Loss of Fas apoptosis inhibitory molecule leads to spontaneous obesity and hepatosteatosis.

Altered hepatic lipogenesis is associated with metabolic diseases such as obesity and hepatosteatosis. Insulin resistance and compensatory hyperinsulinaemia are key drivers of these metabolic imbalances. Fas apoptosis inhibitory molecule (FAIM), a ubiquitously expressed antiapoptotic protein, functions as a mediator of Akt signalling. Since Akt acts at a nodal point in insulin signalling, we hypothesize that FAIM may be involved in energy metabolism. In the current study, C57BL/6 wild-type (WT) and FAIM-knockout (FAIM-KO) male mice were fed with normal chow diet and body weight changes were monitored. Energy expenditure, substrate utilization and physical activities were analysed using a metabolic cage. Liver, pancreas and adipose tissue were subjected to histological examination. Serum glucose and insulin levels and lipid profiles were determined by biochemical assays. Changes in components of the insulin signalling pathway in FAIM-KO mice were examined by immunoblots. We found that FAIM-KO mice developed spontaneous non-hyperphagic obesity accompanied by hepatosteatosis, adipocyte hypertrophy, dyslipidaemia, hyperglycaemia and hyperinsulinaemia. In FAIM-KO liver, lipogenesis was elevated as indicated by increased fatty acid synthesis and SREBP-1 and SREBP-2 activation. Notably, protein expression of insulin receptor beta was markedly reduced in insulin target organs of FAIM-KO mice. Akt phosphorylation was also lower in FAIM-KO liver and adipose tissue as compared with WT controls. In addition, phosphorylation of insulin receptor substrate-1 and Akt2 in response to insulin treatment in isolated FAIM-KO hepatocytes was also markedly attenuated. Altogether, our data indicate that FAIM is a novel regulator of insulin signalling and plays an essential role in energy homoeostasis. These findings may shed light on the pathogenesis of obesity and hepatosteatosis.

Fas apoptosis inhibitory molecule is upregulated by IGF-1 signaling and modulates Akt activation and IRF4 expression in multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B-lymphoid cells. Here, we investigate the role of Fas apoptosis inhibitory molecule (FAIM) in MM. We demonstrate that insulin-like growth factor 1 (IGF-1) treatment upregulated FAIM expression in MM cells in a dose-dependent manner. Silencing of FAIM expression attenuates Akt signaling downstream of IGF-1 and compromises the viability of MM cells. We further showed that IGF-1 stimulation of MM cells leads to enhanced expression of IRF4, a known 'addictive' factor for MM. This upregulation of IRF4 expression by IGF-1 treatment of MM cells is abrogated when FAIM expression is silenced or Akt activation is inhibited. Thus, FAIM modulates IGF-1-induced Akt activation and IRF4 expression and has a role in MM cell survival. Consistent with these findings, FAIM expression is shown to be higher in plasma cells of symptomatic MM patients compared with normal individuals or patients with premalignant conditions. Moreover, a higher level of FAIM expression is shown to correlate with poorer survival outcomes of newly diagnosed MM patients treated with stem cell transplantation or relapsed MM patients treated in clinical trials with Bortezomib. Thus taken together, our study reveals a novel, as well as clinically relevant role for FAIM in MM.

Genetic deletion of faim reveals its role in modulating c-FLIP expression during CD95-mediated apoptosis of lymphocytes and hepatocytes.

Fas-apoptosis inhibitory molecule (FAIM) is inducibly expressed in B lymphocytes and had been shown to antagonize Fas-mediated killing of B-cell lines in vitro. However, its mechanism and role in vivo are unknown. We have generated faim(-/-) mice and found these mutants to be viable. In contrast to fas(-/-) mice, faim(-/-) mice have normal B- and T-cell populations. However, faim(-/-) B cells and thymocytes show increased sensitivity to Fas-triggered apoptosis in vitro, and faim(-/-) mice suffer greater mortality and exhibit exacerbated liver damage in response to Fas (CD95) engagement in vivo. The lack of FAIM results in greater activation of caspase-8 and -3 in Fas-stimulated thymocytes. Detailed biochemical analyses further reveal the decreased expression of c-FLIP(L) and c-FLIP(R) in faim(-/-) thymocytes and increased association of caspase-8 with Fas in Fas-activated mutant cells. Decreased levels of c-FLIP(L) and c-FLIP(R) are also evident in faim(-/-) liver. Thus, FAIM plays a novel role in modulating Fas-mediated apoptosis and acts through influencing the expression of c-FLIP and regulating the physical binding of caspase-8 to Fas.

