Biomedical subjects
J Cerny
Publications and source records attributed to J Cerny.
Precise half-life measurement for the superallowed 0(+)-->0(+) beta emitter (74)Rb: first results from the new radioactive beam facility (ISAC) at TRIUMF.
Presently, the world data for superallowed beta decay leads to a result in disagreement (at the 98% confidence level) with the predictions of the minimal standard model for the unitarity of the Cabibbo-Kobayashi-Maskawa matrix. Precise data for the superallowed 0(+)-->0(+) beta decay of (74)Rb would provide a critical test of the nucleus-dependent isospin symmetry-breaking corrections that must be calculated for these superallowed Fermi beta decays. The present work reports the first precise measurement of the half-life for (74)Rb ( t(1/2) = 64.761+/-0.031 ms). The data were obtained at the radioactive beam facility (ISAC) at TRIUMF using a beam of approximately 4000 (74)Rb ions s(-1).
Canonical germinal center B cells may not dominate the memory response to antigenic challenge.
Spleen and bone marrow (BM) are the major sites of antibody production and anamnestic response in systemically immunized mice. We examined the VDJ segment repertoire of antibody plaque-forming cells (APFC) in those two sites in the course of antibody responses to the hapten nitrophenyl (NP). Individual IgG APFC expressed any one of 10 V(H) segments of the V186.2/V3 (J558) gene family: 186.2, 102, 23, C1H4, 165.l, CH10, 3, 593.3, 24.8 and 671.5. The majority of cells in both spleen and BM expressed the V186.2 gene joined to a D segment with Tyr95. During a 2-month period after a single immunization, the V186.2(+) APFC in BM accumulated 3 times as many somatic mutations than splenic APFC (average 8.5 versus 3 mutations/V(H)); this process was T(h) dependent as shown by in vivo depletion of CD4(+) lymphocytes. However, the V186.2(+) APFC in both spleen and BM shared a recurrent W33L replacement, indicating their common origin from germinal centers. The APFC expressing the other (analogue) V(H) segments were evenly represented in the spleen and BM, but they accumulated few, if any, mutations. The anamnestic V186.2(+) APFC were highly mutated both in the spleen and BM; they represented a new and unexpected clonotype. The V/D segments were joined by Gly95 instead of Tyr95, the W33L was absent and a new shared K58R replacement appeared. The APFC expressing the 'analogue' V(H) genes comprised approximately 20% of the anamnestic response and did not accumulate more mutations, but their affinities were in the range of the memory V186.2(+) cells. These data suggest that the late primary and secondary responses to a hapten may be born by different B cell lineages, and that some clonotypes may reach the memory pool without an extensive mutation and expansion.
Coronary artery bypass grafting in patients with left ventricular ejection fraction of 30% or less.
BACKGROUND: The results of a merely pharmacological therapy in patients with advanced left ventricular dysfunction are unsatisfactory. Coronary artery bypass grafting is frequently the only therapeutic option, but ventricular dysfunction is generally considered to be a risk factor. AIM: To find out the frequency of coronary artery bypass grafting and its outcome in patients with a ejection fraction of 30% or less who were operated on at a single institution. METHODS: Between January 1st, 1996 and October 30th, 1999, 90 patients (4.6% of all patients operated on due to of coronary artery disease) with EF of 30% or less underwent coronary artery bypass grafting. This group consisted of 12 women and 78 men at an average age of 60.2 +/- 9.4 years (range, 33 to 78 years); 75.6% patients were in functional class III or IV and 80.0% had three-vessel disease. On the average, 2.5 grafts per patient were implanted, the left internal mammary artery was used in 24.4% patients, and 10.0% of patients had cardiac surgery without cardiopulmonary bypass. RESULTS: Hospital mortality was 10%. The main cause of death was cardiac or multiorgan failure. Low cardiac output syndrome and supraventricular dysrrhythmias were the most common postoperative complications. Advanced age and low cardiac output syndrome were found to be risk factors of early mortality. Five other patients died during the follow-up (4 to 48 months). One- and three-year survival rate were 83.1% and 81.9%, respectively. Ejection fraction improved during the follow-up from 27.5% to 33.7%. The improvement was more pronounced in patients in whom preoperative end-diastolic diameter of the left ventricle was below 70 mm, and in patients with two and more hibernating segments on dobutamine stress echo. CONCLUSIONS: Successful results of surgical revascularization in patients with severe impairment of left ventricular function can be achieved by careful selection of patients (the presence of viable myocardium is necessary) and management. Early mortality and morbidity was higher than in patients with normal ventricular function. Age and low cardiac output syndrome were revealed as risk factors of early mortality. Long-term prognosis for hospital survivals was satisfactory. (Tab. 5, Fig. 1, Ref. 13.)
