Use of intravenous polymyxin B during continuous venovenous hemodialysis.

Described here is the case of a 23-year-old male with Acinetobacter baumannii sepsis and acute renal failure requiring continuous venovenous hemodialysis treated successfully with intravenous polymyxin B. The drug was administered as a loading dose of 2.5 mg/kg, followed by 2 doses of 1 mg/kg on days 4 and 8, then 0.8 mg/kg daily to complete a 24-day course. A discussion of dosing recommendations for polymyxins in dialysis is presented.

Molting, ecdysis, and reproduction of Trichinella spiralis are supported in vitro by intestinal epithelial cells.

Trichinella spiralis is an obligate parasite of animals that has an unusual intracellular life cycle. Investigation of parasitism at the cellular and molecular levels has been challenging because of a shortage of tools for in vitro cultivation of T. spiralis. We have found that T. spiralis larvae molt, ecdyse, develop to adulthood, and reproduce when they are inoculated onto cultured intestinal epithelial cells. Initially, larvae invade and migrate through cells in a monolayer (T. ManWarren, L. Gagliardo, J. Geyer, C. McVay, S. Pearce-Kelling, and J. Appleton, Infect. Immun. 65:4806-4812, 1997). During prolonged culture in Caco-2 epithelial cells, L1 larvae molted and ecdysed with efficiencies as high as 50%. Molting and ecdysis in vitro required entry of the parasite into cells; conditions that prevented entry into cells also prevented ecdysis. When larvae were inoculated at a low density and cultured for 5 to 9 days, as many as 50% of the larvae developed to adult stages. Low numbers of mature male worms with copulatory appendages were observed in these cultures. The majority of worms that survived for five or more days were unfertilized females. Low-density cultures supported development of female worms with embryos at rates of 4 to 5%. These results show that the intestinal life cycle of T. spiralis can be supported entirely by host epithelial cells. Our model should allow more detailed investigation of intracellular parasitism by T. spiralis.

In vitro and in vivo activities of nitazoxanide against Clostridium difficile.

We have used the hamster model of antibiotic-induced Clostridium difficile intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent. The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) against C. difficile, (ii) toxicity, (iii) ability to prevent C. difficile-associated ileocecitis, and (iv) propensity to induce C. difficile-associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of C. difficile were comparable to those of vancomycin or metronidazole. C. difficile-associated ileocecitis was induced with oral clindamycin and toxigenic C. difficile in a group of 60 hamsters. Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight [gbw]), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw). Animals receiving saline died 3 days post-C. difficile challenge. During the treatment period, NTZ (>/=7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced C. difficile intestinal disease. Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period. Of these surviving animals, all but three died of C. difficile disease by between 3 and 12 days following discontinuation of antibiotic therapy. Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic C. difficile. All of the NTZ-treated animals survived the 15-day postinfection period. Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or C. difficile intestinal disease, nor was C. difficile detected in the cultures of the ceca of these animals. By contrast, vancomycin and metronidazole treatment induced fatal C. difficile intestinal disease in 20 and 70% of recipients, respectively.

Antibodies to tyvelose exhibit multiple modes of interference with the epithelial niche of Trichinella spiralis.

Infection with the parasitic nematode Trichinella spiralis is initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells against T. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941-1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment by T. spiralis.

Participation of parasite surface glycoproteins in antibody-mediated protection of epithelial cells against Trichinella spiralis.

