Teladorsagia circumcincta in the sheep abomasum: defining the role of dendritic cells in T cell regulation and protective immunity.

Parasitic nematodes of the small-ruminant gastrointestinal tract pose a problem worldwide. The impact of these pathogens is worsened by the emergence of anthelmintic resistance to all three available classes of drugs. In addition to causing considerable economic loss, these parasites are detrimental to the health and welfare of sheep and goats. Vaccination offers an alternative approach to drug-based control and a great deal of investment has gone into the investigation of protective antigens for some of these nematode species. However, attempts at vaccination are hindered by a lack of understanding of how best to promote protective immunity to nematode species, such as Teladorsagia circumcincta, which inhabits the abomasum of sheep. This situation contrasts with that in murine models of gastrointestinal nematode infection, where the basis of protective immunity is increasingly well understood. In this review, we discuss the current knowledge of the immune effector mechanisms elicited by T. circumcincta and consider the probable role of dendritic cells in the initiation of both effector and regulatory responses in the abomasum.

The lateral tunnel Fontan procedure for hypoplastic left heart syndrome: results of 100 consecutive patients.

The Fontan procedure for hypoplastic left heart syndrome (HLHS) is well established. Multiple surgical techniques including extracardiac conduits and autologous tissue connections have been developed. We reviewed the results of 100 consecutive patients undergoing the lateral tunnel modification of the Fontan procedure at the University of Michigan. A cross-sectional retrospective study was performed for 100 consecutive patients identified in the University of Michigan Congenital Heart Surgery database with the diagnosis of HLHS. All patients had undergone a lateral tunnel Fontan procedure between June 2000 and August 2004. The medical record was reviewed to assess patient, procedural, and morphologic determinants of outcome. Hospital survival was 97% and intermediate-term survival was 96% with a median follow-up time of 34 months. Preoperative mean pulmonary artery pressure, right ventricular end diastolic pressure, aortic cross-clamp time, and tricuspid valve regurgitation were not associated with late right ventricular function or survival. Three patients required takedown of the lateral tunnel Fontan in the early postoperative period. A positive association was found between protein-losing enteropathy and prolonged (>2 weeks) postoperative pleural drainage (p = 0.035). No patient required cardiac transplantation or late intervention on the Fontan pathway. At the time of follow-up, 100% of patients were New York Heart Association class I or II and 90% were in normal sinus rhythm. The lateral tunnel Fontan procedure for HLHS can be performed with acceptable early and intermediate-term risk. There was a low prevalence of late rhythm disturbances and other complications. Protein-losing enteropathy and prolonged pleural drainage were associated.

Amplatzer closure of atrial septal defect and da Vinci robot-assisted repair of vascular ring.

Technology for minimally invasive approaches to congenital heart disease is a rapidly evolving field. This case report reviews a novel approach to combining two of the newer technologies available to treat a pediatric patient with an atrial septal defect (ASD) and a vascular ring. This report is the first to describe the use of the da Vinci surgical system to assist in a thoracoscopic procedure for a pediatric patient. The da Vinci assisted division of the vascular ring, joined with an Amplatzer closure of the ASD, demonstrates how maximum benefit can be obtained for patients by combining emerging technologies.

Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62.

Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites, Acanthocheilonema viteae and Brugia pahangi. For B. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages of A. viteae differed greatly from each other with a pattern of increased mRNA production with maturation. As a rule A. viteae had higher levels of ES-62 mRNA than B. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, as A. viteae only secreted approximately 3 times as much ES-62 as B. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

Expression of the filarial nematode phosphorylcholine-containing glycoprotein, ES62, is stage specific.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Expression of filarial-specific IgG subclasses under different transmission intensities in a region endemic for loiasis.

Specific IgG subclasses were investigated in two villages (Okoumbi and Ndjokaye) in southeast Gabon with different Loa loa transmission intensities of approximately 9,000 and 1,300 infective larvae (L3) per person per year, respectively. IgG subclasses were measured by an enzyme-linked immunosorbent assay (ELISA) using extracts of L. loa L3, microfilariae (MF), or adult worms. Levels of L3-specific IgG3 were significantly higher in the village with low transmission (Ndjokaye) (P = 0.006). In contrast, MF-specific IgG2 was significantly higher in Okoumbi than in Ndjokaye (P = 0.0009). In the high-transmission village (Okoumbi), levels of both MF- and adult-specific IgG4 were significantly increased in MF carriers compared with amicrofilaremic subjects (P = 0.0015 and P = 0.003, respectively), while levels of L3- and MF-specific IgG1 were significantly higher in amicrofilaremic individuals compared with MF carriers (P = 0.04 and P = 0.03, respectively). Furthermore, among microfilaremic individuals, the level of the specific IgG1 subclass was much lower in Okoumbi than in Ndjokaye (P = 0.036). These results suggest that the expression of antigen-specific IgG3 and IgG2 is more likely to vary with transmission intensity, whereas antigen-specific IgG4 and IgG1 varies with adult worm and MF burden.

