Introduction of an in-house PCR for routine identification of M. tuberculosis in a low-income country.

SETTING: National Tuberculosis (TB) Treatment Centre, Makerere University Medical School and Joint Clinical Research Centre, Kampala, Uganda. OBJECTIVE: To evaluate the introduction of a polymerase chain reaction (PCR) based assay for identification of the Mycobacterium tuberculosis complex (MTC) into routine practice. DESIGN: Routine diagnostic specimens were processed and inoculated into Bactec 12B vials and monitored daily. At a growth index (GI) > or =10, 0.5 ml of the 12B broth was removed and assayed with PCR. The same 12B vial was analyzed using the Bactec NAP method at GI > or =500. Vials at various levels of GI were included. Recurrent cost and time required to perform PCR and NAP were compared. RESULTS: Initially, 71 specimens were analyzed; of these, 68 were NAP-positive while 69 were PCR-positive for MTC. PCR resulted in a 75% reduction in cost for a single test compared with Bactec NAP. PCR has been successfully incorporated into routine practice, and 432 samples have been analyzed. In addition, isolates from solid media were also well identified by PCR. With PCR, more samples can be analyzed at a time, it is faster and is less labor intensive. CONCLUSION: PCR is a reliable and cheaper alternative for the identification of MTC.

Overproduction of SM28GST in a baculovirus expression vector and its use to evaluate the in vivo immune responses of mice vaccinated against Schistosoma mansoni with naked DNA encoding the SM28GST gene.

To evaluate the immune responses of mice vaccinated intramuscularly with naked DNA encoding a single parasite-derived gene, sufficient quantities of protein are necessary for use in the immunological assays. A plasmid carrying the cDNA encoding the entire sequence for the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST) was used as a source of naked DNA to vaccinate mice. Using polymerase chain reaction employing custom primers to add Eco RI and Hind III restriction sites at the 5' and 3' ends, respectively, a 651-bp fragment was amplified from the vaccine plasmid. This product was isolated, ligated into the pFastBac HTb donor plasmid containing a 6X histidine (6X-his) tag, and transposed into the baculovirus expression vector system. Following blue white selection screening, high molecular weight DNA was isolated and transfected in Sf21 insect ovary cells using a liposomal preparation. Culture medium containing infective virus particles was used to infect a series of Sf21 cultures and the cells were lysed after 3-5 days. The lysates were subjected to immobilized metal (Ni-NTA) affinity chromatography from which the 6X-his-tagged recombinant Sm28GST was eluted in 250 mM imidazole. The eluted protein was probed with a polyclonal rabbit antibody specific for the Sm28GST and subsequently recognized using a monoclonal antibody specific for the 6X-his tag following concentration of the pooled fractions. Mice were vaccinated intramuscularly with purified plasmid DNA encoding either the Sm28GST or firefly luciferase. Skin tests performed using recombinant Sm28GST were positive in only those mice vaccinated with naked DNA encoding the Sm28GST gene. In a different group of experimental mice, only sera from mice vaccinated with naked DNA encoding Sm28GST contained IgG-specific anti-Sm28GST antibodies at 14 days postvaccination, and at 42 days the levels were suggestive of an anamnestic response. These results suggest that naked DNA vaccination of mice is capable of inducing both antigen-specific cell-mediated and humoral immune responses against Sm28GST and further strengthen the case for this antigen being a vaccine candidate.

Human toxocariasis and the visceral larva migrans syndrome: correlative immunopathology.

The surface of T. canis is now recognized as a dynamic structure which turns over quite rapidly and serves as a renewable source of large quantities of antigen(s). The major host responses to these antigens include a marked eosinophilia and hyperglobulinemia. Both of these responses are apparently ineffective at ridding the body of infective larvae. Both eosinphils and IgE antibodies are manifestations of the Th2 subset of T helper cells and the cytokines that they secrete. Further, there is reason to believe that the antigens released from T. canis larvae favor the induction of this cellular population. Finally, there is mounting evidence that the chronic production of parasite antigen and its continued stimulation of the host immune system with a concomitant production of eosinophils can lead to a permanent alteration of the normal organization of the cardiopulmonary system. In the absence of any well-documented drugs capable of killing infective larvae, it would seem that immunological intervention may offer the only way to minimize or neutralize this 'gift from man's best friend'. This chapter was not intended to be an exhaustive review of the literature pertaining to toxocariasis. Several other recent publications will hopefully fulfill the need for more detailed information on the biology of this organism and the clinical spectrum of the disease it produces [16, 138-140]. Finally, a MEDLARS search of the current medical literature should bring anyone up to speed in a very short time.

