Nonimmune Guinea Pigs Research Articles

ProSAP-interacting Protein 1 (ProSAPiP1), a Novel Protein of the Postsynaptic Density That Links the Spine-associated Rap-Gap (SPAR) to the Scaffolding Protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.

Analysis of Immunoprotectivity of the Recombinant OmpA of Rickettsia heilongjiangensis

A truncated gene ompA was amplified from Rickettsia heilongjiangensis isolated in China and a 56-kDa truncated OmpA protein was expressed in E. coli cells transformed with the ompA-recombined expression plasmid. High levels of serum antibodies to R. heilongjiangensis and proliferation of the splenic cells were found in mice immunized with the truncated OmpA. After challenge with R. heilongjiangensis or R. rickettsii, fever and pathological damages of the guinea pigs immunized with the truncated OmpA were significantly slighter as compared with those of nonimmunized guinea pigs. These results suggest that the truncated OmpA of R. heilongjiangii is immunogenic for effectively inducing humoral and cell-mediated immune responses against homologous and heterologous species in the spotted fever group.

Induction of histamine release from non-immunized guinea pigs: a possible involvement of lectin-like factor(s) in pollinosis.

To investigate nonimmune pathogenic functions of pollens, vascular permeability enhancement (VPE) activity of pollen extracts was examined using guinea pigs nonimmunized against pollens. Ryegrass, ragweed, mesquite and almond, but not common cattail and sumac, induced VPE which was inhibited primarily by an anti-histamine drug. Ryegrass pollen VPE activity was extracted more at pH 7.3 than at pH 6.5 or 8.0 and the maximal activity was extracted in 30 min. Interestingly, more than 60% of the maximal activity was extracted in 5 min. The maximal VPE activity had a dose-dependency similar to histamine (3 x 10(-5) M) but lasted longer than the histamine activity. The VPE activity was inhibited by oligomannose-glycosylated ovalbumin or avidin, as well as the oligosaccharides but not by the deglycosylated proteins. These results indicate that some pollens contain lectin-like, histamine-releasing factor(s), which may be involved in part in pollinosis, by inducing mast cell degranulation through a nonimmune mechanism and resulting in allergy-like symptoms.

The promotion of patent airways and inhibition of antigen-induced bronchial obstruction by endogenous nitric oxide.

1. The aim of the present study was to investigate the role of nitric oxide (NO), histamine and leukotrienes in bronchial obstruction. For this, guinea-pigs immunised against ovalbumin were studied under anaesthesia during challenge with antigen or agonists. 2. Challenge with nebulised antigen (0.1-1 mg) elicited dose-dependent increases in insufflation pressure which were abolished by combined administration of histamine and leukotriene antagonists. 3. Challenge with nebulised antigen (0.1-1 mg) also elicited dose-dependent increases in the concentration of endogenous nitric oxide in the exhaled air. After an initial peak, exhaled NO concentrations returned to pre-challenge levels. 4. The increase in insufflation pressure and in exhaled NO caused by ovalbumin challenge was inhibited by combined administration of histamine and leukotriene antagonists. 5. In non-immunised guinea-pigs, challenge of the airways with nebulised histamine (10-1000 nmol) or leukotriene C4 (LTC4, 30-300 pmol) elicited dose-dependent increases in insufflation pressure and in concentrations of endogenous NO in exhaled air. 6. The increase in exhaled NO correlated with the increase in insufflation pressure in response to ovalbumin, histamine and LTC4. An inhibitor of endogenous NO synthesis, N omega-nitro-L-arginine methylester (L-NAME, 30 mg kg-1 i.v.) abolished NO exhalation, and markedly augmented the airway responses to ovalbumin, histamine, or LTC4. 7. The potentiation by L-NAME of the increase in insufflation pressure in response to ovalbumin or histamine was prevented by exogenous NO (20 p.p.m.) in the inhaled air. 8. The results indicate that endogenous NO has an inhibitory effect on bronchial obstruction. Increased NO release during allergen challenge is likely to be due to actions of histamine and leukotrienes.

