Common Light Chain Research Articles

Secreted human Ro52 autoantibody proteomes express a restricted set of public clonotypes

Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren’s syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.

Generating Bispecific Human IgG1 and IgG2 Antibodies from Any Antibody Pair

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to Surface Antigen 1 ofToxoplasma gondiiTachyzoites

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.

Development of a Two-part Strategy to Identify a Therapeutic Human Bispecific Antibody That Inhibits IgE Receptor Signaling

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcϵRI on mast cells and basophils by cross-linking FcϵRI with the inhibitory receptor FcγRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.

6506 Predictive markers in patients with upper gastrointestinal cancers treated with erlotinib and bevacizumab in a multicenter phase II trial

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.

Structure and Function of Inter-α-Trypsin Inhibitor Heavy Chains

Inter-α-trypsin inhibitor family proteins are mainly detected in plasma and urine and comprise the common light chain bikunin and at least 6 closely related heavy chains. The bikunin moiety exhibits protease inhibitory activity and has been studied extensively; however, the heavy chains have been largely overlooked. Recent studies clearly indicate that the heavy chain moieties have important biological functions either in association with or independent of bikunin. Because the heavy chains comprise the main part of the protein structure of this family, it is important to understand their functions. This review summarizes the domain structural features of heavy chains, the heavy chain-interacting molecules identified thus far, and the association of heavy chains with diseases to encourage the discovery of novel heavy chains-interacting molecules and to gain a deeper insight into their functions.

Equivalent Involvement of Inter-α-trypsin Inhibitor Heavy Chain Isoforms in Forming Covalent Complexes with Hyaluronan

Inter-α-trypsin inhibitor (IαI) family molecules are composed of a common light chain of chondroitin sulfate proteoglycan, bikunin, and one or two of three genetically distinct heavy chain isoforms (designated HC1, 2, 3) that are bound covalently to the chondroitin sulfate chain. Hyaluronan can substitute for chondroitin sulfate to form a covalent complex with HCs. Important physiological and pathological roles of the formation of HC-hyaluronan complex have been well established. However, the involvement of the three HC isoforms in the assembly of IαI family molecules and the subsequent formation of SHAP-hyaluronan complex has not been studied yet in mice. In this study, we showed that mouse IαI and pre-α inhibitor contain HC1∼HC3 and HC3, respectively. All three HC isoforms are found in the SHAP-hyaluronan complexes of physiological or pathological origins as well as that formed in vitro, indicating that the three HC isoforms are all potential in forming complex with hyaluronan.

372. Passive Immunotherapy for Anthrax Mediated by an Adenovirus Expressing Anti-Protective Antigen Single Chain Antibody

Bacillus anthracis was responsible for 45% of deaths in the 2001 US bioterrorism attacks, despite optimal medical therapy. Any delay in treatment of anthrax significantly affects the chances of survival, therefore, rapid therapy is required. Several vaccines have been developed against B. anthracis, but all require multiple administrations to elicit protective immunity. An anthrax vaccine composed of an adenovirus vector expressing the protective antigen (PA) component of anthrax lethal toxin only requires a single administration to be effective in protecting mice against the anthrax lethal toxin, but it takes >2 wk for anti-anthrax immunity to develop. To address the need for rapid protection, this study describes an in vivo gene transfer-based therapy with a human adenovirus (Ad)-based vector (AdαPAscAb) encoding a single chain antibody that neutralizes protective antigen (PA), a critical component of B. anthracis lethal toxin. To examine the expression and secretion of the anti-PA single chain antibody by AdαPAscAb, A549 cells were infected with AdαPAscAb and, as controls, cells were infected with AdαCTXscAb (an Ad vector encoding an irrelevant single chain antibody) or AdNull (an Ad vector with no transgene). Supernatants from cells infected with AdαPAscAb and AdαCTXscAb both contained antibody reactive with the common human Igκ light chain region. Western analysis of purified protective-antigen or lethal factor proteins with supernatants from AdαPAscAb-infected cells showed that the anti-PA single chain antibody specifically recognized PA. The in vivo expression profile of AdαPAscAb was assessed in serum 0 to 14 days after intravenous administration to BALB/c mice by quantifying anti-PA single chain antibody levels by ELISA and anti-PA neutralizing antibody levels. When measured with a PA-specific ELISA, the anti-PA serum antibody titer in mice receiving AdαPAscAb reached significant levels over a 2 wk period (peak reciprocal titer at day 5 = 12,400 ± 8800). Serum from AdαPAscAb-immunized mice, but not from AdαCTXscAb- or AdNull-immunized mice, had neutralizing activity that paralleled the ELISA titers (peak 50% neutralizing titer at day 5 = 260). Mice that had been injected with AdαPAscAb or AdNull were challenged with lethal toxin at 72 hr post-administration, using naive mice as an additional control. Over a seven day observation period, mice that received AdαPAscAb exhibited a significant survival advantage (100%) relative to naive mice (0%) or mice that received AdαCTXscAb (0%) or AdNull (0%). To determine the timing of protection by AdαPAscAb, mice were given a single intravenous administration with AdαPAscAb, AdαCTXscAb or AdNull at -14 to -1 day before, at the time of, or 0.5 days after challenge with lethal toxin. Although mice immunized at the time of challenge or post toxin administration did not survive, mice that were immunized as early as up to 14 days pre-challenge had a survival advantage relative to control animals (90% protection at day 1, 100% protection at day 2 to day 5, 10% protection at day 14). Passive immunotherapy with AdαPAscAb is a rapid, convenient approach to protect against anthrax or to use in conjunction with antibiotic treatment.