Eosinophilia-induced vascular and airway remodeling and hyperresponsiveness in rat lungs.

We evaluated the effects of pulmonary infiltration of eosinophils without exogenous activators on airway and vascular hyperresponsiveness to muscarinic challenge in the lungs of rats infected with Toxocara [correction of Toxicara] canis, the canine round worm. Bronchoalveolar lavage of infected lungs produced 4.26 x 10(7) cells with 85% eosinophils, 15% mononuclear cells, and essentially no neutrophils. Eosinophils were present in the air spaces and interstitial spaces surrounding airways and vessels. The smooth muscle thickness increased about fourfold in large airways and vessels, and medium and small vessels were muscularized in infected lungs. In the T. canis-infected lungs, baseline airway resistance increased 288%, total vascular resistance (RT) increased 202%, and capillary filtration coefficient increased 208% compared with uninfected control lungs. Lung compliance was 56% of control. The concentration of acetylcholine that produced 50% of maximal response was 18.4 times greater for airway resistance and 18.7 times greater for RT in uninfected controls than in infected lungs. Isoproterenol (10(-4) M) decreased RT and peak airway pressure by 21% in infected lungs but had no significant effect on controls. We conclude that pulmonary interstitial infiltrates of eosinophils cause airway and vascular remodeling and increase baseline resistances and muscarinic reactivities of airways and vessels in rat lungs infected with T. canis.

Toxocara canis: failure to find IgE receptors (Fc epsilon R) on eosinophils from infected mice suggests that murine eosinophils do not kill helminth larvae by an IgE-dependent mechanism.

Eosinophils obtained by bronchoalveolar lavage (BAL) from the lungs of mice infected with Toxocara canis were characterized by flow cytometry with respect to cytophilic antibodies and surface Fc receptors. Freshly harvested BAL eosinophils were negative for sIgM, sIgA, sIgE, and Fc epsilon RII. These eosinophils were positive for sIgG1 and Fc gamma RII, although not all Fc gamma RIIs contained bound ligand. Culturing eosinophils for 24 or 48 hr with exogenous IgE and/or IL-4 did not induce IgE binding capacity or Fc epsilon RII expression. IL-4 did not decrease Fc gamma RII expression but did decrease ligand binding capacity by Fc gamma RII. These findings are in marked contrast to the results of studies characterizing the surface of both human and rat eosinophils and may indicate different functional activities for mouse BAL eosinophils in helminth infections.

Eosinophils increase lung microvascular permeability via the peroxidase-hydrogen peroxide-halide system. Bronchoconstriction and vasoconstriction unaffected by eosinophil peroxidase inhibition.

Eosinophils have been implicated as effector cells in producing vascular and bronchial constriction and increased microvascular permeability in the lung. Hypohalous acids produced by the eosinophil peroxidase (EPO)-hydrogen peroxide (H2O2)-halide system are stable cytotoxic oxidants. We measured the effects of EPO inhibition in activated eosinophils on vascular permeability, assessed using the capillary filtration coefficient (Kf,c), vascular resistance (Rt,vasc), and airway resistance (Raw) in isolated rat lungs perfused with 5% bovine albumin in Kreb's solution. Eosinophils were harvested by bronchoalveolar lavage of Toxicara canis-infected rats. Infusion of 2 x 10(6) phorbol myristate acetate (PMA)-activated cells produced a 3.3-fold increase in Rt,vasc at 30 min, primarily caused by small vessel constriction, a 2.5-fold increase in Raw at 150 min, and a 1.8-fold increase in Kf,c at 90 min. Inhibition of EPO using 3-amino-1,2,4-triazole (3-AT) prevented the increases in Kf,c, but not those in eosinophil superoxide production, Rt,vasc, or Raw. Addition of 2 mM sodium bromide as preferential EPO substrate caused Kf,c, but not Rt,vasc, or Raw, to increase significantly (2.5-fold) compared with activated eosinophils alone. Thus, the acute changes in microvascular permeability were modulated by activity of the EPO-H2O2-Halide system, but the increased vascular and bronchial resistances were mediated through a different pathway.

Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection.