The L1 stage of the parasitic nematode Trichinella spiralis displays on its surface glycoproteins that are immunologically cross-reactive with several larval excretory-secretory (ES) products. The basis for the cross-reactivity is tyvelose, the terminal residue on the complex glycans shared by these surface and ES glycoproteins. In neonatal rats, tyvelose-specific monoclonal antibodies mediate the expulsion of larvae from the intestine. The aim of the studies described in this report was to determine how antibody binding to larval surfaces contributes to expulsion. In these experiments, which involve an in vitro assay of epithelial cell invasion, surface proteins on living larvae were biotinylated to distinguish them from ES products. Biotinylated and nonbiotinylated larvae were cocultured with avidin, biotin-specific antibodies, or anti-tyvelose monoclonal antibodies. Biotinylated larvae cultured with avidin or biotin-specific antibodies invaded Madin-Darby canine kidney (MDCK) cells equally as well as biotinylated larvae cultured with medium alone. Anti-tyvelose monoclonal antibodies were highly protective in this assay; however, biotinylation of larval surfaces hindered the ability of anti-tyvelose monoclonal antibodies to prevent larval invasion of epithelial cells. This correlated with a reduction in the binding of anti-tyvelose antibody to biotinylated larval surfaces. Our results indicate that antibody binding to surface glycoproteins contributes to protection against T. spiralis invasion but that surface binding alone is not sufficient for protection. Our findings support the notion that protection is effected by cross-linking of ES products to surface antigens.

Adoptive transfer of granulomatous inflammation to Brugia antigens in jirds.

Brugia pahangi infections of jirds (Meriones unguiculatus) produce a granulomatous inflammatory response within the lymphatic vessels. Granulomas that form around beads coated with soluble adult antigens embolized in the lungs have been used to measure this response. Similar lesions were observed in naive jirds receiving lymph node cells and splenocytes from animals with acute infections. This was not the case with cells from chronically infected jirds that were hyporesponsive to implanted antigen-coated beads. Passively transferred immune sera collected during the acute and chronic periods of infection did not transfer this response. Lymph node cells but not splenocytes obtained from chronically infected jirds induced a down regulation of this response in animals during the acute period. These results indicate that this inflammatory response in the jird is cell mediated and that adoptive transfer in jirds is feasible. The induction of the down-regulated state may also be mediated by cells, but not serum factors.

Terminal beta-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens.

Indirect evidence that the immunodominant N-glycans of the parasite, Trichinella spiralis are capped by novel beta-linked 3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelose, Tyv) was obtained from immunochemical assays employing monoclonal antibodies and synthetic oligosaccharides. Three of four previously characterized monoclonal antibodies generated from the lymphocytes of T.spiralis infected rats bind BSA glycoconjugates bearing the synthetic epitope beta-D-Tyvp(1-->3)-beta-D-GalNAcp but not to the corresponding alpha-D-Tyvp(1-->3)-beta-D-GalNAcp-BSA glycoconjugate. Monosaccharide and disaccharide glycoside inhibition data mirrors the results of the direct binding experiments. The branched tetrasaccharide beta-D-Tyv(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1 -->3)] beta-D-GalNAcp is the most active synthetic oligosaccharide inhibitor for all four monoclonal antibodies studied, while the corresponding alpha-D-Tyv containing tetrasaccharide and the core trisaccharide beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1-->3)]beta-D-GlcNAcp+ ++ are inactive. The exceptional inhibitory activity of the disaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp with one mAb (18H) compared to that of the branched tetrasaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp( 1-->3)]-beta-D-GlcNAcp is indicative of the presence of linear, nonfucosylated glycan epitopes (beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4) beta-D-GlcNAcp) that lack a fucose residue in one arm of the antigenic, tetra-antennary N-glycan. This observation supports earlier FAB-mass spectrometry evidence for the existence of tetra-antennary, core fucosylated glycans that lack a fucose residue on one of their antennae.

Toxin A secretion in Pseudomonas aeruginosa: the role of the first 30 amino acids of the mature toxin.

Toxin A, one of several virulence factors secreted by the gram-negative bacterium Pseudomonas aeruginosa, is synthesized as a 71 kDa precursor with a typical prokaryotic leader peptide (LP), and is secreted as a 68 kDa mature protein. Evidence from a previous study suggested that a signal required for toxin A secretion in P. aeruginosa may reside within the region defined by the toxin A LP and the first 30 amino acids (aa) of mature toxin A. In the present study, we have used exonuclease Bal31 deletion analysis to examine the specific role of the first 30 aa in toxin A secretion. Four toxA subclones, which encode products containing the toxin A LP and different segments of the 30-residue region fused to a toxin A carboxy-terminal region, were identified. In addition, a gene fusion encoding a hybrid protein consisting of the LP of P. aeruginosa elastase and the final 305 residues of toxin A, was generated. The cellular location of the toxA subclone products in P. aeruginosa was determined by immunoblotting analysis. Toxin A CRMs (cross-reacting material) encoded by different subclones were detected in different fractions of P. aeruginosa including the periplasm and the supernatant. Results from these studies suggest that (1) mature toxin A contains two separate secretion signals one within the N-terminal region and one within the C-terminal region; and (2) the first 30 residues of the mature toxin A form part of the N-terminal secretion signal.

