Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E

Clostridium botulinum neurotoxins (BoNTs) are the most toxic proteins for humans. The current clostridial-derived vaccines against BoNT intoxication have limitations including production and accessibility. Conditions were established to express the soluble receptor binding domain (heavy-chain receptor [HCR]) of BoNT serotypes A and E in Escherichia coli. Sera isolated from mice and rabbits immunized with recombinant HCR/A1 (rHCR/A1) from the classical type A-Hall strain (ATCC 3502) (BoNT/A1) and rHCR/E from BoNT serotype E Beluga (BoNT/E(B)) neutralized the homologous serotype of BoNT but displayed differences in cross-recognition and cross-protection. Enzyme-linked immunosorbent assay and Western blotting showed that alpha-rHCR/A1 recognized epitopes within the C terminus of the HCR/A and HCR/E, while alpha-rHCR/E recognized epitopes within the N terminus or interface between the N and C termini of the HCR proteins. alpha-rHCR/E(B) sera possessed detectable neutralizing capacity for BoNT/A1, while alpha-rHCR/A1 did not neutralize BoNT/E. rHCR/A was an effective immunogen against BoNT/A1 and the Kyoto F infant strain (BoNT/A2), but not BoNT serotype E Alaska (BoNT/E(A)), while rHCR/E(B) neutralized BoNT/E(A), and under hyperimmunization conditions protected against BoNT/A1 and BoNT/A2. The protection elicited by rHCR/A1 to BoNT/A1 and BoNT/A2 and by rHCR/E(B) to BoNT/E(A) indicate that immunization with receptor binding domains elicit protection within sub-serotypes of BoNT. The protection elicited by hyperimmunization with rHCR/E against BoNT/A suggests the presence of common neutralizing epitopes between the serotypes E and A. These results show that a receptor binding domain subunit vaccine protects against serotype variants of BoNTs.     

Monoclonal antibody to human granulocytes: cellular specificity and functional studies

Antigenic specificity and functional studies of G2, a monoclonal antibody to human granulocytes, prepared by fusing spleen cells from immunized Balb/c mice to the nonimmunoglobulin (Ig) secretor line SP2/0, are described. The antibody was reactive with the HL60 and K562 cell lines and with human peripheral blood granulocytes; and unreactive toward human lymphocytes, erythrocytes, a variety of T and B cell lines, as well as toward leukemic cells obtained from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and acute myelocytic leukemia (AML). The G2 monoclonal antibody identified cell surface antigens on cells from cases of acute myelomonocytic leukemia (AMMoL) and on cells from 2 of 12 cases of acute undifferentiated leukemia (AUL). G2 was capable of inhibiting oxygen consumption by human polymorphonuclear leukocytes (PMNL) stimulated with aggregated human immunoglobulin (IgG), opsonized zymosan, f-met-leu-phe (FMLP), and the calcium ionophore A23187. Inhibition of the PMNL response to phorbol myristate acetate (PMA) and digitonin was dependent upon the dose of the stimulant. G2 should facilitate elucidation of the mechanisms of granulocyte membrane perturbation and subsequent activation of various functions via a selective interaction with key cell surface antigens.     

Krankheiten als Folge der Selbstverkennung und Selbstzersetzung von Geweben

Ohne ZusammenfassungVortrag gehalten vor der 105. Versammlung der Gesellschaft Deutscher Naturforscher und Ärzte, Heidelberg, Oktober 1968.     

Kreatinverluste und Kreatinaufnahme durch isolierte Herzen bei Durchströmung mit kreatinhaltigen Lösungen

Zusammenfassung Mit Krebs-Ringerlösung durchströmte Rattenherzen geben um so weniger Kreatin an die Durchströmungsflüssigkeit ab, je schonender sie präpariert wurden.Nach Zugabe von 3 mg bzw. 5 mg Kreatin zu 100 ml Durchstrom nahmen die meisten Herzen innerhalb von 30 min 400 bzw. 830 Kreatin/g Trockengewicht auf. Weitere Steigerung des Kreatinangebotes verbesserte die Kreatinaufnahme nicht, ebensowenig wie Zugabe von Glucose. Enthielt der Durchstrom Insulin, so wurde die Kreatinaufnahme signifikant erhöht.Die Mehrzahl der mit Kreatin durchströmten Herzen nahm aus der Krebs-Ringerlösung Phosphat auf.Kalium wurde von einigen Herzen abgegeben, von anderen aufgenommen. Die Kaliumaufnahme aus dem Durchstrom war bei geschädigten Herzen am größten. Zusammenhänge zwischen dem Verhalten von Kalium einerseits und Kreatin oder Phosphat andererseits wurden in den Versuchen an isolierten Herzen nicht erkennbar.

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Summary Rat hearts perfused with Krebs' Ringer solution, yielded creatine in the perfusion fluid; the more carefully the hearts were dissected, the smaller was the yield.When creatine was added to the perfusion fluid in concentrations of 3 or 5 mg-%, the hearts took up creatine in most cases to the extent of 400g or 830µg respectively, per g dry weight. A further increase in the creatine concentration did not improve the creatine uptake, nor did an addition of glucose to the perfusion fluid. When the latter contained insulin, the creatine uptake was significantly increased.The majority of the creatine-perfused hearts took up phosphate from the Krebs' Ringer solution.During perfusion some of the hearts yielded potassium, others took up potassium. The potassium uptake was greatest with hearts which had been damaged during the dissection. No connection could be observed in the experiments with isolated hearts between the behaviour of potassium on the one hand, and that of creatine or phosphate on the other.