Mechanisms of parasite killing by eosinophils are widely studied and are often implicated in mediating resistance to parasitic infection, especially in conjunction with specific antibodies. Evidence for the eosinophil as an anti-parasite killer cell in vivo is limited and may not justify the belief that eosinophils engage and/or kill infective helminths. We reexamined this question in a mouse model of trichinosis in which antisera to eosinophils were previously used to show the requirement for eosinophils in resistance to this nematode. The current studies used mAb to IL-5 to suppress eosinophil levels in CF1 mice infected with Trichinella spiralis. In mice given a primary infection and injected with an isotype control mAb or left untreated, the medullary and peripheral blood eosinophil numbers peaked at 3 wk postinfection (PI) and returned to baseline levels by 4 wk PI. Peripheral blood eosinophil numbers in infected mice injected with anti-IL-5 were maintained at levels below those of uninfected normal mice through 4 wk of infection. Histologically, there was a prominent eosinophil accumulation in infected, untreated, or control-mAb-treated mice associated with nurse cell complexes containing infective juveniles in skeletal muscle at 3 and 4 wk PI. This was largely eliminated in mice treated with anti-IL-5 mAb. However, the number of muscle stage juvenile worms recovered 3 and 4 wk PI after acid pepsin digestion was unaffected by eosinophil depletion. Challenge infections, in which mice were infected at day 0 with 125 muscle stage worms and challenged at day 28 PI with 350 muscle stage worms, developed peak eosinophil numbers in bone marrow and peripheral blood 3 wk after primary infection and 2 wk after challenge infection in mice receiving either no treatment or control mAb. In challenged mice receiving anti-IL-5 mAb, medullary and peripheral blood eosinophil numbers remained at or below those of uninfected animals. Although all groups exhibited significant resistance measured as muscle stage worm burdens 56 days PI, eosinophil depletion did not affect resistance of muscle worm recovery. These results suggest that eosinophils are not essential in the control of T. spiralis in either primary or challenge infections of CF1 mice. This in vivo study illustrates the questionable value of in vitro killing assays to assign effector function to any single inflammatory cell type.

Oxygen radical scavengers protect against eosinophil-induced injury in isolated perfused rat lungs.

The protective effect of oxygen radical scavengers on lung injury induced by activated eosinophils was examined in isolated perfused rat lungs. Eosinophils were obtained by bronchoalveolar lavage from rats infected with Toxocara canis and activated with phorbol myristate acetate (PMA). There were no changes in pulmonary vascular (RT) and airway (Raw) resistances and only minimal changes in vascular permeability assessed using the capillary filtration coefficient (Kf,c) in PMA control lungs and nonactivated eosinophil-treated lungs. In lungs receiving 3 x 10(6) PMA-activated eosinophils, there were significant increases from baseline of 7.3-fold in RT at 30 min, primarily due to the constriction of small arteries and veins; 3.6-fold in Kf,c at 90 and 130 min; and 2.5-fold in Raw. The lungs also became markedly edematous. Both superoxide dismutase and catalase pretreatment prevented the significant increase in Kf,c and lung wet-to-dry weight ratios and partially attenuated the increase in Raw, but did not significantly inhibit the increase in RT induced by activated eosinophils. Heat-inactivated catalase did not attenuate the eosinophil-induced increases in Kf,c, Raw, or RT. Thus, activated eosinophils acutely increased microvascular permeability primarily through production of oxygen free radicals. The free radical scavengers superoxide dismutase and catalase partially attenuated the bronchoconstriction but had no significant effect on the vasoconstriction induced by activated eosinophils.

Development and characterization of a model of eosinophil-mediated cardiomyopathy in rats infected with Toxocara canis.

This study begins testing the hypothesis that a causal relationship exists between hypereosinophilia and cardiac dysfunction. Rats infected with the nematode Toxocara canis develop marked hypereosinophilia, with peak blood eosinophil levels of approximately 3,500 eosinophils/mm3 whole blood observed approximately 14 days postinfection. Corresponding to the increase in blood eosinophils of infected animals was a decrease in cardiac performance. By 14 days postinfection, cardiac work had declined approximately 25% while negative first derivative of pressure (-dP/dt) fell approximately 10%. As the extent of hypereosinophilia declined from a peak of approximately 3,500 eosinophils/mm3 whole blood to a new steady state of approximately 1,000 eosinophils/mm3, the degree of cardiac dysfunction also was reduced. Cardiac work was 10-15% less in rats 28-42 days postinfection while -dP/dt was 5% depressed in these animals. Myocardial dysfunction was also observed following exposure of perfused hearts obtained from uninfected rats to buffer containing activated eosinophils. The hearts exposed to activated eosinophils exhibited marked histological alterations, characterized by distention of the intermyocyte space, increased pericapillary space, and focal losses of striated staining pattern. These changes were associated with the accumulation of eosinophils within the myocardium, as evidenced by the cytochemical demonstration of eosinophil peroxidase activity within the heart. The data support the hypothesis that hypereosinophilia can lead to cardiac dysfunction.