Equivalence-set genes partitioning using an evolutionary-DP approach.

Computation of transitive-closure equivalence sets has recently emerged as an important step for building static and dynamic models of gene network from DNA sequences. We present an evolutionary-DP approach in which dynamic programming (DP) is embedded into a genetic algorithm (GA) for fitness function evaluation of small equivalence sets (with m genes) within a large-scale genetic network of n genes, where n > m. This approach reduces a computation-intensive optimal problem of high dimension into a heuristic search problem on nCm candidates. The DP computation of transitive closure forms the basic fitness evaluation for selecting candidate chromosomes generated by GA operators. By introducing bounded mutation and conditioned crossover operators to constrain the feasible solution domain, small transitive-closure equivalence sets for large genetic networks can be found with much reduced computational effort. Empirical results have successfully demonstrated the feasibility of our GA-DP approach for offering highly efficient solutions to large scale equivalence gene-set partitioning problem. We also describe dedicated GA-DP hardware using field programmable gate arrays (FPGAs), in which significant speedup could be obtained over software implementation.

Analog and digital FPGA implementation of BRIN for optimization problems.

The binary relation inference network (BRIN) shows promise in obtaining the global optimal solution for optimization problem, which is time independent of the problem size. However, the realization of this method is dependent on the implementation platforms. We studied analog and digital FPGA implementation platforms. Analog implementation of BRIN for two different directed graph problems is studied. As transitive closure problems can transform to a special case of shortest path problems or a special case of maximum spanning tree problems, two different forms of BRIN are discussed. Their circuits using common analog integrated circuits are investigated. The BRIN solution for critical path problems is expressed and is implemented using the separated building block circuit and the combined building block circuit. As these circuits are different, the response time of these networks will be different. The advancement of field programmable gate arrays (FPGAs) in recent years, allowing millions of gates on a single chip and accompanying with high-level design tools, has allowed the implementation of very complex networks. With this exemption on manual circuit construction and availability of efficient design platform, the BRIN architecture could be built in a much more efficient way. Problems on bandwidth are removed by taking all previous external connections to the inside of the chip. By transforming BRIN to FPGA (Xilinx XC4010XL and XCV800 Virtex), we implement a synchronous network with computations in a finite number of steps. Two case studies are presented, with correct results verified from simulation implementation. Resource consumption on FPGAs is studied showing that Virtex devices are more suitable for the expansion of network in future developments.

B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses.

B-cell maturation protein (BCMA) is a member of the tumor necrosis factor (TNF) receptor family and is expressed in B lymphocytes. BCMA binds two TNF family members, BAFF and APRIL, that stimulate cellular proliferation. BAFF in particular has been shown to influence B-cell survival and activation, and transgenic mice overexpressing BAFF have a lupus-like autoimmune disorder. We have inactivated BCMA in the mouse germ line. BCMA(-/-) mice have normal B-cell development, and the life span of mutant B lymphocytes is comparable to that of wild-type B cells. The humoral immune responses of BCMA(-/-) mice to T-cell-independent antigens as well as high and low doses of T-cell-dependent antigens are also intact. In addition, mutant mice have normal splenic architecture, and germinal centers are formed during an ongoing immune response. These data suggest a functional redundancy of BCMA in B-cell physiology that is probably due to the presence of TACI, another TNF receptor family member that is expressed on B cells and that can also bind BAFF and APRIL.

The adaptor protein BLNK is required for b cell antigen receptor-induced activation of nuclear factor-kappa B and cell cycle entry and survival of B lymphocytes.

