Isolation of human monoclonal antibodies that bind to two different antigens and are encoded by germline VH and VL genes.

This paper reports isolation of two monoclonal antibodies (mAbs) that bind to both a membrane protein and a cytoplasmic protein. Most Abs established as markers for autoimmune disease bind to cytoplasmic or nuclear substances. However, it remains unknown how these Abs are produced. On the other hand, there were examples where clones originally isolated as Abs that bind to membrane proteins also showed binding activity to cytoplasmic or nuclear substances. Based on these results, the following hypothesis has been proposed. The Abs that had been originally produced against a membrane protein showed cross-reactivity against cytoplasmic or nuclear substances. In the present study we reported isolation of Abs that bound to both a membrane protein, CADM1, and a cytoplasmic protein, α-actinin-4. The method adopted in the present study could be generally applicable to isolation of Abs showing such dual specificity. Firstly, we constructed a huge human Ab library using various organs including naïve B-cell-rich organs such as bone marrow and umbilical cords. Then, we developed a comprehensive screening method for isolation of Abs that bound to cell surface antigens. Through extensive screenings with many kinds of cell we newly obtained a library composed of around 4000 independent clones that bind to membrane proteins. We screened this library with α-actinin-4 and succeeded in isolating two Abs. They bound to α-actinin-4 and a membrane protein CADM1. Furthermore, they are encoded by naïve heavy and light chain variable genes (VH & VL). These results suggested that cross-reactive Abs to both a membrane protein and a cytoplasmic protein could be present in germline repertoire of Ab in humans. This methodology adopted in the present study could be applied to isolation of cross-reactive Abs possibly involved in autoimmune diseases.

[Dissection of descending aorta with spinal paralysis].

A 56-year-old man had undergone ascending aorta and total arch replacement because of aortic dissection (Stanford type A) in 1997. He had onset of diplegia of the lower limb and vesicorectal disability. Computed tomography (CT) showed serpentine aneurysm in the descending aorta, it was seen between the left subclavian artery and diaphragm level. It was 80 mm of maximum diameter. Magnetic resonance imaging (MRI) was performed for identified Adamkiewicz artery, but we could not identify it. We performed a graft replacement. The 8th intercostal artery was reconstructed with a branch graft. The postoperative course was uneventful. We conclude that graft replacement for spinal ischemia can be effective.

[Surgical treatment with the freestyle bioprosthesis and omentopexy for prosthetic valve endocarditis after aortic root replacement].

A 60-year-old woman, who had undergone aortic root replacement with composite graft 5 months previously, suffered from anemia and slight fever. Transthoracic echocardiography showed pseudoaneurysm in the aortic root, and blood culture was positive. She was diagnosed with prosthetic valve endocarditis, and surgical intervention was planned. Intraoperatively necrotic tissue and dehiscence of the suture line in the aortic annulus were found. Re-aortic root replacement with Freestyle bioprosthesis and re-hemiarch graft replacement were performed with the omentopexy around the aortic root and the new graft. Antibiotics were administered intravenously for 6 weeks postoperatively. At 7 months after the operation, no prosthetic valve infection had recurred. Although the long-term results of Freestyle bioprosthesis have not been determined, it might be a valuable option for aortic root infection as an alternative to an aortic homograft. In addition, omentopexy might also be effective in the prevention of recurrent prosthetic valve infection.

Expression vectors for the introduction of highly diverged sequences into the six complementarity-determining regions of an antibody.

We prepared three kinds of phagemid vector that permit the simultaneous introduction of highly diverged sequences into six complementarity-determining regions (CDRs) of an antibody (Ab) by the polymerase chain reaction (PCR) with degenerate oligodeoxynucleotide (oligo) primers. The phages expressed either the Fv, single-chain Fv (sc Fv) or Fab form of an Ab fused with a half-molecule of cpIII on the surface of M13 phage. A phage-display library, composed of 2 x 10(8) independent clones, was constructed; the phages that were specific for hen egg-white lysozyme (HEL) were selected by three rounds of panning; and 20 clones were isolated. The isolated clones consisted of 17 different clones. Among them, 16 clones expressed proteins that were able to bind to HEL. The association constants for binding of the encoded proteins to HEL ranged from 1.48 x 10(6) to 7.71 x 10(6)/M. These vectors allowed us to prepare many libraries of artificial Ab in which the sequences of six CDRs were very different and reflected the artificial sequences that had been designed for the degenerate oligo that we used as primers for PCR. The libraries should be also useful for the analysis of relationships between the sequences of the CDRs and antigen (Ag) specificity.

Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

Preparation of mouse-human chimeric antibody against pancreatic cancer-associated mucin.

A murine monoclonal antibody, designated Nd2, was generated using a purified mucin from the human pancreatic cancer cell line SW1990 as the immunogen. Administration of Nd2 into the patients with pancreatic cancer has shown that Nd2 is of clinical use for in vivo diagnostics of pancreatic cancer. In this study, starting with a previously established hybridoma producing Nd2, we have established a transfected cell line expressing a recombinant chimeric Nd2 composed of mouse-derived antigen-binding variable regions and human-derived constant regions. The chimeric Nd2 was shown to bind purified mucin from SW1990 cells with the same dose-response curve as the original Nd2. Immunohistochemical experiments have shown that the reactivity of the chimeric Nd2 in normal and malignant pancreatic tissue is also the same as that of the original Nd2. Replacement of a substantial portion of the mouse protein makes this chimeric Nd2 more promising for clinical application than the original mouse-derived Nd2 because its antigenicity is expected to be reduced.

Preparation of mouse-human chimeric antibody to an embryonic carbohydrate antigen, Lewis Y.

Stage-specific embryonic antigen-1 (SSEA-1) is a well-known carbohydrate antigen that is specifically expressed on the surface of cancer cells as well as embryonic cells. In this study, starting with a previously established hybridoma producing a monoclonal antibody (named H18A) to Lewis Y antigen, which is closely related to SSEA-1, we cloned genomic DNA encoding active variable regions both of heavy and light chains of the antibody. Sequence analysis showed that VH and V kappa genes of H18A were in the VH 7183 family and V kappa C1 family, respectively. A transfected cell line named HC-H18A-7 expressing a recombinant chimeric H18A composed of mouse-derived antigen-binding variable regions and human-derived constant regions was established. The chimeric H18A was purified to homogeneity and shown to bind purified Lewis Y antigen with the same dose-response curve as the original H18A. The chimeric H18A looks more promising for clinical application than the original mouse-derived H18A because its antigenicity is expected to be reduced.

Newly developed left main coronary artery lesion after coronary stenting.

Percutaneous transluminal coronary stenting is a proven nonoperative method of direct myocardial revascularization. We encountered a case of iatrogenic significant subacute left main coronary artery stenosis in a patient who had undergone prior percutaneous transluminal coronary artery stenting of the left anterior descending (LAD) artery. Coronary artery bypass grafting was performed. To our knowledge, this is the first report of a surgical case of left main coronary artery stenosis worsened by changes secondary to earlier coronary stenting in the mid portion of left descending coronary artery in Japan.

Effects of levocabastine on lipid mediator release from guinea pig lung fragments.

The effects of levocabastine, a novel histamine H1-receptor antagonist, on lipid mediator release induced by antigen-antibody reaction from actively sensitized guinea pig lung fragments were studied. Levocabastine dose-dependently inhibited the release of leukotriene C4 from guinea pig lung fragments induced by antigen. A significant effect was observed with levocabastine at a concentration of 10(-4) M. On the other hand, levocabastine produced no effect on the release of leukotriene E4 or thromoboxane B2. From these findings, it was concluded that levocabastine may be useful for relieving the nasal obstruction in allergic rhinitis caused by inhibition of leukotriene C4 release.

Participation of mast cells in colitis inflammation induced by dextran sulfate sodium.

We investigated the participation of mast cells in colitis inflammation induced by dextran sulfate sodium (DSS). The damage score and myeloperoxidase (MPO) activity were measured to confirm the occurrence of colitis. Rat mast cell protease (RMPCP) II levels in the serum were estimated as an index of mast cell degranulation. Tissue RMCP I and RMCP II levels in the rectum were also measured as markers of the numbers of connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs), respectively. Administration of 4% DSS resulted in time-related increases in damage score, MPO activity and serum RMCP II levels, which were statistically significant at 7 and 11 days after treatment. Tissue RMCP I and RMCP II levels in the rectum were also increased significantly at 7 and 11 days, and 11 days, respectively after free drinking of 4% DSS. These results suggested that mast cells proliferated or the amount of protease per mast cell increased in the sites of inflammation induced by DSS, and that these mast cells may modulate the disorders observed in DSS-induced colitis.

[Surgical treatment for aortic aneurysms associated with coronary artery disease-selection of bypass conduit].

