Enhancement of influenza virus infections by secalonic acid D.

Secalonic acid D (SAD), a hepatotoxic, teratogenic, and slightly mutagenic metabolite of Penicillium oxalicum has been identified as a natural contaminant of grain dust. Secalonic acid D was administered intraperitoneally to male ICR mice that were exposed to influenza virus aerosols 5 days earlier. The mortality rate was significantly higher (p less than 0.001) in mice subjected to both influenza and SAD than those subjected to influenza alone. Virus titers in lung tissue samples at selected time intervals appeared similar for both influenza and influenza-SAD treated groups of mice for 9 days after exposure to the virus. After 9 days, influenza-SAD treated mice appeared to have higher virus titers. No difference in the pathological progression of pneumonia was discernible between these two groups of mice. The influenza-SAD group, in addition to pneumonia, exhibited severe hepatic necrosis characteristic of SAD administration. Mice infected with influenza virus followed by administration of SAD responded with significantly lower (p less than 0.05) antibody titers to influenza virus than mice exposed to influenza virus alone.

Mansonella ozzardi in Haiti. III. A comparison of the sensitivity of four sampling methods in detecting infections.

A study to compare the sensitivity of four sampling methods in detecting Mansonella ozzardi among 133 inhabitants of Bayeux, Haiti, is reported. The Knott method proved the most sensitive with 40 microfilaria carriers detected. No additional infections were revealed by the other sampling methods. This was followed in order by 20 mm3 thick films of earlobe blood (34 cases), finger-prick blood (32 cases in the first sample), and skin biopsy (14 cases from paired samples). The combination of three finger-prick samples (60 mm3 of blood) detected 38 of 40 (95%) Knott-positive infections. The Knott method detected only one case not observed by at least one of the other three sampling methods. This sampling procedure did not detect more M. ozzardi in the 0-19-year age group than observed by the 20-mm3 finger prick method. Based on these results, a correction factor of 1.25 can be applied to our earlier Bayeux survey.

Mansonella ozzardi in Haiti. IV. Evaluation of antibody reactivity to heterologous antigens.

Sera from individuals in an area of Haiti endemic for Mansonella ozzardi were analyzed for reactivity to antigens of Brugia pahangi, Dirofilaria immitis, Mansonella llewellyni or Ascaris lumbricoides using either an enzyme-linked immunosorbent assay or an indirect immunofluorescent antibody test. IgM and IgG reactivity to all antigens was observed with sera from both microfilaremic and amicrofilaremic individuals when compared to reactivity of sera from individuals from nonendemic areas. Antibody reactivity to B. pahangi was greater than that to other antigens. IgG reactivity of sera from endemic patients to filarial antigens was consistently greater than that of IgM. Antibody reactivity was not correlated with age or microfilarial density.

Ancylostoma larva in a muscle fiber of man following cutaneous larva migrans.

This is a report of a case of massive cutaneous larva migrans in a 20-year-old man who also had pulmonary symptoms and larval invasion of the skeletal muscles. In sections of a muscle biopsy specimen taken 3 months after the initial cutaneous lesions, a third-stage Ancylostoma larva, probably A. caninum, was found within a muscle fiber.

Assessment of Leptoconops bequaerti as a potential vector of Mansonella ozzardi in Haiti.

Experimental studies in Bayeux, Haiti showed that the biting midge, Leptoconops bequaerti, is capable of supporting the complete development of Mansonella ozzardi but only on a very limited scale. This suggests that the species may not be involved in the natural transmission cycle despite its abundance and pestiferous nature in certain areas of Haiti. A midge-holding container is described which markedly enhanced the survival of engorged L. bequaerti in the laboratory.

Uptake and development of Wuchereria bancrofti in Culex quinquefasciatus that fed on Haitian carriers with different microfilaria densities.