Immune Protection Conferred by the Baculovirus-Related Glycoprotein of Thogoto Virus (Orthomyxoviridae)

The coding region of segment 4 of Thogoto (THO) virus, a tick-borne member of the Orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. This construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (MAbs). Immunization of hamsters with Spodoptera frugiperda (Sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against virus challenge; control hamsters developed clinical disease with high-titer viremia 3 days postchallenge. In contrast to hamsters, guinea pigs are comparatively resistant to THO virus infection but support nonviremic transmission between cofeeding infected and uninfected ticks. However, when uninfected ticks fed on guinea pigs immunized with the baculovirus recombinant, only 2% became infected following virus challenge of the animals compared to 26% of ticks on control nonimmune guinea pigs. Furthermore, neutralizing MAbs specific for THO viral glycoprotein protected mice against lethal challenge with THO virus; nonneutralizing MAbs specific for the glycoprotein, which inhibit THO viral agglutinating activity, did not induce a protective response. Thus at least in the murine model, protective immunity is conferred by antibodies directed against the neutralizing epitope(s) of the baculovirus-related glycoprotein of THO virus.

Antibody-Mediated Imbalance of Myocardial Energy Metabolism

The ADP-ATP carrier of the inner mitochondrial membrane is an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain carrier-specific autoantibodies that inhibit the transmembrane nucleotide transport on isolated mitochondria. Guinea pigs immunized with the isolated ADP-ATP carrier protein also generate specific carrier-inactivating antibodies. In this study, we measured the cardiac function of guinea pigs immunized with the ADP-ATP carrier by determining the external heart work (EHW) of their isolated perfused spontaneously beating hearts stimulated by 4.0 mmol/L calcium and aortic ligature. Further, the electrogenic transport activity of the ADP-ATP carrier was estimated by calculating the cytosolic-mitochondrial difference of the phosphorylation potential of ATP [delta G(cyt-mit)] in the freeze-clamped isolated hearts by nonaqueous fractionation. The EHW of immunized guinea pigs was seen to be reduced by 54% (P < .005) compared with nonimmunized control guinea pigs, and delta G(cyt-mit) declined from 4.9 kJ/mol ATP in nonimmunized control hearts to 2.3 kJ/mol ATP in the hearts of the immunized guinea pigs (P < .005). The decisive result of this study, however, is the close relation observed between the magnitude of reduction of delta G(cyt-mit) and the size of the decrease in EHW (r = .87). Therefore, it seems plausible that antibody-mediated carrier dysfunction (creating the observed imbalance in myocardial energy metabolism) is responsible for the impairment of cardiac function. Our data support the hypothesis that immunopathic mechanisms in myocarditis and dilated cardiomyopathy can trigger subsequent heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunopathologic alterations in the bronchi of immunized guinea pigs.

Isolated lungs from guinea pigs actively sensitized to ovalbumin and boosted 2 wk later display an enhanced bronchoconstriction and release larger amounts of secondary mediators as compared with lungs from nonimmunized animals when stimulated by platelet-activating factor or other agonists. We have investigated changes in T lymphocytes and eosinophils found in the bronchial wall of immunized and nonimmunized guinea pigs. The animals received two injections of 10 micrograms ovalbumin in Al(OH)3, at a 2-wk interval. Two studies were performed: (1) the animals were killed 7 days after the booster injection of antigen, (2) they were challenged with ovalbumin at this same day and killed after 2 or 24 h. Lungs were resected and frozen, and cryostat sections stained using monoclonal antibodies that recognize T cells, T cell subsets, or other relevant epitopes. Cyanide-resistant peroxidase activity was used to identify eosinophils. A large number of T cells, mainly of the CD4+ subset, and eosinophils were recruited into the bronchi 7 days after the booster injection of the antigen, compared with nonimmunized or nonboosted animals. In antigen-challenged animals, the numbers of T cells did not change but eosinophils were further increased in number at the 24 h time point. Also at this time point, a population of cells with a dendritic appearance was seen in the bronchial wall, which did not express macrophage markers but was strongly class II positive. Class II positivity was also noted in the bronchial epithelium and on many cells infiltrating the mucosa. These findings suggest that activated T cells and/or their products play an important role in the bronchopulmonary immunopathology associated with this model and possibly with the development of bronchial hyperreactivity.

