Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments.

BACKGROUND: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. RESULTS: In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. CONCLUSION: The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.

In vivo administration of mycobacterial cord factor (Trehalose 6, 6'-dimycolate) can induce lung and liver granulomas and thymic atrophy in rabbits.

Trehalose 6,6'-dimycolate (TDM) is a cell surface molecule of Mycobacterium tuberculosis. TDM induced a loss of body weight and prominent granulomas in the liver and lungs by the intravenous injection of TDM into rabbits. TDM also induced atrophy of the thymus and spleen due to apoptosis. By contrast, sulfolipid (2,3,6, 6'-tetraacyl trehalose 2'-sulfate) induced neither toxicity, nor granuloma formation, nor atrophy of the thymus and spleen. In rabbits the histopathological changes were more dramatic than in mice. The rabbit model may be more sensitive and may provide more information on the beneficial or pathological effects of TDM.

Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection.

The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA tests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WB. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test.

CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli.

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.

Development of the insulin-like growth factor-I assay system.

A high speed full automatic ELISA system for measurement of insulin-like growth factor-I (IGF-I) was established by using magnetic particle-linked monoclonal antibody and enzyme-labeled monoclonal antibody. A standard curve was obtained, and the effect of dilution on the assay system was investigated. An IGF-I spike recovery test of human serum samples and a study of the correlation with a radioimmunoassay system were performed, and good results were obtained from all studies. The assay range was 0.5-50 ng/ml, and the time required for the full automatic measurement was 15 minutes. This assay system will play a central role in the clinical approach to IGF-I.

Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium.

An ELISA with cord factor (trehalose-6,6'-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6'-tetraacyl trehalose 2'-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria.

Ouabain-like immunoreactivity in the medulla oblongata of rats.

An isomer of ouabain, the ouabain-like compound (OI,C), may participate in the regulation of body fluid volume and vascular tone. Forebrain regions, especially the hypothalamus, are reported to be sites of OLC action in the central nervous system. The medulla oblongata is another critical area involved in central cardiovascular regulation. We reported that the microinjection of either monoclonal antibody to ouabain T8B11 or Fab fragment of digoxin-specific antibody into the rostral ventrolateral medulla significantly decreased mean arterial pressure and renal sympathetic nerve activity in anesthetized normotensive rats (TERUYA et al.: J. Clin. Invest. 99: 2791-2798, 1997). Using T8B11, we examined the ouabain-like immunoreactivity in the medulla oblongata of normotensive rats. In periodate-lysine-paraformaldehyde fixed tissues, ouabain-like immunoreactive neurons were detected in the nuclei and regions in the medulla oblongata including the ventrolateral medulla, ventromedial medulla, nucleus ambiguus, caudal raphe nuclei, nucleus of solitary tract, and dorsal motor nucleus of the vagus. When an Fab fragment of digoxin-specific antibody was used as a first antibody, the digoxin-like immunoreactive neurons were distributed in almost the same pattern as those observed with the use of T8B11. In the brain fixed with the "three-step" procedure developed by YAMADA et al. (1987), which was used in a previous ouabain immunohistochemical study of the hypothalamus, ouabain-like immunoreactivity in the medulla oblongata was much weaker in intensity and less restricted in distribution than that in the hypothalamus. These findings suggest that ouabain-like immunoreactivities are present in the medulla oblongata with a manner of distribution different from that seen in the hypothalamus. Some ouabain-immunopositive nuclei and regions in the medulla oblongata, especially the rostral ventrolateral medulla, may be other OLC action sites.

Elevation of ouabainlike compound levels with hypertonic sodium chloride load in rat plasma and tissues.

A major biologically active endogenous digitalis-like factor in the mammalian body may be an isomer of ouabain (ouabainlike compound, OLC). However, the exact role of OLC in sodium homeostasis is still unclear, and acute isotonic volume expansion does not enhance the secretion of OLC. We tested the hypothesis that OLC may be more important in the response to acute hypertonic NaCl load rather than isotonic volume expansion. We injected intraperitoneally 2 mL of 20% NaCl solution into male Wistar rats (n=34) and measured OLC levels in plasma, hypothalamus, pituitary, and adrenal at baseline (n=10) and 1, 2, and 4 hours (n=8 for each). In response to hypertonic NaCl loading, plasma Na-K ratio was elevated at 2 and 4 hours (P<.01). OLC levels in pituitary increased (P<.01) at 1 hour. Thereafter, plasma OLC levels increased at 2 and 4 hours (P<.05; basal, 75+/-11 pmol/L [+/-SEM]; 1 hour, 55+/-11; 2 hours, 130+/-24; 4 hours, 156+/-20). Concomitantly, OLC levels in adrenal increased at 2 and 4 hours (P<.01; basal, 1.7+/-0.2 pmol/g; 1 hour, 4.5+/-0.9; 2 hours, 5.0+/-0.7; 4 hours, 6.8+/-2.2). A significant correlation was observed between OLC levels in plasma and adrenal (P<.05). Plasma Na-K ratio positively correlated with OLC levels in plasma (r=.51, P<.01) and adrenal (r=.48, P<.01). Similar injection of physiological saline solution or hypertonic sucrose solution in physiological saline did not increase OLC levels in plasma and tissues. These findings indicate the elevation of OLC levels in plasma, pituitary, and adrenal in response to acute hypertonic NaCl load in rats and suggest that OLC may be involved in the response to the hypernatremic state.