In studies conducted between 1984 and 1986, the vector competency of Culex quinquefasciatus was assessed after bloodfeeding on 61 Haitian volunteers with different densities of Wuchereria bancrofti microfilariae (mf) and on 11 that were amicrofilaremic. Infected volunteers included persons previously given diethylcarbamazine citrate for 12 consecutive days and some that were untreated. Mosquitoes, derived from field-collected larvae, were released under bed nets and fed upon the volunteers while they slept. The mean mf uptake in mosquitoes that fed on 21 carriers with low to ultralow densities (1-28 mf/ml blood) was 0.2-4 mf/mosquito (mean = 1.7 mf). The observed infectivity rate of greater than 3,000 mosquitoes that fed on high (56-7,500 mf/ml blood; median = 525), low (11-49 mf/ml blood; median = 20), or ultralow (1-10 mf/ml blood; median = 3) density carriers was 4, 11, and 30 times higher, respectively, than the expected rate calculated from the estimated volume of the imbibed bloodmeal. These results indicate that "hidden" carriers (less than 50 mf/ml blood) may serve as a source of infection for mosquitoes, and support the increasing evidence that mosquitoes ingest more microfilariae than expected.

Bancroftian filariasis in Haiti: preliminary characterization of the immunological responsiveness of microfilaremic individuals.

Patent infections with the lymphatic filariae, Wuchereria bancrofti and Brugia malayi, are associated with suppressed in vitro cellular responsiveness to filarial antigens. In studies of bancroftian filariasis in Haiti, a significant number of microfilaremic individuals can be characterized as "responders" to filarial antigens. Cells from 37/74 untreated microfilaremic subjects responded to B. pahangi antigen (stimulation ratio greater than 2) as detected by in vitro blastogenesis. A comparison of responders to nonresponders revealed a significant difference in mean B. pahangi reactivity (15,822 vs. 4,538 cpm, P less than 0.001), but no significant differences with respect to age, microfilaremia, PPD or PHA reactivity, or B. pahangi-specific antibody levels. Subtle differences may exist between these groups with respect to recognition of specific antigens on Western blots.

Epidemiology of Wuchereria bancrofti in Leogane, Haiti.

A survey for Wuchereria bancrofti in Leogane, Haiti, revealed that 140 of 421 individuals (33%) had a patent infection, of which 40% lived in the suburban outskirts of the city. The median microfilaria density was 19.1 per 20 mm3 of blood for suburban dwellers compared with only 8.8 for those living in the city. The vector, Culex quinquefasciatus (Say), breeds mostly in and around numerous rum distilleries, located exclusively around the periphery of the city, and this undoubtedly accounts for the higher prevalence and intensity of infection among suburban dwellers.

The effect of diethylcarbamazine treatment of Bancroftian filariasis on the immunological reactivity of microfilaraemic individuals.

Patent filarial infection has been correlated with a profound suppression of humoral and cellular responses to filarial antigens. In the present study, the filarial antigen-specific humoral and cellular reactivity of 30 Haitian subjects with patent Wuchereria bancrofti infection was monitored before and after treatment with diethylcarbamazine. Microfilarial density was reduced from a pre-treatment mean of 1778/ml to 9/ml, with residual microfilaraemias detectable in 10 subjects. Peripheral blood mononuclear cells from 18 of the 30 patients responded to an extract of Brugia pahangi before treatment, and this number increased to 25 after treatment. There was no significant change in the mean level of response to B. pahangi in patients who were responsive to filarial antigen before treatment; however, the mean responsiveness to B. pahangi of individuals who were classified as nonresponders before treatment was significantly increased following treatment. Cellular reactivity to purified protein derivative and geometric mean titres to soluble B. pahangi, measured by enzyme-linked immunosorbent assay, were unaffected by treatment. Similarly, most post-treatment sera did not recognize new B. pahangi bands on Western blots, compared to pre-treatment controls. These observations imply that the relationship between microfilariae and immunosuppression is complex.

