Longitudinal Assessment of Lipoprotein(a) Levels in Perinatally HIV-Infected Children and Adolescents.

HIV is an independent risk factor of cardiovascular disease (CVD); therefore, perinatally HIV-infected (PHIV) children potentially have a greater CVD risk at older age. Lipoprotein(a) (Lp(a)) is an established risk factor for CVD in the general population. To evaluate a potential increased CVD risk for PHIV children, we determined their lipid profiles including Lp(a). In the first substudy, we assessed the lipid profiles of 36 PHIV children visiting the outpatient clinic in Amsterdam between 2012 and 2020. In the second substudy, we enrolled 21 PHIV adolescents and 23 controls matched for age, sex and ethnic background on two occasions with a mean follow-up time of 4.6 years. We assessed trends of lipid profiles and their determinants, including patient and disease characteristics, using mixed models. In the first substudy, the majority of PHIV children were Black (92%) with a median age of 8.0y (5.7-10.8) at first assessment. Persistent elevated Lp(a) levels were present in 21/36 (58%) children (median: 374 mg/L (209-747); cut off = 300). In the second substudy, the median age of PHIV adolescents was 17.5y (15.5-20.7) and of matched controls 16.4y (15.8-19.5) at the second assessment. We found comparable lipid profiles between groups. In both studies, increases in LDL-cholesterol and total cholesterol were associated with higher Lp(a) levels. A majority of PHIV children and adolescents exhibited elevated Lp(a) levels, probably associated with ethnic background. Nonetheless, these elevated Lp(a) levels may additionally contribute to an increased CVD risk.

Elevated Lipoprotein(a) in Perinatally HIV-Infected Children Compared With Healthy Ethnicity-Matched Controls.

BACKGROUND: HIV-associated cardiovascular disease (CVD) risk in combination antiretroviral therapy (cART)-treated perinatally HIV-infected patients (PHIV+) remains unknown due to the young age of this population. Lipoprotein(a) (Lp(a)) has been established as an independent causal risk factor for CVD in the general population but has not been well established in the population of PHIV+. METHODS: We cross-sectionally compared lipid profiles, including nonfasting Lp(a), together with total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides between 35 cART-treated PHIV+ children aged 8-18 years and 37 controls who were matched for age, sex, ethnicity, and socioeconomic status. We explored associations between Lp(a) and disease- and treatment-related factors (inflammation, monocyte activation, and vascular), biomarkers, and neuroimaging outcomes using linear regression models. RESULTS: PHIV+ children had significantly higher levels of Lp(a) compared with controls (median, 43.6 [21.6-82.4] vs 21.8 [16.8-46.6] mg/dL; P = .033). Other lipid levels were comparable between groups. Additional assessment of apolipoprotein B, apolipoprotein CIII, apolipoprotein E, and APOE genotype revealed no significant differences. Higher Lp(a) levels were associated with higher plasma apoB levels and with lower monocyte chemoattractant protein-1 and TG levels in PHIV+ children. Lp(a) was not associated with HIV- or cART-related variables or with neuroimaging outcomes. CONCLUSIONS: cART-treated PHIV+ children appear to have higher levels of Lp(a) compared with ethnicity-matched controls, which may implicate higher CVD risk in this population. Future research should focus on the association between Lp(a) and (sub)clinical CVD measurements in cART-treated PHIV+ patients. DUTCH TRIAL REGISTER NUMBER: NRT4074.

Effects of the cholesteryl ester transfer protein inhibitor, TA-8995, on cholesterol efflux capacity and high-density lipoprotein particle subclasses.

