Preventive Anti-CD2 Treatment does not Impair Parasite Control in a Murine Toxoplasmosis Model.

Targeting human CD2 with the monoclonal antibody (mAb) CB.219 reduces intestinal inflammation in a colitis model where T cells carry human CD2. Here, we asked whether this mAb has adverse effects on infection control. Mice expressing human CD2 on T cells (huCD2tg) were orally infected with Toxoplasma (T.) gondii and treated with the human CD2-specific mAb CB.219 in a preventive setting. The intestinal T. gondii loads in CB.219 treated mice did not differ from the control group. Histologically, huCD2tg mice showed moderate ileal inflammation that did not change with CB.219 treatment. In the ileum, CB.219 treatment reduced the protein levels of interferon-γ, transforming growth factor ß and interleukin-6, whereas interleukin-18 mRNA was slightly increased. The infiltration of neutrophils, macrophages, and T cells into the ileum was unaffected by CB.219 treatment. However, CB.219 treatment decreased the numbers of forkhead box P3(+) regulatory T cells (Treg) in ileum and liver of huCD2tg mice. This was confirmed in vitro using human peripheral blood mononuclear cells. Taken together, targeting CD2(+) T cells by the human CD2 mAb CB.219 does not prevent beneficial immune reactions necessary for pathogen control. Further experiments will address gut specificity, underlying mechanisms, and general applicability of CB.219 treatment.

Mesenteric fat - control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro.

Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 ameliorates acute DSS colitis.

In experimental models of and humans with intestinal inflammation, increased levels of the matrix-degrading gelatinases MMP-2 and -9 in inflamed tissues can be detected. The synthetic collagen analogue (Gly-Pro-Hyp)10, (GPO)10, has been identified as a relevant binding structure for proMMP-2/-9 and promotes enzymatic activity of proMMP-2. Since targeted MMP strategies might offer promising anti-inflammatory treatment options, we for the first time studied in vivo actions exerted by (GPO)10 applying an acute dextrane sulfate sodium (DSS) induced colitis model. Seven-day intraperitoneal (GPO)10 treatment ameliorated clinical symptoms and histopathological colonic changes as compared to placebo controls with severe colitis. (GPO)10-treated mice displayed a diminished influx of neutrophils, and T- and B-lymphocytes into their colonic mucosa whereas numbers of regulatory T-cells and regenerative cells were higher as compared to placebo controls. Furthermore, IL-6 secretion was down-regulated in ex vivo colonic biopsies derived from (GPO)10-treated mice whereas higher concentrations of the anti-inflammatory cytokine IL-10 in extra-intestinal compartments such as MLN and spleen could be detected. Strikingly, influx of inflammatory cells into lungs was abolished following (GPO)10 application. We therefore propose (GPO)10 as a promising effective and safe treatment option of intestinal and extra-intestinal inflammatory conditions in humans.

Histone deacetylases: novel targets for prevention of colitis-associated cancer in mice.

OBJECTIVE: Inhibition of histone deacetylases, well known for its antiproliferative efficacy in vivo, was recently shown to ameliorate inflammation in experimental colitis. Since inflammatory bowel disease is associated with an increased risk of developing colon cancer, here the combined anti-inflammatory and proapoptotic efficacy of histone deacetylase inhibitors was studied in mouse models. METHODS: The novel histone deacetylase inhibitor ITF2357 was compared with suberoylanilide hydroxamic acid in models of experimental colitis. Effects on tumour growth were studied after treatment of mice with azoxymethane and dextran sulphate sodium, and in interleukin 10 (IL10) knockout mice, respectively. Possible underlying mechanisms involving apoptosis and nuclear factor (NF)-kappaB activation were addressed by flow cytometry and western blot. RESULTS: In dextran sulphate sodium- and trinitrobenzene sulphonic acid-induced colitis, treatment with ITF2357 was superior to suberoylanilide hydroxamic acid as shown by macroscopic and histological amelioration of inflammation, by reduced production of interferon gamma (IFN gamma) and by increased production of IL10. In both models of inflammation-mediated tumourigenesis, inhibition of histone deacetylases resulted in a significant suppression of tumour growth in terms of size and number, along with reduced signs of inflammation. As for potential mechanisms of ITF2357 action, increased acetylation of histone 3, reduced production of IFN gamma and enhanced apoptosis in lamina propria mononuclear cells were found to accompany a histone deacetylase-dependent activation of NF-kappaB. CONCLUSIONS: These results indicate that inhibition of histone deactylases can attenuate inflammation-mediated tumour growth, which is paralled by an inhibition of NF-kappaB. Thus histone deacetylase inhibitors provide a promising strategy that combines anti-inflammatory and proapoptotic modes of action.