Inhibition by azelastine of the effects of platelet-activating factor in lungs from actively sensitised guinea-pigs

The effect of azelastine on platelet-activating factor (PAF)-induced bronchoconstriction and mediator release in isolated lungs from actively sensitised guinea-pigs was investigated. Guinea-pigs were actively sensitised with two s.c. injections of 10 μg ovalbumin in 1 mg Al (OH) 3 at a 2-week interval. One week after the second injection, the lungs were removed and challenged intra-arterially with PAF or arachidonic acid. In some experiments lungs from non-immunised guinea-pigs were injected with PAF or histamine. Bronchoconstriction, the release of thromboxane (TX)B 2 or leukotriene (LT)-like material and the histamine content of the effluent were evaluated. Azelastine was given s.c. at 10 or 100 μg/kg, 4 h before lung removal. Azelastine (100 μg/kg) did not inhibit PAF-induced bronchoconstriction and mediator release from lungs from non-immunised guinea-pigs. In contrast, the hyperresponsiveness to 1 ng PAF observed in lungs from actively sensitised animals was dose dependently inhibited by azelastine. Azelastine did not reduce the histamine-induced bronchoconstriction and consequent TXB 2 release from lungs from immunised guinea-pigs, indicating that the protective effect exerted against hyperresponsiveness to PAF was not due to histamine antagonism. Azelastine also reduced arachidonic acid-induced bronchoconstriction and LT-like material release from sensitised lungs, regardless of the presence of indomethacin. These results suggest that inhibition of lung hyperresponsiveness to PAF by azelastine may result from an interference with leukotriene synthesis.

The role of complement and murine monoclonal antibodies in binding and ingestion of Haemophilus influenzae type b by murine macrophages.

Complement proteins C3b and C3bi are important opsonins for a variety of human pathogens. Recent work from our laboratory has demonstrated a role for C3 in mediating interactions of encapsulated Haemophilus influenzae type b (Hib) with macrophages [1]. We observed that <15% of organisms bound via C3 were ingested; however, when subopsonic concentrations of monoclonal antibody to Hib capsule were added, 537c of organisms bound to macrophages via C3 were ingested [1]. On the basis of these observations we proposed that C3 acts chiefly to bind H. influenzae to macrophages, and under certain conditions of low antibody concentration, complement is required for antibody-directed ingestion. To examine further how this C3mediated binding influences the opsonic effect of antibody, we measured the ability of six murine monoclonal antibodies to mediate binding and ingestion of Hib. Binding and ingestion of bacteria by resident peritoneal murine macrophages was measured as previously described [1]. This assay does not assess killing of bacteria by macrophages. Bacteria were added to macrophages adherent to glass coverslips and incubated for 60 min at 370C in 300 jliI of buffer containing 8% nonimmune serum, 20 ^1 of a 1:20 dilution of antibody, or 8% nonimmune serum and 20 /ul of a 1:20 dilution of antibody. Antibodies El 17-5, 5M1H9, PCP61-1, G56-5, and G102-5 were provided by D. Madore (Praxis Biologies, Rochester, NY). Antibody 7F3 was provided by T. Murphy (School of Medicine, State University of New York at Buffalo). Concentrations of antibodies in undiluted fluids were determined by radial immunodiffusion assay (ICN Biomedicals, Costa Mesa, CA). Bactericidal activity was assessed by methods previously used in our laboratory. These assays were done to demonstrate the ability of antibodies to recognize bacterial antigens and to fix complement. Recent work has suggested that these assays cannot be used to predict in vivo effects of antibody and complement [2]. Bacteriolysis was measured using both human agammaglobulinemic serum and nonimmune guinea pig serum as complement sources. Murine serum does not mediate bacteriolytic activity. Only monoclonal antibody El 17-5 was capable of mediating binding and ingestion in the absence of nonimmune murine serum (table 1). No binding or ingestion was seen with the five other monoclonals tested without serum, even when undiluted preparations were added to the system. Addition of serum to

