Oral administration of antibodies as prophylaxis and therapy in Campylobacter jejuni-infected chickens.

Passive immunity against gastrointestinal infections has recently been successfully applied as prophylaxis and therapy in patients in a variety of virally and bacterially induced infections. Campylobacter jejuni is frequently associated with acute diarrhoea in humans, and several species of animals have been shown to transmit the disease, although birds have been implicated as the main source of infection. We used bovine and chicken immunoglobulin preparations from the milk and eggs, respectively, of immunized animals for prophylactic and therapeutic treatment of chickens infected with C. jejuni. A marked prophylactic effect (a >99% decrease in the number of bacteria) was noted using either antibody preparation, whereas the therapeutic efficacy, i.e. when antibodies were given after the infection was established, was distinctly lower (80-95%) as judged by faecal bacterial counts. These observations may serve as a starting point for experiments aimed at elimination of the infection in an industrial or farm setting. It may also encourage future attempts to treat, prophylactically or therapeutically, patients with Campylobacter-induced diarrhoea.

Two chicken B cell lines resistant to ouabain for the production of chicken monoclonal antibodies.

Two chicken B cell lines, termed MuH1 and MuH4, which are resistant to ouabain, were established as fusion partners for production of chicken monoclonal antibody from the parental cell lines R27H1 and R27H4 which were deficient in thymidine kinase activity. The MuH1 synthesized, but did not secrete mu chain, whereas the MuH4 secreted non-specific IgM. Fusions of the MuH1 and MuH4 with spleen cells from chickens immunized with human IgG resulted in successful preparations of hybridomas secreting antibodies specific to human IgG. Transformed cells insensitive to HAT appeared in many culture wells after cell hybridizations with R27H4, but on hybridization with MuH1 or MuH4 the appearance of such unfavorable cells could be avoided by the inclusion of ouabain in HAT medium. Thus, in comparison with the R27H4 line, the MuH1 and MuH4 lines did not provide higher fusion efficiencies, but gave increased opportunities to obtain hybridomas producing specific antibodies. Among a total of eight hybridomas, four derived from HuM1 and four derived from MuH4, seven secreted both IgG and IgM, and the remaining one derived from MuH1 secreted only IgG. In all eight hybridoma lines IgG was specifically reactive to human IgG.

Comparative evaluation of DQ-2511, a novel gastroprokinetic agent, with cisapride in ameliorative action on experimentally induced delayed gastric emptying.

We compared the main pharmacological effect of DQ-2511 (3-[[[2-(3,4-dimethoxyphenyl)- ethyl]carbamoyl]methyl]amino-N-methylbenzamide), a novel gastroprokinetic agent, with that of cisapride. Single oral administration of DQ-2511 (3-10 mg kg-1) caused similar significant improvements to delays in gastric emptying of semi-solid meals evoked by cholecystokinin-octapeptide (CCK8: 5 micrograms kg-1, i.v.) in monkeys, to that with cisapride (3 mg kg-1). A 2-week oral treatment of unilaterally vagotomized rats with DQ-2511 (1-10 mg kg-1) lessened delays in gastric emptying, whereas cisapride (0.3-10 mg kg-1) had no effect under the same experimental protocols. In anesthetized rats, bolus intravenous injection of either compound (60 micrograms kg-1) enhanced gastric motility determined by means of strain gauge force transducers. Electrophysiological investigations revealed that bolus injection of DQ-2511 (6-60 micrograms kg-1) depressed the afferent discharge rate of the ventral gastric branch of the vagus nerve, while cisapride showed no effect. These results suggest that the mechanism of ameliorative action of DQ-2511 on delayed gastric emptying may differ from that of cisapride.

Pharmacological characteristics of DQ-2511 as a prokinetic agent.

