Local Immune Changes in Early Stages of Inflammation and Carcinogenesis Correlate with the Collagen Scaffold Changes of the Colon Mucosa.

Continuous activation of the immune system inside a tissue can lead to remodelling of the tissue structure and creation of a specific microenvironment, such as during the tumour development. Chronic inflammation is a central player in stimulating changes that alter the tissue stroma and can lead to fibrotic evolution. In the colon mucosa, regulatory mechanisms, including TGF-ß1, avoid damaging inflammation in front of the continuous challenge by the intestinal microbiome. Inducing either DSS colitis or AOM colorectal carcinogenesis in AVN-Wistar rats, we evaluated at one month after the end of each treatment whether immunological changes and remodelling of the collagen scaffold were already in development. At this time point, we found in both models a general downregulation of pro-inflammatory cytokines and even of TGF-ß1, but not of IL-6. Moreover, we demonstrated by multi-photon microscopy the simultaneously presence of pro-fibrotic remodelling of the collagen scaffold, with measurable changes in comparison to the control mucosa. The scaffold was significantly modified depending on the type of induced stimulation. These results suggest that at one month after the end of the DSS or AOM inductions, a smouldering inflammation is present in both induced conditions, since the pro-inflammatory cytokines still exceed, in proportion, the local homeostatic regulation of which TGF-ß1 is a part (inflammatory threshold). Such an inflammation appears sufficient to sustain remodelling of the collagen scaffold that may be taken as a possible pathological marker for revealing pre-neoplastic inflammation.

Immune activation by microbiome shapes the colon mucosa: Comparison between healthy rat mucosa under conventional and germ-free conditions.

Germ-free animals (GF) are those without a microbiome since birth. This particular biological model has become one of special interest with the growing evidence of importance of the microbiome in the life, development, adaptation, and immunity of humans and animals in the environments in which they live. Anatomical differences observed in GF compared with conventionally-reared animals (CV) has given rise to the question of the influence of commensal microflora on the development of structure and function (even immunological) of the bowel. Only recently, thanks to achievements in microscopy and associated methods, structural differences can be better evaluated and put in perspective with the immunological characteristics of GF vs. CV animals. This study, using a GF rat model, describes for the first time the possible influence that the presence of commensal microflora, continuously stimulating mucosal immunity, has on the collagen scaffold organization of the colon mucosa. Significant differences were found between CV and GF mucosa structure with higher complexity in the CV rats associated to a more activated immune environment. The immunological data suggest that, in response to the presence of a microbiome, an effective homeostatic regulation in developed by the CV rats in healthy conditions to avoid inflammation and maintain cytokine levels near the spontaneous production found in the GF animals. The results indicated that collagen scaffold adapted to the immune microenvironment; therefore, it is apparent that the microbiome was able to condition the structure of the colon mucosa.

Crucial Role of Microbiota in Experimental Psoriasis Revealed by a Gnotobiotic Mouse Model.

Psoriatic patients have altered microbiota, both in the intestine and on the skin. It is not clear, however, whether this is a cause or consequence of the disease. In this study, using an experimental mouse model of psoriasis induced by imiquimod (IMQ), we show that oral treatment with a broad spectrum of antibiotics (MIX) or metronidazole (MET) alone mitigates the severity of skin inflammation through downregulation of Th17 immune response in conventional mice. Since some antibiotics, including MET, can influence immune system reactivity, we also evaluated the effect of MIX in the same model under germ-free (GF) conditions. GF mice treated with MET did not show milder signs of imiquimod-induced skin inflammation (IISI) which supports the conclusion that the therapeutic effect is mediated by changes in microbiota composition. Moreover, compared to controls, mice treated with MIX had a significantly higher abundance of the genus Lactobacillus in the intestine and on the skin. Mice treated with MET had a significantly higher abundance of the genera Bifidobacterium and Enterococcus both on the skin and in the intestine and of Parabacteroides distasonis in the intestine. Additionally, GF mice and mice monocolonized with either Lactobacillus plantarum or segmented filamentous bacteria (SFB) were more resistant to IISI than conventional mice. Interestingly, compared to GF mice, IMQ induced a higher degree of systemic Th17 activation in mice monocolonized with SFB but not with L. plantarum. The present findings provide evidence that intestinal and skin microbiota directly regulates IISI and emphasizes the importance of microbiota in the pathogenesis of psoriasis.

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis.

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMß2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b- cells. The nonhemolytic AC+ Hly- bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly- mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b- cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

Intestinal Microbiota Promotes Psoriasis-Like Skin Inflammation by Enhancing Th17 Response.

