Atomic force microscopic investigation of respiratory syncytial virus infection in HEp-2 cells.

Respiratory syncytial virus (RSV) primarily causes bronchiolitis and pneumonia in infants. In spite of intense research, no safe and effective vaccine has been developed yet. For understanding its pathogenesis and development of anti-RSV drugs/therapeutics, it is indispensable to study the RSV-host interaction. Although, there are limited studies using electron microscopy to elucidate the infection process of RSV, to our knowledge, no study has reported the morphological impact of RSV infection using atomic force microscopy. We report the cytoplasmic and nuclear changes in human epidermoid cell line type 2 using atomic force microscopy. Human epidermoid cell line type 2 cells, grown on cover slips, were infected with RSV and fixed after various time periods, processed and observed for morphological changes using atomic force microscopy. RSV infected cells showed loss of membrane integrity, with degeneration in the cellular content and cytoskeleton. Nuclear membrane was disintegrated and nuclear volume was decreased. The chromatin of the RSV infected cells was condensed, progressing towards degeneration via pyknosis and apoptosis. Membrane protrusions of ~150-200 nm diameter were observed on RSV infected cells after 6 h, suggestive of prospective RSV budding sites. To our knowledge, this is the first study of RSV infection process using atomic force microscopy. Such morphological studies could help explore viral infection process aiding the development of anti-RSV therapies.

Expression and characterization of a multivalent human respiratory syncytial virus protein.

Respiratory syncytial virus (RSV) has been recognized as one of the most common causes of severe respiratory tract infection in infants worldwide. As yet, a safe and effective vaccine has not been developed to protect humans from RSV. The F and G surface proteins have been widely investigated due to their potential to induce protective immunity. In addition, the M2 protein has been shown to be important in inducing a T-cell response. Our project involved the cloning of the immunodominant regions of the RSV F, M2 and G proteins into a bacterial vector, pET-32a (+). The recombinant RFM2G protein was expressed in Escherichia coli and purified using His Bind columns. The purified rRFM2G protein was analyzed by polyacrylamide gel electrophoresis and Western blotting. The predicted structure of the recombinant protein built by the Swiss PDB Viewer program suggested a rod shape with a distinct swollen head and neck which was confirmed by transmission electron microscopy and atomic force microscopy. BALB/c female mice were immunized with either RSV, rRFM2G alone, or rRFM2G in combination with flagellin as a mucosal adjuvant. Serum was collected on days 0, 14, 28 and 49 to assess the immune response by Enzyme-linked immunosorbent assay. Intranasal immunization of mice with the rRFM2G protein yielded significantly high serum IgG titers. Co-administration of the rRFM2G protein with flagellin did not augment the serum antibody response.

CD19(+) cells produce IFN-gamma in mice infected with Borrelia burgdorferi.

We have recently shown that production of IFN-gamma and IL-10, but not IL-4 is specifically induced in the lymph nodes of C3H/HeJ (disease susceptible) and C57BL/6J (disease resistant) mice 1 week after infection with Borrelia burgdorferi spirochetes. The present study was conducted to determine the phenotypes of ex vivo lymph node cells obtained from infected mice of both strains at this time point. The percentages of CD3(+), CD4(+), CD8(+), TCRalpha/beta (+) and TCRgamma/delta (+) cells decreased in both strains of mice compared to LN from naive mice. In contrast, there was a threefold increase in the proportion of CD19(+) cells. In view of this expansion of the B cell proportion, we examined the ability of purified CD19(+) cells and CD43(+) cells to produce both IL-10 and IFN-gamma when the cells were restimulated in vitro with B. burgdorferi freeze-thawed spirochetes. As expected, CD43(+) cells were able to produce both cytokines, but not IL-4. Surprisingly, CD19(+) (B) cells also were able to produce IFN-gamma in comparable amounts, in addition to IL-10. Intracellular staining of CD19(+) cells with anti-IFN-gamma antibody confirmed this finding. We discuss this novel phenomenon in terms of its possible underlying mechanisms and its relevance, both in the context of the immunology of Lyme disease and that of other infectious diseases.