B lymphocytes lacking the adaptor protein B cell linker (BLNK) do not proliferate in response to B cell antigen receptor (BCR) engagement. We demonstrate here that BCR-activated BLNK(-)/- B cells fail to enter the cell cycle, and this is due to their inability to induce the expression of the cell cycle regulatory proteins such as cyclin D2 and cyclin-dependent kinase 4. BCR-stimulated BLNK(-)/- B cells also do not up-regulate the cell survival protein Bcl-x(L), which may be necessary for the cells to complete the cell cycle. In addition, BLNK(-)/- B cells exhibit a high rate of spontaneous apoptosis in culture. Examination of the various BCR-activated signaling pathways in mouse BLNK(-)/- B cells reveals the intact activation of Akt and mitogen-activated protein kinases but the impaired activation of nuclear factor (NF)-kappaB that is known to regulate genes involved in cell proliferation and survival. The inability to activate NF-kappaB in BCR-stimulated BLNK(-)/- B cells is due to a failure to induce the degradation of the inhibitory kappaB protein. In all these aspects, BLNK(-)/- B cells resemble xid B cells that have a mutation in Bruton's tyrosine kinase (Btk). Recently, phospholipase C (PLC)-gamma2 has also been demonstrated to be essential for NF-kappaB activation. Since BLNK has been shown separately to interact with both Btk and PLC-gamma2, our finding of normal Btk but impaired PLC-gamma2 activation in BCR-stimulated BLNK(-)/- B cells strongly suggests that BLNK orchestrates the formation of a Btk-PLC-gamma2 signaling axis that regulates NF-kappaB activation. Taken together, the NF-kappaB activation defect may be sufficient to explain the similar defects in BCR-induced B cell proliferation and T cell-independent immune responses in BLNK(-)/-, Btk(-)/-, and PLC-gamma2(-)/- mice.

Memory B-cell persistence is independent of persisting immunizing antigen.

Immunological memory in the antibody system is generated in T-cell-dependent responses and carried by long-lived memory B cells that recognize antigen by high-affinity antibodies. But it remains controversial whether these B cells represent true 'memory' cells (that is, their maintenance is independent of the immunizing antigen), or whether they are a product of a chronic immune response driven by the immunizing antigen, which can be retained in the organism for extended time periods on the surface of specialized antigen-presenting cells (follicular dendritic cells). Cell transfer experiments provided evidence in favour of a role of the immunizing antigen; however, analysis of memory cells in intact animals, which showed that these cells are mostly resting and can persist in the absence of detectable T-cell help or follicular dendritic cells, argued against it. Here we show, by using a genetic switch mediated by Cre recombinase, that memory B cells switching their antibody specificity away from the immunizing antigen are indeed maintained in the animal over long periods of time, similar to cells retaining their original antigen-binding specificity.

Cutting edge: B cell linker protein is dispensable for the allelic exclusion of immunoglobulin heavy chain locus but required for the persistence of CD5+ B cells.

The pre-B cell receptor (pre-BCR) and the BCR are required for B lymphopoiesis and for the allelic exclusion of Ig genes. Mice lacking B cell linker (BLNK) protein that is a component of the BCR signaling pathway have impaired B cell development. In this report, we show that allelic exclusion is intact in BLNK(-/-) mice harboring a V(H)12 transgene. This differs from mice lacking the tyrosine kinase Syk that is upstream of BLNK in BCR signaling and contrasts with mice lacking SLP-76 that is the equivalent adaptor molecule in TCR-signal transduction. We also show that, whereas most wild-type V(H)12-expressing B cells are CD5(+), the majority of the splenic V(H)12-expressing BLNK(-/-) B cells are CD5(-). A small population of V(H)12-expressing, BLNK(-/-) CD5(+) B cells is detectable in the peritoneal cavity of younger but not older mice. This suggests that BLNK deficiency affects not only the generation but also the persistence of B-1 cells.

B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice.

