Nematodes and human therapeutic trials for inflammatory disease.

Helminth infections likely provide a protective influence against some immune-mediated and metabolic diseases because helminth infection dramatically decreased in developed countries shortly before the explosive rise in the prevalence of these diseases. The capacity of helminths to activate immune-regulatory circuits in their hosts and to modulate the composition of intestinal flora appears to be the mechanisms of protective action. Animal models of disease show that various helminth species prevent and/or block inflammation in various organs in a diverse range of diseases. Clinical trials have demonstrated that medicinal exposure to Trichuris suis or small numbers of Necator americanus is safe with minor, if any, reported adverse effects. This includes exposure of inflamed intestine to T. suis, asthmathic lung to N. americanus and in patients with atopy. Efficacy has been suggested in some small studies, but is absent in others. Factors that may have led to inconclusive results in some trials are discussed. To date, there have been no registered clinical trials using helminths to treat metabolic syndrome or its component conditions. However, the excellent safety profile of T. suis or N. americanus suggests that such studies should be possible.

Clinical trials of helminth therapy in autoimmune diseases: rationale and findings.

Some helminths are major human pathogens. Recently, however, increased understanding of the immunoregulatory responses induced by this class of parasites, in combination with epidemiologic and animal studies, suggests that helminths may have therapeutic potential in autoimmune diseases (AD) and other conditions. This article reviews the rationale for and results of clinical trials to test the safety and efficacy of helminth therapy in AD. Also discussed are future prospects for investigation and the possibility that helminth treatment may serve as a probe to help reveal the pathogenesis of AD.

Substance P and the regulation of inflammation in infections and inflammatory bowel disease.

Substance P (SP) and its natural analogue hemokinin-1 (HK1) are produced by lymphocytes and macrophages, and at times B cells. These peptides are an important component of the immune response during several infections and in inflammatory bowel disease (IBD). The synthesis of SP and HK1 in leucocytes is subject to immune regulation. IL12 and IL23 stimulate T cells and macrophages to make SP respectively. The cytokines driving HK1 production are not presently defined. These peptides act through a shared receptor called neurokinin-1. T cells, macrophages and probably other immune cell types can express this receptor. Several cytokines IL12, IL18 and TNFα as well as T-cell antigen receptor activation induce neurokinin-1 receptor expression on T cells, while IL10 blocks receptor display. TGFß delays internalization of the SP/neurokine-1R complex on T cells resulting in stronger receptor signalling. One of the functions of SP and neurokinin-1 receptor is to enhance T cell IFNγ and IL17 production, amplifying the proinflammatory response. Thus, SP and HK1 have overlapping functions and are part of a sophisticated immune regulatory circuit aimed at amplifying inflammation at mucosal surfaces and in other regions of the body as shown in animal models of infection and IBD.

Tryptophan-kynurenine metabolism and insulin resistance in hepatitis C patients.

Chronic hepatitis C virus (HCV) infection is associated with 50% incidence of insulin resistance (IR) that is fourfold higher than that in non-HCV population. IR impairs the outcome of antiviral treatment. The molecular mechanisms of IR in HCV are not entirely clear. Experimental and clinical data suggested that hepatitis C virus per se is diabetogenic. However, presence of HCV alone does not affect IR. It was proposed that IR is mediated by proinflammatory cytokines, mainly by TNF-alpha. TNF-alpha potentiates interferon-gamma-induced transcriptional activation of indoleamine 2,3-dioxygenase, the rate-limiting enzyme of tryptophan- (TRP-) kynurenine (KYN) metabolism. Upregulation of TRP-KYN metabolism was reported in HCV patients. KYN and some of its derivatives affect insulin signaling pathways. We hypothesized that upregulation of TRP-KYN metabolism might contribute to the development of IR in HCV. To check this suggestion, we evaluated serum concentrations of TRP and KYN and HOMA-IR and HOMA-beta in 60 chronic HCV patients considered for the treatment with IFN-alpha. KYN and TRP concentrations correlated with HOMA-IR and HOMA-beta scores. Our data suggest the involvement of KYN and its metabolites in the development of IR in HCV patients. TRP-KYN metabolism might be a new target for prevention and treatment of IR in HCV patients.

Randomised clinical trial: the safety and tolerability of Trichuris suis ova in patients with Crohn's disease.