Role of ouabain-like compound in the rostral ventrolateral medulla in rats.

To determine whether ouabain-like compound (OLC) exerts modulatory influences on the activity of vasomotor neurons in the rostral ventrolateral medulla (RVLM), we examined the effects of microinjecting ouabain, digoxin-specific antibody Fab fragments, and mAb against ouabain on the rat RVLM. Microinjection of ouabain into the unilateral RVLM of anesthetized normotensive rats elicited dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). The pressor and sympathoexcitatory effects of ouabain in the RVLM were reversed by microinjections of an M2 muscarinic antagonist, gallamine, or digoxin-specific antibody Fab fragments. Furthermore, a prior microinjection in the RVLM of gallamine, digoxinspecific antibody Fab fragments, or kainic acid or intravenous injection of hexamethonium all prevented the pressor and sympathoexcitatory effects induced by a subsequent microinjection of ouabain. Microinjections of either digoxinspecific antibody Fab fragments or gallamine per se significantly decreased baseline MAP and RSNA. Injection of digoxin-specific antibody Fab fragments attenuated the effects of a subsequent injection of gallamine. Microinjection of mAb against ouabain, but not nonspecific IgG, also significantly decreased baseline MAP and RSNA. These results suggest that OLC in the RVLM contributes to the tonic activity of vasomotor neurons in anesthetized normotensive rats, and the action of OLC in the RVLM is at least partly mediated by M2 muscarinic mechanisms.

Rapid and highly sensitive enzyme immunoassay for quantitative determination of tetrodotoxin.

A monoclonal antibody against tetrodotoxin (TTX) was obtained from Balb/c mice immunized with TTX-bovine serum albumin (BSA) conjugate. The monoclonal antibody was highly specific for TTX and had no cross-reaction to tetrodonic acid, which is a TTX derivative, or gonyautoxins, although a minor cross-reaction to anhydro-tetrodotoxin was observed. The monoclonal antibody neutralized the lethal activity of TTX. By using the monoclonal antibody, a rapid and highly sensitive competitive enzyme immunoassay (EIA) for quantitative analysis of TTX was developed. By the competitive EIA system, TTX can be determined quantitatively in about 30 min (90 min are required if the time for preparation of the solid-phase antigen was included), and the working range for quantitative analysis of TTX was 2-100 ng/ml. In recovery tests and examinations of TTX samples, results of the mouse bioassay and EIA analyses correlated well (r = 0.987). Moreover, it was demonstrated that low concentrations of TTX, which could not be detected by the mouse bioassay, could be determined quantitatively by the competitive EIA.

Stress-induced elevation of ouabainlike compound in rat plasma and adrenal.

Recent observations demonstrate the presence of neurosteroids and their rapid increase in response to acute stress. In view of a steroidal nature of ouabainlike compound, we tested the hypothesis that ouabainlike compound may participate in a homeostatic response to acute stress. Male Wistar rats were subjected to acute stress by swimming in water (22 degrees C) for 10 minutes. The levels of ouabainlike compound in plasma, hypothalamus, pituitary, and adrenal at 10, 40, and 70 minutes (n = 8 for each) after the end of swim stress were compared with nonstressed control levels (n = 10). Ouabainlike compound was measured by a radioimmunoassay for ouabain. Plasma levels of corticosterone and catecholamines were also measured. Plasma corticosterone concentrations increased rapidly at 10 minutes (P < .01) and then declined. A trend for a rise in plasma catecholamines was found at 10 minutes. Adrenal levels of ouabainlike compound concomitantly increased at 10 minutes (P < .01, control: 58.9 +/- 5.9 pmol ouabain equivalents per gram; 10 minutes: 92.5 +/- 4.8; 40 minutes: 47.3 +/- 9.6; 70 minutes: 45.1 +/- 6.3). In contrast, the response of plasma ouabainlike compound was slow and doubled at 40 minutes (P < .01, control: 115 +/- 12 pmol ouabain equivalents per liter; 10 minutes: 132 +/- 23; 40 minutes: 226 +/- 53; 70 minutes: 117 +/- 16). Ouabainlike compound levels in hypothalamus and pituitary remained unaltered. These findings suggest that ouabainlike compound may function as a stress hormone.

Studies of radioimmunoassay for measurement of insulin-like growth factor-I (IGF-I) by using anti-IGF-I monoclonal antibody.