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Ausgeführt mit Unterstützung des Landes Nordrhein-Westfalen (Landesamt für Forschung).

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Herrn Prof. Dr.E. Lehnartz zum 65. Geburtstag.     

Lymphocytic choriomeningitis virus

Lymphocytic choriomeningitis (LCM) virus-specific complement-fixing (CF) antigen (ECFA) has been solubilized, concentrated, and partially purified. When inoculated together with Freund's adjuvant, ECFA induced CF antibody but not neutralizing antibody or protective immunity. By itself it boosted pre-existing CF antibody but not neutralizing antibody. In double diffusion tests one line developed between ECFA and its antiserum, and a corresponding line became visible when ECFA interacted with an antiserum directed against all LCM virus-specific antigens. Absorption of either serum with ECFA abolished all ECFA-precipitating qualities. Ouchterlony tests also revealed that ECFA prepared from cells and tissues of various species is immunologically identical. By a variety of procedures ECFA was not found to be represented on the surface of either the virion or the infected cell. When purified infectious LCM virus was disrupted, a CF antigen corresponding immunologically to ECFA was set free. In double diffusion tests this antigen gave a line of identity with ECFA. Thus, ECFA appears to be an internal component of the infectious LCM virus.Part of the work reported here has been published in preliminary communications [8, 10, 24, 25].     

The Kallikrein-Kinin system and muscle metabolism—Clinical aspects

The influence of synthetic bradykinin (BK) on disturbed protein and carbohydrate metabolism was studied in chemical and manifest maturity-onset diabetics, in surgical patients and in alloxan diabetic rats. BK,mixed with insulin and injected subcutaneously twice daily in alloxan diabetic rats lowered the morning blood glucose concentration in a dose-dependent way, whereas in a control group treated with insulin only no decrease was seen. Accelerated local blood flow or enhanced vascular permeability as a cause of increased glucose uptake could be ruled out by control experiments using papaverine and eledoisin. Better metabolic control in the BK/insulin-treated group was also indicated by lower arterial levels of free fatty acids and of -hydroxybutyrate, normalized hepatic glycogen content and better growth of body weight. In healthy man an intravenous infusion of BK (80 g/h) did not influence normal fasting blood glucose concentrations, whereas elevated glucose levels in maturity-onset diabetics were continuously reduced within 100 min by 12.2±1.4%. A comparable diabetic group receiving saline alone showed no spontaneous drop of blood glucose concentration. An improvement of pathological carbohydrate metabolism by infusion of BK i.v. could also be demonstrated using the intravenous glucose tolerance test in chemical and manifest maturity-onset diabetics and in surgical patients: in all groupsk values of the glucose tolerance test were significantly increased by BK. This effect was neither due to stimulated insulin release nor to changed glucose pool or to increased renal glucose loss, which was even reduced by BK. Interestingly, normalk values in healthy volunteers were not further improved by BK. A stimulated protein breakdown, which occurs after surgery due to peripheral insulin resistance, can also be restricted by intravenous infusion of BK: in surgical patients urinary nitrogen excretion was reduced by 50% during infusion of BK and was accelerated again after cessation of the infusion. These results indicate that BK can improve the efficacy of exogenous insulin in insulin-deficient animals and depressed insulin sensitivity in maturity-onset diabetics and surgical patients.     

Demineralized bone matrix as a biological scaffold for bone repair

Experimental models were created in rat fibula to represent impaired bone healing so that biological deficiencies that cause bone repair to fail or to be delayed may be investigated. These models consist of a 4-mm-long segmental defect, created in rat fibula by osteotomy, and fitted with a 7-mm-long tubular specimen of demineralized bone matrix (DBM) over the cut ends of the fibula. The experiments in this study involved various modifications of the DBM scaffold designed to reduce its osteoinductive activity: steam sterilization (sDBM), ethylene oxide sterilization (eoDBM), trypsin digestion (tDBM), and guanidine hydrochloride extraction (gDBM). Bone healing was evaluated by bending rigidity of the fibula and mineral content of the repair site at 7 weeks post-surgery. The sDBM scaffolds resorbed completely by 7 weeks and hence this model was a nonhealing negative control. Rigidities in the unmodified DBM and tDBM groups were comparable, whereas in the gDBM and eoDBM groups it was significantly reduced. Histologically, in the 4-mm defects repaired with unmodified DBM, direct and endochondral bone formation in the scaffold and the defect resulted in a neocortex consisting of woven and lamellar bone uniting the broken bone by 7 weeks post-surgery. We conclude that the eoDBM and gDBM groups represent failure or delay of the bone repair process when compared with the unmodified DBM group in which the process is analogous to normal bone healing.     

Race as a moderator in a model of sexual harassment: an empirical test

L. F. Fitzgerald, C. L. Hulin, and F. Drasgow (1995) proposed that victim characteristics, such as race, might moderate the relationships between sexual harassment and its job, psychological, and health status outcomes. This study describes 2 theoretical positions, tokenism and double jeopardy, that could account for this possible moderation by race, as well as the alternative view that no moderating effects exist. The effects of race are empirically examined through simultaneous path analysis. Results indicate that whereas mean levels of harassment differ across race, the phenomenon of sexual harassment unfolds similarly across races; race is not a moderator of the relationships between sexual harassment and the variables proposed as its antecedents and outcomes.