Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).

The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene.

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

Immunoglobulin gene rearrangements and the pathogenesis of multiple myeloma.

The ability to rearrange the germ-line DNA to generate antibody diversity is an essential prerequisite for the production of a functional repertoire. While this is essential to prevent infections, it also represents the "Achilles heel" of the B-cell lineage, occasionally leading to malignant transformation of these cells by translocation of protooncogenes into the immunoglobulin (Ig) loci. However, in evolutionary terms this is a small price to pay for a functional immune system. The study of the configuration and rearrangements of the Ig gene loci has contributed extensively to our understanding of the natural history of development of myeloma. In addition to this, the analysis of Ig gene rearrangements in B-cell neoplasms provides information about the clonal origin of the disease, prognosis, as well as providing a clinical useful tool for clonality detection and minimal residual disease monitoring. Herein, we review the data currently available on both Ig gene rearrangements and protein patterns seen in myeloma with the aim of illustrating how this knowledge has contributed to our understanding of the pathobiology of myeloma.

Incomplete DJH rearrangements of the IgH gene are frequent in multiple myeloma patients: immunobiological characteristics and clinical implications.

DH-JH rearrangements of the Ig heavy-chain gene (IGH) occur early during B-cell development. Consequently, they are detected in precursor-B-cell acute lymphoblastic leukemias both at diagnosis and relapse. Incomplete DJH rearrangements have also been occasionally reported in mature B-cell lymphoproliferative disorders, but their frequency and immunobiological characteristics have not been studied in detail. We have investigated the frequency and characteristics of incomplete DJH as well as complete VDJH rearrangements in a series of 84 untreated multiple myeloma (MM) patients. The overall detection rate of clonality by amplifying VDJH and DJH rearrangements using family-specific primers was 94%. Interestingly, we found a high frequency (60%) of DJH rearrangements in this group. As expected from an immunological point of view, the vast majority of DJH rearrangements (88%) were unmutated. To the best of our knowledge, this is the first systematic study describing the incidence of incomplete DJH rearrangements in a series of unselected MM patients. These results strongly support the use of DJH rearrangements as PCR targets for clonality studies and, particularly, for quantification of minimal residual disease by real-time quantitative PCR using consensus JH probes in MM patients. The finding of hypermutation in a small proportion of incomplete DJH rearrangements (six out of 50) suggests important biological implications concerning the process of somatic hypermutation. Moreover, our data offer a new insight in the regulatory development model of IGH rearrangements.

The Myosin Heavy Chain specific A4.1025 antibody discriminates different cardiac segments in ancient groups of gnathostomes: Morphological and evolutionary implications

The pan-Myosin Heavy Chain (pan-MyHC) marker MF20 have been reported to show similar, homogeneous signal in the myocardial segments of the heart of teleosts and tetrapods. However, in an ongoing study of the myocardial structure of the dogfish (Scyliorhinus canicula; Chondrichthyes), we observed differential immunostaining of the cardiac segments using another pan-MyHC, the A4.1025 antibody. In order to investigate the relevance of this finding for better understanding of the morphology and evolution of the vertebrate heart, we performed immunohistochemistry, slot blot and western blot in several species of chondrichthyans, actinopterygians and mammals using the above mentioned antibodies. In the dogfish heart, A4.1025 and MF20 specifically recognized MyHC isoforms, although with different degree of affinity. MF20 reactivity was homogeneous and high in all the myocardial segments. However, A4.1025 reactivity was heterogeneous. It was high in the sinus venosus (external layer), atrium and atrioventricular region, low in the ventricle and conus arteriosus, and null in the internal layer of the sinus venosus. A heterogeneous pattern of A4.1025 immunoreactivity was also detected in two other elasmobranchs, a holocephalan, a polypteryform and an acipenseriform. In all of these species, MF20 immunoreactivity was homogeneous. In addition, both markers showed a homogeneous immunoreactivity pattern in teleosts and mammals. Our results indicate that in the hearts of ancient gnathostomes, in all of which a conspicuous conus arteriosus exists, one or more MyHC isoforms with low affinity for A4.1025 show segment-specific distributions. Thus, A4.1025 appears to be an appropriated marker to identify the cardiac segments and their boundaries. We propose that the segmentspecific distribution of MyHC isoforms may generate a particular type of myocardial contractility associated with the presence of a conus arteriosus.

Immunogenetic characteristics of immunoglobulin E in allergic disease

Patients with allergic diseases produce an excess of allergen-specific IgE, the specific effector molecule that triggers allergic reactions. The provocation for this excess IgE production is still uncertain. Current ideas include oligoclonal expansion of allergen-specific B cells emanating from germinal centres, activation by superantigen of a subset of B cells, or polyclonal B cells class switching to IgE due to an IL-4 predominance. Additionally, genetic elements contribute to a propensity for increased allergen-specific IgE production. The procedure of RT-PCR allows for amplification of infrequent IgE mRNA transcripts from B cells of atopic individuals, and so facilitates examination of expressed Ig cDNA sequences. Better knowledge of the molecular characteristics of IgE produced by patients with allergic diseases would elucidate the immunogenetic basis for elevated allergen-specific IgE levels. The 'immunogenetic footprint' of IgE transcripts may elucidate the origin and activation of IgE-producing B cells in allergic disease. Here we review studies of the immunogenetic features of IgE in allergic diseases, highlighting the major advances and the experimental limitations.