Immunocytochemical studies on Schistosoma mansoni. II. Soluble cercarial antigens in cercarial and schistosomules.

The localization of soluble, cercarial antigen preparation (CAP) in cercariae and schistosomules of Schistosoma mansoni was performed at the light and electron microscope levels using the unlabeled antibody method. Reaction produce was observed associated with the contents of the pre- and postacetabular glands and with the filamentous coat of mature cercariae. No reaction product was observed associated with the glycoacalyx of schistosomules. However, several schistosomules did retain remnants of their filamentous coats and reaction product was observed associated with those remains. CAP components were also observed in the area surrounding the intrasporocyst cercariae.

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi.

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.

Immunoregulation in experimental filariasis. II. Responses to parasite and nonparasite antigens in jirds with Brugia pahangi.

Chronic B. pahangi infection (greater than or equal to 5 mo) in the jird, Meriones unguiculatus, leads to the induction of adherent nonspecific suppressor cells that are capable of modulating the in vitro mitogen responsiveness of spleen cells. In the present studies, a correlation between suppression of mitogen responsiveness and lack of reactivity to B. pahangi antigens was observed in vitro with splenic lymphocytes from chronically infected animals. However, the ability of jirds with a chronic B. pahangi infection to develop in vivo humoral responsiveness to SRBC and DTH to DNFB was comparable to that of uninfected controls. Analysis of the relationship between the development of antigen-specific and nonspecific immunoregulatory activity over the course of the infection was undertaken, too. Altered in vitro responsiveness of spleen cells from infected jirds to mitogens and B. pahangi antigens was associated with the onset of microfilaremia (8 wk post-infection). A transient lack of reactivity to SRBC was observed after the development of a patent infection in jirds. However, nonspecific suppressor cells capable of modifying the in vitro mitogen responsiveness of normal lymphocytes were not observed in the spleens of B. pahangi-infected animals exhibiting a lack of reactivity to SRBC. The relationship of antigen-specific suppressor cells to immunoregulation in experimental filariasis is discussed.

Induction of cellular and humoral immunological responsiveness to a soluble cercarial antigen preparation from Schistosoma mansoni.

The development of immunological responsiveness to a soluble cercarial antigenic preparation (CAP) from Schistosoma mansoni was analyzed in inbred CBA/J mice infected with cercariae, one or multiple times, or sensitized using CAP. Repeated exposure to 75 cercariae at three weekly intervals (3X-75) or subcutaneous administration of 20 mug of CAP (CAP/complete Freund adjuvant [CFA]) stimulated the development of specific anti-CAP lymph node cell blastogenesis. The degree of responsiveness was dependent upon the concentration of CAP in the culture system and was optimal in the dose range of 20 to 30 mug of protein of CAP per culture. Animals exposed once to 75 or 225 cercariae or to two sequential weekly infections with 75 cercariae exhibited a minimal response to CAP in comparison to the responsiveness of 3X-75 or CAP/CFA lymph node cells. Assessment of anti-CAP agglutinating antibody by application of a microtiter passive hemagglutination technique revealed that both 3X-75 and CAP/CFA animals possessed low titers of activity. In addition, both 3X-75 and CAP/CFA sera contained reagin-like antibodies to CAP as detected by the heterologous (rat), 72-h latent period, passive cutaneous anaphylaxis technique.

Analysis of the intradermal response against a soluble cercarial antigenic preparation from Schistosoma mansoni.