BACKGROUND: TA-8995 is a potent inhibitor of cholesteryl ester transfer protein (CETP) with beneficial effects on lipids and lipoproteins. The effect of TA-8995 on cholesterol efflux capacity (CEC), a measure of high-density lipoprotein (HDL) function, and HDL subparticle distribution is largely unknown. OBJECTIVE: To assess the effect of the CETP inhibitor TA-8995 on ABCA1- and non-ABCA1-driven CEC and on HDL particle distribution. METHODS: Total, non-ABCA1-, and ABCA1-specific CEC from J774 cells and HDL subclass distribution assessed by two-dimensional gel electrophoresis were measured at baseline and after 12-week treatment in 187 mild-dyslipidemic patients randomized to placebo, 1 mg, 5 mg, 10 mg TA-8995, or 10 mg TA-8995 combined with 10 mg rosuvastatin (NCT01970215). RESULTS: Compared with placebo, total, non-ABCA1-, and ABCA1-specific CEC were increased dose dependently by up to 38%, 72%, and 28%, respectively, in patients randomized to 10 mg of TA-8995. PreBeta-1 HDL, the primary acceptor for ABCA1-driven cholesterol efflux, was increased by 36%. This increase in preBeta-1 HDL correlated significantly with the total and the ABCA1-driven CEC increase, whereas the high-density lipoprotein cholesterol (HDL-C) increase did not. CONCLUSION: TA-8995 dose dependently increased not only total and non-ABCA1-specific CEC but also ABCA1-specific CEC and preBeta-1 HDL particle levels. These findings suggest that TA-8995 not only increases HDL-C levels but also promotes functional properties of HDL particles. This CETP inhibitor-driven preBeta-1 HDL increase is an important predictor of both ABCA1 and total CEC increase, independent of HDL-C increase. Whether these changes in HDL particle composition and functionality have a beneficial effect on cardiovascular outcome requires formal testing in a cardiovascular outcome trial.

Cholesterol ester transfer protein inhibition by TA-8995 in patients with mild dyslipidaemia (TULIP): a randomised, double-blind, placebo-controlled phase 2 trial.

BACKGROUND: Dyslipidaemia remains a significant risk factor for cardiovascular disease and additional lipid-modifying treatments are warranted to further decrease the cardiovascular disease burden. We assessed the safety, tolerability and efficacy of a novel cholesterol esterase transfer protein (CETP) inhibitor TA-8995 in patients with mild dyslipidaemia. METHODS: In this randomised, double-blind, placebo-controlled, parallel-group phase 2 trial, we recruited patients (aged 18-75 years) from 17 sites (hospitals and independent clinical research organisations) in the Netherlands and Denmark with fasting LDL cholesterol levels between 2·5 mmol/L and 4·5 mmol/L, HDL cholesterol levels between 0·8 and 1·8 mmol/L and triglyceride levels below 4·5 mmol/L after washout of lipid-lowering treatments. Patients were randomly allocated (1:1) by a computer-generated randomisation schedule to receive one of the following nine treatments: a once a day dose of 1 mg, 2·5 mg, 5 mg, or 10 mg TA-8995 or matching placebo; 10 mg TA-8995 plus 20 mg atorvastatin; 10 mg TA-8995 plus 10 mg rosuvastatin or 20 mg atorvastatin or 10 mg rosuvastatin alone. We overencapsulated statins to achieve masking. The primary outcome was percentage change in LDL cholesterol and HDL cholesterol from baseline at week 12, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01970215. FINDINGS: Between Aug 15, 2013, and Jan 10, 2014, 364 patients were enrolled. At week 12, LDL cholesterol levels were reduced by 27·4% in patients assigned to the 1 mg dose, 32·7% in patients given the 2·5 mg dose, 45·3% in those given the 5 mg dose, and 45·3% in those given the 10 mg dose (p<0·0001). LDL cholesterol levels were reduced by 68·2% in patients given 10 mg TA-8995 plus atorvastatin, and by 63·3% in patients given rosuvastatin plus 10 mg TA-8995 (p<0·0001). A daily dose of 1 mg TA-8995 increased HDL cholesterol levels by 75·8%, 2·5 mg by 124·3%, 5 mg by 157·1%, and 10 mg dose by 179·0% (p<0·0001). In patients receiving 10 mg TA-8995 and 20 mg atorvastatin HDL cholesterol levels increased by 152·1% and in patients receiving 10 mg TA-8995 and 10 mg rosuvastatin by 157·5%. We recorded no serious adverse events or signs of liver or muscle toxic effects. INTERPRETATION: TA-8995, a novel CETP inhibitor, is well tolerated and has beneficial effects on lipids and apolipoproteins in patients with mild dyslipidaemia. A cardiovascular disease outcome trial is needed to translate these effects into a reduction of cardiovascular disease events. FUNDING: Dezima.