BACKGROUND: Recent evidence suggests that embryonated eggs of the porcine whipworm Trichuris suis ova (TSO) may be an effective treatment for inflammatory bowel disease (IBD). AIM: To assess the safety and tolerability of TSO following a single dose in patients with Crohn's disease. METHODS: This was a sequential dose-escalation (500, 2500 and 7500 viable embryonated TSO), randomised, double-blind, placebo-controlled study to evaluate the safety of a single dose of oral suspension TSO in patients with Crohn's disease. Twelve patients were randomised into each of three cohorts. Patients were assessed 1, 3, 5, 7, 9, 11 and 14 days following dosing (via a telephone call and diary symptom collection through 14 days postdose) for adverse events, changes to concomitant medications and gastrointestinal (GI) signs and symptoms. Patients were again assessed at Months 1, 2 and 6. RESULTS: Eighteen males and 18 females were enrolled, ages 20 to 54 years. All patients were dosed and completed the initial 2-month follow-up period (five patients did not attend their 6-month study visit). GI disorders were reported with the highest frequency; 7 (25.9%) TSO-treated patients and 3 (33.3%) placebo-treated patients. No dose-dependent relationship was observed, with 3 (33.3%) placebo, 4 (44.4%) TSO 500, 0 (0.0%) TSO 2500 and 3 (33.3%) TSO 7500 patients experiencing at least one GI event, and no clinically meaningful changes in GI signs and symptoms. CONCLUSIONS: A single dose of Trichuris suis ova up to 7500 ova was well tolerated and did not result in short- or long-term treatment-related side effects. Clinicaltrials.gov NCT01576461.

Substance P receptor antagonist reverses intestinal pathophysiological alterations occurring in a novel ex-vivo model of Cryptosporidium parvum infection of intestinal tissues derived from SIV-infected macaques.

BACKGROUND: Cryptosporidium infection leads to life-threatening diarrhea in AIDS patients. Pathogenesis of cryptosporidiosis is due to intestinal physiological alterations. We devised an ex-vivo model using ex-vivo Cryptosporidium parvum infection of jejunal tissues derived from SIV-infected macaques and studied the role of substance P (SP) in the pathogenesis of cryptosporidiosis. METHODS: We measured jejunal SP protein levels using ELISA, and electrophysiological alterations using the Ussing chamber technique in an ex vivo model of Cryptosporidium infection. Paraformaldehyde-fixed jejunum from SIV-infected macaques with and without naturally occurring cryptosporidiosis was studied for SP protein expression by immunohistochemistry and fluorescence deconvolution microscopy. RESULTS: Ex-vivo Cryptosporidium-infected tissues and tissues from SIV-infected macaques with naturally occurring cryptosporidiosis demonstrated elevated SP protein levels compared with tissues from SIV-infected animals without ex-vivo C. parvum infection or tissues from SIV-infected animals that have no evidence of cryptosporidiosis. In our ex-vivo model of Cryptosporidium infection, we demonstrated pathophysiological alterations that were blocked by SP-receptor antagonist treatment. CONCLUSIONS: These studies suggest that SP-receptor antagonists could prove useful for treatment of AIDS-related cryptosporidiosis.

Use of multidimensional separation protocols for the purification of trace components in complex biological samples for proteomics analysis.

The routine detection of low abundance components in complex samples for detailed proteomics analysis continues to be a challenge. Whilst the potential of multidimensional chromatographic fractionation for this purpose has been proposed for some years, and was used effectively for the purification to homogeneity of trace components in bulk biological samples for N-terminal sequence analysis, its practical application in the proteomics arena is still limited. This article reviews some of the recent data using these approaches, including the use of microaffinity purification as part of multidimensional protocols for downstream proteomics analysis.

Preclinical analysis of the analinoquinazoline AG1478, a specific small molecule inhibitor of EGF receptor tyrosine kinase.

