Evaluation of an orally administered vaccine, using hydrogels containing bacterial exotoxins of Pasteurella haemolytica, in cattle.

Poly(methacrylic acid) hydrogels were tested for oral delivery of a vaccine against Pasteurella haemolytica infection in cattle. Culture supernatants of P haemolytica, the most common bacterium associated with pneumonia in cattle, were used as the antigens in the vaccine. Hydrogels containing culture supernatants were administered orally to calves. Calves were then challenge-exposed with virulent P haemolytica. Calves were euthanatized 3 days after challenge exposure. The lungs of each calf were scored for severity and size of pneumonic lesions. Results indicated that vaccinated calves had smaller, less severe pneumonic lesions and lived longer than nonvaccinated calves. These results indicated that hydrogels can be used to deliver vaccines orally to calves to enhance resistance to pneumonia caused by P haemolytica.

Stimulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine.

Human neutrophil Fc receptor-mediated phagocytosis can be markedly enhanced by a low m.w. (less than 10,000) heat-labile cytokine(s) derived from specifically stimulated human mononuclear cells and from a human T cell line, MO(t). PMN incubated with supernatant from control mononuclear leukocyte (MNL) culture bound EIgG (percentage of rosettes = 73.7% +/- 7.1) but did not ingest the attached targets (phagocytic index, PI = 40.7 +/- 9.5) as efficiently as PMN incubated with supernatant from adherent MNL, which had ingested EIgG and were then cocultured with nonadherent MNL (PI = 264.3 +/- 46.3). Cytokine-containing supernatants were fractionated on YM-10 Centricon microconcentrators, and the effluent (YM-10E) was found to contain the phagocytosis-enhancing activity. Optimal Fc receptor-mediated ingestion by YM-10E-stimulated PMN required a critical level of target-bound IgG; stimulation was dose dependent and detectable after 5 min at 37 degrees C with a maximal response by 15 min. Monoclonal antibody 3G8 (anti-PMN Fc receptor) inhibited in a dose-dependent fashion both Fc receptor-mediated rosette formation and ingestion by nonstimulated and YM-10E-stimulated PMN. Solid-phase 3G8 Fab had the same effect. A previously undescribed monoclonal antibody, 1C2, exhibited a different pattern of inhibition. It had no effect on rosetting or ingestion of EIgG by nonstimulated PMN; however, it inhibited EIgG phagocytosis by YM-10E-stimulated PMN down to the level of nonstimulated ingestion without affecting rosette formation. Solid-phase 1C2 had the same effect. These data indicate that phagocytosis mediated by 3G8-positive Fc receptors may be enhanced by cytokine(s) stimulation in a manner requiring the molecule recognized by 1C2. Monoclonal antibodies to the alpha-chain of CR3 had only minimal effects on YM-10E-stimulated ingestion. Fluorescence flow cytometry of YM-10E-stimulated PMN, indirectly stained with 3G8 or 1C2, indicated that cytokine enhancement of EIgG ingestion occurred without an increase in either 3G8 or 1C2 binding sites. These data show that the low avidity Fc receptor, which binds immune complexes, may be functionally modulated at sites of inflammation where PMN and macrophages mediate clearance and destruction of immune complexes and opsonized particles.

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin.

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence.

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.

Identification of neutrophil subpopulations with monoclonal antibodies.

Two monoclonal antibodies have been produced by the hybridoma technique that recognize subpopulations of human neutrophils. The antibodies, termed 1B5 and 4D1, react with a mean percentage of 57% and 51% of peripheral blood granulocytes, respectively. The antigens recognized appear to be neutrophil specific in that these antibodies do not react with eosinophils, platelets, erythrocytes, monocytes, or nonadherent peripheral blood mononuclear cells. Although the neutrophil subpopulations recognized by these antibodies are nearly identical (coinclusive), the antigenic determinants recognized appear to be different. These monoclonal antibodies to neutrophil subpopulations may prove useful to studying functional heterogeneity among neutrophils as well as for investigations of normal and abnormal myeloid differentiation.

Evidence for an IgD homologue on chicken lymphocytes.

Chicken lymphocyte membrane immunoglobulins (Ig), were precipitated with mouse monoclonal antibodies specific for heavy and light chain isotypes and analyzed by polyacrylamide gel electrophoresis. Very little or no membrane-bound IgG and IgA was detected. After sequential precipitation and removal of IgM reactive with any of three monoclonal anti-mu antibodies, anti-light chain antibody precipitated residual Ig with a relative electrophoretic mobility similar to that of IgM. Under reducing conditions, these surface Ig molecules had a heavy chain that appeared slightly larger (approximately 81,000 daltons) than mu-chain (approximately 79,000 daltons), and light chains of approximately 25,000 daltons. Complete clearance of membrane-bound IgM reactive with an anti-mu allotype antiserum left similar molecules precipitate by monoclonal anti-light chain antibody. These non-IgM molecules could be detected on the surface of lymphocytes from blood, spleen, bursa and the B cell line RAV-1, but not from thymus or blood from an agammaglobulinemic chicken. After capping of B cell surface IgM with anti-mu, immunofluorescent staining with anti-light chain antibody revealed residual Ig molecules disturbed across the surface of more than 90% of the IgM-bearing cells. The data suggest the existence of an avian homologue of mammalian IgD. Affinity-purified goat anti-mu antibodies and a fourth monoclonal anti-mu antibody reacted with both IgM and the putative IgD molecules, which suggests that the IgD homologue shares at least one common determinant with chicken IgM.