Heat shock and developmental expression of hsp83 in the filarial nematode Brugia pahangi.

hsp83 was cloned from the filarial nematode Brugia pahangi. The mRNA was constitutively expressed at 37 degrees C in life cycle stages that live in the mammalian host (microfilariae and adult worms). Heat shock resulted in only a minimal increase in levels of transcription. A genomic copy of hsp83 was isolated and was shown to contain 11 introns while sequencing of the 5' upstream region revealed several heat shock elements. Using a chloramphenicol acetyltransferase (CAT) reporter gene construct the expression of hsp83 from B. pahangi (Bp-hsp83) was studied by transfection of COS-7 cells. Similar to the expression pattern in the parasite, CAT activity was detected at 37 degrees C and was not influenced by heat shock. When the free-living nematode Caenorhabditis elegans was transfected with the same construct, CAT activity was not observed at normal growth temperatures (21 degrees C) or under moderate heat shock conditions (28 degrees C). However exposure to more severe heat shock (35 degrees C) resulted in an increase in CAT activity. These results suggest that Bp-hsp83 has a temperature threshold > or = 35 degrees C for expression.

Esophagectomy for achalasia: patient selection and clinical experience.

BACKGROUND: In 1989, we predicted an increasing number of esophagectomies for megaesophagus and for recurrent symptoms after prior esophagomyotomy or balloon dilatation for achalasia. Patient selection in this group is challenging, as the potential operative morbidity of an esophagectomy must be weighed against the expected clinical outcome after a redo esophagomyotomy or alternative procedures designed to salvage the native esophagus. METHODS: The hospital records of 93 patients undergoing esophagectomy for achalasia during the past 20 years were reviewed retrospectively and the results of operation assessed using our prospectively established Esophageal Resection Database and follow-up information obtained through personal contact with the patients. RESULTS: Patient age averaged 51 years. Indications for esophagectomy included tortuous megaesophagus (64%), failure of prior myotomy (63%), and associated reflux stricture (7%). Ninety-four percent of the patients underwent a transhiatal esophagectomy. Stomach was used as the esophageal substitute in 91% cases. Intraoperative blood loss averaged 672 mL. Postoperative length of stay averaged 12.5 days. Major complications included anastomotic leak (10%), recurrent laryngeal nerve injury (5%), delayed mediastinal bleeding requiring thoracotomy (2%), and chylothorax (2%). There were 2 hospital deaths (2%) from respiratory insufficiency and sepsis. Follow-up has averaged 38 months. In all, 95% of patients eat well; nearly 50% have required an anastomotic dilatation; troublesome regurgitation has been rare; and 4% have refractory postvagotomy dumping. CONCLUSIONS: Esophagectomy, preferably through a transhiatal approach, is generally safe and effective therapy in selected patients with achalasia. Unique technical considerations include difficulty encircling the dilated cervical esophagus, deviation of the esophagus into the right chest, large aortic esophageal arteries, and adherence of the exposed esophageal submucosa to the adjacent aorta after prior myotomy.

Lymphatic filariasis: parallels between the immunology of infection in humans and mice.

Mouse models of Brugia infection have provided much useful quantitative and qualitative information on the immune response elicited by different life cycle stages of filarial worms. Many parallels exist between the immune response in the mouse and the infected human and in this review we highlight areas of topical interest, including the induction of specific cytokine responses and their role in immunomodulation and protective immunity. These studies have reinforced the concept that different life cycle stages of filarial parasites each have their own mechanism of modulating responses so that potentially inflammatory IFN-gamma responses are downregulated. While the precise mechanisms of protective immunity remain to be defined, studies in the mouse have suggested novel pathways, including a possible role for granulocytes.

Temperature is a cue for gene expression in the post-infective L3 of the parasitic nematode Brugia pahangi.

