trigger cell cycle progression. To address the mechanism underlying the varied proliferative responses of B-1 and B-2 cells, we evaluated the expression and activity of the G1 cell cycle regulator, cyclin D2, and its associated cyclin-dependent kinases (Cdks). Cyclin D2 expression was upregulated rapidly, within 2-4 h, in phorbol ester-stimulated B-1 cells, in a manner dependent on intact transcription/translation, but was not increased in phorbol ester- stimulated B-2 cells. Phorbol ester-stimulated cyclin D2 expression was accompanied by the formation of cyclin D2-Cdk4, and, to a lesser extent, cyclin D2-Cdk6, complexes; cyclin D2- containing complexes were found to be catalytically functional, in terms of their ability to phosphorylate exogenous Rb in vitro and to specifically phosphorylate endogenous Rb on serine780 in vivo. These results strongly suggest that the rapid induction of cyclin D2 by a normally nonmitogenic phorbol ester stimulus is responsible for B-1 cell progression through G1 phase. The ease and rapidity with which cyclin D2 responds in B-1 cells may contribute to the proliferative features of this subset.

Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes.

Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

Antibody expression stability in CHO clonally derived cell lines and their subclones: Role of methylation in phenotypic and epigenetic heterogeneity.

Routine CHO cell line development practices involve a lengthy process of iteratively screening clonally derived cell lines to identify a single line suitable for IND filing and clinical manufacture. Paramount in this process is development of a stable production cell line having consistent growth, productivity and product quality for the entire generational length of the manufacturing process. Scale-down stability models used to screen clones for consistency are time consuming and often a rate-limiting step in clone selection. To investigate CHEF1 production stability in CHO cells we analyzed genotypic and phenotypic attributes of monoclonal primary clones and their respective subclones over time in standard antibody production models. The main finding of this work indicates that monoclonal cell lines derived from single cell progenitors grow into populations of cells with varied phenotypic heterogeneity, as revealed in their subclones, from either stable or unstable cell lines. Investigation of the subclones demonstrates that clonally derived cell lines grow out into populations with variable phenotypes and genotypes, even if the primary clone shows consistency in both over many generations in a stability study. Phenotypic and genotypic heterogeneity mostly did not correlate, but growth and productivity appear driven in part by cytosine methylation heterogeneity in both primary and secondary clones. This work presents evidence that epigenetic analysis may be useful for early detection of stability traits, but emphasizes the continued importance of rigorous cell line stability screening to identify primary clones that have consistent phenotypic characteristics, especially growth and productivity, throughout the in vitro lifecycle of the cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:635-649, 2018.

Lymph-node infarction in two young men.

Benisch, Barry M., and Howard Rudolf G.: Lymph-node infarction in two young men. Am J Clin Pathol 63: 818-823, 1975. Two cases of massive lymph-node infarction in young men are described. Both patients had fever and lymphadenopathy and findings that suggested the possibility of viral infection. Follow up has revealed both patients to be asymptomatic, with lymphadenopathy, and with normal laboratory findings.

Micromolar Ca2+ requiring protease from human platelets: purification, partial characterization and effect on the cytoskeletal proteins.

A calcium-activated neutral protease (CANP) has been purified 2,800 fold, to near homogeneity, from human platelets. The purification procedure involved ammonium sulfate fractionation of the platelet cytosol followed by chromatography on Sephacryl S-200, DEAE-Sephacel, Agarose-Hexylamine, Agarose-Octylamine and alpha-casein-Sepharose 4B affinity gel. The protease consisted of two polypeptides of Mr = 74,000 and 28,000 as judged on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It hydrolyzed [methyl-14C] alpha-casein at a significant rate of 37 degrees C which was, therefore, used as an exogenous substrate. Microtubules and intermediate filament proteins were also susceptible to hydrolysis by the purified protease. It attained maximum activity at 0.06 uM CaCl2 and displayed two pH maxima: one at 5.5 and the other at 6.5. The protease was fully active in the presence of MnCl2 and was about 75% active with BaCl2 and SrCl2. Among the actinomycete protease inhibitors, leupeptin, antipain and pepstatin, the order of inhibition was: leupeptin greater than antipain greater than pepstatin. The protease was also inhibited by sulfhydryl modifying agents.

How sensitive is ultrasound in the detection of renal scars?