Adenosine triphosphate-binding cassette transporter genes up-regulation in untreated hepatocellular carcinoma is mediated by cellular microRNAs.

UNLABELLED: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are drug efflux pumps responsible for the multidrug resistance phenotype causing hepatocellular carcinoma (HCC) treatment failure. Here we studied the expression of 15 ABC transporters relevant for multidrug resistance in 19 paired HCC patient samples (16 untreated, 3 treated by chemotherapeutics). Twelve ABC transporters showed up-regulation in HCC compared with adjacent healthy liver. These include ABCA2, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC10, ABCC11, ABCC12, and ABCE1. The expression profile and function of some of these transporters have not been associated with HCC thus far. Because cellular microRNAs (miRNAs) are involved in posttranscriptional gene silencing, we hypothesized that regulation of ABC expression in HCC might be mediated by miRNAs. To study this, miRNAs were profiled and dysregulation of 90 miRNAs was shown in HCC compared with healthy liver, including up-regulation of 11 and down-regulation of 79. miRNA target sites in ABC genes were bioinformatically predicted and experimentally verified in vitro using luciferase reporter assays. In total, 13 cellular miRNAs were confirmed that target ABCA1, ABCC1, ABCC5, ABCC10, and ABCE1 genes and mediate changes in gene expression. Correlation analysis between ABC and miRNA expression in individual patients revealed an inverse relationship, providing an indication for miRNA regulation of ABC genes in HCC. CONCLUSION: Up-regulation of ABC transporters in HCC occurs prior to chemotherapeutic treatment and is associated with miRNA down-regulation. Up-regulation of five ABC genes appears to be mediated by 13 cellular miRNAs in HCC patient samples. miRNA-based gene therapy may be a novel and promising way to affect the ABC profile and overcome clinical multidrug resistance.

Genetically Modified Lactococcus lactis for Delivery of Human Interleukin-10 to Dendritic Cells.

Interleukin-10 (IL-10) plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs) to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L. lactis(IL-10)) on DC function in vitro. Monocyte-derived DC incubated with L. lactis(IL-10) induced effector Th-cells that markedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L. lactis(IL-10)-derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 during DC-Th-cell interaction and coculturing L. lactis(IL-10)-derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130 pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases.

Induction of antigen-specific tolerance by oral administration of Lactococcus lactis delivered immunodominant DQ8-restricted gliadin peptide in sensitized nonobese diabetic Abo Dq8 transgenic mice.

Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (LL) provides a novel therapeutic approach for the induction of tolerance. Celiac disease is associated with either HLA-DQ2- or HLA-DQ8-restricted responses to specific antigenic epitopes of gliadin, and may be treated by induction of Ag-specific tolerance. We investigated whether oral administration of LL-delivered DQ8-specific gliadin epitope induces Ag-specific tolerance. LL was engineered to secrete a deamidated DQ8 gliadin epitope (LL-eDQ8d) and the induction of Ag-specific tolerance was studied in NOD AB degrees DQ8 transgenic mice. Tolerance was assessed by delayed-type hypersensitivity reaction, cytokine measurements, eDQ8d-specific proliferation, and regulatory T cell analysis. Oral administration of LL-eDQ8d induced suppression of local and systemic DQ8-restricted T cell responses in NOD AB degrees DQ8 transgenic mice. Treatment resulted in an Ag-specific decrease of the proliferative capacity of inguinal lymph node (ILN) cells and lamina propria cells. Production of IL-10 and TGF-beta and a significant induction of Foxp3(+) regulatory T cells were associated with the eDQ8d-specific suppression induced by LL-eDQ8d. These data provide support for the development of effective therapeutic approaches for gluten-sensitive disorders using orally administered Ag-secreting LL. Such treatments may be effective even in the setting of established hypersensitivity.