The temporal expression pattern of two genes, Bp-cdd and Bp-S3, was studied at defined points throughout the life cycle of Brugia pahangi. Both mRNAs were up-regulated to coincide with the transition of the L3 from the vector to the mammalian host. Bp-cdd was expressed almost exclusively in the post-infective (p.i.) L3 and L4 stages of the life cycle while Bp-S3 was also expressed in adult worms, but at a much lower level than in the larval stages. Immunogold labelling with an antiserum raised to the recombinant Bp-CDD localised the native antigen to the hypodermis in the p.i. L3 and L4. Specific labelling was not detected in the adult worm. The expression of both mRNAs could be triggered by exposure of the vector-derived L3 to a simple mammalian culture system. Analysis of the factors, which induced expression suggested that the temperature shift which accompanies the transition from mosquito to mammal was the most important cue for expression of both genes.

NO contributes to proliferative suppression in a murine model of filariasis.

Infection of BALB/c mice with microfilariae (mf) of Brugia pahangi leads to the suppression of antigen (Ag)-specific proliferative responses in the spleen. The proliferative defect is dependent on inducible nitric oxide synthase (iNOS) activity, since inhibition of iNOS with either L-N-monomethyl arginine (L-NMMA) or aminoguanidine reversed defective proliferation. Splenocytes from mf-infected animals produce high levels of gamma interferon (IFN-gamma) upon in vitro restimulation with Ag, and experiments in IFN-gamma receptor-deficient (IFN-gamma R(-/-)) mice demonstrated that signaling via the IFN-gamma R is essential in the induction of NO production and subsequent proliferative suppression. Restimulation of splenocytes from mf-infected animals with an extract of Acanthocheilonema viteae, a related filarial worm which lacks endosymbiotic bacteria, also resulted in NO production and proliferative suppression, demonstrating that lipopolysaccharide of bacterial origin is not essential to the induction of iNOS activity. These results extend previous observations that infection with different life cycle stages of Brugia leads to the development of differentially polarized immune responses and demonstrate one method by which these differences may exert their effects on the proliferative potential of cells from infected animals.

The third-stage larva (L3) of Brugia: its role in immune modulation and protective immunity.

In this review, we focus on the role of the L3 (third-stage larva) of lymphatic filarial nematodes in immunomodulation and in the development of protective immunity. Studies in the mouse models of Brugia have been fundamental to our understanding of the mechanisms by which infection with L3 results in Th2 responses and the active suppression of Th1 responses. The relevance of these phenomena to the human infection is discussed.

Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism.

Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.

Identification of abundant mRNAs from the third stage larvae of the parasitic nematode, ostertagia ostertagi.

Reverse transcriptase-PCR (RT-PCR) was carried out on total RNA prepared from the third-stage larvae (L3) of Ostertagia ostertagi in order to clone and characterize the major transcripts expressed in this larval stage, as an initial investigation of arrested larval development in the parasite. Distinct bands were visible on an agarose gel and four of these were cloned and sequenced. Three of the bands represented multiple transcripts, while the fourth band encoded the enzyme GTP cyclohydrolase I (GTP-CH), which catalyses the first and rate-limiting step in pteridine biosynthesis. Northern blot analysis and RT-PCR demonstrated that GTP-CH is highly up-regulated in the L3 stage and undetectable in either the L2 or adult stages. Using immunogold electron microscopy, GTP-CH was shown to be predominantly localized to the cell body of the body wall muscles and the cells of the intestine in the L3.

Hippocampus development and generation of dentate gyrus granule cells is regulated by LEF1.

Lef1 and other genes of the LEF1/TCF family of transcription factors are nuclear mediators of Wnt signaling. Here we examine the expression pattern and functional importance of Lef1 in the developing forebrain of the mouse. Lef1 is expressed in the developing hippocampus, and LEF1-deficient embryos lack dentate gyrus granule cells but contain glial cells and interneurons in the region of the dentate gyrus. In mouse embryos homozygous for a Lef1-lacZ fusion gene, which encodes a protein that is not only deficient in DNA binding but also interferes with (beta)-catenin-mediated transcriptional activation by other LEF1/TCF proteins, the entire hippocampus including the CA fields is missing. Thus, LEF1 regulates the generation of dentate gyrus granule cells, and together with other LEF1/TCF proteins, the development of the hippocampus.

Cloning and characterization of two nuclear receptors from the filarial nematode Brugia pahangi.