Measurement of excitation functions in the reactions 197Au(11C, xn)208-xAt using a radioactive 11C beam
A light-element radioactive ion-beam capability has been developed at the LBNL 88-Inch Cyclotron. The system is based on the coupled-cyclotrons method and utilizes short-lived species, e.g., 11C, 14O, 13N produced by (p,n) and (p,alpha) reactions at the LBNL Biomedical Isotope Facility Cyclotron. In a first experiment, 197Au(11C,xn)208-xAt excitation functions have been measured for energies ranging from the Coulomb barrier up to 110 MeV using a beam of 11C with intensities up to (1-2)x10(8) ions/sec on target. The results of this experiment are compared to measurements of 197Au(12C, xn)209-xAt excitation functions.
Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice.
Umbilical cord blood transplantation is considered an alternative to traditional bone marrow transplantation for patients who do not have matched sibling donors. In this study, we examined the effects of ex vivo treatment of human cord blood cells with cytokine mixtures and assessed the ability of treated cells to engraft in NOD-scid mice. We incubated the cord blood with a four-factor cytokine mixture of interleukin (IL)-3, IL-6, IL-11 and stem cell factor, or with a two-factor cytokine mixture of thrombopoietin and flt-3. Incubation of cord blood for 48 h with either cytokine mixture did not affect progenitor cell number or proliferative potential as measured by the high proliferative potential (HPP) assay. Cytokine-treated cord blood injected into irradiated NOD-scid mice resulted in multilineage human engraftment. Overall, incubation with cytokines resulted in variable levels of engraftment with different cord blood samples. Incubation of cord blood with the four-factor cytokine mixture resulted in increased survival of irradiated NOD-scid recipients. These results demonstrate that short-term ex vivo treatment of human progenitor cells gives variable results on in vivo multipotential capabilities.
Postdoctoral patterns, career advancement, and problems.
Postdoctoral appointments can have different functions and meanings, depending on the field and whether the postdoc is a man or a woman. The Ph.D.'s-Ten Years Later study confirmed that in biochemistry, the postdoc, not the Ph.D., has become the general proving ground for excellence both in academia and industry. Because they spent a longer time in these "mandatory" postdocs, biochemists had the largest proportion of untenured faculty 10 to 13 years after the Ph. D. In mathematics, where substantially fewer postdoctoral positions are available, Ph.D.'s taking postdocs are more likely to obtain faculty positions, but this is true only for men. University administrators should be accountable for monitoring the total time spent in these positions and should provide administrative assistance for skills training, career growth, and the job search. In addition, creative solutions concerning the dual-career couple phenomenon are necessary.
Characterization of CD4+ T cells in mouse bone marrow. I. Increased activated/memory phenotype and altered TCR Vbeta repertoire.
A significant proportion of memory B cells home to bone marrow (BM) which is a major site of anamnestic antibody responses in mice. We hypothesized that memory T cells likewise accumulate in BM perhaps to provide help for antibody production, and that the compartment of CD4+ T cells in BM of unimmunized mice would be enriched for memory phenotype cells that might have been activated by environmental antigens. The phenotype of activated/memory CD4+ lymphocytes has been defined as CD44hi CD45RBlo CD62L-. Conversely, the phenotype of immunologically naive cells is CD44lo CD45RBhi CD62L+. Flow cytrometric analysis of tissue from normal, adult C57BL/6 mice identified 1-2 % CD3+CD4+ cells in BM. Up to 40 % of CD3+CD4+ cells in the BM expressed the activated/memory phenotype compared with < or = 10% in the spleen and lymph nodes. Analysis of TCR Vbeta repertoire revealed that expression of Vbeta3 and Vbeta7 genes was increased as much as fourfold in BM compared to the periphery; most of this increase was within the CD44hi T cells. The accumulation of activated/memory T cells and clonotypic expansion(s) was not seen in the BM of germ-free mice, indicating that it reflects the history of the animal's exposure to antigens. Finally, immunization of mice which express a transgenic T cell receptor specific for ovalbumin peptide resulted in appearance of antigen-specific T cells with activated/memory phenotype in the BM.
CD4 segregates into specific detergent-resistant T-cell membrane microdomains.
In T cells, glycolipids, glycoproteins attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor, and Src-like tyrosine kinases are highly enriched in a membrane fraction resistant to solubilization by nonionic detergents. We have investigated the distribution of CD4 in T-cell membranes and found that approximately 10% of the CD4 co-receptor is associated with detergent-insoluble membrane microdomains, whilst the remaining 90% is in soluble membranes. Moreover, approximately 60% of the "insoluble CD4" is present in membrane microdomains containing GPI-anchored proteins and high glycolipid-dependent kinase activity, whereas the remaining 40% displays no association with GPI-anchored proteins and lacks glycolipid-associated kinase activity These results indicate that CD4 segregates at least into three different membrane microenvironments: 1) soluble membranes; 2) insoluble membrane microdomains containing GPI-anchored proteins; and 3) insoluble membrane microdomains devoid of GPI-anchored proteins. The level of CD4 in insoluble membranes was not modified upon triggering activation by T-cell receptor-crosslinking but detectable amounts of CD3 subunits were recruited into these specialized membranes under those conditions. The physical separation of CD4 into different membrane microenvironments raises the possibility of that some of the multiple functions of CD4 might segregate into distinct types of lipid microenvironment. The fact that components of T-cell receptor/CD3 complex were recruited into insoluble membranes upon stimulation is consistent with the CD4 present in this membrane fraction might participate in T-cell receptor-triggered activation events.