Intradermal testing was performed with a soluble cercarial antigenic preparation (CAP) from Schistosoma mansoni cercariae in CBA/J mice multiply infected with S. mansoni or sensitized with CAP. Both an early (5-h) response and a late (24- to 48-h) reaction to CAP, as measured by increase in dermal thickness, was elicited after injection of antigen into the ears of either multiply infected (3X-75) or CAP-sensitized (CAP/complete Freund adjuvant [CFA]) mice. Histopathological examination showed that the early response was primarily vascular in nature and involved a polymorphonuclear cell infiltrate in and around dilated capillaries. The late reaction to CAP consisted of a perivascular cellular infiltrate of polymorphonuclear and mononuclear cell types. Passive transfer of 3X-75-infected or CAP/CFA-sensitized serum (0.4 ml) to normal mice conveyed the ability to mount an early (5-h) response to CAP which was marked histopathologically by a prominent polymorphonuclear cell infiltrate. The majority of the responsiveness in normal mice after administration of lymph node cells (40 X 10(6)) from multiply infected or CAP/CFA-sensitized mice was observed 24 to 48 h after injection of CAP and was mononuclear in nature.

Effect of cyclophosphamide on the immune responsiveness of jirds infected with Brugia pahangi.

The in vitro immune responsiveness of lymphocytes from Brugia pahangi-infected jirds was examined after serial administration of cyclophosphamide (20 mg/kg). Cyclophosphamide had no effect on parasite burdens, anti-B. pahangi antibody titers, or suppressed spleen cell reactivity to B. pahangi antigens. Cyclophosphamide restored cellular responsiveness to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen.

Immunoregulation in experimental filariasis. IV. Induction of non-specific suppression following in vitro exposure of spleen cells from infected jirds to Brugia pahangi antigen.

Studies with inbred jirds chronically infected (greater than 5 months) with Brugia pahangi have demonstrated splenic suppressor cells which modulate in vitro responsiveness to mitogens and parasite antigens. The stimuli which induce suppression were characterized by analysing the effect of activated cells from inbred normal or B. pahangi-infected jirds on the PHA and PWM responsiveness of cultures on normal cells. Regulatory cells were stimulated in vitro with concanavalin A (Con A; 5 micrograms/ml) or an extract of adult B. pahangi (20 micrograms/ml) for 72 hr and irradiated (1500 rads) prior to cocultivation with normal cells. Addition of Con A-activated normal spleen cells to normal cells produced moderate suppression of PHA and enhancement of PWM responsiveness. However, Con A-stimulated spleen cells from infected animals consistently suppressed both the PHA and PWM responsiveness of normal cells by 80-90%. Spleen cells from chronically infected jirds were also induced by B. pahangi antigen to suppress both the PHA and PWM responsiveness of normal lymphocytes. In contrast, spleen cells from animals 3-15 weeks after infection and lymph node cells from all time points were capable of suppressing only PWM responses when stimulated by antigen. Normal spleen cells were not induced by B. pahangi antigens to exhibit immunoregulatory activity. The suppression mediated by antigen-induced spleen cells from chronically infected jirds was partially or totally alleviated by removal of non-specific suppressor cells which are plastic adherent and cyclophosphamide-sensitive, or by removal of antigen-specific suppressor cells which bear receptors for histamine. the results suggest the involvement of regulatory cell circuits in experimental filarial infections.

Immunoregulation in experimental filariasis. III. Demonstration and characterization of antigen-specific suppressor cells in the spleen of Brugia pahangi-infected jirds.

Antigen specific immunoregulatory phenomena in both human and experimental filariasis are correlated with the presence of circulating microfilariae. Previous studies of inbred jirds infected with Brugia pahangi have demonstrated that the onset of microfilaremia (8-10 weeks post-infection) is associated with a loss of responsiveness to parasite antigens in the spleen, but not the lymph nodes of infected animals. The present experiments defined immunoregulatory phenomena responsible for altered antigen-specific in vitro lymphocyte blastogenesis in the spleen of B. pahangi-infected jirds. Diminished splenic responsiveness to B. pahangi antigens was associated with the presence of a cell capable of suppressing the responsiveness of lymph node cells from infected jirds to parasite antigens. antigen-induced in vitro blastogenic responsiveness of spleen cells from microfilaremic , but not normal animals was restored by depletion of cells adherent to nylon wool or bearing receptors for histamine, peanut agglutinin, soybean agglutinin or jird Fc. The responsiveness of spleen cells from chronically infected (greater than or equal to 20 weeks) animals to mitogens, but not parasite antigens, was enhanced by removal of plastic-adherent cells. The results suggest the involvement of antigen-specific suppressor T cells, in the spleen of B. phangi -infected jirds which are distinct from non-specific suppressor cells previously described in this system.