Radioimmunoassay system for measurement of insulin-like growth factor-I (IGF-I) was established by using anti-recombinant human IGF-I monoclonal antibody (MAb). This MAb is capable of recognizing not only human but also rat IGF-I. It was thus suggested that this RIA system can quantify IGF-I in human and rat sera. MAb used in this paper was clarified to be an antibody against the common epitope of C-region of human and rat IGF-I. Gly32-Ser33-Ser34 sequence of C-region of IGF-I is discussed to be an antigenic determinant to which this antibody might specifically bind. MAb does not cross-react with proinsulin and insulin as well as anti-IGF-I polyclonal antibody (PAb). And it is general that PAb has almost 2% cross-reactivity with IGF-II. But this MAb did not cross-react with IGF-II. Actually, the value of IGF-I measured by this system was lower than that measured by RIA using PAb.

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia.

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10(-3), 10(-4), 10(-5) M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.

An anti-platelet activating factor antibody and its effects on platelet aggregation.

Production of antibodies against platelet activating factor (PAF) has been difficult, probably because of the low antigenicity of PAF, a low-molecular-weight phospholipid. We therefore used colloidal gold as a hapten carrier to produce anti-PAF polyclonal and monoclonal antibodies. Both antibodies reacted with PAF, lyso PAF, and L-alpha-lysophosphatidyl choline palmitoyl (lyso PCP), but they did not react phosphorylcholine chloride (PCC). Their affinities were higher for PAF than for lyso PAF and lyso PCP. When the antibodies were tested on PAF-induced platelet aggregation, they suppressed aggregation in a dose-dependent manner.

Distribution and characterization of tumor necrosis factor-alpha-like immunoreactivity in the murine central nervous system.

Tumor necrosis factor-alpha (TNF alpha) is a protein released from macrophages during infection and inflammation. Recent studies suggest that it has several effects within the central nervous system, including generation of fever, enhancement of slow wave sleep, and stimulation of pituitary hormone secretion. We have proposed that TNF alpha may be synthesized by neurons in the CNS and used as a neuromodulator in the pathways involved in the central control of these activities. To test this hypothesis, we have used an antiserum raised against recombinant murine (rm) TNF alpha with an indirect immunoperoxidase technique to stain the murine CNS immunohistochemically. Western blot analysis of mouse brain homogenates revealed one band with electrophoretic mobility identical to that of rmTNF alpha. We identified TNF alpha-like immunoreactive (ir) neurons in the hypothalamus, in the bed nucleus of the stria terminalis, in the caudal raphe nuclei, and along the ventral pontine and medullary surface. TNF alpha ir innervation was widespread within the CNS, particularly in areas involved in autonomic and endocrine regulation, including the hypothalamus, amygdala, bed nucleus of the stria terminalis, parabrachial nucleus, dorsal vagal complex, nucleus ambiguus, and thoracic sympathetic preganglionic cell column. Our data suggest that TNF alpha may serve as a neuromodulator in central pathways involved in the regulation of the autonomic, endocrine and behavioral components of the acute-phase response to inflammation and infection.

Characterization of catch-relaxing peptide (CARP) immunoreactive neurons in the Helix nervous system.

Both small and large diameter of CARP immunoreactive neurons could be observed in the different ganglia of CNS of Helix but not in the foot musculature. The immunoreactivity is the strongest in the varicose segments of immunoreactive fibers. The present findings suggest a transmitter or modulatory role of CARP in both central and peripheral regulatory processes.

Drug-specific T cells derived from patients with drug-induced allergic hepatitis.

Drug-induced allergic hepatitis is a tissue-specific inflammatory disease caused by hypersensitivity to a particular drug. Although the frequency of drug-induced allergic hepatitis appears to increase in proportion to the medicine, the mechanism by which tissue specificity is determined is still to be elucidated. In this study, we established CD4+ T cell clones specific for particular drugs from patients with drug-induced allergic hepatitis accompanied with mild blood eosinophilia and analyzed the possible role of liver protein as a directing factor of liver-specific inflammatory reactions. All CD4+ T cell clones obtained from two patients with this disease proliferated in response to a combination of the particular drug plus liver specific protein (LSP), which consists of over 30 proteins. Some T cell clones were responsive to an antigenic conformation consisting of the 200-kDa glycoprotein (partly purified LSP), a component of LSP, plus the causal drug. In contrast, all CD4+ T cell clones from a patient with simple drug-induced eosinophilia responded to the causal drug in the absence of LSP and partly purified LSP. These data suggested that LSP or partly purified LSP of the appropriate Ag is the target that leads to liver-specific inflammation in drug-induced allergic hepatitis. Furthermore, T cell lines derived from patients with drug-induced allergic hepatitis and simple drug-induced eosinophilia produced large amounts of IL-5 after the appropriate antigenic stimulation, whereas CD4+ T cell clones from donors with a normal amount of peripheral blood eosinophils secreted a much less IL-5. Taken together, these results indicate that overproduction of IL-5 by the allergen-sensitized T cells may result in blood eosinophilia.