Alterations in lipoprotein homeostasis during human experimental endotoxemia and clinical sepsis.

Cell wall constituents of bacteria are potent endotoxins initiating inflammatory responses which may cause dramatic changes in lipid metabolism during the acute phase response. In this study, the sequential changes in lipoprotein composition and lipid transfer and binding proteins during clinical sepsis and during low-dose experimental endotoxemia were followed. In addition, the effect on (phospho)lipid homeostasis by administration of reconstituted HDL (rHDL) prior to low-dose LPS administration was investigated. Changes in (apo)lipoprotein concentrations typical of the acute phase response were observed during clinical sepsis and experimental endotoxemia with and without the rHDL intervention. During clinical sepsis negative correlations between the acute phase marker C-reactive protein (CRP) and lecithin:cholesterol acyltransferase (LCAT) and cholesterylester transfer protein (CETP) activities were seen, whereas positive correlations between plasma phospholipid transfer protein (PLTP) activity and acute phase markers such as CRP and LPS binding protein were observed. Plasma lipid changes upon rHDL/LPS infusion were comparable with the control group (low-dose LPS only). PLTP activity decreased upon LPS infusion and transiently increased during rHDL infusion, whereas LCAT activity slightly decreased upon both LPS infusion and LPS/rHDL infusion. However, long-lasting increases of circulating HDL cholesterol, apo A-I and a high initial processing of both phosphatidylcholine (PC) and lyso-PC, were indicative for extensive rHDL and LDL remodelling. Both sepsis and experimental endotoxemia lead to a disbalance of lipid homeostasis. Depending on the magnitude of the inflammatory stimulus, LCAT and PLTP activities reacted in divergent ways. rHDL infusion did not prevent the lipid alterations seen during the acute phase response. However profound changes in both HDL and LDL phospholipid composition occurred upon rHDL infusion. This may be explained, at least in part, by the fact that PLTP as a positive acute phase protein, can accelerate the alterations in (phospho)lipid homeostasis thereby playing a role in the attenuation of the acute phase response.

Bone morphogenetic protein signaling suppresses tumorigenesis at gastric epithelial transition zones in mice.

Bone morphogenetic protein (BMP) signaling is known to suppress oncogenesis in the small and large intestine of mice and humans. We examined the role of Bmpr1a signaling in the stomach. On conditional inactivation of Bmpr1a, mice developed neoplastic lesions specifically in the squamocolumnar and gastrointestinal transition zones. We hypothesized that the regulation of epithelial cell fate may be less well defined in these junctional zones than in the adjacent epithelium and found that the mucosa at the squamocolumnar junction in mice shows a lack of differentiated fundic gland cell types and that foveolar cells at the gastrointestinal junctional zone lack expression of the foveolar cell marker Muc5ac. Precursor cell proliferation in both transition zones was higher than in the surrounding epithelium. Our data show that BMP signaling through Bmpr1a suppresses tumorigenesis at gastric epithelial junctional zones that are distinct from the adjacent gastric epithelium in both cellular differentiation and proliferation.

Induction of ovalbumin-specific tolerance by oral administration of Lactococcus lactis secreting ovalbumin.