Delayed Type Hypersensitivity (DTH): Studies on Necrotising DTH-Reactions Against Listerial Antigen in the Skin of Guinea Pigs

Delayed type hypersensitivity (DTH) to facultative intracellular bacteria which leads to destructive skin reactions was so far only investigated against mycobacterial antigens in guinea pigs whereas this work investigates destructive DTH-reactions in the skin of guinea pigs which are directed against listerial antigens. Toxic factors of viable listerias induced an enhancement of destructive skin reactions in non-immunised guinea pigs as compared to immunised ones. In contrast, heat killed listerias (HKL) induced necrotising skin reactions in immunised and non-immunised guinea pigs which were significantly enhanced by DTH in immunised guinea pigs. 5 days after immunisation, necrotising reactivity was maximal and increased in a dose-dependent mode with higher amounts of HKL. Listeria-specific T-cells were able to interact specifically with allogeneic macrophages in vitro. By means of adoptive transfer of listeria-specific T-cells it was possible to transfer successfully Listeria-specific DTH-reactivity from immunised donors to non-immunised recipients.

Delayed type hypersensitivity (dth): quantitative studies of mycobacteria-induced destructive and non-destructive dth-reactions in the skin of guinea pigs

Research on delayed type hypersensitivity (DTH) to facultative intracellular bacteria in guinea pigs focused especially on intracutaneous DTH reactions. However, phenomena of erythematous and necrotic skin reactions were only evaluated qualitatively, dose-response relationships not investigated, differences between early and late reactions overlooked and the development of unspecific skin necrosis ignored. In this work, these aspects were investigated by characterisation of DTH-mediated reactions against mycobacterial antigens in the skin of guinea pigs. Intracutaneous injection of mycobacterial antigen into the skin of immunised and non-immunised guinea pigs induced an erythematous reaction which reached its peak after 24 h. On days two and three after challenge, the centre of the erythema blanched and necrotised. During the next days, skin necrosis increased and reached a peak on days six and seven. In immunised guinea pigs, DTH led to a specific enhancement of erythematous and necrotising skin reactions which also appeared in non-immunised guinea pigs. Thus, an early erythematous and a late necrotising DTH-reaction were identified by their morphology and time course. Both types of DTH-mediated allergy revealed dose-dependency from the intracutaneously injected antigen doses. Erythematous and necrotising DTH reactions against viable and killed mycobacteria showed a similar time course and morphology. DTH reactivity which was induced by immunisation with Mycobacterium tuberculosis showed cross-reactivity with antigens of Mycobacterium bovis.

Development of an Immunoassay for Human Serum Osteoclastic Tartrate-Resistant Acid Phosphatase*

