Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

OBJECTIVE: To compare serum CA 125 assays with computed tomography (CT) and transvaginal ultrasound (TVUS) for early detection of disease recurrence in patients with ovarian cancer. METHODS: Sixty-two patients with non-mucinous epithelial ovarian cancer who had positive CA 125 levels (> 35 U/ml) were studied. We performed a retrospective review to determine the usefulness of serum CA 125 measurements. Setting the cut-off limit at either 35 U/ml or 16 U/ml, the accuracy of CA 125 measurements was compared with that of CT scanning, TVUS and operative findings at second-look laparotomy (SLL) in the early detection of recurrent tumors. RESULTS: Compared with SLL, both the specificity and the positive predictive value of CA 125 measurements were 100% at 16 and 35 U/ml. The sensitivity and the negative predictive value were 30.8 and 71.9%, respectively, below 35 U/ml and 53.8 and 79.3%, respectively, below 16 U/ml. The false-negative rate of CT was 36.1%. When the cut-off limit was reduced from 35 to 16 U/ml, 57.1% of patients considered to be in remission were reclassified as having persistent disease. A complete response confirmed by CT did not represent remission: CA 125 levels were 7.5-fold higher at the time of re-evaluation by CT. TVUS also lagged behind CA 125 assays in detecting disease recurrence. The sensitivity of ultrasound appeared to be lower than that of CT because it failed to detect extrapelvic lesions. CONCLUSION: A screening threshold (cut-off level) of 16 U/ml for CA 125 should be used to detect recurrent serous ovarian adenocarcinoma. Although ultrasound is a convenient method of detecting intrapelvic lesions, and has cost benefit, CT is necessary to detect extrapelvic recurrence. Neither CT nor ultrasound are more accurate than serial CA 125 assays in detecting disease recurrence.     

Mucin-related epitopes distinguish M cells and enterocytes in rabbit appendix and Peyer's patches

The biochemical composition of the apical membranes of epithelial M cells overlying the gut-associated lymphoid tissues (GALT) is still largely unknown. We have prepared monoclonal antibodies (MAbs) directed against carbonate-washed plasma membranes from epithelial cells detached with EDTA from rabbit appendix, a tissue particularly rich in GALT. As determined by immunofluorescence microscopy, several MAbs specifically recognized either M cells or enterocyte-like cells of the domes from rabbit appendix, sacculus rotundus, and Peyer's patches. M cells were identified by their large ventral pocket containing lymphoid cells and by specific labeling with antivimentin. Among various characterized MAbs, MAb 104 recognized rabbit immunoglobulins and was used as an apical marker for M cells in the rabbit appendix, MAb 58 selectively stained an integral membrane glycoprotein of greater than 205 kDa located at the apex of M cells, and MAb 214 stained a smaller soluble glycoprotein associated with the apical surfaces from neighboring enterocytes. In addition, both MAbs 58 and 214 also labeled luminal mucus and secretory granules in goblet cells. The selective association of mucin-related molecules at the surfaces of either M cells or enterocyte-like cells of the follicle-associated epithelium suggests that specific carbohydrate antigens are differentially expressed by epithelial cells and could account for the differential binding properties of pathogens.     

Interactions of the bacterial pathogen Listeria monocytogenes with mammalian cells: bacterial factors, cellular ligands, and signaling

Risedronate ([1-hydroxy-2-(3-pyridinyl)-ethylidene[bis]phosphonic acid] monosodium salt) was evaluated for induction of hepatic microsomal drug metabolizing enzymes in male and female Sprague Dawley rats (N = 4/sex/dose group). Main study animals received water (vehicle control), risedronate (0.1, 0.8, 4, or 16 mg/kg/day) or phenobarbital (80 mg/kg/day, positive control) by daily oral gavage for 14 consecutive days. Recovery study animals received water, risedronate (16 mg/kg/day) or phenobarbital (80 mg/kg/day) by daily oral gavage for 14 consecutive days and then were maintained drug-free for 14 days to evaluate the reversibility of any observed effects. At the conclusion of each study the animals were sacrificed, the liver removed, weighed and the microsomal subcellular fraction prepared. The hepatic microsomal fraction was then evaluated for protein content, cytochrome P450, and the activities of aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase. Risedronate was well tolerated during the dosing phase of the study as evidenced by clinical observations, body weight gain and food consumption which were not significantly different from the vehicle controls. Risedronate did not significantly increase (P > 0.05) liver weight, liver/body weight ratio, protein content, P450, aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase or p-nitrophenol UDP-glucuronosyltransferase in rats of either sex when compared to vehicle controls. As expected, the hepatic microsomal enzyme inducer phenobarbital significantly increased (P < 0.05) liver weight, liver/body weight ratio, protein content (males only), P450, aniline hydroxylase (males only), aminopyrine N-demethylase (males only), ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase in rats relative to vehicle controls. Following the 14 day drug-free recovery period the induction parameters increased by phenobarbital reversed to vehicle control levels. The results obtained in this well controlled study indicate that risedronate is not an inducer of hepatic microsomal drug metabolizing enzymes in the rat.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