Psoriasis is a chronic inflammatory skin disease in which Th17 cells play a crucial role. Since indigenous gut microbiota influences the development and reactivity of immune cells, we analyzed the link among microbiota, T cells and the formation of psoriatic lesions in the imiquimod-induced murine model of psoriasis. To explore the role of microbiota, we induced skin inflammation in germ-free (GF), broad-spectrum antibiotic (ATB)-treated or conventional (CV) BALB/c and C57BL/6 mice. We found that both mice reared in GF conditions for several generations and CV mice treated with ATB were more resistant to imiquimod-induced skin inflammation than CV mice. The ATB treatment dramatically changed the diversity of gut bacteria, which remained stable after subsequent imiquimod application; ATB treatment resulted in a substantial increase in the order Lactobacillales and a significant decrease in Coriobacteriales and Clostridiales. Moreover, as compared to CV mice, imiquimod induced a lower degree of local and systemic Th17 activation in both GF and ATB-treated mice. These findings suggest that gut microbiota control imiquimod-induced skin inflammation by altering the T cell response.

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms.

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.

The structure-dependent toxicity, pharmacokinetics and anti-tumour activity of HPMA copolymer conjugates in the treatment of solid tumours and leukaemia.

Polymer drug carriers that are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used in the development and synthesis of high-molecular-weight (HMW) drug delivery systems for cancer therapy. In this study, we compared linear (Mw ~27kDa, Rh ~4nm) and non-degradable star (Mw ~250kDa, Rh ~13nm) HPMA copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazone bond. We determined the in vitro and in vivo toxicity of both conjugates and their maximum tolerated dose (MTD). We also compared their anti-tumour activity in mouse B-cell leukaemia (BCL1) and a mouse T-cell lymphoma (EL4) model. We found that MTD was higher for the linear conjugate (85mgDOX/kg) and lower for the star conjugate (22.5mgDOX/kg). An evaluation of the intestinal barrier integrity using FITC-dextran as a gut permeability tracer proved that no pathology was caused by the MTD of either conjugate. However, free DOX showed some damage to the gut barrier. The therapy of BCL1 leukaemia by both of the polymeric conjugates using the MTD or its fraction (i.e., equitoxic dosage) showed better results in the case of the star conjugate. On the other hand, treatment of EL4 lymphoma seemed to be more efficient when the linear conjugate was used. We suppose that the anti-cancer treatment of solid tumours and leukaemias requires different types of drug conjugates. We hypothesise that the most suitable HPMA copolymer-DOX conjugate for the treatment of solid tumours should have an HMW structure with increased Rh that would be stable for three to four days after the conjugate administration and then rapidly disintegrate in the short polymer chains, which are excretable from the body by glomerular filtration. On the other hand, the treatment of leukaemia requires a drug conjugate with a long circulation half-life. This would provide an active drug, whilst slowly degrading to excretable fragments.

[IgA Nephropathy. Facts, uncertainties, and potential causal therapy approaches]. / IgA nefropatie--jistoty, pochybnosti a výhledy kauzální lécby.

IgA nephropathy is currently the most frequently investigated glomerulonephritis. The disease is defined by the presence of dominant or co-dominant deposits of IgA1 in the glomerular mesangium. Circulating immune complexes are most likely the source of the deposited IgA1. However, it is also possible that the aggregates of structurally altered IgA1 or enhanced binding to IgA receptors expressed on mesangial cells lead to deposition. The cause of the formation of immune complexes responsible for IgA nephropathy lies in the incomplete O-linked oligosaccharide side chains, which, due to the deficiency of corresponding glycosyltransferases, lack terminal galactose residues leading to the exposure of N-acetylgalactosamine. Naturally occurring antibodies of the IgG or IgA1 isotype bind to this sugar antigen. In the clinical course, we differentiate between the early stage usually characterized by hematuria, and a variable late stage characterized either by a clinical remission, by persistence of hematuria, or by increasing proteinuria and blood pressure and decreasing renal function in one third of the patients. In the early stage, it is difficult to predict the prognosis of IgA nephropathy, either on the basis of clinical presentation and morphological findings, or according to the level of galactose-deficient IgA1 in the circulation. The reliable criteria of serious prognosis emerge only in the later stages of the disease and include proteinuria, hypertension, and histologically apparent tubular atrophy and interstitial sclerosis. The dominant trend in the treatment of IgA nephropathy is the emphasis on administration of ACE inhibitors/sartans, which are introduced into the treatment at the time of microalbuminuria. If proteinuria does not decrease below 1 g/24 h, treatment with prednisone is justifiable. New findings concerning the molecular/cellular mechanism involved in the pathogenesis of IgA nephropathy suggest the possible therapeutical interference with the generation of nephritogenic immune complexes by a selective blocking of the IgA1 molecules with altered glycan structures using monovalent reagents.