Severe Fletcher factor (plasma prekallikrein) deficiency with partial deficiency of Hageman factor (factor XII): report of a case with observation on in vivo and in vitro leukocyte chemotaxis.

A case of cross-reacting material-negative Fletcher trait with additional partial deficiency of Hageman factor (HF, Factor XII) is described. Although the patient presented with a recent history of frequent epistaxis, he had no other personal or family history of a tendency toward bleeding or infection. Similar to other cases of Fletcher trait, his plasma showed a markedly prolonged partial thromboplastin time which could be corrected by prolonged incubation with the surface-activator kaolin. Surface-induced fibrinolysis, amidolysis of alpha-N-benzoyl-proline-L-phenylalanine-L-arginine-p-nitroanilide, and cold-promoted enhancement of factor VII activity, reactions requiring the presence in the plasma of fletcher factor (prekallikrein), in addition to Hageman factor and Fitzgerald factor (high-molecular weight kininogen), were also defective. In vivo chemotaxis of polymorphonuclear leukocytes and monocytes (Rebuck's skin window technique) in response to skin abrasions was defective, but was normal when diphtheria-tetanus toxoid was also applied. In vitro leukocyte chemotaxis (Boyden chamber technique) in response to normal or patient's own serum activated with zymosan was normal. Together with previous observations that kallikrein generated chemotactic activity, possibly via activation of C5, the present observations suggest that prekallikrein activation may be important for in vivo leukocyte chemotactic response to skin abrasion. The inheritance of Fletcher trait in this patient is unclear.l Although the father was an apparent heterozygote, the mother was completely normal for Fletcher factor procoagulant activity and antigen. The mild Hageman factor deficiency in the patient did not contribute significantly to the plasma defects described and was likely inherited from the father who had a low HF procoagulant activity.

Phagocytosis and killing of salmonella typhimurium by peritoneal exudate cells.

Normal peritoneal cells from conventional, germfree, or nu/nu mice readily killed opsonized salmonellae, an observation that suggests that this activity in the normal peritoneal cavity may not be dependent on either environmental antigenic stimulation or T-cell mediation. In contrast, peritoneal cells elicited 4 days after injection with thioglycolate medium failed to kill opsonized salmonellae but appeared to be highly phagocytic. Peritoneal cells from thioglycolate-treated mice could be induced to kill opsonized salmonellae by giving the mice a primary footpad injection and a secondary intraperitoneal injection of Corynebacterium parvum. This activation by C. parvum appeared to be thymus dependent, since it did not occur in nu/nu mice.

Neutrophil oxidative metabolism in sickle cell disease.

Bacterial infections are the most frequent cause of death and an important factor of morbidity in sickle cell disease. A defect in oxidative metabolism of neutrophils from these patients has been reported as a possible cause for these infections. Since normal neutrophil functions are essential in the defense against bacteria, it seemed important to reassess the capacity of neutrophils from patients with sickle cell disease to undergo the metabolic events associated with phagocytic bactericidal activity. Accordingly, neutrophils from patients and controls were compared for their ability to reduce nitroblue tetrazolium dye, to activate the hexose monophosphate shunt, and to generate superoxide anion, hydrogen peroxide, and chemiluminescence. Patients' cells performed normally in each of these assays and, in addition, killed Staphylococcus aureus as well as did cells from controls. Thus, an abnormality of neutrophil oxidative metabolism cannot explain the propensity to bacterial infections in sickle cell disease.

Generation of superoxide anion and chemiluminescence by human monocytes during phagocytosis and on contact with surface-bound immunoglobulin G.

Extent of O-2-release and chemiluminescence, attributed to singlet oxygen, has been compared in human monocytes and neutrophils during phagocytosis, stimulation by the surface-active agent phorbol myristate acetate, or contact with aggregated IgG in a model of immune complex disease. Monocytes generated O-2-and chemiluminescence with each of the three stimuli, although values were significantly less than those of neutrophils from the same individuals. Lymphocytes had no significant activity in either assay with any stimulus. Oxygen metabolites released from mononuclear phagocytes are highly reactive and could play a part in both the beneficial and detrimental aspects of chronic inflammation.

Elaboration of toxic oxygen by-products by neutrophils in a model of immune complex disease.

Contact between human neutrophils and aggregated immunoglobulin G bound to micropore filters has been studied as a model of the pathogenesis of tissue damage in immune complex disease. Contact with this surface, as well as with plain filters and polystyrene petri dishes, induced neutrophils to elaborate superoxide anion and hydrogen peroxide and to generate chemiluminescence, which has been attributed to singlet oxygen. Pretreatment of the cells with cytochalasin B decreased these activities but increased release of lysosomal beta-glucuronidase, suggesting that degranulation and the burst of oxygen metabolism that characterizes phagocytes are independently regulated functions. Toxic oxygen metabolites released from neutrophils are highly reactive and could mediate tissue injury at sites of inflammation.

The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes.

The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. However, O2- generation by intact cells during phagocytosis was markedly diminished in nine patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.OH). A requirement for .OH in the phagocytic bactericidal event might explain the apparent requirement for both O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from O2- and .OH also might explain these findings. It would seem clear from these and other studies that the granulo cyte elaborates O2- as a concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbialdeath through O2- itself, hydrogen peroxide, .OH, singlet oxygen, or some other agent remains to be clearly defined.