Myosin-cell wall interactions during cytokinesis in fission yeast: a framework for understanding plant cytokinesis?

Myosins are associated with the division machinery in plants, animals and fungi. In the fission yeast, Schizosaccharomyces pombe, cytokinesis involves a contractile actomyosin ring (CAR). Constriction of the CAR on the inside of the cell membrane is coordinated with the deposition of the septum, the new cell wall that partitions the two newly created daughter cells, on the outside of the cell membrane. S. pombe possesses five myosins, two type II myosins, Myo2 and Myp2, two type V myosins, Myo51 and Myo52, and a single type I myosin, Myo1 (Win et al., 2002). Myo2, Myp2 and Myo51 are components of the cytokinetic actin ring (CAR) while Myo1 and Myo52 are involved in the deposition of the septum. The accumulation of all five myosins at the division plane in M-phase is a function of the septation initiation network (SIN), a signal transduction pathway that couples cytokinesis to mitotic exit. The targets of SIN pathway remain to be defined but probably include Myo2 (Mulvihill et al., 2001) and the 1-3-glucan synthase Cps1 which is involved in the deposition of the septum (Liu et al., 2002). We have developed a new procedure to directly examine the spatial and temporal relationships between CAR formation and contraction and septum formation in living fission yeast cells expressing GFP-tagged Myo2 in combination with the cell wall stain Calcofluor. In wild type cells, CAR contraction is a continuous, smooth process and the centripetal deposition of the septum precisely follows the constricting ring. The dynamics of CAR contraction is identical in cells lacking either Myp2 or Myo51. In the absence of the type V myosin, Myo52, however, CAR contraction is slower and discontinuous. These findings demonstrate the interdependence of CAR contraction and septum deposition and show that Myo52 links the two processes. The class VIII myosins in plants may play a comparable role in the organisation and deposition of new cell wall material at the cell plate (Reichelt et al., 1999). Myo2 and Myp2 have no role other than cytokinesis. The myo2 is an essential gene. Germinating myo2 spores go through a few cell divisions forming short filaments with septa that fail to cleave (May et al., 1997). Cells lacking Myp2 show only a mild cytokinetic defect (Mulvihill et al., 2000). Myo2 and Myp2 contain 1527 and 2104 amino acids, respectively, the difference being entirely due to the length of the -helical tail. The two type II myosins share common essential and regulatory light chains, Cdc4 and Rlc1, respectively. Myo2 is believed to be a conventional two-headed dimer while Myp2 is thought to be monomeric (Bezanilla and Pollard, 2000). The Myo2 tail contains nine proline residues dispersed throughout the structure whereas the Myp2 tail contains 25 prolines, 19 of which are concentrated at the centre, dividing the tail into two subdomains which are thought to fold back on one another to form an anti-parallel coiled coil (Bezanilla and Pollard, 2000). Since we were unable to find an obvious function for Myp2 we wondered whether other fungi that divide by formation of a cytokinetic septum also possessed a second type II myosin. We therefore screened the genome projects of Aspergillus fumigatus, Neurospora crassa for myosin sequences. In each case three myosin genes were found, one each of myosin types I, II and V. This might represent the minimum number of myosins required to sustain cell function eukaryotic cells other than plants which have their own unique myosin inventory (Reddy and Day, 2001). Inspection of the tails of the type II myosins revealed that both were Myp2-like in both length and in the number and disposition of proline residues. A. fumigatus Myo2 contains 2409 amino acids and has 15 prolines within its * Corresponding author. Tel.: +44-20-7679-7343; fax: +44-20-7679-7343. Cell Biology International 27 (2003) 239–240 Cell Biology International