A tartrate-resistant acid phosphatase (TrACP), which has been suggested to be very similar to the osteoclastic TrACP, was partially purified from the spleen of a patient with hairy cell leukemia. The purification procedure consisted of carboxymethyl-Sepharose, phosphocellulose, Sephacryl S-200, and phenyl-Sepharose chromatographies. Polyclonal antibodies were generated in guinea pigs with a titer of at least 1:6000. Immunohistochemical staining of fetal rat tibia with the antisera revealed that only the lysosomes of osteoclasts, but not osteoblasts, were stained. An enzyme-linked immunosorbent assay (ELISA) was developed with the antisera. There was no cross-reactivity with 1) partially purified acid phosphatases (ACPs) from normal human and beef spleens, 2) ACPs in extracts of human osteoblastic cells, 3) purified bovine bone matrix TrACP, or 4) commercial prostatic ACP. However, extracts of giant cell bone tumors, containing large amounts of bona fide osteoclasts, showed large amounts of cross-reactive material, which diluted in parallel with the partially purified hairy cell leukemic TrACP in the ELISA. Commercial serum band 5b TrACP also displaced in parallel with the partially purified hairy cell leukemic TrACP. Immunoblotting studies revealed that the antiserum, but not nonimmune guinea pig serum, reacted with the homogeneous hairy cell leukemia splenic band 5 TrACPs, which were recently purified by our laboratory. Preliminary application of the ELISA to sera of patients with metabolic bone diseases revealed that normal healthy individuals had measurable amounts of the immunoreactive material, and patients with Paget's disease or hyperparathyroidism, who should have high bone turnover, had elevated levels of this immunoreactive material in their sera. In contrast, the level of serum osteoclastic TrACP in a patient with an acute lymphatic leukemia was normal. In summary, 1) we have shown that hairy cell leukemia splenic TrACP shares significant immunological similarity with the osteoclastic TrACP and with the serum band 5b TrACP, and 2) the ELISA holds promise for a sensitive and specific assay for bone resorption.

Limited interference of specific Paf antagonists with hyper‐responsiveness to Paf itself of lungs from actively sensitized guinea‐pigs

1. The interference of various platelet-activating factor (Paf) antagonists with Paf- and antigen-induced effects in isolated lungs from actively sensitized guinea-pigs was investigated. 2. WEB 2086 and BN 52021, two antagonists structurally unrelated to Paf, at concentrations 10-100 fold above those exerting a potent and selective inhibition of the effects of Paf in lungs from non-immunized guinea-pigs, failed to inhibit bronchoconstriction and mediator release evoked by the phospholipid when administered into lungs from actively sensitized animals. 3. In contrast to WEB 2086 and BN 52021, antagonists such as Ro 19-3704 and Ro 19-1400, structurally related to the Paf molecule, at concentrations which abrogate the effects of Paf in lungs from non-immunized guinea-pigs, inhibited bronchoconstriction and release of histamine and leukotriene-like material evoked by the intra-arterial administration of Paf into lungs from actively sensitized animals. 4. Ro 19-3704 and Ro 19-1400 also inhibited markedly the release of leukotriene C4 (LTC4)-like material and, to a smaller extent, the histamine secretion induced by 10 micrograms arachidonic acid. 5. CV 6209, another Paf antagonist structurally related to the phospholipid, failed to antagonize its bronchopulmonary and secretory effects in sensitized lungs. 6. All the antagonists used, irrespective of their ability to interfere or not with bronchoconstriction and mediator release triggered by Paf, suppressed oedema formation as measured by the increase in lung wet weight induced by either Paf or ovalbumin. 7. Our results indicate that: (i) the increase in vascular permeability and the subsequent oedema formation on one hand and the bronchopulmonary effects of Paf on the other hand are mediated by different mechanisms; and (ii) active sensitization provokes a marked modification of the lung reactivity to Paf.

Immunological regulation of colonic ion transport

Challenge of distal colonic epithelium from Trichinella spiralis-infected guinea pigs with parasite-derived antigen elevated short-circuit current (Isc) for approximately 60 min. The maximum elevation (delta Isc) was approximately 250 microA/cm2 at 5 min after the addition of trichinella antigen. The antigen-induced alterations in Isc were of greater magnitude and duration than those evoked in jejunum. Colonic electrical resistance was transiently reduced after exposure to antigen. There was no significant effect of antigen on electrical parameters of colon from nonimmunized (uninfected) guinea pigs. The antihistamine pyrilamine (10(-5) M) and the prostaglandin synthesis inhibitor indomethacin (10(-6) M) reduced the colonic Isc response to antigen by 40% when used in combination but had insignificant effects when used singly. In contrast, the jejunal Isc response to antigen was totally eliminated by the combined use of those inhibitors. Antigenic stimulation of sensitized colon released histamine and prostaglandin E2 (PGE2). However, the histamine released was only about one-tenth that stimulated by antigen in the jejunum, and PGE2 released was only one-tenth of that stimulated by bradykinin in the colon. PGE2 was not released after antigenic stimulation of jejunum. The antigen-induced colonic delta Isc was reduced approximately 50% by either furosemide or tetrodotoxin. Although histamine- and indomethacin-sensitive factors contribute greatly to the mediation of the antigen-induced delta Isc in jejunum, these autacoids contribute to a lesser extent to the antigen-induced delta Isc in guinea pig colon.