Eosinophil peroxidase levels in hearts and lungs of mice infected with Toxocara canis.

Toxocara canis infection of abnormal hosts results in a condition in which infective larvae migrate through the soft tissues of the body, exclusive of the skin. This condition is known as visceral larva migrans (VLM) and causes a syndrome characterized by hepatosplenomegaly, hyperglobulinemia, hypereosinophilia, and transient pulmonary infiltrates. Because of the known association between hypereosinophilia and eosinophilic heart disease, we have been studying the hearts of mice infected with T. canis for evidence of myocardial damage and have previously described a severe eosinophilic myocarditis that leads to a marked myocardial fibrosis. We have measured eosinophil peroxidase (EPO) levels (a marker enzyme for specific granules of eosinophils) in homogenized lungs, homogenized hearts, and eosinophils recovered from the lungs of mice infected with T. canis over a 6-wk period. A marked accumulation of EPO was observed in the lungs of infected mice from day 14 postinfection (PI) to at least 6 wk of infection. Most of the EPO was associated with eosinophils that comprise the bulk of the pulmonary infiltrates associated with the VLM syndrome. However, following bronchoalveolar lavage, cytochemical localization of EPO activity in lungs from infected mice suggested that eosinophil degranulation had resulted in this marker enzyme being deposited within the pulmonary parenchyma. Peak levels of EPO were found in the myocardium by day 14 PI and declined over the 6-wk period. These levels equaled about 1/3 of the levels seen in the lungs of the same mice. These studies suggest that in mice infected with T. canis, the presence of increased numbers of eosinophils may lead to marked peroxidatic cardiopulmonary damage.

Human anisakiasis transmitted by marine food products.

Seafood-transmitted parasitic diseases represent an emerging area of interest to the U.S. Food and Drug Administration. Human infections with marine parasites are generally the result of ingesting uncooked seafood products. Over 50 species of helminthic parasites are known to infect humans worldwide. Recently, the number of infections with one of these helminths, the juvenile stage of the marine nematode, Anisakis simplex, has increased in the United States. Raw fish dishes such as lomi lomi salmon and sashimi are known to transmit the parasite to unsuspecting citizens and the most frequently implicated fish in the transmission of this zoonotic disease is the Pacific salmon (Oncorhynchus spp.). The risk of infection from fishes caught in Hawaiian waters is slight; however, a juvenile Anisakis simplex infected one patient from either locally caught aku or ahi. We report 4 new cases, which brings the total number of known cases in Hawaii to 7. Five of the 7 cases were diagnosed and treated by means of an endoscope and biopsy forceps. Serological profiles are presented in several of these cases. One case represents the first known instance of reinfection; the initial infection occurred 2 years prior. The second infection gave an opportunity to compare the human response to a challenge infection and to investigate the validity of the "double hit" theory. Increased awareness by physicians to the clinical features of this disease is warranted. The zoonotic disease, anisakiasis, should be considered in patients presenting with intense abdominal pain, if these patients admit they have recently eaten raw or undercooked seafoods.

Activated eosinophils increase vascular permeability and resistance in isolated perfused rat lungs.

The effects of eosinophils activated with phorbol myristate acetate (PMA) on isolated perfused rat lungs were examined. Eosinophils were obtained from lungs of rats infected with Toxocara canis by bronchoalveolar lavage, incubated with PMA, and administered to an isolated perfused rat lung preparation. Vascular endothelial permeability was assessed by measuring the capillary filtration coefficient (Kf,c) in the perfused lungs. In lungs receiving either no eosinophils (control) or nonactivated eosinophils, there were no changes in pulmonary hemodynamics or Kf,c. However, in lungs receiving 2 x 10(6) eosinophils activated with PMA, there was a transient 4.8-fold increase in pulmonary vascular resistance that peaked at 30 min, primarily due to the constriction of small arteries and veins. After the initial pressor response, Kf,c was increased to 7.5 times control at 130 min and resulted in marked lung edema, increased wet-dry weight ratios, and edema on histologic examination. Pulmonary arterial pressure and Kf,c responses were dose related for eosinophil numbers between 1 x 10(6) and 4 x 10(6) cells. Peak airway pressure (Paw) during constant tidal volume ventilation also increased in lungs receiving activated eosinophils compared to the control and nonactivated eosinophil groups. These findings indicate that activated eosinophils are potent effector cells and can cause pulmonary vasoconstriction, bronchoconstriction, and vascular endothelial injury without widespread plugging of capillaries by aggregated eosinophils.