Microtubule-associated protein 1 subunit expression in primary cultures of rat brain

Microtubule-associated protein 1A (MAP1A) and MAP1B are developmentally regulated proteins linked to axon formation. They each consist of a unique heavy chain and three common light chains. We used immunofluorescence microscopy to qualitatively assess the variability in MAP1 subunit expression between individual cells. The ratio of light chain 1 to MAP1 heavy chain varies greatly between cells with some cells expressing MAP1A heavy chain in the apparent absence of light chain 1. The results imply the existence of MAP1 molecules that differ in light chain composition. Transfection experiments indicate that the light chains differ in microtubule binding activity and subcellular targeting activity. This further suggests that the regulation of MAP1 light chain content can control MAP1 function.

Molecular mechanism of myosin-II assembly at the division site in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells divide by virtue of the F-actin-based contractile ring (F-actin ring). Two myosin-II heavy chains, Myo2 and Myp2/Myo3, have been localized to the F-actin ring. Here, we investigated the mechanism of myosin-II assembly at the division site in S. pombe cells. First, we showed that Cdc4, an EF-hand protein, appears to be a common myosin light chain associated with both Myo2 and Myo3. Loss of function of both Myo2 and Myo3 caused a defect in F-actin assembly at the division site, like the phenotype of cdc4 null cells. It is suggested that Myo2, Myo3 and Cdc4 function in a cooperative manner in the formation of the F-actin ring during mitosis. Next, we investigated the dynamics of myosin-II during mitosis in S. pombe cells. In early mitosis when accumulation of F-actin cables in the medial region was not yet observed, Myo2 was detected primarily as dots widely located in the medial cortex. Myo2 fibers also became visible following the appearance of the dots. The Myo2 dots and fibers then fused with each other to form a medial cortical network. Some Myo2 dots appeared to be localized with F-actin cables which are also accumulated in the medial region. Finally these structures were packed into a thin contractile ring. In mutant cells that cannot form the F-actin ring such as cdc3(ts), cdc8(ts) and cdc12(ts), Myo2 was able to accumulate as dots in the medial cortex, whereas no accumulation of Myo2 dots was detected in cdc4(ts) cells. Moreover, disruption of F-actin in the cell by applying latrunculin-A did not affect the accumulation of Myo2 dots, suggesting that F-actin is not required for their accumulation. A truncated Myo2 which lacks putative Cdc4-binding sites (Myo2dIQs) was able to rescue myo2 null cells, myo3 null cells, cdc4(ts) mutant cells and cdc4 null cells. The Myo2dIQs could assemble into a normal-shaped ring in these cells. Therefore, its assembly at the division site does not require the function of either Cdc4 or Myo3.

An efficient route to human bispecific IgG.

Production of bispecific IgG (BsIgG) by coexpressing two different antibodies is inefficient due to unwanted pairings of the component heavy and light chains. To overcome this problem, heavy chains were remodeled for heterodimerization using engineered disulfide bonds in combination with previously identified "knobs-into-holes" mutations. One of the variants, S354C:T366W/Y349'C:T366'S:L368'A:Y407++ +'V, gave near quantitative (approximately 95%) heterodimerization. Light chain mispairing was circumvented by using an identical light chain for each arm of the BsIgG. Antibodies with identical light chains that bind to different antigens were identified from an scFv phage library with a very restricted light chain repertoire for the majority (50/55) of antigen pairs tested. A BsIgG capable of simultaneously binding to the human receptors HER3 and cMpI was prepared by coexpressing the common light chain and corresponding remodeled heavy chains followed by protein A chromatography. The engineered heavy chains retain their ability to support antibody-dependent cell-mediated cytotoxicity as demonstrated with an anti-HER2 antibody.