The pharmacological characteristics of DQ-2511, a substituted benzamide (3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl]methyl] amino-N-methylbenzamide), as a prokinetic agent were studied. Cholecystokinin-octapeptide, dopamine, and alpha-calcitonin gene-related peptide, all suppressed gastric emptying in mice. Reversal of the depressed emptying occurred when DQ-2511 was administered by the oral or intraperitoneal route. When the action of eight proposed metabolites of DQ-2511 on the mouse cholecystokinin-octapeptide model was investigated, the main metabolite in plasma, MA-2, showed no effect, although two minor metabolites ameliorated or aggravated the delayed gastric emptying. This finding implies that DQ-2511, as the parent compound itself, exerts the ameliorative action. In dogs treated with cisplatin or copper sulfate, DQ-2511 had no antiemetic activity, as assessed by the number of vomiting episodes. The concern that the mechanism of action of DQ-2511 was blockade of receptors for cholecystokinin-octapeptide, dopamine, serotonin, alpha-calcitonin gene-related peptide, nicotine or muscarine, was resolved by results of radioligand binding studies showing the absence of a DQ-2511 binding to any of these receptor types. Evidence is accumulating that the mechanism of the prokinetic action of DQ-2511 involves the intrinsic and extrinsic autonomic innervation.

Enhancing effect of DQ-2511 on gastric emptying of spontaneously hypertensive rats.

The present study was designed to clarify the potential of DQ-2511 (3-[[[2-(3,4-dimethoxyphenyl)ethyl]carbamoyl]carbamoyl]methyl] amino-N-methylbenzamide: ecabapide) as a gastroprokinetic agent in spontaneously hypertensive rats (SHR). The gastric emptying of SHR was clearly retarded relative to that of weight-matched normotensive Wistar-Kyoto (WKY) rats when evaluated by the acetaminophen (APAP) method with the semi-solid test meal. There was, however, no significant difference between both strains in gastric mucosal blood flow (GMBF) determined by means of a laser doppler flowmetry. A 2-week treatment of SHR with DQ-2511 (1 mg/kg, oral) stimulated gastric emptying without affecting body weight gain or indirect systolic blood pressure (SBP), whereas cisapride (3 and 10 mg/kg, oral) had no effect under the same conditions. The pharmacological characteristics of DQ-2511 as a gastroprokinetic agent are discussed on the basis of these results.

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii.

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.

Mass-spectrometric identification of trehalose 6-monomycolate synthesized by the cell-free system of Bacterionema matruchotii.

The fluffy layer fraction prepared from Bacterionema matruchotii was found to possess high activity for the biosynthesis of mycolic acids which were bound to an unknown compound by an alkali-labile linkage [T. Shimakata, M. Iwaki, and T. Kusaka (1984) Arch. Biochem. Biophys. 229, 329-339]. To determine the structure of the mycolate-containing compound, it was purified and analyzed by field desorption (FD) and secondary ion mass spectrometry (SI-MS). When non-labelled palmitic acid was used as a precursor in the in vitro biosynthetic system, the underivatized product had a cationized molecular ion, [M + Na]+, at m/z 843 in FD-MS and a protonated ion, [M + H]+, at m/z 821 in SI-MS, corresponding to the quasimolecular ion of trehalose monomycolate (C32:0). In SI-MS, characteristic fragment ions due to cleavage of glycosidic linkages were clearly detected in addition to the molecular ion. If [1-13C]palmitic acid was the precursor, 2 mass unit increases in both the quasimolecular and fragment ions were observed, indicating that two molecules of palmitate were incorporated into the product. alpha-Trehalose was found in the aqueous phase after saponification of the product. By the electron impact mass spectrometry of the trimethylsilylated product, the mycolate was found to be esterified with an hydroxyl group at position 6 of the trehalose molecule. These results clearly demonstrated that the predominant product synthesized by the fluffy layer fraction with palmitate as substrate was 6-monomycolate (C32:0) of alpha-D-trehalose. Because newly synthesized mycolic acid was mainly in the form of trehalose monomycolate instead of free mycolate or trehalose dimycolate, the role of trehalose in the biosynthesis of mycolic acid is discussed.