The English language medical literature was reviewed to determine the strength of the published evidence for the assertion that dimercaptosuccinic acid scintigraphy (DMSA) is superior to ultrasound (US) in the detection of established renal scarring in children. The MEDLINE database was used to identify papers published between 1985 and 1997 that claimed to be concerned with the detection of scars, and contained sufficient information to permit calculation of the sensitivity of US relative to DMSA. Only 10 studies were identified. The sensitivity of US for scarring, using DMSA as a gold standard, ranged from 37% to 100%, and its specificity from 65% to 99%. These wide ranges mean that evaluation of the role of US in the detection of scarring remains controversial. All papers contained methodological flaws. Allowing for these, the sensitivity of US appears to be acceptable. Further research that avoids these methodological problems is required.

Vesicoureteric reflux and renal scarring in Chinese children.

Vesicoureteric reflux (VUR) and renal scarring are commonly found in children with urinary tract infection (UTI). The prevalence of VUR and scarring may vary between racial groups. There are no published data on the prevalence of VUR and scarring in Chinese children with UTI. A retrospective, single-institution study was made of Hong Kong Chinese children aged less than 5 years with a documented UTI investigated by both micturating cystourethrography and dimercaptosuccinic acid scintigraphy. VUR was identified in 39% of 93 Chinese children with UTI. Renal scarring was present in 28% of boys, which is comparable with published data on Western children. Scarring appears to be less common in Chinese girls with UTI (11%) than in Western girls (30-38% from published data), and its severity is poorly related to VUR grade. There is a significant dependency relationship between grade of VUR and degree of scarring in Chinese boys (p < 0.05). In conclusion, renal scarring appears to be relatively uncommon in Chinese girls. The correlation between grade of VUR and degree of scarring in Chinese boys suggests a relationship, but provides no evidence about the direction of causation.

Improved recombinant protein yield using a codon deoptimized DHFR selectable marker in a CHEF1 expression plasmid.

The CHEF1 expression plasmid utilizes regulatory domains of the Chinese Hamster Elongation Factor 1 (CHEF1) housekeeping gene to drive stable recombinant protein expression in Chinese Hamster Ovary (CHO) cells. Rapid development of stable CHEF1 cell lines is possible, in part, because high titer pools are produced in the absence of methotrexate-induced DHFR amplification. Inhibiting DHFR activity with methotrexate increases the selection pressure on transfected cells and can result in a compensatory event to ensure cell survival, such as dhfr gene amplification or integration into a transcriptionally active genomic site. Methotrexate amplification often results in improved cell line productivity but it is a time-consuming process. Herein, we describe a novel mechanism to increase selection stringency via codon deoptimization of the mouse dhfr selectable marker utilizing hamster codon preference that results in improved expression of a linked recombinant protein compared to wild-type DHFR selection. We show that deoptimizing the translatability of the dhfr gene reduces the expression level of the DHFR protein in CHO transfection pools and derived clones. Lower DHFR expression increases the transfection selection stringency, shown as lower transfection efficiency in pools, and correlates with increased recombinant protein expression in both pools and clones. These results demonstrate a new mechanism for increasing selection stringency and improving recombinant protein production during cell line development without time-consuming gene amplification steps.

Disulfiram inhibits the in vitro growth of methicillin-resistant staphylococcus aureus.

Several antibiotics have disulfiram-like effects; we evaluated disulfiram for its antibiotic-like effects. Disulfiram inhibited the in vitro growth of methicillin-resistant Staphylococcus aureus, with an MIC of 1.33 micrograms/ml, but was not effective against members of the family Enterobacteriaceae or Pseudomonas species.

Adult-type osteopetrosis presenting as carpal tunnel syndrome.

We describe a patient with adult-type osteopetrosis presenting as carpal tunnel syndrome. Radiographs demonstrated sclerosis of the carpal bones, bone biopsy revealed wide bone spicules containing areas of cartilage, and electrophysiologic studies confirmed the diagnosis of median nerve entrapment in the carpal tunnel. Any condition which alters the size or shape of the carpal canal or its contents may result in median nerve compression.

Induction of bcl-x by CD40 engagement rescues sIg-induced apoptosis in murine B cells.