BACKGROUND AND AIMS: Obtaining antigen-specific immune suppression is an important goal in developing treatments of autoimmune, inflammatory, and allergic gastrointestinal diseases. Oral tolerance is a powerful means for inducing tolerance to a particular antigen, but implementing this strategy in humans has been difficult. Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (L lactis) provides a novel therapeutic approach for inducing tolerance. METHODS: We engineered the food grade bacterium L lactis to secrete ovalbumin (OVA) and evaluated its ability to induce OVA-specific tolerance in OVA T-cell receptor (TCR) transgenic mice (DO11.10). Tolerance induction was assessed by analysis of delayed-type hypersensitivity responses, measurement of cytokines and OVA-specific proliferation, phenotypic analysis, and adoptive transfer experiments. RESULTS: Intragastric administration of OVA-secreting L lactis led to active delivery of OVA at the mucosa and suppression of local and systemic OVA-specific T-cell responses in DO11.10 mice. This suppression was mediated by induction of CD4(+)CD25(-) regulatory T cells that function through a transforming growth factor beta-dependent mechanism. Restimulation of splenocytes and gut-associated lymph node tissue from these mice resulted in a significant OVA-specific decrease in interferon gamma and a significant increase in interleukin-10 production. Furthermore, Foxp3 and CTLA-4 were significantly up-regulated in the CD4(+)CD25(-) population. CONCLUSIONS: Mucosal antigen delivery by oral administration of genetically engineered L lactis leads to antigen-specific tolerance. This approach can be used to develop effective therapeutics for systemic and intestinal immune-mediated inflammatory diseases.

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

Infliximab use in children and adolescents with inflammatory bowel disease.

Infliximab is a chimeric monoclonal antibody (75% human, 25% murine) against tumor necrosis factor-alpha, a cytokine with a central role in the pathogenesis of inflammatory bowel disease. Large randomized controlled trials have shown the efficacy and safety of infliximab for the induction and maintenance of remission in adult patients with active Crohn disease (CD). In children and adolescents, mostly small, nonrandomized, non-placebo-controlled studies have supported the notion that infliximab is a potent drug in a population that does not respond to standard therapies. The safety of infliximab is of major concern, and the most frequent severe adverse events are related to severe infections and reactivation of tuberculosis. Non-life-threatening infusion reactions occur rather frequently and seem to be related to the formation of antibodies. The indications for infliximab treatment are therapy-resistant luminal CD (no efficacy or insufficient efficacy of conventional treatment) and therapy-resistant fistulas. An efficient remission induction strategy consists of 3 initial infliximab infusions at 0, 2, and 6 weeks in a dosage of 5 mg/kg to sustain remission. Patients needing maintenance therapy are subsequently treated with an infliximab infusion every 8 weeks. There are indications that the early stages of CD may be more susceptible to immunomodulation, and the natural history of CD may be altered by the introduction of infliximab early in the disease process instead of waiting until conventional therapy has failed. Major points of discussion are whether infliximab maintenance treatment should be episodic (on demand) or scheduled and when infliximab therapy can be discontinued.

An improved protocol for generation of immuno-potent dendritic cells through direct electroporation of CD14+ monocytes.

In this study we demonstrate a novel protocol showing that electroporation of CD14+ monocytes directly isolated from blood with green fluorescent protein (GFP) RNA results in a 3-fold higher yield of antigen presenting dendritic cells (DCs) when compared to conventional methods employing immature DCs for electroporation. We further show a stable electroporation efficacy resulting in 60% of GFP positive cells. Expression of co-stimulatory molecules and maturation markers such as CD80, CD86, CD83 as well of the chemokine receptor 7 (CCR7) was found in 90% of the mature DCs. Importantly, production of IL-12p70 was 10 times higher in cells electroporated at the monocyte stage compared to cells electroporated at the immature DC stage. Stimulation of autologous naïve lymphocytes by DCs electroporated at monocytes stage elicited proliferation of CD8+ T-cell with 7-fold increase in IFN-gamma release. Blocking of the MHC-Class I molecules significantly inhibited the IFN-gamma release, indicating that antigen presentation was MHC-Class I mediated. In summary, electroporation of CD14+ monocytes with RNA results in a high yield of antigen presenting DCs with high immuno-stimulatory capacity and antigen presentation on MHC-Class I molecules. This improved method may represent an attractive approach for RNA-based DC immunotherapy.