Regulation of VH gene repertoire and somatic mutation in germinal centre B cells by passively administered antibody.
Immunization with T-dependent antigens induces a rapid differentiation of B cells to plasmacytes that produce the primary immunoglobulin M (IgM) and IgG antibodies with low affinities for the immunogen. It is proposed that the IgG antibody forms immune complexes with the residual antigen which provide an important stimulus for the formation of germinal centres (GC) and the activation of somatic mutation. This hypothesis was tested by passive administration of hapten-specific antibody into mice shortly after the immunization with nitrophenyl (NP) coupled to chicken gamma globulin (NP-CGG) in an environment of limited T-cell help. Athymic mice that received normal T helper cells at 72 hr after the administration of antigen produced low levels of anti-NP antibody and the splenic GC formation was delayed until day 12 after the antigen administration. The analysis of VDJ segments from NP-reactive GC B cells showed very few mutations in the VH genes. Passive injection of anti-NP IgG1 monoclonal antibody - but, not IgM - stimulated the GC formation up to normal levels and the somatic mutation activity in the GC B cells was fully restored. In addition, GC B cells in the recipients of IgG1 antibody demonstrated a change in the usage of germline-encoded VH genes which was not apparent among the primary antibody-forming cells. These results suggest the existence of a specific feedback mechanism whereby the IgG antibody regulates the GC formation, clonotypic repertoire and somatic mutation in GC B cells.
Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation.
The pattern of somatic mutations of shark and frog Ig is distinct from somatic hypermutation of Ig in mammals in that there is a bias to mutate GC base pairs and a low frequency of mutations. Previous analysis of the new antigen receptor gene in nurse sharks (NAR), however, revealed no bias to mutate GC base pairs and the frequency of mutation was comparable to that of mammalian IgG. Here, we analyzed 1023 mutations in NAR and found no targeting of the mechanism to any particular nucleotide but did obtain strong evidence for a transition bias and for strand polarity. As seen for all species studied to date, the serine codon AGC/T in NAR was a mutational hotspot. The NAR mutational pattern is most similar to that of mammalian IgG and furthermore both are strikingly akin to mutations acquired during the neutral evolution of nuclear pseudogenes, suggesting that a similar mechanism is at work for both processes. In yeast, most spontaneous mutations are introduced by the translesion synthesis DNA polymerase zeta (REV3) and in various DNA repair-deficient backgrounds transitions were more often REV3-dependent than were transversions. Therefore, we propose a model of somatic hypermutation where DNA polymerase zeta is recruited to the Ig locus. An excess of DNA glycosylases in germinal center reactions may further enhance the mutation frequency by a REV3-dependent mutagenic process known as imbalanced base excision repair.
Antibody feedback and somatic mutation in B cells: regulation of mutation by immune complexes with IgG antibody.
In response to an appropriate antigenic stimulus, and with help from T lymphocytes, naive B cells differentiate into plasmacytes which produce the primary (germline-encoded) IgM and IgG antibody with low affinity for the antigen. The isotype switch from IgM to IgG coincides with the burst of germinal center reaction and the onset of somatic hypermutation. Here we propose that formation of immune complexes between the residual antigen and the primary IgG antibody, which activate complement and localize specifically in the network of follicular dendritic cells, provides an important signal for triggering the mutation mechanism in germinal center B cells. This hypothesis has been supported by studies on immunogenicity of immune complexes in vivo. The experiments have included an immunization with pre-formed antigen/IgG antibody complex and/or an administration of IgG antibody shortly after the antigen injection. Either of these strategies, which are known to augment the germinal center formation, resulted in earlier onset of somatic mutation and increased mutation frequency in VDJ rearrangements in antigen-reactive B cells, provided that help from T cells was also present. It is presumed that the antigen/antibody/complement complex is able to deliver this important signal by cross-linking of antigen receptor with the CD21/CD19/CD81 molecules on B cells. As a corollary, the signaling by immune complexes may lower the threshold of cell activation determined by receptor affinity for antigen and stimulate diverse V-gene repertoire of B-cell clones in germinal centers.