Impact of microfilaremia on maintenance of a hyporesponsive cellular immune response in Brugia-infected gerbils (Meriones unguiculatus).

The purpose of these experiments was to define the significance of the microfilarial stage to the hyporesponsive condition seen in lymphatic filariasis. Two types of experiments were conducted with Brugia pahangi-infected gerbils. In one, in vitro lymphocyte blastogenesis and in vivo granuloma formation in response to parasite antigen were correlated to microfilaremia in chronically infected individuals. In a second set of experiments, the level of in vivo granuloma formation was assessed following chemotherapeutic removal of microfilariae with ivermectin. The results indicated that the microfilarial stage alone is not responsible for the maintenance of the low cellular responses seen during chronic infections in this model. Furthermore, the data suggest that the degree of downregulation of these responses may be related to parasite burden.

Brugia pahangi: differential granulomatous reactivity of infected jirds (Meriones unguiculatus) to fractions of adult worm extract.

Soluble extracts of adult Brugia pahangi (SSE) were fractionated by lectin affinity chromatography, followed by reversed phase HPLC. The immunologic and in vivo inflammatory reactivity of the resulting fractions were compared in jirds with acute and chronic infections of B. pahangi. When separated by SDS-PAGE, all fractions possessed bands which were recognized in Western blots by antibodies from jirds with both acute and chronic infections. Fractions were coupled to sized Sepharose beads that were subsequently embolized into the lungs of infected and uninfected control jirds. Granulomas were induced by SSE, the lectin column eluate, and HPLC fractions E, F, and G in acutely infected jirds. These reactions were significantly reduced in chronically infected jirds. HPLC fractions B, C, and D did not elicit an in vivo inflammatory response. A perivascular infiltrate of eosinophils and mononuclear cells was also observed in lungs of acutely infected jirds which received granuloma-inducing coated beads but not in lungs of similar jirds which received beads that did not induce this inflammatory response. Proliferative responses of splenocytes stimulated with SSE or the lectin eluate and lymph node cells and splenocytes stimulated with HPLC fractions B, C, or D corresponded to the in vivo granulomatous response to homologously coated beads. Correlations between in vivo inflammatory responses and in vitro proliferative responses were not seen using other fractions in these assays. These data indicate that varying degrees of granulomatous inflammation are induced by different filarial proteins mixtures and that the in vivo granuloma induction by antigen-coated beads will be useful in the identification of specific proteins involved in the induction, maintenance, and regulation of filariae-elicited inflammatory reactions. Although the size of these granulomas corresponds to severity of granulomatous inflammatory responses visualized within the jird lymphatics during the course of infection, the reaction does not correlate in all instances to lymphoproliferative responses of cells from peripheral lymph nodes or the spleen. Distinct differences between antibody and granulomatous reactivity to some fractions were noted.

Murine tissues exposed to cytotoxic drugs display altered patterns of Candida albicans adhesion.

An ex vivo adhesion assay was used to examine the binding of Candida albicans yeast cells to tissues from mice treated with cytotoxic drugs such as lipopolysaccharide and the clinically used anticancer drugs doxorubicin, cisplatin, and vincristine. No major differences were observed in binding of the fungal cells to liver and kidney tissues from treated or untreated animals. All drug-treated spleens displayed altered patterns of C. albicans adhesion compared with the control group, with yeast cells bound not only to the marginal zone but also to the white and red pulp. Immunostaining for macrophages, which are proposed as the site of normal adhesion, showed no apparent differences between the control and the experimental spleens that could account for the change in adhesion patterns. Scanning electron microscopy images suggested that yeast binding to the white pulp of treated tissue is mediated through fibers, perhaps extracellular matrix components exposed as result of the cytotoxic treatment. Exposure of new attachment sites for C. albicans in treated tissues may facilitate initiation of infection.