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific inhibitor of EGFR tyrosine kinase whose favourable preclinical profile supports progression towards clinical trials. Microphysiometric evaluation revealed a short (<24 min) effective inhibition of cellular receptor response to EGF challenge in BaF/ERX cells indicating a need to maintain sustained levels of inhibitor. Initial pharmacokinetic evaluation in mice of novel AG1478 formulations in a beta-cyclodextrin (Captisol) showed monoexponential elimination from plasma (half-life 30 min) following subcutaneous administration. A two-fold dose escalation gave a 2.4-fold increase in the total AUC. Bolus i.v. and 6 h continuous infusion were investigated in rats to mimic a more clinically relevant administration regimen. Drug elimination following bolus i.v. administration was biphasic (terminal elimination half-life 30-48 min). The linear relationship between dose and AUC(0-->infinity) (r2=0.979) enabled the prediction of infusion rates and doses for sustained delivery using continuous 6 h infusions, where steady state was reached in 120 min. Plasma levels of AG1478>10 microM were achieved over the duration of the infusion. At the lowest dose, plasma drug levels after the cessation of infusion declined with a half-life of approximately 43 min. EGFR activity, measured both by autophosphorylation and downstream signalling, was inhibited in a dose-dependent manner by injection of AG1478 in mice bearing xenografts of the human glioblastoma cell line U87MG.delta2-7, which expresses a constitutively active variant of the EGF receptor. Taken together, these experiments provide essential data to assess the anti-tumour efficacy of AG1478 and will assist in the rational design of dose regimens for clinical studies.

Trichuris suis therapy in Crohn's disease.

BACKGROUND: Crohn's disease is common in highly industrialised Western countries where helminths are rare and uncommon in less developed areas of the world where most people carry worms. Helminths diminish immune responsiveness in naturally colonised humans and reduce inflammation in experimental colitis. Thus exposure to helminths may help prevent or even ameliorate Crohn's disease. AIMS: The aim of the study was to determine the safety and possible efficacy of the intestinal helminth Trichuris suis in the treatment of patients with active Crohn's disease. PATIENTS: Twenty nine patients with active Crohn's disease, defined by a Crohn's disease activity index (CDAI) > or =220 were enrolled in this open label study. METHODS: All patients ingested 2500 live T suis ova every three weeks for 24 weeks, and disease activity was monitored by CDAI. Remission was defined as a decrease in CDAI to less than 150 while a response was defined as a decrease in CDAI of greater than 100. RESULTS: At week 24, 23 patients (79.3%) responded (decrease in CDAI >100 points or CDAI <150) and 21/29 (72.4%) remitted (CDAI <150). Mean CDAI of responders decreased 177.1 points below baseline. Analysis at week 12 yielded similar results. There were no adverse events. CONCLUSIONS: This new therapy may offer a unique, safe, and efficacious alternative for Crohn's disease management. These findings also support the premise that natural exposure to helminths such as T suis affords protection from immunological diseases like Crohn's disease.

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12.

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.

Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease.

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.

High-performance liquid chromatographic analysis of the tyrphostin AG1478, a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in mouse plasma.

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is undergoing evaluation as a potential new anticancer agent. We have developed a specific and sensitive reversed-phase HPLC assay for AG1478 in mouse plasma. The method involves a rapid and simple extraction process followed by separation on a Symmetry C8 stationary phase with a gradient of acetonitrile in ammonium acetate buffer. A linear response was achieved over the concentration range of 0.2-100 microM using multilevel calibration with internal standard method of calculation. Inter- and intra-assay accuracy and precision were better than +/-10%. The limit of quantitation was 0.2 microM. We have used this method to study the preclinical pharmacokinetics of this new agent in mice.

Interleukin 12 and antigen independently induce substance P receptor expression in T cells in murine schistosomiasis mansoni.

Substance P (SP) regulates interferon-gamma (IFN-gamma) production through interaction with the SP receptor NK1 (SPr) on T cells at sites of inflammation. Using murine schistosomiasis, we evaluated whether SPr expression was subject to immunoregulation. Splenocytes from schistosome-infected mice cultured for < or =18 h did not express SPr, as determined by quantitative polymerase chain reaction assay. However, exposure to schistosome egg antigen (SEA) for < or =4 h induced strong receptor expression. Experiments using splenocytes fractionated with antibody-coupled, paramagnetic beads showed that induction localized exclusively to T cells. Receptor protein expression was confirmed with Western blot. Interleukin 12 (IL-12) also induced strong T-cell SPr expression. Both SEA and IL-12 remained strong inducers of T-cell SPr in lymphocytes from the IL-12 (p40) and IFN-gamma R double-knockout mouse, which suggested that SEA did not require IL-12 to induce SPr and that both worked independently of IFN-gamma. Splenocytes from wild-type mice cultured with SEA and neutralizing anti-IL-12 monoclonal antibody (mAb) also showed SPr induction. However, anti-Ia mAb inhibited SEA induction of SPR: Thus, SPr is inducible on T cells. SEA induces SPr through interaction with T-cell receptor (TCR), independently of IL-12 and IFN-gamma. IL-12 induces SPr independently of TCR activation and IFN-gamma expression. SP and its receptor, which regulate IFN-gamma production, are probably part of the IL-12-Th1 circuit.