The carboxyl terminus of bradykinin and amino terminus of the light chain of kininogens comprise an endothelial cell binding domain.

Bradykinin (BK), a potent vasoactive nonapeptide formed by proteolytic cleavage of kininogen, mediates its activity by binding to specific cell surface receptors. Delivery of BK to these receptors is limited by cell-bound and plasma kininases that degrade BK to inactive fragments. Binding of its parent compound, kininogen, to specific endothelial cell receptors may provide an environment in which the degradation of BK by kininases is restricted. The determinants that mediate kininogen binding to endothelial cells have not been fully elucidated. The present studies suggest that part of BK and the amino-terminal amino acids of kininogens' common light chain constitute part of this recognition sequence. Human umbilical vein endothelial cells (HUVEC) in culture at 37 degrees C express 2-3-fold more binding sites for biotinylated high molecular weight kininogen (biotin-HK) containing BK than for biotin-HK from which BK has been liberated by plasma kallikrein. Binding of BK-free biotin-HK was not restored by preincubating HUVEC with BK, arguing against the possibility that BK released from biotin-HK stimulated expression of additional HK receptors. The extent of biotin-HK binding to HUVEC at 37 degrees C directly correlated with the amount of BK retained within the protein. Four lines of evidence suggest that part of BK and the amino terminus of the light chain of kininogens are part of the sequence recognized by the endothelial cell kininogen receptor. First, monoclonal antibodies to the carboxyl terminus of BK (MBK3) and the common light chains of the kininogens (HKL6, HKL8) inhibited biotin-HK binding to HUVEC. Second, a synthetic, dimeric bradykinin receptor antagonist blocked biotin-HK (IC50 = 9 microM) binding to HUVEC as did two synthetic tissue kallikrein inhibitors modeled after the carboxyl-terminal sequence of BK. Third, synthetic peptides containing the carboxyl-terminal portion of BK and the amino terminus of kininogens' common light chain, MKRPPGFSPFRSSRIG and GFSPFRSSRIG, blocked binding of biotin-HK (IC50 = 2-3 mM), whereas an overlapping peptide, SPFRSSRIGEIKEETT, at 5 mM and a scrambled peptide, FSGPKRSPIMGRPSFR, did not. Fourth, biotinylated GMKRPPGFSPFRSSRIG specifically bound to HUVEC, and its binding was blocked by HK. Since the presence of the nonapeptide BK in HK contributes to maximal binding of HK to HUVEC, there is a novel type of BK receptor in which part of the nonapeptide is recognized within the context of its parent molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

The MHC Class I Associated Beta2-microglobulin (beta2m) Light Chain is Expressed in a Molar Excess Over HLA-ABC and CD1 on the Membrane of Leukaemic B Cells but not Leukaemic T Cells: Evidence for Further beta2m-Associated Molecules

Beta 2-microglobulin (beta 2m) constitutes the common light chain of both the MHC-encoded HLA-ABC molecules and a group of structurally related glycoproteins recognized by antibodies of the first cluster of differentiation (CD1a, CD1b and CD1c). These CD1 antigens appear similar to murine T1 and Qa molecules in terms of structure and tissue distribution, although the question of inter-species homology is controversial. A further group of alloantigens expressed predominantly on T cells has been reported however, with immunogenetic characteristics more closely analogous to the murine T1/Qa system than the CD1 antigens, although their precise identity remains ill-defined. Having previously shown that malignant B cells may express membrane CD1c, we examined leukaemic B-cells corresponding to early lymphoblastic differentiation (null- and common acute lymphoblastic leukaemia) through to the terminal plasma cell stage for the expression of other non-HLA class I beta 2m-associated molecules. It was found that leukaemic B-cells at intermediate/late stages of differentiation, represented by non-Hodgkin's lymphoma (B-NHL) and 'hairy-cell' leukaemia (HCL), had significantly higher beta 2m:HLA-ABC ratios than did the cells from other types of B-cell malignancy. Although leukaemic B cells with a demonstrable non HLA-ABC-associated beta 2m component expressed detectable levels of CD1c, and insignificant levels of CD1a and CD1b, the antigen density was insufficient to account for the excess beta 2m. In vitro stimulation of leukaemic B cells by phorbol ester substantially increased the expression of HLA-ABC and CD1c, but also accentuated further the difference between the expression of these molecules and that of beta 2m. There was no detectable beta 2m other than that associated with HLA-ABC and CD1 on the surface of malignant T cells by contrast. Our findings strongly support the existence, at certain stages of leukaemic B-cell differentiation, of an additional beta 2m component(s) other than that associated with HLA-ABC and CD1.

Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin.

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

Heterogeneous malignant non Hodgkin's lymphomas as a causative disorder in lethal midline granuloma

The present report describes the results of a combined morphological, enzyme- and immunohistochemical analysis of nine cases of malignant non Hodgkin's lymphomas (NHL) clinically presenting as lethal midline granuloma. In a previous report written before antibodies directed against B and T lymphocytes were available, a histiocytic origin of such neoplasms had been suggested. A panel of antibodies reactive with most B cells (L26, MB1, KiB3) and a majority of T cells (MT1, UCHL1) was applied on paraffin sections of formalin fixed tissues as well as antibodies directed against leukocyte common antigen (LCA), myeloid/histiocyte antigen (MAC 387), lysozyme, alpha-1-antitrypsin, alpha-1-antichymotrypsin, S-100 protein, prekeratin and immunoglobulin light chains. Enzyme histochemistry included tests for non-specific acid esterase, acid phosphatase, beta-glucuronidase and chloroacetate esterase. As a result, five T, two B and two unclassified (malignant histiocytosis probable) NHL were identified, indicating distinct heterogeneity of NHL as causative disorders in lethal midline granuloma.

A common myosin light chain is expressed in chicken embryonic skeletal, cardiac, and smooth muscles and in brain continuously from embryo to adult.

We have isolated cDNA clones of the mRNA for chick embryonic myosin light chain (MLC), L23, by cross-hybridization with chicken skeletal muscle MLC1 cDNA. The identification of the isolated cDNAs was carried out by in vitro translation of hybrid-selected mRNA. Sequence analysis of the cloned cDNAs revealed that the cDNA insert contained 832 nucleotides and predicted a polypeptide of 185 amino acids with a calculated molecular weight of 20,687. The deduced amino acid sequence for L23 showed high sequence similarities to those of adult alkali type MLCs from various tissues, indicating that L23 belongs to the alkali MLC group. Using the cloned cDNA as a hybridization probe, we have revealed by RNA blot analysis that the expression of L23 mRNA was regulated in temporal and tissue-specific manners. The L23 mRNA of 1.1 kilobases is transiently expressed in embryonic skeletal, cardiac, and smooth muscles of chickens. It is also found in the brain of chickens during all stages of development so far investigated. Only a single gene for L23 was detected by Southern blot of chick genomic DNA. We therefore suggest that L23 is expressed from a single gene in both embryonic muscles and brain.

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.

Multiple VH gene segments encode murine antistreptococcal antibodies.

Most mouse strains are able to mount a diverse antibody response against group A streptococcal carbohydrate (GAC). We have previously reported that murine anti-GAC antibodies are for the most part restricted to IgM and IgG3 subclasses. In addition, despite extensive heterogeneity in their isoelectric focusing patterns, greater than 50% of A/J anti-GAC antibodies share a common light chain defined by spectrotypic and idiotypic (VK1GAC) criteria. We have used protein and DNA sequencing strategies to examine the genetic basis of diversity in murine anti-GAC antibodies. In particular, we report that, (a) multiple, closely homologous VH gene segments contribute to the generation of anti-GAC antibodies, (b) a common framework sequence, related to the VK27 subgroup, probably defines VK1GAC, and (c) the A/J anti-GAC VH regions and BALB/c anti-inulin VH sequences are 95% homologous at the protein level and are likely encoded by overlapping VH gene families. Lastly, we discuss the genetic mechanisms that might permit the evolution of multiple, closely homologous germline VH gene segments in the context of highly divergent flanking region sequences.

Restricted structural heterogeneity in antibodies: might different heavy chains have a common light chain?

MOST structural studies of immunoglobulins have utilized homogeneous myeloma proteins because antibodies elicited during conventional immunization are a heterogeneous population of molecules. Hyperimmunization with streptococcal vaccines1 has been used to obtain large quantities of antibody of known specificity and “restricted structural heterogeneity” for studies on the structural basis of antibody function and on the genetic basis of the immune response1,2. In the course of these studies we have characterized an antibody which seems to have a single light chain associated with two or more different heavy chains.