The cation-conducting channel of the nicotinic acetylcholine (ACh) receptor is lined by the first (M1) and second (M2) membrane-spanning segments of each of its five subunits. Six consecutive residues, alphaS239 to alphaT244, in the alpha subunit M1-M2 loop and at the intracellular end of M2 were mutated to cysteine. The accessibility of the substituted cysteines were probed with small, cationic, sulfhydryl-specific reagents added extracellularly and intracellularly. In the closed state of the channel, there is a barrier to these reagents added from either side between alphaG240 and alphaT244. ACh induces the removal of this barrier, which acts as an activation gate. The residues alphaG240, alphaE241, alphaK242, and alphaT244 line a narrow part of the channel, in which this gate is located.     

Combined effect of gamma radiation and heating on the destruction of Listeria monocytogenes and Salmonella typhimurium in cook-chill roast beef and gravy

The effect of heating alone (60, 65 or 70 degrees C), heating after irradiation (0.8 kGy) and heating after irradiation and storage for 14 days at 2-3 degrees C on the destruction of Listeria monocytogenes and Salmonella typhimurium in artifically inoculated minced cook-chill roast beef and gravy was investigated. Inoculated minced roast beef samples (5 g) were heated in Stomacher bags completely immersed in a water bath at each of the test temperatures. Survivors were enumerated and D and z values were determined for each of the pathogens. Observed thermal D values for two strains of L. monocytogenes at 60, 65 and 70 degrees C in the absence of pre-irradiation were 90.0-97.5 s, 34.0-53.0 s and 22.4-28.0 s, respectively, whereas thermal D values after pre-irradiation were 44.0-46.4 s, 15.3-16.8 s and 5.5-7.8 s at 60, 65 and 70 degrees C, respectively. This reduction in D values provides evidence for radiation-induced heat-sensitisation in L. monocytogenes. There was some evidence of heat-sensitisation of S. typhimurium at 60 degrees C, but not at either 65 or 70 degrees C. The z value also decreased as a consequence of pre-irradiation to a dose of 0.8 kGy (11.0-12.7 degrees C). The radiation-induced heat-sensitivity in L. monocytogenes was found to persist for up to 2 weeks storage at 2-3 degrees C prior to heating. As cook-chill products are intended to be reheated prior to consumption the results of the present study suggest that any L. monocytogenes present in a cook-chill product would be more easily killed during reheating if it were to be treated with a low dose of gamma radiation during manufacture.     

Alteration of HLA-B27 peptide presentation after infection of transfected murine L cells by Shigella flexneri

Shigella flexneri is a triggering agent for reactive arthritis in HLA-B27-susceptible individuals. Considering the intracellular multiplication of bacteria, it seems likely that bacterial peptides may be presented by the major histocompatibility complex (MHC) class I pathway. To examine this hypothesis, we infected HLA-B*2705- and/or human beta2-microglobulin-transfected murine L-cell lines with M90T, an invasive strain of S. flexneri. Bacterial infection induced no detectable modifications in the biosynthesis and expression level of HLA-B27, as assessed by immunoprecipitation, Northern blot analysis, and flow cytometry. Using confocal microscopy, we observed that bacterial infection induced a clustering of HLA-B27 molecules during macropinocytosis and before bacterial dissemination from cell to cell. Peptides naturally bound to HLA-B27 molecules were acid eluted from infected cells and separated by high-performance liquid chromatography. Major differences were observed in high-performance liquid chromatography profiles and in the nature of peptides presented following bacterial infection. Although most of the antigens presented were not accessed by Edman degradation, we obtained two sequences partially homologous to bacterial proteins. These peptides lacked the major HLA-B27 peptide anchor (Arg) at position 2, and one had an unusual length of 14 amino acids. These data suggest that alterations in the peptide presentation by HLA-B27 occur during infection, which could be relevant to the pathogenesis of HLA-B27-related arthritis.     