Seroprevalence of Schistosoma mansoni in Puerto Ricans with inflammatory bowel disease.

BACKGROUND: The etiology of Inflammatory Bowel Diseases, Crohn's disease (CD) and ulcerative colitis (UC), is unknown. These diseases have a higher incidence in industrialized countries and their pathogenesis involves an over-reaction of the immune system. A genetic factor is believed to predispose to the development of chronic inflammation in response to an unidentified stimulus. Exposure to infections in childhood may modulate future immune responses. Parasitosis, particularly Schistosomiasis, stimulate Th2 immune responses. It has been hypothesized that the absence of these parasitic infections, as seen in economically developed countries, favors a Th1 response that may result in the clinical appearance of Crohn's disease later in life. OBJECTIVE: To determine the prevalence of Schistosoma mansoni antibodies in Puerto Ricans with Inflammatory Bowel Disease and controls. METHODS: Serum from 92 Puerto Ricans with IBD and 106 controls was screened for S. mansoni adult microsomal antigens (MAMA) using the FAST:ELISA assay. Those positive were confirmed with an enzyme-linked immunoelectrotransfer blot test. RESULTS: Seven serum samples (3 UC and 4 controls) were positive for S. mansoni antibodies. There was no significant difference between groups in gender, municipality of origin or seroprevalence of Schistosomiasis. The control group was slightly older than the IBD group. CONCLUSIONS: Our study did not demonstrate an inverse relation between Schistosomiasis and IBD. However, the decreasing prevalence of Schistosomiasis in the general population of Puerto Rico may account for this result.

The somatostatin immunoregulatory circuit present at sites of chronic inflammation.

Somatostatin is part of an immunoregulatory circuit that helps limit interferon-gamma (IFNgamma) production at sites of chronic inflammation. In murine schistosomiasis. parasite eggs induce focal, chronic granulomatous inflammation in the liver and intestines. These granulomas produce somatostatin 1-14 and express somatostatin receptor subtype number 2 (SSTR2), which is the exclusive somatostatin receptor present in this inflammation. Granuloma and splenic macrophages as well as macrophage cell lines make somatostatin. There appears to be no other inflammatory cell source of the peptide. Various inflammatory mediators induce this expression, whereas substance P inhibits somatostatin production. Somatostatin can suppress IFN-gamma secretion from T cells via interaction with the SSTR2 receptor expressed on these cells. Other cells within the granuloma also display SSTR2. The effect of somatostatin on these other cell types remains unknown. The thymus of normal mice has a complete somatostatin regulatory circuit. The thymic epithelial and dendritic cells make somatostatin. Like the granulomas of murine schistosomiasis, the thymus expresses only SSTR2. Somatostatin likely has an important role in thymic T cell education and selection.

The Listening Partners program: an initiative toward feminist community psychology in action.

The Listening Partners intervention is described and analyzed as a synthesis of feminism and community psychology, within a developmental framework. Working from an empowerment perspective, this social action, peer group intervention supported a community of poor, rural, isolated, young, White mothers to gain a greater voice, claim the powers of their minds, and collaborate in developmental leadership--creating settings that promote their own development and that of their families, peers, and communities. High quality dialogue, individual and group narrative, and collaborative problem-solving were emphasized, in a feminist context affirming diversity, inclusiveness, strengths, social-contextual analyses, and social constructivist perspectives. The power of enacting a synergy of feminism and community psychology is highlighted.

Does the failure to acquire helminthic parasites predispose to Crohn's disease?