Detection of serum IgG antibodies specific for Wolbachia surface protein in rhesus monkeys infected with Brugia malayi.

The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood. To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys. Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys. It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic. WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development. These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.

Borrelia burgdorferi stimulates in vitro a selective expansion of CD3-CD4-CD8- (triple negative) autoreactive cells in naive rhesus monkey lymphocytes.

Autoreactive cell lines were generated from cell suspensions of freshly isolated naive monkey lymph node (LN) cells and peripheral blood mononuclear cells by cocultivation with freeze-thawed Borrelia burgdorferi spirochetes (Bb/FT). These cells produced interleukin (IL)-6 when cocultured with autologous antigen-presenting cells (APCs) alone without Bb/FT. IL-6 production was not observed when control cell lines were stimulated in the same fashion. CD4+-enriched T cell populations obtained from the LN autoreactive cell line also produced IL-6 when cultivated with APCs alone. When these cells were cultivated further in the presence of APCs, a population of cells whose phenotype was CD56+/-CD4-CD8-CD3- was predominantly selected. These cells both proliferated and produced IL-6 when cocultured with APCs alone. The possible relevance of these cells to Lyme disease pathogenesis remains to be determined.

Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins.

We determined previously that lipoproteins of Borrelia burgdorferi stimulate inflammatory and anti-inflammatory cytokines (interleukin-10 [IL-10]) in monocytes. IL-10 could have an effect on innate and acquired immune responses to B. burgdorferi and influence the magnitude of the infectious inoculum and disease outcome. To understand the mechanism(s) of IL-10 action during early infection, when innate immunity expressed chiefly by skin macrophages is key, we investigated the effect of exogenous and endogenous IL-10 on the production of the macrophage-derived cytokines IL-6, IL-1beta, IL-12, and tumor necrosis factor alpha (TNF-alpha). We used the THP-1 human monocytic cell line and recombinant lipidated OspA (L-OspA) as the model target cell and stimulant, respectively. To determine the kinetics of cytokine production by THP-1 cells, we stimulated them with L-OspA and also with heat-killed B. burgdorferi cells (HBb) and lipopolysaccharide (LPS). Exogenous IL-10 dampened production of inflammatory cytokines, as elicited by lipoproteins. The inhibition of endogenous IL-10 function by anti-IL-10 antibody reduced the production of IL-12 and IL-6 but not that of IL-1beta and TNF-alpha. An inspection of the kinetics of cytokine production clarified this finding. TNF-alpha was produced prior to, and IL-beta was produced at the same time as, IL-10, whereas IL-6 and IL-12 were produced later. HBb, LPS, and L-OspA yielded similar kinetics of cytokine production. This result reinforces the notion that lipoproteins are the functional molecules in HBb and perhaps in vivo. It indicates also that signaling pathways utilized by LPS and lipoproteins may be extensively shared. The results are consistent with a major role played by IL-10 in controlling the initial phase of infection with this spirochete.

Early induction of gamma interferon and interleukin-10 production in draining lymph nodes from mice infected with Borrelia burgdorferi.

Lymph node (LN) cells from C3H/HeJ mice (Lyme disease susceptible) infected for 1 week with Borrelia burgdorferi strain JD1 produced higher levels of gamma interferon (IFN-gamma) when stimulated in vitro with B. burgdorferi spirochetes than equivalent cells from B. burgdorferi-infected C57BL/6J mice (disease resistant). The interleukin-10 (IL-10) levels were comparable in the two strains, whereas the IL-4 levels were below detection limits. B. burgdorferi-stimulated LN cells from C57BL/6J mice produced significantly higher levels of IFN-gamma in the presence of neutralizing anti-IL-10 antibody than cells cultured with B. burgdorferi alone. No effect of IL-10 neutralization on IFN-gamma production by LN cells from C3H/HeJ mice was observed. Neutralizing antibody to IFN-gamma had no effect on the production of IL-10 by LN cells from C57BL/6J mice. A slight decrease in IL-10 production was detected in culture supernatants of equivalent cells from C3H/HeJ mice. The differential effect of IL-10 on IFN-gamma production in C57BL/6J and C3H/HeJ mice suggests that IL-10 is probably involved in the regulation of IFN-gamma production by LN cells during infection and may be at the root of the differential susceptibility to Lyme arthritis in these two strains of mice.

Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14.

We previously showed that heat-killed Borrelia burgdorferi spirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691-2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killed B. burgdorferi. B. burgdorferi similarly induced an upregulation of the IL-1beta and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) of B. burgdorferi lipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced by B. burgdorferi lipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.

Diminished production of T helper 1 cytokines and lack of induction of IL-2R+ T cells correlate with T-cell unresponsiveness in rhesus monkeys chronically infected with Brugia malayi.

The relationship between antigen-specific responsiveness, parasitic burden, and lymphatic pathology was investigated in nine rhesus monkeys with chronic Brugia malayi infections. Specifically, in vitro proliferation, cytokine gene expression and production, IL-2R expression on T cells, microfilaria (mf) densities, and lymphedema were evaluated. PBMC from three animals (two mf- one mf+) proliferated in response to filarial antigen (responder monkeys, RM) and cells from six animals (5 mf+; one mf-) did not (nonresponder monkeys, NRM). All RM showed lymphedema and none of the NRM did. Antigen-specific IL-2 and IFN-gamma (mRNA and protein) were induced in PBMC from all RM whereas PBMC from only one of six NRM responded with IL-2 and IFN-gamma expression. IL-4 transcripts were induced in PBMC from two of three RM and in cells from all six NRM. IL-10 mRNA expression and protein production were induced in PBMC from two of three RM and in cells from five of six NRM. A marked increase in the frequency of IL-2R+ T cells was observed in antigen-stimulated PBMC cultures of RM but not in those of NRM. The data show that diminished production of Th1 cytokines and lack of induction of IL-2R+T cells may contribute to the unresponsiveness of PBMC from NRM to filarial antigen. They also show that the polarization of immune responses and lymphatic pathology observed in rhesus monkeys is similar to that generally described in human lymphatic filariasis patients.

Histopathological, lymphoscintigraphical, and immunological changes in the inguinal lymph nodes of rhesus monkeys during the early course of infection with Brugia malayi.

The relationship of the early lymphatic pathophysiological alterations with those of tissue inflammatory and cellular responses in the inguinal lymph nodes of Brugia malayi-infected rhesus monkeys was examined. Each of five animals was inoculated subcutaneously in the right calf with 200 third stage larvae (L3) and 5 weeks later, before the onset of patency [10 to 12 weeks postinoculation (PI)], their right inguinal nodes began to show signs of enlargement, becoming most prominent between weeks 10 to 16 PI. Histopathologically, the right nodes had eosinophilic lymphadenitis, lymphoid hyperplasia, and pronounced germinal centers. Lymphoscintigraphy using 99mTc-antimony trisulfide colloid showed pathophysiological alterations of the lymph flow rate in the right leg but not in the left leg at weeks 7 and 15 PI. In vitro blastogenesis to B. malayi antigens at week 10 PI showed the inguinal lymph node cells proliferated more vigorously than did peripheral blood cells early in infection. However, at week 24 PI both lymph node and peripheral blood cells proliferated to antigens. Flow cytometry showed an upregulation of HLA-DR+ lymphocytes in right lymph node cells from infected animals when compared to those from control animals. No changes in CD2, CD4, CD8, CD20, CD29, and CD45R cell numbers in lymph node of infected animals were seen when compared to control animals. Our results show that lymphatic pathology occurs early before the onset of patency, correlating with a marked tissue inflammatory and cellular responses of lymph node cells in B. malayi-infected rhesus monkeys. The rhesus could be an extremely useful model for understanding the evolution of pathology and pathogenesis of the disease.