Engagement of the B cell receptor (BCR) leads to the activation of tyrosine kinases and other signaling molecules that ultimately determine the type and magnitude of the B lymphocyte's cellular response. The adaptor protein BLNK/SLP-65 plays a pivotal role in BCR signal transduction by coupling Syk activation to downstream elements such as Grb2, phospholipase C-gamma, Vav and Nck. We have generated BLNK(-/-) mice to determine the physiological role of this protein in B cell development and activation. BLNK(-/-) mice exhibit an incomplete block in B cell development with a severe inhibition of pro-B to pre-B cell differentiation. BLNK(-/-) sIgM(+) cells can develop, seed the peripheral lymphoid tissues and accumulate in numbers overtime. However, these mutant B cells failed to mature and are non-responsive to BCR cross-linking in terms of proliferation and up-regulation of activation markers such as CD69 and CD86 (B7-2). In addition, the CD5(+) subset of B cells is absent. The immune response to T cell-independent antigen but not T cell-dependent antigen is also impaired. Overall, the phenotype of BLNK(-/-) mice bears a striking resemblance to that of xid mice which is the murine model of human XLA that has a mutation in Bruton's tyrosine kinase. This raises the interesting possibility that mutation in BLNK/SLP-65 may be responsible for certain human immunodeficiencies.

B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development.

Mice expressing the immunoglobulin (Ig) heavy (H) chain variable (V) region from a rearranged V(H)12 gene inserted into the IgH locus generate predominantly B-1 cells, whereas expression of two other V(H) region transgenes (V(H)B1-8 and V(H)glD42) leads to the almost exclusive generation of conventional, or B-2, cells. To determine the developmental potential of B cells bearing two distinct B cell antigen receptors (BCRs), one favoring B-1 and the other favoring B-2 cell development, we crossed V(H)12 insertion mice with mice bearing either V(H)B1-8 or V(H)glD42. B cells coexpressing V(H)12 and one of the other V(H) genes are readily detected in the double IgH insertion mice, and are of the B-2 phenotype. In mice coexpressing V(H)12, V(H)B1-8 and a transgenic kappa chain able to pair with both H chains, double H chain-expressing B-2 cells, and B-1 cells that have lost V(H)B1-8 are generated, whereas V(H)B1-8 single producers are undetectable. These data suggest that B-1 but not B-2 cells are selected by antigenic stimuli in whose delivery BCR specificity and surface density are of critical importance.

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles.

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.

Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity in vivo.

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-loxP-mediated gene inversion is used to change the specificity of the BCR on mature IgM+ IgD+ B cells in vivo to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive bcl-2 transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.

In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death.

Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system also depends on BCR expression, we have generated a transgenic mouse in which the BCR can be inducibly ablated through V region gene deletion. Ablation leads to rapid death of mature B lymphocytes, which is preceded by down-regulation of MHC antigens and up-regulation of CD95 (Fas) and can be delayed by constitutive bcl-2 expression.

B-1 cells: unique origins and functions.

B-1 cells represent a distinct population of B lymphocytes with unique phenotypic, developmental and functional characteristics. We present evidence that the expression of MHC class II antigens differentiates two distinct developmental pathways which define the fetal-type (FT) and adult-type B-cell lineages. Further in-vivo and in-vitro analyses suggest that B-1 cells are derived primarily if not exclusively from the FT lineage. Combining these results with other studies suggesting that signalling plays a role in the development of B-1 cells, we propose a modified dual lineage model for the generation of B-1 cells. In this model fetal-type B cells are uniquely 'born' with the capacity to be B-1 cells, however, they must be properly educated (i.e. receive the appropriate signals) before they can be 'made' functional, phenotypic B-1 cells. With respect to the function of B-1 cells, we have used PerC/BM allotype chimeric animals to investigate the capacity of B-1 cells to participate in the formation of germinal centers. In these mice we were unable to detect any significant involvement of B-1 cells in the generation of germinal centers. These results are discussed in the context of the known characteristics of B-1 responses including the low frequency of hypermutation and isotype class switching.

Major histocompatibility complex class II expression distinguishes two distinct B cell developmental pathways during ontogeny.