Two polarized patterns (Th1 and Th2) of cytokines regulate inflammatory responses. Each cytokine pattern inhibits production of the opposing pattern. Lymphocytes from inflamed intestine due to Crohn's disease secrete a Th1 pattern of cytokines. Crohn's disease is most prevalent in highly industrialized countries with temperate climates. It occurs rarely in tropical third world countries with poor sanitation. We propose that exposure to an environmental agent predisposes individuals to Crohn's disease. Parasitic worms (helminths) are common in tropical climates and in populations subject to crowding and poor sanitation. Children are most subject to helminthic colonization. Many helminths live within or migrate through the human gut where they interact with the mucosal immune system. The host mounts a mucosal response that includes Th2 cytokine production limiting helminthic colonization. Helminths and their eggs probably are the most potent stimulators of mucosal Th2 responses. The Th2 response provoked by parasitic worms can modulate immune reactions to unrelated parasitic, bacterial, and viral infections. Many people in developed countries now live in increasingly hygienic environments, avoiding exposure to helminths. Perhaps failure to acquire these parasites and experience mucosal Th2 conditioning predisposes to Crohn's disease, which is an overly active Th1 inflammation.

Complications and cost associated with parenteral nutrition delivered to hospitalized patients through either subclavian or peripherally-inserted central catheters.

BACKGROUND AND AIMS: Total parenteral nutrition (TPN) is typically delivered through catheters inserted into the superior vena cava (SVC) via a subclavian or internal jugular vein approach. A peripherally-inserted central venous catheter (PICC), utilizing a cephalic or basilic venous approach, may provide a safe alternative to the standard catheter approach and, because non-physician providers can insert the PICC, may introduce a potential cost-savings to health care institutions. We sought to determine if PICC lines are safer and more cost-effective than the standard central venous catheter approach for hospitalized patients who require TPN. METHODS: One hundred and two hospitalized patients (age range, 18-88 years) who required TPN were prospectively randomized to receive therapy via a centrally-inserted subclavian catheter (n=51) or a peripherally-inserted PICC line (n=51). The primary end-point was the development of a complication requiring catheter removal. Other end-points included catheter infection and thrombophlebitis. Cost associated with insertion and maintenance of each catheter was also studied. RESULTS: Complication-free delivery rate (without the need to remove or replace the catheter) was 67% for subclavian catheters and 46% for PICC lines (P<0.05). The overall infection rate was 4.9 per 1000 catheter days and was similar for each catheter type (P=0.68). PICC lines were associated with higher rates of clinically-evident thrombophlebitis (P<0.01), difficult insertion attempts (P<0.05), and malposition on insertion (P<0.05). No catheter complications resulted in significant long-term morbity or mortality. No significant difference was noted between the two catheter types in terms of aborted insertion attempts (P=0.18), dislodgement (P=0.12), or line occlusion (P=0.25). After standardizing costs for each hospital, the direct institutional costs for insertion and maintenance of PICC lines (US$22.32+/-2.74 per day) was greater than that for subclavian lines (US$16.20+/-2.96 per day;P<0.05). CONCLUSION: PICC catheters have higher thrombophlebitis rates and are more difficult to insert into certain patients when compared to the standard subclavian approach for central venous access in hospitalized patients who require TPN. Because of this, PICCs may be less cost-effective than currently believed because of the difficulty in inserting and maintaining the catheter.

IL-4 regulates VIP receptor subtype 2 mRNA (VPAC2) expression in T cells in murine schistosomiasis.

In murine schistosomiasis, granuloma T cells express VPAC2 mRNA, whereas there is none in splenocytes. This suggests that T cell VPAC2 mRNA is inducible. To address this issue, splenocytes from schistosome-infected mice were incubated with anti-CD3 to induce VPAC2 mRNA, which only appeared when cell cultures also contained anti-IL-4 mAb. Granuloma cells expressed VPAC2 mRNA. This natural expression decreased substantially when cells were cultured 3 days in vitro. However, granuloma cells cultured with anti-IL-4 mAb strongly expressed VPAC2 mRNA. IL-4 KO mice were examined to further address the importance of IL-4 in VPAC2 regulation. Splenocytes and dispersed granuloma cells from IL-4 KO animals had substantially more VPAC2 mRNA than those in wild-type controls. VPAC2 mRNA content decreased when cells were cultured with rIL-4. VPAC2 mRNA localized to CD4+ T cells. Th1 cell lines expressed VPAC2 mRNA much stronger than Th2 cells. Anti-IL-4 mAb increased VPAC2 mRNA expression in Th2 cells cultured in vitro. However, rIL-4 could not suppress VPAC2 mRNA expression in Th1 cells. Thus, VPAC2 is an inducible CD4+ T cell receptor, and IL-4 down-modulates VPAC2 mRNA expression in Th2 cells.