Borrelia burgdorferi stimulates the production of interleukin-10 in peripheral blood mononuclear cells from uninfected humans and rhesus monkeys.

Heat-killed Borrelia burgdorferi spirochetes stimulate in vitro production of interleukin-10 (IL-10) at both mRNA and protein levels in peripheral blood mononuclear cells (PBMC) of uninfected rhesus monkeys. A concomitant down-modulation of IL-2 gene transcription was observed. Neither IL-4 nor gamma interferon gene expression was ostensibly affected by B. burgdorferi spirochetes. These phenomena were observed regardless of whether the stimulating spirochetes belonged to the B. burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii genospecies, the three main species that cause Lyme disease. B. burgdorferi also induced production of IL-10 in uninfected human PBMC, indicating that this effect might play a role in human Lyme disease. Purified lipidated outer surface protein A (OspA), but not its unlipidated form, induced the production of high levels of IL-10 in uninfected human PBMC. Thus, the lipid moiety is essential in the induction of IL-10 in these PBMC. B. burgdorferi M297, a mutant strain that lacks the plasmid that encodes OspA and OspB, also induced IL-10 gene transcription in PBMC, indicating that this phenomenon is not causally linked exclusively to OspA and its lipid moiety. These results demonstrate that B. burgdorferi can stimulate the production of an antiinflammatory, immunosuppressive cytokine in naive cells and suggest that IL-10 may play a role both in avoidance by the spirochete of deleterious immune responses and in limiting the inflammation that the spirochete is able to induce.

The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey.

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa.

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa-inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10-35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression of CD4+ and CD8+ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4+ and CD8+ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4+ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8+ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals (P < 0.0001), coinciding with an increase in CD8+ T cells numbers in these cultures. The data show that factors besides IL-2; and probably an imbalance in the percentages of CD4+ and CD8+ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa-inoculated rhesus monkeys.

Cellular immune responses of jirds to extracts of life cycle stages and adult excretory secretory products during the early development of Brugia pahangi.

The Brugia-jird model of lymphatic filariasis was used to examine the induction of cellular immune responses during the early premicrofilaremic phases of the infection. The intensity of the pulmonary granulomatous inflammatory response (PGRN) was determined by measuring granuloma areas around Sepharose beads coated with parasite extracts which were embolized in the lungs of jirds prior to necropsy. Necropsies were performed at 7, 14, 28, 56, and 150 days postinfection (DPI). These time points correspond to specific developmental changes in the life cycle. Lymphocyte blastogenesis assays were performed using cells from draining renal lymph nodes and splenocytes at 14 and 150 DPI. Soluble extracts of third stage larvae (L3), fourth stage larvae (L4), adult females, adult males, microfilariae (MF), and excretory secretory products (ES) of males and females were used in both measurements of cellular responsiveness. A marked granulomatous response to parasite extracts peaked at 7 DPI or 14 DPI followed by a gradual decrease to a hyporesponsive state at 120 DPI. The response of renal lymph node cells also was significantly elevated at 14 DPI and significantly decreased at > 150 DPI. The splenocyte responses were erratic and did not follow this pattern. Significant differences in PGRN responses to somatic extract preparations were not seen during the early stages of the infection (7, 14, 28 DPI), but those to MF and L3 were significantly less at 56 and 120 DPI. Although PGRN responses to ES followed a similar pattern, these were less than those to the somatic extract. The data indicated that a rapid, intense cell-mediated inflammatory response is induced early during a primary infection and that this response is rapidly downregulated. This downregulation begins prior to the maturation of adult parasites and microfilarial production. The early phase of the cellular response appears to be compartmentalized in that this response was consistently observed in the renal lymph nodes but not in the spleen. Soluble protein components of the parasites responsible for these responses are likely multiple and shared by all life cycle stages.