Interactions of a nonneutralizing IgM antibody and complement in parainfluenza virus neutralization

While many viruses activate the complement cascade directly, this is generally not a neutralizing event in the absence of antibody. We used a nonneutralizing IgM monoclonal antibody to parainfluenza virus type 3 (PIV3) hemagglutinin-neuraminidase (HN) to explore the role of antibody in complement-dependent neutralization of PIV3. Neither the antibody nor nonimmune guinea pig serum (GPS) neutralized PIV3 significantly, but a more than 100-fold reduction in titer was found when antibody and GPS were combined. Heat-inactivated GPS or GPS lacking either of two different complement proteins were all inactive with or without antibody. Specific repletion of the deficient sera with highly purified complement proteins restored neutralizing activity, indicating an absolute requirement for the classical pathway of complement activation and lytic terminal complement components, and viral lysis was confirmed by electron microscopy. The presence of antibody before complement activation was essential; later addition had no effect. Spontaneous complement activation by PIV3 occurred via the classical pathway in the absence of antibody. Addition of antibody did not alter the overall rate or extent of complement component C3 binding to PIV3 in these experiments. We conclude that certain nonneutralizing antibodies may support complement-dependent PIV3 neutralization by facilitating viral lysis. This process does not, however, involve enhanced activation through the C3 step. Lysis may require antibody-dependent localization of the membrane attack complex or reorganization of the viral envelope structures to facilitate attack complex insertion and lysis.

Mitogenic action ofLegionella pneumophila: Ratio of proliferating T and B cell changes after immunization

The splenic lymphocyte proliferative response of guinea-pigs immunized with L. pneumophila antigen (ALP) was studied. The immune animals were shown to express enhanced reactivity in response to E. coli lipopolysaccharide, as compared to the controls, while the response to concanavalin A was markedly decreased. After 3 days in the culture ALP induced a significant nonspecific lymphocyte transformation that was expressed to a similar degree in both experimental and control group. However, after a 5-day incubation ALP induced a specific proliferative response of the immunized guinea-pig splenocytes. The experiments on fractionation by passing through the column with nylon wool revealed that T cells are activated in non-immunized guinea-pigs. The results obtained prove that T cells mediate the immune response to ALP in guinea-pigs.

Host-Clonal Interactions in the Generation of Proviral Gene Deletion Variants

A nonproducer clone (clone A1) (from a retrovirus-infected guinea pig fibrosarcoma) has been described that under conditions of in vivo immunologic selection forms variants that lack the proviral gene. One trivial explanation for the apparent loss of the provirus from clone A1 is that clone A1 did not originate from a single cell. For evaluation of this possibility, subclones were derived from clone A1 and tested for tumor recurrence in nonimmune and virus-immune animals. Each of four subclones contained the A1 provirus and exhibited specific viral interference; tumor recurrences formed from each of these four subclones lacked the clone A1 provirus. Possible, when heterogeneous populations of retrovirus-infected cells are injected into nonimmune animals, some clones will elicit immunologic responses to retroviral antigens and subject other clones in the population to immunologic selective pressures. For testing this concept, clone A1 was injected in admixture with a producer clone (clone A4) into nonimmune Sewall Wright strain 2 guinea pigs. Tumors formed in nonimmune guinea pigs inoculated with clone A1 in admixture with clone A4 were shown to lack a detectable clone A1 provirus. The results supported the concept that a somatic mutational event (deletion of the proviral gene) occurs during growth of clone A1. When heterogeneous populations of retrovirus-infected cells are injected into animals, host-clonal interactions may occur leading to outgrowth of proviral gene deletion variants. These results supported the notion that interactions between tumor clones and the host can change the dominant clonal type of the tumor and provide a genetic basis for this change.