Targeting of a B7-1 (CD80) immunoglobulin G fusion protein to acute myeloid leukemia blasts increases their costimulatory activity for autologous remission T cells.

Transfection of tumor cells with the gene encoding the costimulatory molecule B7-1 (CD80), the ligand for CD28 and cytotoxic T lymphocye antigen-4 on T cells, has been shown to result in potent T-cell-mediated antitumor immunity. As an alternative approach, this study analyzed the costimulatory capacity of a human B7-1 immunoglobulin G (IgG) fusion protein targeted to the cell membrane of human acute myeloid leukemia (AML) blasts. Flow cytometric analysis revealed a low constitutive expression of B7-1 on human AML blasts (on average, 3.0 +/- 4.3%; n = 50). In contrast, the expression of B7-2 (CD86) was highly heterogeneous and higher in AML blasts of French-American-British classification types M4 and M5 (P <.0001). The B7-1 IgG fusion protein used in this study efficiently costimulated the proliferation of resting and preactivated T cells when immobilized on plastic. After preincubation with B7-1 IgG, specific binding of the fusion protein to the high-affinity Fcgammareceptor I (CD64) on leukemic cells was demonstrated and was found to increase the proliferation of both allogeneic and autologous T cells in costimulation experiments. Furthermore, targeting of B7-1 IgG to the tumor membrane resulted in increased proliferation of autologous remission T cells and had the potential to generate an enhanced redirected cytotoxic T-cell response against autologous AML blasts. In summary, the targeting of B7-1 IgG fusion protein described in this study represents a strategy alternative to gene therapy to restore the expression of the costimulatory molecule B7-1 on human AML blasts, thereby enhancing their immunogenicity for autologous T cells. This new approach may have implications for T-cell-mediated immunotherapy in AML.

Differential effects of a stem cell factor-immunoglobulin fusion protein on malignant and normal hematopoietic cells.

We genetically connected the extracellular domain of human stem cell factor to the Fc-portion of human IgG1. The chimeric recombinant stem cell factor IgG1 fusion protein (rSCF-IgG1) had an apparent approximately Mr 190,000 and consisted of three identical covalently linked subunits. It specifically bound to c-kit and the high affinity Fc gamma receptor, respectively. Liquid phase rSCF-IgG1 was, on a molar basis, about eight times more potent than native human rSCF in stimulating the proliferation of c-kit-positive leukemic cell lines and of nonmalignant CD34-positive hematopoietic progenitor cells. Although the effective dose conferring half maximum of [methyl-3H]thymidine uptake by liquid phase and solid phase-bound rSCF-IgG1 were comparable, the plateau level of [methyl-3H]thymidine uptake by malignant cells was decreased by the latter, whereas proliferation of nonmalignant progenitor cells was supported. Liquid phase rSCF-IgG1 had a 2-fold increased potential to maintain primitive nonmalignant progenitor cells in stroma-free long-term culture compared with rSCF. Liquid phase rSCF-IgG1 caused enhanced and prolonged receptor phosphorylation and a more rapid down modulation of c-kit. Our data support the concept that solid phase-attachment of rSCF-IgG1 is sufficient for alteration of biological function and that rSCF-IgG1 partially blocks SCF-stimulated malignant cell growth while supporting normal progenitor cells.

[Histological findings of the small intestinal mucosa in rotavirus infections in infants and young children]. / Histologische Befunde der Dünndarmschleimhaut bei Rotavirusinfektionen im Säuglings- und Kleinkindalter.

In a prospective study we performed small intestinal mucosal biopsies in 40 children with acute rotavirus diarrhoea. Biopsies were taken from these children between the 2nd to 10th day of acute phase. The children were at an age of less than 18 month. 95% of the patients had normal histological findings of the small intestinal mucosa. Only 2 children (5%) had well defined injuries of intestinal mucosa. Correlations were not found between clinical findings, morphological results and therapy. In one child with gastroenteritis and meningitis rotaviruses were found in cerebrospinal fluid. In 31.25% of all children rotavirus infection was acquired by a nosocomial infection. The treatment of rotavirus infection is symptomatic. Usually intestinal mucosal biopsy as a routine diagnostic method isn't recommended and it should be used only in intractable courses.