Microfilarial densities, hematologic changes, and serum antibody levels in Loa loa-infected rhesus monkeys (Macaca mulatta).

To better define Loa loa infection in the rhesus monkey and assess the potential usefulness of this host as a model for studies of human loiasis, 12 monkeys (four splenectomized and eight nonsplenectomized) were inoculated with L. loa infective larvae. Microfilaremia and hematologic changes as well as parasite-specific antibody were assessed as a function of time in these animals. Eleven of 12 inoculated monkeys became microfilaremic. Splenectomized animals had moderate (250-1,000) to low (< 250) numbers of microfilariae per milliliter (mf/ml), whereas the mf/ml in nonsplenectomized animals varied from high (> 1,000) to low. A significant increase in total leukocyte and lymphocyte numbers was seen in animals with moderate-to-low mf/ml but not in animals with high mf/ml mainly because of variations between animals in the latter group, rather than a direct consequence of increased mf numbers. All infected animals developed an eosinophilia before patency, suggesting the adult worms most likely contribute to this phenomenon. As the infection progressed, the eosinophil numbers decreased significantly. Although splenectomized animals overall had slightly higher numbers of total leukocytes (lymphocytes and eosinophils), these hematologic changes as a function of time were not significantly different from those in nonsplenectomized animals. Parasite-specific IgG antibody was increased significantly before patency in all animals and with the exception of the one amicrofilaremic animal, it decreased after patency. This study shows that splenectomy of rhesus monkeys prior to L. loa inoculation does not enhance the microfilarial density nor does it adversely affect eosinophilia or antibody production.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation and partial characterization of an eosinophil chemotactic cytokine produced by sensitized equine mononuclear cells stimulated with Strongylus vulgaris antigen.

Supernatants generated by stimulation of peripheral blood mononuclear cells (PBMC) from Strongylus vulgaris sensitized or immunized ponies were assayed in vitro for eosinophil chemotactic activity (ECA) using the filter system in blind well chambers. The supernatants from these cultures were chemotactic for eosinophils, but not for neutrophils. Supernates from cultures of unsensitized PBMC stimulated with S. vulgaris antigen were not chemotactic for eosinophils. ECA was first detected in culture supernatants after 1.5 h of incubation and was dependent on both antigen and PBMC concentrations, but independent of serum concentrations. Both female and male S. vulgaris worm antigens stimulated ECA production from sensitized PBMC. ECA was not induced by in vitro stimulation of sensitized S. vulgaris PBMC by female Strongylus edentatus worm antigen. Partial characterization of the eosinophil chemotactic cytokine showed it to be nondialyzable, greater than 8000 molecular weight (MW), and sensitive to heating (56 and 95 degrees C), trypsin, and sodium metaperiodate treatments, suggesting that the cytokine is a protein containing some essential carbohydrate moieties. The cytokine described in this paper could partially contribute to the in vivo blood and tissue eosinophilia in experimental S. vulgaris infection.

Early and early disseminated phases of Lyme disease in the rhesus monkey: a model for infection in humans.

We demonstrate that Borrelia burgdorferi infection in the rhesus monkey mimics the early and early disseminated phases of human Lyme disease. Clinical, bacteriological, immunological, and pathological signs of infection were investigated during 13 weeks after inoculation of the spirochete. Three animals were given B. burgdorferi (strain JD1) by needle inoculations, six animals were exposed to the bite of B. burgdorferi-infected Ixodes dammini ticks, and three animals were uninfected controls. B. burgdorferi could be recovered from all animals that were given the spirochete. Bacteria were detectable until week 6 postinoculation (p.i.) in blood, until week 8 p.i. in skin biopsies, and at 10 weeks p.i. in the conjunctiva of one of two animals which developed conjunctivitis. Erythema migrans (EM) appeared in one of the three animals infected by needle inoculation and in five of the six animals infected by ticks. Deep dermal perivascular lymphocytic infiltrations (characteristic of human EM) were observed in all animals showing EM clinically. Both EM and conjunctivitis were documented concomitantly with the presence of the spirochete. Lethargy, splenomegaly, and cerebrospinal fluid pleocytosis were also noted in some animals, but the direct connection of these signs with the infection was not shown. The appearance rate of immunoglobulin M and immunoglobulin G antibodies to B. burgdorferi, as well as the antigen spectra recognized, were remarkably similar to those seen in humans. Serum antibodies from infected animals were able to kill B. burgdorferi in vitro in the presence of rhesus complement. The rhesus monkey model appears to be useful for the investigation of the immunology and pathogenesis of Lyme disease and for the development of immunoprophylactic, diagnostic, and chemotherapeutic protocols.