Nuclear receptors (NRs) encompass a superfamily of cytoplasmic/nuclear localized receptors that on ligand binding (or by phosphorylation) directly regulate the transcription of target genes. NRs are involved in many developmental processes, including moulting in insects and dauer larva formation in Caenorhabditis elegans. Here we report the isolation of two genes related to NRs from the filarial nematode Brugia pahangi. Bp-nhr-1 is a member of the NGF1-B subfamily of NRs and is expressed at very low levels in post-infective larval stage 3 (L3) after their transmission to the mammalian host. The second gene, Bp-nhr-2, is related to XR78E/F of Drosophila, a gene involved in the ecdysone response, over the region of its DNA-binding domain. cDNA and genomic clones have been isolated that correspond to Bp-nhr-2. The most striking feature of the encoded protein is that, although there is a DNA-binding domain similar to that of other NRs, the ligand-binding domain is absent. To investigate the pattern of transcription of Bp-nhr-2 in the filarial life cycle, semi-quantitative reverse-transcriptase-mediated PCR was performed; this analysis demonstrated that the gene is expressed in early stages after infection and in the adult and microfilariae, and is up-regulated just before the moult between L3 and L4 but is not expressed during the moult between L4 and adult. Antibodies raised against a peptide corresponding to the transactivation domain of Bp-nhr-2 demonstrate that the protein is expressed in microfilariae and adult samples and that another cross-reactive protein is expressed in these life-cycle stages.

The isolation of differentially expressed cDNA clones from the filarial nematode Brugia pahangi.

A cDNA library constructed from 3 day post-infective L3 of the filarial nematode Brugia pahangi was screened by differential hybridization with cDNA probes prepared from different life-cycle stages. Five cDNA clones hybridizing selectively to the mosquito-derived L3 probe were isolated and characterized. Northern blot analysis of 4 of the clones confirmed that each was most highly expressed in the mosquito-derived L3. The expression of each mRNA during parasite development in the mosquito vector was investigated using RT-PCR, and all were shown to be abundant in the immature L3. Four of the 5 cDNAs cloned coded for structural proteins: 2 cuticular collagens, and the muscle proteins tropomyosin and troponin. Further studies on troponin using an antiserum raised to the recombinant protein demonstrated that the protein, unlike the mRNA, was present in all life-cycle stages examined, while immunogold labelling demonstrated that it was localized to the muscle blocks.

cut-1-like genes are present in the filarial nematodes, Brugia pahangi and Brugia malayi, and, as in other nematodes, code for components of the cuticle.

A fragment of a cut-1 like gene from the filarial nematode Brugia pahangi (designated Bp-cut-1) was isolated by PCR from genomic DNA. The sequence was used to design primers for use in RT-PCR and resulted in the isolation of a cDNA fragment from larvae in the process of the L3-L4 moult. Screening of a B. malayi genomic library identified a single clone, Bm-cut-1. Using primers designed from the Brugia sequences, semi-quantitative RT-PCR was carried out on 11 different life cycle stages chosen to cover periods around the moult and inter-moult periods. This analysis demonstrated that the cut-1 mRNA was most abundant preceding the moult, consistent with its function as a cuticular protein. Immuno-gold electron microscopy using an affinity purified antiserum raised to the highly conserved region of Ascaris CUT-1 confirmed that the protein was restricted to a tight band in the median layer of the cuticle. Despite the fact that no transcripts could be detected in mature adult worms by RT-PCR, immuno-gold microscopy revealed staining of the microfilarial cuticle within the uterus of the adult female worm, suggesting that other cut-1-like genes are present in Brugia.

Interleukin-10 and antigen-presenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi.

Infection with the third-stage larvae (L3) of the filarial nematode Brugia results in a Th2-biased immune response in mice and humans. Previously we have shown that the production of interleukin 4 (IL-4) is critical for down-regulating polyclonal Th1 responses in L3-infected mice. However, the in vitro neutralization of IL-4 did not fully recover the defective polyclonal Th1 responses, nor did it result in the production of any antigen (Ag)-specific Th1 cytokines, suggesting that perhaps infection with L3 does not result in priming of Th1 cells in vivo. In this study, we analyzed the role of IL-10 and Ag-presenting cells (APCs) in the spleen as additional factors controlling the Th2 bias in infected mice. Our data show that IL-10 and APCs also contribute to the suppression of mitogen-driven Th1 responses of spleen cells from infected mice. In addition, the neutralization of IL-10 or the replacement of the resident APC population from spleen cell cultures resulted in the production of Ag-specific Th1 cytokines. Irradiated spleen cells from either L3-infected or uninfected mice were able to restore Ag-specific Th1 responses in vitro. Therefore, it appears that Brugia-reactive Th1 cells are primed following infection with L3, but are actively suppressed in vivo by a mechanism that involves IL-10 and the resident APC population, but not IL-4. These results indicate that a complex interplay of cytokines and cell populations underscores the Th2-polarized response in L3-infected mice.