Eosinophilic myocarditis in CBA/J mice infected with Toxocara canis.

In humans, chronic eosinophilia has been associated clinically with endomyocardial fibrosis and myocardial damage. Mice infected with Toxocara canis have a marked eosinophilia, and develop eosinophil-rich granulomatous lesions in the soft tissues of the body, especially the lungs, liver, brain, and skeletal muscle. Few reports have described myocardial lesions associated with T. canis infections in mice. We examined the hearts of CBA/J mice killed at weekly intervals over an 8-week period for evidence of myocardial damage that might be attributable to eosinophils. Total white blood cell counts and eosinophil counts were obtained during this period, and revealed a peak white blood cell count of approximately 28,000 cells/mm3 at day 7 after infection and a peak eosinophil count of approximately 4,000 cells/mm3 at day 14 after infection. Myocardial lesions in the ventricular wall began as focal infiltrates of eosinophils and histiocytes, then progressed into granulomata containing necrotic debris. Collagen deposition was noted by day 21 after infection. By day 42 after infection, the lesions had contracted greatly because of a loss of cellularity, and consisted mainly of fibroblasts and hemosiderin-laden macrophages. Myocyte damage, characterized by increased eosinophilia and necrosis, was observed. T. canis-infected CBA/J mice thus offer a useful model to study eosinophil-dependent myocardial damage.

Anisakis simplex: histopathological changes in experimentally infected CBA/J mice.

Third-stage juveniles (L3) of Anisakis simplex, surgically implanted into the abdominal cavity of CBA/J mice and necropsied at 7, 14, or 21 days postinfection (PI), embedded in the gut mesentery and only rarely invaded viscera. Histologically, intense aggregations of neutrophils adjacent to the parasites were noted at Day 7 PI. At Day 14 PI, mature granulomata consisting mostly of eosinophils and large numbers of fibroblasts and associated collagen were observed. Granulocytes and occasionally multinucleate giant cells occupied the host-parasite interface. At 21 day PI, lesions displayed the predominance of connective tissue. Multinucleate giant cells were found adjacent to the L3 with eosinophils adjacent to parasite remnants or scattered within the walls of the granulomata. Most L3 were viable at Days 7 and 14 PI; however, at Day 21 PI the L3 were dead and invaded by inflammatory cells. Hematological findings indicated that infected mice had a neutrophilia of varying magnitude regardless of the number of worms implanted. Eosinophil levels as a percentage of the total leukocyte pool in peripheral blood always remained at or below normal limits. On Days 7 and 14 PI, the peripheral blood showed an increase in neutrophils that began to return to normal values at 21 day PI. Conversely, peripheral blood eosinophils decreased on Days 7 and 14 PI and returned to normal values on Day 21 PI. Surgical implantation of A. simplex L3 into mice produced both a hematological and histological picture consistent with that seen in human anisakiasis.

Effects of phorbol myristate acetate-stimulated human leukocytes on rat lung.

Human blood was separated into polymorphonuclear (PMN) and mononuclear (MN) leukocyte fractions, and 3 x 10(7) cells (PMN or MN) were added to isolated rat lungs perfused with 5% human albumin in buffer and stimulated with phorbol myristate acetate (PMA). Lungs perfused with either albumin alone, PMN, or MN but not stimulated with PMA showed no change in vascular resistance or endothelial permeability measured as the capillary filtration coefficient (Kf,c). Lungs that were stimulated with PMA with no cells showed no change in Kf,c (0.34 +/- 0.07 vs. 0.37 +/- 0.7), but vascular resistance increased in all segments of the circulation. Capillary pressure, the major force responsible for edema formation, nearly doubled in the absence of cells 40 min after PMA. Lungs perfused with either PMN or MN and stimulated with PMA were injured. Kf,c increased from 0.41 +/- 0.03 to 0.87 +/- 0.10 (PMN) and from 0.36 +/- 0.07 to 0.81 +/- 0.23 (MN) 90 min after PMA. In addition to the increased endothelial permeability, vascular resistances and pressures also increased in the cell-perfused PMA-stimulated lungs. These results demonstrate that cells other than granulocytes are capable of producing severe acute lung injury and cannot be ignored when the effects of PMA on neutrophil-depleted lungs are studied.