Role of the eosinophil in serum-mediated adherence of equine leukocytes to infective larvae of Strongylus vulgaris.

The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent eosinophils from eosinophilic ponies immobilized L3. Neutrophils were less adherent and did not immobilize L3. Mononuclear cells failed to adhere. Normal eosinophils from strongly-naive ponies did not immobilize S. vulgaris L3 in the presence of immune serum, suggesting the in vivo activation of eosinophils in eosinophilic animals. Immune serum promoted less adherence of buffy coat cells to Strongylus edentatus or mixed species of Cyathostominae L3, suggesting that the serum-mediated cellular adherence phenomenon was species-specific. Normal serum promoted less cellular adherence to S. vulgaris L3 than immune serum. The adherence mediated by normal serum was removed by heat inactivation, suggesting that this nonspecific phenomenon was a complement-mediated reaction. Immune globulins promoted reactions similar to that seen using heat-inactivated immune serum, whereas normal globulins did not promote adherence. Immune globulins absorbed with pieces of S. vulgaris adult worms did not promote the adherence of buffy coat cells to S. vulgaris L3, suggesting that adult and L3 stages share antigens important in this phenomenon that resulted in the removal of specific adherence antibody during absorption.

Immune responses of pony foals during repeated infections of Strongylus vulgaris and regular ivermectin treatments.

Ten helminth-free pony foals divided into three groups were used in this study. Eight foals were each experimentally infected per os with 50 Strongylus vulgaris infective larvae weekly for 4 weeks, at which time one foal died of acute verminous arteritis. The remaining seven foals subsequently received 50 S. vulgaris infective larvae every 2 weeks for an additional 20 weeks. Four of the infected foals remained untreated (Group 1) and three of the infected foals were given ivermectin at 8, 16 and 24 weeks post initial infection (Group 2). Two foals served as controls (Group 3). Foals in Group 1 developed eosinophilia, which was sustained throughout the course of infection. A mild eosinophilia also developed in Group 2 foals; however, the eosinophil numbers were markedly reduced for 3 weeks after each ivermectin treatment. Only foals in Group 1 developed significant (P less than 0.05) hyperproteinemia, hyperbetaglobulinemia and a reversal of the albumin/globulin (A/G) ratio 4 weeks after initial infection. Significant (P less than 0.05) IgG anti-S. vulgaris ELISA titers developed in foals in Groups 1 and 2 3 weeks after infection and were sustained for the duration of the experiment. Western blot analysis of soluble somatic antigens of S. vulgaris adult female and male worms probed with sera from foals in Groups 1 and 2 revealed only subtle differences between these animals. The blastogenic reactivity of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin and concanavalin A was not significantly different between groups. Peripheral blood mononuclear cells from foals in Groups 1 and 2 developed significant (P less than 0.05) blastogenic reactivity to S. vulgaris soluble adult somatic antigen when examined at 25 weeks after infection. Mesenteric lymph node cells from foals in Group 2, although not statistically significant, were more reactive to antigen than were the mesenteric lymph node cells from foals in Group 1 when examined at 27 weeks after infection. These results suggest that significant alterations in the immune response of ponies to S. vulgaris does not occur after intravascular killing of larvae by ivermectin treatments.