Comparative analysis of colonic gene expression of three experimental colitis models mimicking inflammatory bowel disease.

BACKGROUND: Mouse models of inflammatory bowel diseases (IBD) are used to unravel the pathophysiology of IBD and to study new treatment modalities, but their relationship to Crohn's disease (CD) or ulcerative colitis (UC) is speculative. METHODS: Using Agilent mouse TOX oligonucleotide microarrays, we analyzed colonic gene expression profiles in three widely used models of experimental colitis. In 2 of the models (TNBS and DSS-induced colitis), exogenous agents induce the colitis. In the third model the colitis is induced after transfer of a T-cell population (CD4(+)CD45RB(high) T cells) that lacks regulatory cells into an immunodeficient host. RESULTS: Compared with control mice, in DSS, TNBS, and the CD45RB transfer colitis mice, 387, 21, and 582 genes were more than 2-fold upregulated in the intestinal mucosa. Analyses of exclusively shared gene expression profiles between the different models revealed that DSS/transfer colitis share 69 concordantly upregulated genes, DSS/TNBS 6, and TNBS/transfer colitis 1. Seven genes were upregulated in all three models. The CD45RB transfer model expression profile included the most genes that are known to be upregulated in IBD. Of 32 genes that are known to change transcriptional activity in IBD (TNF, IFN-gamma, Ltbeta, IL-6, IL-16, IL-18R1, IL-22, CCR2, 7, CCL2, 3, 4, 5, 7, 11, 17, 20, CXCR3, CXCL1, 5, 10, Mmp3, 7,9, 14, Timp1, Reg3gamma, and Pap, S-100a8, S-100a9, Abcb1, and Ptgs2), 2/32 are upregulated in TNBS, 15/32 are upregulated or downregulated in DSS and 30/32 are upregulated or downregulated in the CD45RB transfer colitis. CONCLUSION: The pattern of gene expression in the CD45RB transfer model most closely reflects altered gene expression in IBD.

Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomodulatory molecule.

BACKGROUND: Filamentous haemagglutinin (FHA) of Bordetella pertussis subverts host immune responses by inhibiting interleukin (IL)12 and enhancing IL10 production by macrophages and dendritic cells, and promoting the induction of regulatory T cells. HYPOTHESIS: Injection of FHA would ameliorate disease in a T cell-dependent model of colitis via the induction of anti-inflammatory cytokines and regulatory T cells. METHODS: Colitis was induced by injection of CD4CD45RB(high) naive T cells into severe combined immunodeficient (SCID) mice. Mice were treated with four subcutaneous injections of FHA or buffer alone. RESULTS: Parenteral injection of FHA stimulated IL10 and/or transforming growth factor beta production in local and mesenteric lymph nodes and Peyer's patches of mice 2-6 h after administration. Compared with phosphate-buffered saline-treated mice, FHA-treated SCID mice had significantly (p<0.01) less weight loss, lower colon weight, less colon shrinkage and reduced inflammatory lesions. The therapeutic effect of FHA was associated with enhanced IL10 and reduced type 1 and type 2 T helper cytokine production by spleen cells. Finally, FHA also attenuated the symptoms of colitis in SCID mice transferred with CD4CD45RB(high) T cells from IL10-deficient mice. CONCLUSIONS: Our finding shows that FHA suppresses type 1 T helper and pro-inflammatory cytokines, and ameliorates disease activity in a chronic T cell-dependent model of colitis, an effect that was not dependent on IL10 production by T cells, but was associated with induction of anti-inflammatory cytokines in vivo. Having already been used as a pertussis vaccine component in children, FHA is a promising candidate for clinical testing in patients with Crohn's disease.