Mutations in Drosophila Myosin Rod Cause Defects in Myofibril Assembly

The roles of myosin during muscle contraction are well studied, but how different domains of this protein are involved in myofibril assembly in vivo is far less understood. The indirect flight muscles (IFMs) of Drosophila melanogaster provide a good model for understanding muscle development and function in vivo. We show that two missense mutations in the rod region of the myosin heavy-chain gene, Mhc, give rise to IFM defects and abnormal myofibrils. These defects likely result from thick filament abnormalities that manifest during early sarcomere development or later by hypercontraction. The thick filament defects are accompanied by marked reduction in accumulation of flightin, a myosin binding protein, and its phosphorylated forms, which are required to stabilise thick filaments. We investigated with purified rod fragments whether the mutations affect the coiled-coil structure, rod aggregate size or rod stability. No significant changes in these parameters were detected, except for rod thermodynamic stability in one mutation. Molecular dynamics simulations suggest that these mutations may produce localised rod instabilities. We conclude that the aberrant myofibrils are a result of thick filament defects, but that these in vivo effects cannot be detected in vitro using the biophysical techniques employed. The in vivo investigation of these mutant phenotypes in IFM development and function provides a useful platform for studying myosin rod and thick filament formation generically, with application to the aetiology of human myosin rod myopathies. (C) 2012 Elsevier Ltd. All rights reserved.

Structure-Guided Design and Biophysical Characterization of Novel Anti-HIV Reagents

Despite over 30 years of effort, an HIV-1 vaccine that elicits protective antibodies still does not exist. Recent clinical studies have identified that during natural infection about 20% of the population is capable of mounting a potent and protective antibody response. Closer inspection of these individuals reveal that a subset of these antibodies, recently termed potent VRC01-like (PVL), derive exclusively from a single human germline heavy chain gene. Induced clonal expansion of the B cell encoding this gene is the first step through which PVL antibodies may be elicited. Unfortunately, naturally occurring HIV gp120s fail to bind to this germline, and as a result cannot be used as the initial prime for a vaccine regimen. We have determined the crystal structure of an important germline antibody that is a promising target for vaccine design efforts, and have set out to engineer a more likely candidate using computationally-guided rational design.

In addition to prevention efforts on the side of vaccine design, recently characterized broadly neutralizing anti-HIV antibodies have excellent potential for use in gene therapy and passive immunotherapy. The separation distance between functional Fabs on an antibody is important due to the sparse distribution of envelop spikes on HIV compared to other viruses. We set out to build and characterize novel antibody architectures by incorporating structured linkers into the hinge region of an anti-HIV antibody b12. The goal was to observe whether these linkers increased the arm-span of the IgG dimer. When incorporated, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen binding domains, by contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein, and tetratricopeptide repeats (TPRs). This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the modification and construction of antibodies and other fusion proteins.

T-cell-rich histiocyte-rich B-cell lymphoma of parotid gland

We describe a malignant lymphoma of the parotid gland arising in a patient with Sjögren's syndrome. Diagnosis was established on needle biopsy which showed a mixed population of lymphoid cells. Immunohistochemistry revealed B-lymphoid cells, T-lymphoid cells and histiocytes. Clonal immunoglobulin heavy chain gene rearrangement was demonstrated using the polymerase chain reaction. Within the confines of the small biopsy, the lesion qualifies for the designation T-cell-rich histiocyte-rich B-cell lymphoma. The value of molecular techniques in the diagnosis of malignant lymphoma on limited tissue samples is highlighted by this case.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

Detecção do rearranjo da prpoteína BCL2/JH em carcinomas epidermóides de boca e faringe

The BCL2 protein found in the internal mothocondrial membrana regulates the apoptosis preventing the programmed cell death. The translocation (14:18), detected in 70 to 85% of the follicular lymphoma, lead the super expression of BCL2 protein, by juxtaposition of BCL2 gene to the JH segment of the immunoglobulins' heavy chain gene. However, the found of the BCL2 expression in head and neck carcinoma are contradictious. OBJECTIVE: To investigate the presence of the translocation (14:18) of the BCL2 gene in head and neck carcinoma. METHOD: Sixteen DNA samplers were examinated being 13 of squamous cells carcinoma (SCC) and 3 of epidermoid (CE), y means of chain reaction of the polymerase (PCR). RESULTS: The BCL2/JH rearrangement in 2 (15%) of the CCE 13 cases and in none of the 3 cases of CE. The average of the frequency of molecules with rearrangement was 46,44x107. Was not observed association between the rearrangement presence and the exhibition to tobacco and alcohol (p=0, 6545). CONCLUSION: Different from the results found in follicular lymphoma, the presence of the translocation (14; 18) in head and neck carcinomas is not common and, when it occurs, it can be an occasional mutation not associated to exhibition to the tobacco and alcohol.

Humoral immune response to ADAMTS13 in acquired thrombotic thrombocytopenic purpura

The apparently spontaneous development of autoantibodies to ADAMTS13 in previously healthy individuals is a major cause of thrombotic thrombocytopenic purpura (TTP). Epitope mapping studies have shown that in most patients antibodies directed towards the spacer domain of ADAMTS13 are present. A single antigenic surface comprising Arg(660) , Tyr(661) and Tyr(665) that contributes to the productive binding of ADAMTS13 to unfolded von Willebrand factor is targeted by anti-spacer domain antibodies. Antibodies directed to the carboxyl-terminal CUB1-2 and TSP2-8 domains have also been observed in the plasma of patients with acquired TTP. As yet it has not been established whether this class of antibodies modulates ADAMTS13 activity. Inspection of the primary sequence of human monoclonal anti-ADAMTS13 antibodies suggests that the variable heavy chain germline gene segment VH1-69 is frequently incorporated. We suggest a model in which 'shape complementarity' between the spacer domain and residues encoded by the VH1-69 gene segment explain the preferential use of this variable heavy chain gene segment. Finally, a model is presented for the development of anti-ADAMTS13 antibodies in previously healthy individuals that incorporates the recent identification of HLA DRB1*11 as a risk factor for acquired TTP.