Penetration of M cells and destruction of Peyer's patches by Yersinia enterocolitica: an ultrastructural and histological study

Yersinia enterocolitica is enteropathogenic for man and rodents. Previous studies provided evidence that Y. enterocolitica invades the lymphoid follicles of the Peyer's patches (PP) of the small intestine. In this study Y. enterocolitica-induced tissue alterations of the follicle-associated epithelium (FAE) and the underlying PP tissue were analysed by scanning (SEM) and transmission electron microscopy (TEM) as well as by conventional histological examination. For this purpose, an experimental mouse infection model including orogastric infections as well as ileal loop experiments were used. A rapid and selective colonisation of the FAE after orogastric yersinia infection was observed by SEM. TEM studies confirmed that Y. enterocolitica adhered closely to the FAE including M cells and enterocytes. Histological studies and TEM revealed that Y. enterocolitica selectively invaded the PP via M cells but not via other cells of the FAE. One day after Y. enterocolitica infection the FAE was altered and small micro-abscesses comprising yersiniae expressing the major outer-membrane protein YadA were observed immediately beneath the FAE. Adjacent villi were dilated from lymphangiectasis and transmigrating polymorphonuclear leucocytes (PMNL) were found within the epithelium. At 5-7 days after infection the FAE and parts of PP were destroyed. Profound alterations of the cyto-architecture of the PP were due to the enormous recruitment of PMNL. By day 5 after infection, abscesses were found in the mesenteric lymph nodes. However, TEM studies revealed evidence that Y. enterocolitica may disseminate from the PP not only via the lymphatics but also by invasion of blood vessels. Taken together, the results of this study demonstrate that the FAE is the primary site of host-pathogen interaction in Y. enterocolitica infection and that this pathogen penetrates M cells and subsequently induces destruction of the PP.     

Bacterial surface proteins recognized by CD4+ T cells during murine infection with Listeria monocytogenes

Optimal immunity to the Gram-positive pathogen Listeria monocytogenes (LM) requires both CD8+ and CD4+ antigen-specific T cell responses. Understanding how CD4+ T cells function in an immune response to LM and how bacterial proteins are processed to peptide/MHC class II complexes in infected cells requires identification of these proteins. Using LacZ-inducible, LM-specific CD4+ T cells as probes, we identified two immunogenic LM proteins by a novel expression cloning strategy. The antigenic peptides contained within these proteins were defined by deletion analysis of the genes, and their antigenicity was confirmed with synthetic peptides. The nucleotide sequences of the genes showed that they encode previously unknown LM proteins that are homologous to surface proteins in other bacterial species. Consistent with their surface topology, mild trypsin treatment of LM protoplasts ablated T cell recognition of these Ags. These findings establish a general strategy for identifying unknown CD4+ T cell Ags and demonstrate that LM surface proteins can provide the peptides for presentation by MHC class II molecules that are specific targets for CD4+ T cells during murine LM infection.     

Interactions of itraconazole with amphotericin B in the treatment of murine invasive candidiasis

The interactions of amphotericin B and itraconazole were studied in murine invasive candidiasis. Candida albicans-infected mice were treated for 10 consecutive days, 24 h after infection. Survival was monitored over 30 days and kidney cultures were done. Mice treated with amphotericin B (0.2 mg/kg/day intraperitoneally) or itraconazole (100 mg/kg/day by oral gavage in two divided doses/ day) had a 30-day survival of 20% or 40%. Concomitant administration of both drugs resulted in 100% mortality; 90% of mice treated with amphotericin B (1 mg/kg/day) survived. With the combination, 100% were dead by day 28 (P < or = .001 vs. amphotericin B). With sequential therapy (i.e., 5 days with one drug and then 5 days with the other), survival was inferior to that with amphotericin B alone but similar to that with itraconazole alone. Kidney culture results confirmed the antagonism of the combination compared with amphotericin B alone. In treatment of murine invasive candidiasis, the concomitant or sequential use of amphotericin B and itraconazole results in a negative interaction.     

Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma

A live attenuated AroA- auxotrophic mutant of Salmonella typhimurium (SL7207) has been used as carrier for the pCMVbeta vector that contains the beta-galactosidase (beta-gal) gene under the control of the immediate early promoter of Cytomegalovirus (CMV). We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether beta-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the beta-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen. After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to beta-gal. Mice vaccinated with the Salmonella harboring pCMVbeta, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells. These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs. To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter. Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter. These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells. Finally, approximately 19% of the splenocytes expressed GFP. Among them, F4/80(+) macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression. Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens. We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors.     