Prediction of antitumour necrosis factor clinical efficacy by real-time visualisation of apoptosis in patients with Crohn's disease.

BACKGROUND: The human anti-tumour necrosis factor (TNF) antibody infliximab binds to the membrane TNF and subsequently induces apoptosis of activated lamina propria T lymphocytes in patients with Crohn's disease in vitro. AIM: To test whether the ability of rapid anti-TNF-induced apoptosis in the gut predicts the efficacy of anti-TNF treatment in inflammatory bowel disease. METHODS: (99m)Technetium-annexin V single-photon emission computer tomography (SPECT) was performed in 2 models of murine experimental colitis and in 14 patients with active Crohn's disease as assessed by the Crohns Disease Activity Index (CDAI) to study the effect of anti-TNF treatment on apoptosis in the intestine during active colitis. Disease activity was evaluated 2 weeks after infliximab infusion using the CDAI (definition response: drop of >100 points). RESULTS: Colonic uptake of (99m)Tc-annexin V significantly increased in 2,4,6-trinitrobenzene sulphonate-induced colitis as well as in transfer colitis on administration of anti-TNF antibodies compared with a control antibody as determined with dedicated animal pinhole SPECT. In addition, uptake of (99m)Tc-annexin V significantly increased in patients with active Crohn's disease responding to infliximab treatment. Colonic (99m)Tc-annexin V uptake ratio (mean (SEM)) increased from 0.24 (0.03) to 0.41(0.07) (p<0.01), 24 h after infliximab infusion (5 mg/kg). A mean increase of 98.7% in colonic uptake of (99m)Tc-annexin V could be detected in 10 of the 14 responding patients (CDAI >100 points at week 2) compared with 15.2% in non-responding patients (p = 0.03). Analysis of the mucosal biopsy specimens identified lamina propria T cells as target cells undergoing apoptosis. CONCLUSIONS: These in vivo observations support the notion that colonic uptake of (99m)Tc-annexin V correlates with clinical benefit of anti-TNF treatment and might be predictive of therapeutic success.

Treatment and prevention of Staphylococcus epidermidis experimental biomaterial-associated infection by bactericidal peptide 2.

Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices and are predominantly caused by staphylococci, especially Staphylococcus epidermidis. We investigated the in vitro microbicidal activity of the synthetic antimicrobial peptide bactericidal peptide 2 (BP2) and its efficacy in a murine model of S. epidermidis BAI. BP2 showed potent microbicidal activity at micromolar concentrations against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The staphylocidal activity of BP2 was not affected by physiological salt concentrations and was only slightly affected by the presence of human plasma. In the BAI model, injection of BP2 (5 mg/kg of body weight) 1 h after challenge with S. epidermidis resulted in an 80% reduction in the number of culture-positive implants and a 100-fold reduction in survival of S. epidermidis in peri-implant tissue at 24 h postchallenge. When BP2 was injected along implants 3 h prior to bacterial challenge, the median numbers of CFU cultured from biomaterial implants and peri-implant tissue were reduced by 85% and 90%, respectively. In conclusion, BP2 has potent, broad-spectrum in vitro microbicidal activity and showed potent in vivo activity in a murine model of S. epidermidis biomaterial-associated infection.

A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease.

BACKGROUND & AIMS: The use of living, genetically modified bacteria is an effective approach for topical delivery of immunomodulatory proteins. This strategy circumvents systemic side effects and allows long-term treatment of chronic diseases. However, treatment of patients with a living, genetically modified bacterium raises questions about the safety for human subjects per se and the biologic containment of the transgene. METHODS: We treated Crohn's disease patients with genetically modified Lactococcus lactis (LL-Thy12) in which the thymidylate synthase gene was replaced with a synthetic sequence encoding mature human interleukin-10. Ten patients were included in a placebo-uncontrolled trial. Patients were assessed daily for the presence of potential adverse effects by direct questioning and assessment of disease activity. We evaluated the presence and kinetics of LL-Thy12 release in the stool of patients by conventional culturing and quantitative polymerase chain reaction of LL-Thy12 gene sequences. RESULTS: Treatment with LL-Thy12 was safe because only minor adverse events were present, and a decrease in disease activity was observed. Moreover, fecally recovered LL-Thy12 bacteria were dependent on thymidine for growth and interleukin-10 production, indicating that the containment strategy was effective. CONCLUSIONS: Here we show that the use of genetically modified bacteria for mucosal delivery of proteins is a feasible strategy in human beings. This novel strategy avoids systemic side effects and is biologically contained; therefore it is suitable as maintenance treatment for chronic intestinal disease.