The alpha cell response to glucose change during perfusion of anti-insulin serum in pancreas isolated from normal rats.

To determine the effect of neutralization of endogenous insulin upon the glucagon response to a rise and fall of glucose concentration, pancreata isolated from normal rats were perfused with either a potent anti-pork insulin guinea pig serum or a nonimmune guinea pig serum for 30 min. During this period glucose concentration was changed from 100 mg/dl to either 130, 180 or 80 mg/dl for 10 min. Antiserum perfusion at 100 mg/dl caused an approximately two-fold increase in glucagon which was not suppressed by an increase in glucose concentration to either 130 or 180 mg/dl, although glucagon secretion was significantly suppressed in the control experiments in which nonimmune serum was perfused. However, the 0.38 +/- 0.21 ng/min rise in glucagon secretion in response to a reduction in glucose concentration to 80 mg/dl in the control experiments was not abolished by antiserum perfusion but, instead, was enhanced (2.66 +/- 0.60 ng/min). These findings suggest that insulin may be required for glucose-mediated suppression of glucagon in the isolated pancreas of normal rats but not for stimulation of glucagon secretion by mild glucopenia. Alternatively, neutralization of insulin-mediated release-inhibition of glucagon secretion may simply have altered alpha cell responsiveness in a direction that desensitized it nonspecifically to suppression and sensitized it to stimulation.

Insulin within islets is a physiologic glucagon release inhibitor.

To determine if glucagon secretion is under physiological control of intra-islet insulin, pancreata from normal rats were perfused at a 100 mg/dl glucose concentration with either guinea pig antiinsulin serum or normal guinea pig serum in a nonrecirculating system. Perfusion of antiserum was followed within 3 min by a significant rise in glucagon that reached peak levels three times the base-line values and assumed a hectic pattern that returned rapidly to base-line levels upon termination of the antiserum perfusion. Nonimmune guinea pig serum had no effect. To gain insight into the probable site of insulin neutralization, 125I-labeled human gamma-globulin was added to antiserum or nonimmune serum and perfused for 3 min. More than 83% of the radioactivity was recovered in the effluent within 3 min after termination of the infusion, and only 0.05 +/- 0.015% of the radioactivity injected was present in the pancreas 10 min after the perfusion. The maximal amount of insulin that could be completely bound to insulin antibody at a dilution and under conditions simulating those of the perfusion experiments was 20 mU/min. It is concluded that insulin maintains an ongoing restraint upon alpha cell secretion and in its absence causes hectic hypersecretion of glucagon. This restraint probably occurs largely in the intravascular compartment. Loss of this release-inhibiting action of insulin may account for initiation of hyperglucagonemia in insulin-deficient states.

Protective effects of antibody against intestinal invasion by Escherichia coli.

Seven Escherichia coli isolates from newborn calves with diarrhea were examined for enteropathogenic properties. One isolate penetrated into HeLa cells, four produced enterotoxin(s) and the remaining two possessed neither of these properties. Penetration of E. coli into HeLa cells was inhibited by antibody in bovine colostrum and in bovine and rabbit immune sera. The effective antibodies appeared to be mostly of the IgM class. The invasion by E. coli isolates was also examined by inoculation of the bacteria into the small intestine of E. coli-immunized and non-immunized guinea pigs. The isolate which penetrated into HeLa cells could penetrate the intestinal mucosa to be disseminated into various organs of non-immunized guinea pigs, but not of immunized guinea pigs, whereas no other isolates showed such pathogenicity in vivo. The inhibition of the invasion was observed when non-immunized guinea pigs were inoculated with the bacteria together with colostral or serum antibody. The results show the importance of antibody in the local defense mechanism against E. coli invasion.