Pulmonary granuloma formation in murine toxocariasis: transfer of granulomatous hypersensitivity using bronchoalveolar lavage cells.

A major drawback to studying granuloma formation in murine toxocariasis is the ability of the second-stage larva of Toxocara canis to escape from a developing granuloma, migrate elsewhere, and initiate granuloma formation anew. In an attempt to circumvent this difficulty, 2 different T. canis-derived antigenic preparations were covalently attached to Sepharose 4B beads and embolized into the microvasculature of the lungs of CBA/J mice that had been infected 10 days previously with 25 T. canis ova. Both T. canis egg extract (TEE) and T. canis exoantigens (TEX) were able to elicit antigen-specific granulomas 6 days postembolization as determined by both histologic and morphologic criteria. Histologically, the eosinophil-rich granulomas forming around antigen-coated beads embolized into infected mice resembled the developing granuloma previously described forming around the second-stage larva. Attempts to transfer granulomatous reactivity in this model using either immune spleen cells or immune serum were unsuccessful. Successful transfer of granulomatous hypersensitivity was achieved using cells obtained by bronchoalveolar lavage of previously infected mice. The results suggest the feasibility of using this embolic model of granuloma formation in murine toxocariasis.

Use of bronchoalveolar lavage to compare local pulmonary immunity with the systemic immune response of Toxocara canis-infected mice.

Following infection of mice with larvae of the canine roundworm Toxocara canis, there is a persistent pneumonitis. Heretofore, nothing was known about the immunologic potential of the cells that constitute this inflammatory exudate. By performing bronchoalveolar lavage (BAL), enough inflammatory cells were obtained to compare the local pulmonary immune response to T. canis infection with the systemic immune response as reflected in the peripheral blood and spleen cells of the same mice. Groups of C57BL/6J female mice were given 100 infective ova and BAL, peripheral blood, and spleen cells collected on days 8, 11, 14, and 17 postinfection. The percentage of eosinophils in the BAL averaged about 80% and was four to five times as great as that in the peripheral blood at all times assayed. Use of concanavalin A (ConA)-elicited lymphocyte blastogenesis to evaluate T-lymphocyte activity revealed that BAL T-cell activity was low on day 8 and peaked on day 11. When the B-cell mitogen lipopolysaccharide was used in the assay, there appeared to be far less BAL cell reactivity compared with BAL T-cell activity. Both B- and T-cell responses of the BAL cells were only a fraction of the responses seen concurrently in spleen cells. Use of Toxocara exoantigens in the blastogenesis assay revealed that Toxocara exoantigens could elicit between 20 and 95% of the ConA response in BAL cells, while in spleen cells Toxocara exoantigens could only elicit 1 to 5% of the ConA response. These results suggest that BAL is a useful method for recovering local inflammatory cells that possess detectable immunologic activity. In the case of pulmonary toxocariasis, eosinophils account for the majority of the cells that are present, with most of the remaining cells being T. canis antigen-specific T lymphocytes.

Factors affecting the infection of the D variant of encephalomyocarditis virus in the B cells of C57BL/6J mice.

The D variant of encephalomyocarditis virus is capable of infecting most inbred strains of mice. However, only certain strains are susceptible to the diabetogenic effect of this virus. In order to understand why some inbred strains do not become diabetic, the pathogenesis of infection was studied in diabetes-resistant C57BL/6J mice. It was the purpose of the investigation to ascertain whether specific host defense factors might play a crucial role in the mechanism of resistance. To determine whether perturbations of the immune response would alter the resistance of these animals, mice were treated with a high dose (1.15 mmol/kg body weight) of the T- and B-cell toxin cyclophosphamide prior to infection with the D variant. This treatment did not induce overt diabetes or glucose intolerance in the mice tested 7 days after infection. Based on this finding, it appeared likely that resistance to the D variant is conveyed by some factor other than cell-mediated immunity. A likely candidate to control this viral infection is the interferon system. To investigate this possibility, C57BL/6J mice were infected with the D variant and the concentrations of serum interferon titred at various intervals thereafter. In contrast to previous reports with diabetes susceptible mice, C57BL/6J mice were found to generate a substantial interferon response against this variant, with peak levels found in the serum at 24 h following infection. Additional studies were performed in which mice were treated with antibody to mouse interferon alpha/beta at the time of infection and again 3 days after infection with the D variant.(ABSTRACT TRUNCATED AT 250 WORDS)