CD40L, a membrane protein of activated T cells, interacts with the B cell receptor CD40. This interaction has been implicated in the rescue of germinal center B cells from apoptosis and in the rescue of WEHI-231 B lymphoma cells from sIg-induced apoptosis. In this report, we have demonstrated that the signal mediated by CD40L acts upon bcl-x, a bcl-2 homologue. bcl-x expression is strongly enhanced by CD40 receptor engagement, while there is little or no induction by sIg cross-linking. The expression of bax and bcl-2 is not significantly affected by either CD40L or sIg cross-linking. Antisense but not sense phosphorothioate oligonucleotide for bcl-x can partially block this CD40-mediated apoptotic rescue. This result suggests that the up-regulation of bcl-x by CD40L plays an important role in CD40-mediated apoptotic rescue in murine B cells.

IL-4 induces Fas resistance in B cells.

Activated B cells express Fas (CD95) and are targets for apoptosis induced by CD4+ Th1 effector cells that kill in a Fas-dependent fashion. We report here that IL-4 reverses the susceptibility to Fas-mediated apoptosis that characterizes CD40-stimulated primary B cells. IL-4-induced Fas resistance is not associated with an alteration in the elevated level of Fas expression produced by CD40 ligand and does not depend on additional receptor-mediated signals. However, IL-4-induced resistance to Th1 cell-mediated cytotoxicity (Th1-CMC) develops more slowly than resistance mediated by surface Ig and is not affected by protein kinase C depletion, unlike anti-Ig-induced Fas resistance. By these two criteria, IL-4-and anti-Ig-induced resistance to Th1-CMC appear to be driven through distinct mechanisms; in keeping with this, suboptimal doses of IL-4 and anti-Ig act in synergy to induce marked protection against Th1-CMC. An important role for IL-4-induced Fas resistance is suggested by the observation that sera from IL-4-overexpressing transgenic mice contain autoreactive Abs.

Imaging findings of paediatric oncology patients presenting with acute neurological symptoms.

Paediatric oncology patients are prone to central nervous system (CNS) complications due to multiple factors including disorders of the blood cell counts (which include neutropenia, thrombocytopenia or hyperleukocytosis), immunosuppression, neurotoxicity of the treatment, CNS dysfunction due to failure of other organ systems, disease progression of the primary malignancy or metastases. Imaging plays an important role in the management of paediatric oncology patients presenting with acute neurological symptoms. This pictorial review is from our institutional experience on imaging children who are under the care of the Child Cancer Centre. The review consists of a spectrum of neurological complications in paediatric oncology patients. The complications can be classified as (1) cerebrovascular complications, (2) treatment-elated complications, (3) opportunistic infections and (4) tumoural involvement of the CNS. Computed tomography (CT) is the initial choice of investigation, which is easily available and helps to exclude major intracranial abnormality such as haemorrhage. If the CT is negative, magnetic resonance imaging (MRI) should be performed, which is more sensitive for detection of CNS lesions.

Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen.

Covalent attachment of activated products of the third component of complement to antigen enhances its immunogenicity, but the mechanism is not clear. This effect is mediated by specific receptors, mCR1 (CD35) and mCR2 (CD21), expressed primarily on B cells and follicular dendritic cells in mice. To dissect the role of mCR1 and mCR2 in the humoral response, we have disrupted the Cr2 locus to generate mice deficient in both receptors. The deficient mice (Cr2-/-) were found to have a reduction in the CD5+ population of peritoneal B-1 cells, although their serum IgM levels were within the range of normal mice. Moreover, Cr2-/- mice had a severe defect in their humoral response to T-dependent antigens that was characterized by a reduction in serum antibody titers and in the number and size of germinal centers within splenic follicles. Reconstitution of the deficient mice with bone marrow from MHC-matched Cr2+/+ donors corrected the defect, demonstrating that the defect was due to B cells themselves. These results indicate an obligatory role of B cell complement receptors in responses of the B cells to protein antigens.

Regulation of the B cell response to T-dependent antigens by classical pathway complement.

Mice deficient in complement components C3 (C3 -/-) and C4 (C4 -/-) were found to have a profound defect in their Ab response to a T-dependent Ag (bacteriophage (phi X174). Characterization of the deficient mice demonstrated a diminished level of peanut agglutinin+ germinal centers and a failure in isotype switching despite normal B cell signaling in vitro. The nature of the defect was found to lie at the B cell level, as the T cells were primed in C3- and C4-deficient mice as well as those in wild-type mice. These results, and the finding that the defect could be partly reversed by a 10-fold increase in Ag dose, support the hypothesis that covalent attachment of complement ligands, i.e., C3b and C3d to the Ag-Ab complex, increases its immunogenicity.