Brugia pahangi: effects of maternal filariasis on the responses of their progeny to homologous challenge infection.

Granulomatous lesion formation and immune responses to Brugia pahangi infections were compared in age-matched male progeny of homologously infected and uninfected female jirds. Infections initiated in 2-week-old offspring yielded mean +/- SD adult worm recoveries of 6.0 +/- 5.7 and 4.2 +/- 5.4 in offspring from infected or uninfected mothers, respectively. Infections initiated in 4-week-old offspring resulted in an mean +/- SD recovery of adult worms of 11.3 +/- 11.3 and 10.2 +/- 5.8 in offspring from infected and uninfected mothers, respectively. The ratio of intralymphatic thrombi per intralymphatic worm was similar between infected offspring from infected or uninfected mothers within experiments. Areas of granulomas around B. pahangi antigen-coated beads embolized in the lungs were not significantly affected by maternal origin in infected or uninfected progeny. Offspring infected at 2 or 4 weeks of age from infected mothers exhibited significantly reduced titers of serum IgG antibodies to Brugia antigens at 5-8 weeks postinfection compared to infected offspring of uninfected mothers. Infected offspring from infected mothers also had significantly fewer splenic IgG plaque-forming cells to B. pahangi antigens at 5 weeks postinfection than similarly infected offspring from uninfected mothers. Western immunoblot analysis indicated qualitative and quantitative reductions in serum antibody reactivity to adult B. pahangi antigens in infected progeny of infected females compared to age-matched infected controls. Reduced homologous serum antibody responses in progeny exposed to maternal B. pahangi infection suggest that maternal immunoregulation to filarial antigens may occur. Reduced antibody responsiveness to B. pahangi antigens observed in infected offspring from infected mothers, however, had no demonstrable effect on adult worm burdens, microfilaremias, lymphatic lesion formation, or antigen-specific granulomatous inflammatory responses compared to infected progeny of uninfected mothers.

Qualitative characterization of antibody responses to single and multiple Brugia pahangi infections in jirds.

Antibody responses of jirds, singly and multiply inoculated with Brugia pahangi infective larvae (L3), to soluble somatic extracts of adult parasites were characterized by western blot analysis. Forty-two protein bands ranging in molecular weight from 12 to 160 kDa were recognized by sera from infected jirds. Antibody recognition of individual B. pahangi antigen bands in this assay appears to be independent of antibody enzyme-linked immunosorbent assay (ELISA) titers to crude parasite extract, severity of lymphatic lesions, levels of microfilaremia, numbers of L3 inoculated, or numbers of adult parasites in individual jirds. Antibody recognition of protein bands with molecular weights of 37 kDa, 21 kDa, and 17 kDa, however, did temporally correspond with certain parasitological and pathologic events. Antibody against the 37-kDa protein band first was identified at the onset of patency, reaching a 90% prevalence rate by 90 days postinfection (DPI). The prevalence of this antibody remained high. Antibody recognition of the 21-kDa protein band first occurred at 90 DPI and gradually increased in prevalence during the course of infection temporally similar to the increase in microfilaremia. Recognition of the 17-kDa protein band first occurred at 48 DPI, reached a maximum prevalence of 80% at 90 DPI, and decreased to a minimal prevalence by 160 DPI. Prevalence of antibody responses to the 17-kDa protein band corresponded temporally with the kinetics of the rise and fall of numbers of intralymphatic thrombi. The patterns of antibody response to these 3 bands were similar in both singly and multiply inoculated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Brugia pahangi: circulating antibodies to adult worm antigens in uninfected progeny of homologously infected female jirds.