Intravenous pamidronate in combination with calcium and vitamin D: highly effective in the treatment of low bone mineral density in inflammatory bowel disease.

OBJECTIVE: Decreased bone mineral density (BMD) is common in inflammatory bowel disease (IBD) and an increased risk of fractures has been reported. Guidelines state bisphosphonate treatment for IBD patients with decreased BMD, but orally available bisphosphonates have been associated with gastrointestinal side effects and the absorption is poor. We investigated whether intravenous pamidronate is a safe and effective treatment. MATERIAL AND METHODS: Forty-nine IBD patients with decreased BMD as assessed by DEXA scan were treated with calcium 1000 mg and vitamin D 400 IU daily. In addition, 30 mg of pamidronate was administered intravenously every 3 months. DEXA scanning was performed prior to treatment, after 6 months and after 1 year. RESULTS: Of 49 IBD patients, 40 were osteoporotic and 9 were osteopenic. Twenty-six patients were female (mean age 40.8) and 23 were male (mean age 43.3). Treatment was discontinued in one patient because of fever after infusion. Otherwise, tolerability was excellent, and no adverse events were documented. A mean 0.51 increase of lumbar spine (L1-L4) T-scores was observed (CI 95% 0.35-0.67; p<0.0004). The effect of treatment on left femoral neck T-scores was less pronounced: 0.39 (CI 95% 0.24-0.53; p<0.0004). CONCLUSIONS: We conclude that intravenous pamidronate in combination with calcium and vitamin D is a well tolerated strategy for treating Crohn's disease associated osteopenia and osteoporosis. Although uncontrolled, treatment results in a significant increase of BMD in the lumbar spine.

Pharmacokinetics of 6-thioguanine in patients with inflammatory bowel disease.

6-Thioguanine (6-TG) seems to be an attractive alternative in both AZA- and 6-MP-intolerant and -resistant IBD populations. However, little is known of 6-TG pharmacokinetics, metabolite levels, and their correlation with drug efficacy and toxicity in IBD patients. This study reports the 6-TG pharmacokinetics in a population of IBD patients and the predictive value of metabolite concentrations. Red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) concentrations were measured in 28 IBD patients at t = 1, 2, 4, and 8 weeks after starting 6-TG, 20 mg once daily. Outcome measures included mean 6-TGN concentrations (+/-95% confidence interval [CI95%]) and their associations with TPMT genotype, 6-TG dose, and hematological, hepatic, pancreatic, and efficacy parameters during the 8 week period. Steady-state 6-TGN concentrations were reached after 4 weeks, indicating a half-life of approximately 5 days, and measured 856 (CI95% 715-997) pmol/8 x 10 RBCs. Large interpatient variability occurred at all time-points. No correlation was found between steady-state 6-TGN concentrations and drug dose per kilogram body weight. No significant differences in 6-TGN concentrations were found between patients with adverse events and patients without any event. Also, mean 6-TGN concentrations did not differ in patients with active disease versus patients in remission. In IBD patients on 6-TG treatment, large interindividual differences in metabolite concentrations occur. In our population, we could not demonstrate a clear relationship between 6-TGN concentrations on one hand and toxicity and efficacy on the other, as exist in AZA- and 6-MP-treated patients.