Induction of germinal center B cell markers in vitro by activated CD4+ T lymphocytes: the role of CD40 ligand, soluble factors, and B cell antigen receptor cross-linking.
Following primary immunization, B cells differentiate to memory cells with help from T cells. The specialized path to B cell memory takes place in lymphoid germinal centers (GC), where mouse B cells up-regulate peanut agglutinin receptor (PNA-R), B7-2 (CD86), and MHC class II expression. Using an in vitro culture system, we have studied how different stimuli can enhance the expression of these markers. We show that PNA-R is up-regulated when splenic B cells are cocultured with anti-CD3-stimulated CD4+, but not CD8+, T cells and that this process requires CD40-CD40 ligand engagement. Increased expression of PNA-R is also inducible with supernatants of activated CD4+, but not CD8+, T cells in combination with mitogenic signals, such as anti-Ig, anti-CD40, or LPS, but not by either supernatants or mitogenic signals alone. Unlike with PNA-R, increased expression of B7-2 and I-A occurs in response to activated T cells of either CD4+ and CD8+ subsets or their supernatants, does not require CD40 costimulation, and is readily induced with mitogenic signals alone. Taken together, these results indicate that PNA-R up-regulation has more restricted signaling requirements than B7-2 or I-A, and that it can be induced/maintained by Ag receptor cross-linking or CD40 engagement, as long as there is an appropriate cytokine milieu.
Immunization with immune complex alters the repertoire of antigen-reactive B cells in the germinal centers.
The differentiation of memory B cells in germinal centers (GC) is selectively enhanced upon administration of antigen-antibody complexes. To characterize the repertoire of this response, we examined the rearranged immunoglobulin heavy chain variable (V(H)) genes from mouse splenic GC after a single immunization with either antigen, nitrophenyl (NP) hapten coupled to keyhole limpet hemocyanin, or with a preformed complex of antigen with a monoclonal anti-NP antibody of gamma1 isotype. Among antigen-immunized mice, NP-reactive GC B cell populations in the antigen-induced GC consisted mostly of cells expressing the canonical V186.2 gene which contained, on average, 0.8 point mutations/V(H) gene by day 8 after immunization. These results are indicative of the beginning of somatic hypermutation and consistent with previously published analyses of NP antigen-driven GC. In contrast, the NP-specific B cells in GC that were elicited by administration of immune complex represented a heterogeneous cell population expressing nine different germ-line segments of the V186.2/V3 (J558) gene family, i.e. V23, V24.8, C1H4, V3, CH10, V165.1, V102, V671.5 and V186.2. Moreover, the average frequency of mutations in these genes was 1.7, reaching up to 4 mutations/V(H) in some GC. Administration of the antigen NP in complex with specific antibody apparently alters the process of interclonal competition in the GC and results in loss of dominance by V186.2+ cells and nearly stochastic representation of diverse clonotypes. These results suggest an important feedback regulation of the B cell repertoire by antibody and indicate a role for immune complexes in the activation of somatic hypermutation.
Age-related changes in antibody repertoire: contribution from T cells.
The immune system of aged mice produces antibodies that are characterized by low affinity, diminished protection against infections and autoreactivity. It has been shown that these antibodies may be encoded by different immunoglobulin V genes and that the mechanism of somatic hypermutation in the V genes is inefficient. Studies on scid mice reconstituted with B and T cells from donors of different ages suggested that both lymphocyte subsets may contribute to the age-related changes in antibody repertoire. With help provided by T cells from young mice, the response to a hapten, nitrophenyl(acetyl), became gradually dominated by B-cell clones that rearranged a particular germline VH gene (V186.2). However, help from the aged T cells resulted in a heterogeneous response of B cells expressing many different V segments. Analysis of discrete foci of primary antibody-forming cells suggested that the aged T-helper cells are unable to govern the normally-occurring competition between the B-cell clones that have different affinities for the hapten. It is proposed that a signaling disequilibrium from the aged T cells, which provide less efficient help in quantitative terms, supports the growth of low-affinity B cells. This process may be exacerbated due to the apparent hyperactivity of aged B cells to CD40-mediated mitogenic signal.
Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice.
The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.
Low response to high-dose intravenous immunoglobulin in the treatment of acquired factor VIII inhibitor.
Prednisone is the classic first-line therapy to suppress an acquired factor VIII inhibitor and may achieve complete remission in about 30% of patients. More recently, promising results have been reported with high-dose intravenous immunoglobulin (IVIg). However, after an extensive review of the literature, we found only three complete remissions (12%) among the 26 assessable patients treated by IVIg. These data are in agreement with the low response to IVIg that we experienced in our series of patients. This study suggests that steroids should still be preferred to IVIg, an expansive therapy, to suppress an acquired factor VIII inhibitor.
Additional results from the beta -delayed proton decays of 27P and 31Cl.
Explore the source record for details and available documents.