Serum IgG antibody levels to adult Brugia pahangi antigens were measured in uninfected offspring from uninfected and B. pahangi-infected female jirds. Antibody titers to B. pahangi antigens in sera of offspring from infected females mimicked the maternal titer during the suckling period. Neonate titers peaked at 2 weeks of age at levels as high as 1:4100, then decreased to levels well below maternal titers by 8-12 weeks of age. Concurrent maternal and 2-week-old neonate sera recognized identical B. pahangi antigens in Western blots. Spleen cells from 2-week-old filariae-exposed and unexposed offspring failed to produce measurable antibody to B. pahangi in vitro. Progeny of uninfected mothers nursed by B. pahangi-infected females showed circulating IgG antibody titers to adult worm antigens similar to those of homologously reared offspring. Conversely, offspring born to B. pahangi-infected females and nursed by an uninfected female had no serum antibodies to B. pahangi antigens. Blastogenic responses of spleen cells to the mitogens phytohemagglutinin and pokeweed mitogen, and adult B. pahangi antigens, were not different between offspring groups. Mean areas of pulmonary granulomas induced by the intravenous inoculation of B. pahangi antigen-coated beads also did not differ between 4- and 8-week-old progeny of uninfected or infected females. These results suggest that the circulating IgG antibodies to adult B. pahangi antigens demonstrated in offspring of infected female jirds are maternally derived via the milk and do not alter the cellular responses of uninfected offspring to B. pahangi antigens as measured by antigen-stimulated blastogenesis or pulmonary granulomatous inflammatory response.

Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus).

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.

A comparison of host responses of the Mongolian jird to infections of Brugia malayi and B. pahangi.

Host responses of jirds receiving a single subcutaneous inoculation of subperiodic Brugia malayi were compared with those of jirds similarly infected with B. pahangi. Parasite burdens, lymphatic lesion severity, granulomatous reactivity, antibody responses to parasite antigens, and complete blood cell counts were assessed at 60 and 150 days post-inoculation. At 60 days post-inoculation, percentages of adults recovered at necropsy and lymphatic lesion severity were greater in B. pahangi-infected jirds. At 150 days post-inoculation, lesion severity and percentages of worms recovered were similar in both infections. No significant differences were noted in either infection in reactivity to homologous or heterologous parasite antigens in any parameter measured. Similarities in the kinetics of the inflammatory reactivities of the 2 infections suggest that previous observations made in the jird-B. pahangi model could be utilized in designing studies using B. malayi. Further, the more marked lesion severity observed in B. pahangi-infected jirds and the relative ease of maintaining B. pahangi in the laboratory support the continued use of this system as a conceptual model for the study of lymphatic lesion pathogenesis.

Circulating parasite antigen in Brugia pahangi-infected jirds.

The Mongolian jird is used widely in filariasis research for studies of protective immunity, pathogenesis, and therapy. The purpose of this study was to evaluate parasite antigen detection as a means of noninvasively monitoring Brugia pahangi infection in jirds. A parasite antigen with Mr of 105-110 kDa was identified in sera from i.p.- and s.c.-infected jirds by immunoblot with a monoclonal antibody to phosphorylcholine. The same antibody was used in a direct sandwich enzyme immunoassay to measure antigen in jird sera. Parasite antigen was detectable as early as 2 wk after i.p. or s.c. injection of L3. Antigen titers increased between 2 and 12 wk and stabilized between 12 and 36 wk after infection in s.c.-infected animals. A different pattern was seen in i.p.-infected jirds with antigen titers peaking at 16 wk and falling significantly between 16 and 32 wk after infection. Parasite antigen titers correlated significantly with adult worm infection intensities in jirds with mature i.p. and s.c. infections. Antigenemia was also detectable in sera from jirds after i.p. implantation of adult parasites of either sex. However, antigen was not detected in sera from infant offspring of antigenemic infected mothers. We conclude that parasite antigen detection allows B. pahangi development and survival as well as infection intensity to be monitored in living animals with unprecedented sensitivity and accuracy. This technique should facilitate drug and vaccine studies in this important experimental filariasis model.