Graft replacement for arch aneurysms and concomitant coronary artery bypass grafting (CABG) were performed in four consecutive patients over a three-year period between March 1995 and October 1998. The etiology of the aneurysms was atherosclerosis in all the patients. One early death as a result of a cerebellar infarction occurred on the 74th postoperative day. In all cases, respiratory failure frequently occurred after surgery. In a recent case, the internal mammary artery was used as a graft conduit to the left anterior descending artery (LAD). Both artery and vein grafts were anastomosed to coronary arteries during the initial core cooling. Selective cerebral perfusion was carried out during the reconstruction of the transverse aortic arch and its branches. The left subclavian artery was anastomosed first to secure perfusion to the LAD. To achieve sufficient myocardial protection and obtain good postoperative hemodynamics, CABG was simultaneously performed at the time of aortic aneurysm repair in cases complicated with coronary artery disease.

[A case of protruding atherosclerotic plaque with mobile projections in the sino-tubular junction].

A 66-year-old woman who received maintenance hemodialysis for the last 14 years was referred to our hospital due to the sudden onset of ischemic stroke and systemic emboli. Transthoracic echocardiography before surgery showed large protruding masses measuring 2 cm which projected into the lumen of the aortic root. The plaque originated on a wide base of the posterior aspect of the ascending aorta at its junction with the sinus of Valsalva. The mobile plaque was surgically removed by endarterectomy from the posterior wall of sino-tubular junction under cardiopulmonary bypass. Pathologic examination of the masses removed at the time of surgery showed that they were atherosclerotic plaque with superimposed thrombi. During operation, transesophageal echocardiography appears to be useful for the rapid detection of a protruding aortic atheroma especially in the initial period of cardiopulmonary bypass until aortic cross clamp.

Molecular characterization of Borrelia persica, the agent of tick borne relapsing fever in Israel and the Palestinian Authority.

The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5'end of the 16S rRNA gene to the 5'end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5' fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area.

Comparison of strategies for the construction of libraries of artificial antibodies.

Construction of libraries of artificial antibodies has been reported by several groups of investigators. Various forms of antibody fused to surface protein, cpII, are expressed on the surface of filamentous phage. Since phages that encode desired antibodies can be easily grown in Escherichia coli after selection with target antigens, the phage-display antibody system appears to be very useful for various biological purposes. In this brief review, recent progress in research into the production of artificial antibodies is summarized and the strategies used for the construction of libraries of artificial antibodies are compared.

Effects of substitutions of closely related amino acids at the contact surface in an antigen-antibody complex on thermodynamic parameters.

We constructed a library of 512 kinds of Fv fragment, derivatives of a monoclonal antibody, D1.3, specific for hen egg-white lysozyme, in which a total of nine of the original amino acids were replaced by closely related amino acids at positions in the complementarity-determining regions of the H chain. More than 80% of the clones in the library produced Fv fragments in Escherichia coli. Two wild-type and 13 mutant Fv fragments were prepared in large quantities and subjected to analysis by differential titration calorimetry. The association constants of the 15 Fv fragments with hen egg-white lysozyme were distributed between 0.12 x 10(7) and 1.59 x 10(8) M-1. The changes in delta H0 and -T delta S0 caused by one-point mutation at each position did not have intrinsic values for each change. The same changes at one position had different effects on KA, delta H0, and -T delta S0 when differences had been introduced in other regions. The delta(delta G0) caused by a single-point mutation ranged from -0.56 to 1.56 kcal/mol. By contrast, the delta(delta H0) and delta(-T delta S0) caused by a single-point mutation ranged from -3.5 to 3.4 and from -3.8 to 3.4 kcal/mol, respectively. When antibodies gain the binding energy contributed by the effects of enthalpy, they lose the binding energy contributed by the effects of entropy and vice versa. In general, changes in entropy compensate for changes in enthalpy.

L-leucine and its analogue: specific inhibitors for S-benzyl-L-cysteine-p-nitroanilide-hydrolyzing enzyme in Escherichia coli B.

An enzyme that catalyzes hydrolysis of S-benzyl-L-cysteine-p-nitroanilide was purified from E. coli B. The enzyme was a monomer with a molecular weight of 82,000. In addition to L-cysteinylglycine, the enzyme hydrolyzed various glycine-containing dipeptides most efficiently at pH 7.0. The enzyme required no metal ions for activity and was specifically inhibited by L-leucine and its analogue with free carboxyl group at the physiological concentrations.