All mature B cells coexpress major histocompatibility complex (MHC) class II molecules, I-A and I-E, which are restriction elements required for antigen presentation to CD4+ T cells. However, the expression of class II during the early stages of B cell development has been unclear. We demonstrate here that there is a difference in the expression of class II during murine B cell development in the fetal liver and adult bone marrow (BM). These differences define two distinct B cell developmental pathways. The Fetal-type (FT) pathway is characterized by pre-B and immature IgM+ B cells generated in the fetal liver which initially lack all class II expression. In contrast, the Adult-type (AT) pathway is typified by B cells developing in the adult BM which express class II molecules from the pre-B cell stage. In vitro stromal cell cultures of sorted fetal liver and adult BM pro-B cells indicated that the difference in I-A expression during B cell development is intrinsic to the progenitors. In addition, we show that FT B cell development is not restricted to the fetal liver but occurs in the peritoneal cavities, spleens, liver, and BM of young mice up to at least 1 mo of age. The AT B cell development begins to emerge after birth but is, however, restricted to the BM environment. These findings indicate that there are two distinct B cell developmental pathways during ontogeny, each of which could contribute differentially to the immune repertoire and thus the functions of B cell subsets and lineages.

A high frequency of hybridomas from M54 mu heavy chain transgenic mice initially co-express transgenic and rearranged endogenous mu genes.

The M54 transgenic mouse line, which carries the 17.2.25 Ig mu heavy chain gene, rearranges Ig heavy chains and expresses both transgenic and endogenous mu. B cell lineage development is selectively impaired in these mice and cells that simultaneously express transgenic and endogenous mu ('double-producers') are common amongst the B cells and plasma cells that do develop. Weaver, Imanishi-kari, Baltimore and colleagues failed to obtain double-producing hybridomas from M54 mice; however, molecular and serologic studies presented here show that such hybridomas are readily generated. These hybridomas are extremely unstable and rapidly yield variants producing either transgenic or endogenous mu. Therefore the stable cloned lines we obtained, like Weaver et al., were almost all single or non-producers. We also found that the VH gene usage in our hybridomas was skewed towards the JH proximal (VHQ52, VH81X) families, supporting the idea that the expression of the M54 transgene alters the endogenous Ig repertoire.

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.

Radiological progression and lung function in silicosis: a ten year follow up study.

Chest radiographs and spirometric tests were performed on 81 patients who had silicosis from two granite quarries in 1975, 73 of whom were followed up for two to 10 (mean 7.2) years. Each patient's initial and most recent chest radiographs were assessed independently by three experienced readers, and the yearly declines in forced expiratory volume in one second and forced vital capacity were estimated from two to four (mean 3.45) serial spirometric readings. Estimates of individual dust exposure were based on extensive historical data on hygiene. All but 11 patients were no longer exposed to dust by the start of follow up, but 24 (45%) of 53 patients who had simple silicosis and 11 (55%) of 20 who had the complicated disease showed radiological evidence of disease progression. In patients who had simple silicosis and showed no radiological progression the yearly declines in forced expiratory volume in one second and forced vital capacity were modest (64 ml/year and 59 ml/year, respectively), whereas significantly greater declines in lung function were seen in those who showed radiological evidence of progression (97 ml/year and 95 ml/year, respectively). In addition to radiological progression the previous average dust concentration to which patients had been exposed also influenced declines in both forced expiratory volume in one second and forced vital capacity after allowing for the effects of age, smoking, duration of exposure, history of tuberculosis, initial state of disease, and baseline lung function. The probability of radiological progression was most strongly influenced by the average dust concentration previously exposed to. The progression of simple silicosis is thus accompanied by appreciable declines in lung function and is strongly affected by previous levels of exposure to dust.

Contour map registration using fourier descriptors of gradient codes.

To estimate the position of a subpicture P1 (with unknown angular misaligment) in a contour map P2, a method based on Fourier descriptors of multidirectional gradient codes is suggested. It is assumed that P2 is characterized by a set of magnitudes at equally spaceddiscrete points over a rectangular area; and P1 is described by a set of magnitudes at discrete points on directional axes emanated from a point with magnitude c*. Using the measurements of P1, the multidirectional gradient or successive-gradient codes and their Fourier descriptors are generated. A contour map for P2 having c* as one of the isopleth values is then obtained. For each point on all c*-isopleths, a two-level classifier, utilizing information derived from the Fourier descriptors and the phase correlation function, is used to estimate the possible location of P1 in P2. Simulation has indicated that in many cases the angular misalignment and the position of P1 with respect to P2 can be determined.