Catheptic carboxypeptidase B as a major component in "T-cell activating factor" of macrophages.

TAF (T-cell activating factor), produced by peritoneal macrophages and assayed by its ability to potentiate DNA synthesis in macrophage-depleted lymph node cells stimulated with Phytochemagglutinin-concanavalin A, was shown to contain catheptic carboxypeptidase B (CPB) which accounted almost quantitatively for its potentiating activity. TAF action was inhibited by CPB inhibitors and could be mimicked by commercial pig pancreas CPB. The activity was absorbed from active supernatants on EACA-Sepharose and could be eluted with free EACA (epsilon aminocaproic acid).

Serosuppression in experimental filariasis.

Both antigen specific and non-specific immunoregulation by cells have been described in jirds infected with Brugia pahangi, but the contribution of serum factors to immunoregulatory phenomena in this infection has not been examined. The present study determined the effect of serum from normal or B. pahangi infected jirds on the mitogen responsiveness of spleen cells from uninfected animals and on the antigen responsiveness of lymph node cells (LNC) from infected jirds. Addition of heat-inactivated jird serum to cultures of cells supplemented with 1% fetal bovine serum demonstrated that serum from chronically infected (greater than or equal to 20 weeks post-infection), but not normal jirds consistently suppressed responsiveness of LNC from infected jirds to B. pahangi extracts in a dose-dependent manner (0.25%-1% concentration). A comparison of sera from jirds at different times post-infection demonstrated that sera (1%) from chronically infected (30 weeks; 100% suppression), but not acutely infected (4 weeks; 0% suppression) or recently microfilaremic (10 weeks; 11% suppression) animals were capable of suppressing antigen reactivity of LNC. In contrast, the inhibitory effect of serum on lymphocyte reactivity to the mitogens, PHA and PWM, was observed intermittently throughout the course of the infection and was less than the effect of chronic serum on antigen responsiveness. The B. pahangi antigen response of spleen cells from infected jirds depleted of suppressor cells by fractionation over nylon wool was also inhibited by chronic sera. Following fractionation of chronic sera by Sephadex G-200 chromatography, suppressor activity was observed in the void volume and IgG peaks. Suppressor activity was not associated with protein A, anti-jird Ig, or B. pahangi antigen bound fractions, nor with polyethylene glycol precipitable material.

Growth cycle-dependent generation of complement-resistant Leishmania promastigotes.

The ability of in vitro grown Leishmania promastigotes to resist lysis by complement and survive in undiluted human serum was related to the species of Leishmania and the growth phase in culture. Promastigotes from log phase cultures were always killed in undiluted serum, whereas survival of stationary phase promastigotes varied among species. All L. major and L. m. amazonensis were killed, while up to 30% of L.b. panamensis and 10% of L. donovani survived. Lysis of promastigotes by human serum was inhibited in heat-inactivated serum and EDTA-chelated serum, indicating that activation of complement was responsible for killing. Therefore, during growth in vitro, some strains of Leishmania promastigotes can undergo development from a complement-susceptible to -resistant stage. Stationary phase promastigotes of L.b. panamensis which survived in undiluted human serum were capable of subsequent growth in culture, and were also able to initiate infection in Mystromys albicaudatus. Organisms selected on the basis of complement resistance were more infective for M. albicaudatus than either log phase promastigotes or unselected promastigotes from stationary cultures. These data support the notion that the life cycle of Leishmania includes an infective stage promastigote which is generated during growth within the sandfly and which, on inoculation, is able to survive the potentially lethal effect of normal serum before uptake by host macrophages.