Influences of everninomicin, vancomycin and teicoplanin on chemical mediator release from rat peritoneal mast cells.

We examined influences of certain antibiotics on the release of chemical mediators from isolated rat peritoneal mast cells in vitro. Isolated peritoneal mast cells were obtained from male Wistar strain rats. Everninomicin (0.06-1.2 mg/ml), vancomycin (0.05-1.0 mg/ml), teicoplanin (0.07-1.4 mg/ml) and concanavalin A (0.01 mg/ml) were used. Isolated mast cells were incubated in the presence of various concentrations of the test compound at 37 degrees C for 10 min. Histamine contents of the supernatant and cell pellet were measured by an automated fluorometric method. Prostaglandin D2 (PGD2) contents of the supernatant were determined by enzyme immunoassay. Everninomicin (0.06-1.2 mg/ml) had no influence on histamine and PGD2 release from mast cells. On the other hand, vancomycin significantly released both histamine (0.5 and 1.0 mg/ml) and PGD2 (1.0 mg/ml) from mast cells, but vancomycin did not affect concanavalin A-induced histamine and PGD2 release. Teicoplanin (more than 0.07 mg/ml) significantly stimulated histamine and PGD2 release from mast cells and it also significantly potentiated concanavalin A-induced histamine and PGD2 release. These results suggest that everninomicin causes no chemical mediator release from mast cells, different from vancomycin and teicoplanin.

Papillary fibroelastoma in association with thrombosis on a mechanical valve.

A patient with a thrombosed mechanical valve underwent valve re-replacement during which a tumor of the left ventricular outflow tract with the typical macroscopic and microscopic characteristics of a papillary fibroelastoma was successfully removed surgically. The 60-year-old woman had undergone isolated mitral valve replacement with a St Jude Medical 29-mm valve for mitral regurgitation 15 years ago. The present admission was for investigation of dyspnea on exertion. Two-dimensional transthoracic echocardiography demonstrated a posteroseptal, pedunculated mass, measuring 1.3x1.0 cm, in the outflow tract of the left ventricle, mild mitral regurgitation and slight aortic stenosis.

Pruritus-associated response mediated by cutaneous histamine H3 receptors.

BACKGROUND: Histamine is one of the most common chemical mediators causing pruritus, and H1 receptor antagonists have been used as a first choice in its treatment. On the other hand, although the presence of H3 receptors has been identified in the skin, few studies have investigated the involvement of H3 receptors on pruritus. OBJECTIVE: The purpose of this study was to examine whether H3 receptor agonist or antagonist influences the incidence of scratching behaviour in ICR or mast cell-deficient WBB6F1-W/WV mice. METHODS: The mice were given an intradermal injection of H3 receptor agonist or antagonist into the rostral part of the back, and the occurrence of scratching behaviour at the injected site by the hind paws was counted over 60 min. RESULTS: H3 receptor antagonists, thioperamide and AQ0145 significantly increased the incidence of scratching behaviour in ICR mice. H3 receptor agonist, (R)-alpha-methylhistamine, had no effect. On the other hand, (R)-alpha-methylhistamine significantly inhibited thioperamide or AQ0145-induced scratching behaviour. In addition, both thioperamide and AQ0145 elicited scratching behaviour in mast cell-deficient WBB6F1-W/WV mice. CONCLUSION: From these results, it may be concluded that H3 receptors are involved in the modulation of pruritus in the skin, and mast cells are not essential in this response. In addition, H3 receptor agonists can be useful as a novel therapeutic approach against pruritus.

Recurrent aortic valve endocarditis caused by Gemella morbillorum--report of a case and review of the literature.

Gemella morbillorum (G. morbillorum) is part of the commensal flora of the oropharynx and intestinal tract, and on rare occasions causes infective endocarditis. A 55-year-old man with massive aortic regurgitation caused by recurrent infective endocarditis with G. morbillorum had a history of prior endocarditis caused by alpha-hemolytic streptococcus and multiple antibiotic allergies 5 years prior, and was successfully treated by aortic valve replacement. Almost all the reported cases of endocarditis caused by G. morbillorum have been bacteriologically cured with antibiotics and this is the first reported case of recurrent endocarditis caused by G. morbillorum in which the initial infection was bacteriologically cured by antibiotics and the secondary infection treated with valve replacement. This organism can be one of the causes of infective endocarditis and prompt surgical repair is mandatory if the infection is refractory or there is progression of congestive heart failure under antibiotic cover.