Detection of Listeria monocytogenes in humans, animals and foods

The Listeria monocytogenes-carrying rates were 100% for listeriosis patients and 1.3% for healthy humans. The L. monocytogenes contamination rates for retail sliced beef (34.2%) and pork (36.4%) were significantly higher (p < 0.05) than those for cattle (2.0%) and pigs (0.8%) and for cattle (4.9%) and swine (7.4%) carcasses. The percentages of serotypes 1/2a, 1/2b and 4b which are most dominant in human patients were high in isolates from fresh (90.0%) and processed (100%) fish and shellfish and imported natural cheese (96.7%).     

Interactions of surfactant protein D with pathogens, allergens and phagocytes

Surfactant protein D (SP-D) is considered to play an important role in innate immunity in the lungs by binding, via its multiple C-type lectin domains, to carbohydrate structures present on a range of viruses, bacteria, yeasts and fungi. The resulting agglutination of the target pathogens provides host defence which can be further enhanced by killing and clearance mechanisms mediated by phagocytic cells which carry receptors for SP-D. Recent findings suggest that SP-D, and the structurally related lung surfactant protein A (SP-A), may also modulate allergic reactions by binding certain glycosylated allergens. The finding of SP-D at a variety of other sites besides the lungs, such as the gastric mucosae, is suggestive that it may play a general protective role in several secretions.     

Intracellular distribution of ampicillin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles

A performance evaluation of the DIABETES rule-based expert system prototype for clinical decision making is presented. The system facilitates multiple insulin regimen and dose adjustment of insulin dependent Type I or II diabetic patients. The study was performed on 600 subjects from two diabetological centres and three diabetological offices of Greek hospitals. The responses of the attendant medical doctors were compared with those of the DIABETES system, with the aid of a specifically devised valuation range (0-5 degrees, 0 indicating full agreement and 5 full disagreement). The capabilities and the weakness of the system in terms of its practicality for decision support in assisting therapy of diabetes mellitus by blood glucose monitoring and subsequent insulin dose adjustment are discussed. The potential benefits of decision support systems for diabetic patient management are seen to be the cost saving they provide in terms of man-hours of verbal instruction by medical experts, the support in terms of objective and consistent decision making, as well as the recording of medical knowledge in the ill-defined field of insulin administration, thus aiding the education and training of medical personnel.     

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.     

Inactivation of DsbA, but not DsbC and DsbD, affects the intracellular survival and virulence of Shigella flexneri

In this study, three mutants, dsbA::kan, dsbC-kan, and dsbD-kan, of Shigella flexneri serotype 5 were constructed and characterized to investigate the role of the periplasmic thiol:disulfide oxidoreductases in pathogenicity. In gentamicin protection assays and the Serény test, the dsbA mutant showed reduced virulence while the dsbC and dsbD mutants were similar to the wild type. That inactivation of dsbA was responsible for the reduced virulence was verified by complementation with the cloned wild-type gene in in vitro and in vivo assays. Despite the changed virulence behavior, the dsbA mutant could penetrate HeLa cells 15 min postinfection, consistent with the fact that it actively secretes Ipa proteins upon Congo red induction. Furthermore, the dsbA mutant was able to produce actin comets and protrusions, indicating its capacity for intra- and intercellular spread. However, a kinetic analysis of intracellular growth showed that the dsbA mutant barely grew in HeLa cells during a 4-h infection whereas the wild type had a doubling time of 41 min. Electron microscopy analysis revealed that dsbA mutant bacteria were trapped in protrusion-derived vacuoles surrounded by double membranes, resembling an icsB mutant reported previously. Moreover, the trapped bacteria appeared to be lysed simultaneously with the double membranes, resulting in characteristic empty vacuoles in the host cell cytosol. Thus, the attenuation mechanism for dsbA mutant appears to be more complicated than was previously suggested.     

Induction of the phoE promoter upon invasion of Salmonella typhimurium into eukaryotic cells

Live attenuated Salmonella typhimurium strains expressing foreign antigens can be used for vaccination purposes. Due to deleterious effects of constitutive, high-level expression of the heterologous antigens, there is often strong selection pressure against plasmids encoding these antigens, resulting in rapid segregation in vivo. In vivo-inducible promoters may be a good alternative for constitutive promoters. The outer membrane protein PhoE of Escherichia coli is being used as a carrier for foreign antigenic determinants. Here we studied whether its expression from a plasmid is induced in S. typhimurium upon invasion of eukaryotic cells. This appeared to be the case. Furthermore, a S. typhimurium phoE mutant was constructed and the effects of the mutation on invasion, intracellular survival and virulence were studied. Survival in HEp-2 cells or in the macrophage-like cell line J744 was not, or only slightly, affected. Furthermore, the mutant appeared to be as virulent for mice as the wild-type strain.     

The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.     

Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10-13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and beta-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.     

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.