The effect of dibenzo-p-dioxin- and dibenzofuran-contaminated soil on the earthworm Eisenia andrei.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) belong to the group of persistent organic pollutants, highly toxic environmental pollutants that include hydrophobic compounds with the tendency to bioaccumulate. Earthworms (Eisenia andrei) were exposed to PCDD/Fs-contaminated soil, and changes in their lipophilic structures and the gene expression of their defense molecules were followed. Damage to the intestinal wall and adjacent chloragogenous tissue was observed. Further, the up-regulation of the expression of several genes was detected. On the basis of these results, the mechanism of the impact of PCDD/Fs on earthworms has been proposed. Dioxins that accumulate in the lipophilic structures cause an increase in reactive oxidative species that triggers oxidative stress followed by the gene expression of two molecules that play a role in protection against oxidant toxicity, calreticulin (CRT) and Hsp70. Moreover, the effect of microbial biomass on the expression of coelomic cytolytic factor (CCF), a pattern recognition receptor, was also observed.

Immunocompatibility evaluation of hydrogel-coated polyimide implants for applications in regenerative medicine.

Immunocompatibility of gelatin-based hydrogels to be applied as implant coatings for local regenerative treatment has been studied. First, the bio- and immuno-acceptability of the methacrylamide-modified gelatin hydrogels per se was screened. The results indicated that the hydrogels support cell growth. Metabolic activity of normal cells and permanent cell lines representing various cell types (endothelial, epithelial, fibroblast, and monocyte/macrophage) cultivated on the gelatin hydrogels was moderately lower compared to cells cultivated on tissue culture plastic. The cells cultivated on the hydrogels produced identical cytokines as the control cells although at lower levels. Importantly, no inflammatory activity, measured by nitric oxide and pro-inflammatory cytokine (IL-1α, IL-6, and TNFα) production, was observed in peritoneal cells and monocyte/macrophage RAW 264.7 cell line cultivated on the hydrogels. Finally, polyimide (PI) implantable membranes were surface-modified with gelatin hydrogels and screened for their in vivo immunocompatibility. Their histological examination performed after subcutaneous implantation in mice produced a sound proof of immunoacceptability. Normal tissue repair, mild cellular infiltration and edema mainly induced by the surgery were observed after 2 and 6 days. No adverse tissue responses were induced by the implants. Analysis performed after 4 and 9 weeks indicated areas of foreign body granuloma without formation of a fibrous capsule.

Evaluation of tumor suppressor gene expressions and aberrant methylation in the colon of cancer-induced rats: a pilot study.

Altered expression and methylation pattern of tumor suppressor and DNA repair genes, in particular involved in mismatch repair (MMR) pathway, frequently occur in primary colorectal (CRC) tumors. However, little is known about (epi)genetic changes of these genes in precancerous and early stages of CRC. The aim of this pilot study was to analyze expression profile and promoter methylation status of important tumor suppressor and DNA repair genes in the early stages of experimentally induced colorectal carcinogenesis. Rats were treated with azoxymethane (AOM), dextran sodium sulphate (DSS) or with their combination, and sacrificed 1 or 4 months post-treatment period. The down-regulation of Apc expression in left colon, detectable in animals treated with DSS-AOM and sacrificed 1 month after the end of treatment, represents most early marker of the experimental colorectal carcinogenesis. Significantly reduced gene expressions were also found in 5 out of 7 studied MMR genes (Mlh1, Mlh3, Msh3 Pms1, Pms2), regarding the sequential administration of DSS-AOM at 4 months since the treatment. Strong down-regulation was also discovered for Apc, Apex1, Mgmt and TP53. Tumors developed in rectum-sigmoid region displayed significantly lower Apc and Pms2 expressions. The decreased expression of studied genes was not in any case associated with aberrant methylation of promoter region. Present data suggest that down-regulation of Apc and MMR genes are prerequisite for the development of CRC. In this study we addressed for the first time early functional alterations of tumor suppressor genes with underlying epigenetic mechanisms in experimentally induced CRC in rats.

Altered gut microbiota promotes colitis-associated cancer in IL-1 receptor-associated kinase M-deficient mice.

BACKGROUND: Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model. METHODS: Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and ß-glucuronidase activity was measured in intestinal content by fluorescence assay. RESULTS: ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased ß-glucuronidase activity. However, the ß-glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in ß-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue. CONCLUSIONS: We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.

Colostrum of healthy mothers contains broad spectrum of secretory IgA autoantibodies.

PURPOSE: Human colostrum and milk provide a newborn with immunomodulatory components, ensuring protection and proper development of the immune system. Secretory IgA antibodies in colostrum represent the first line of defence against harmful substances, but their potential spectra of reactivity with autoantigens remains unclear. Here, we characterised the repertoire of natural sectretory IgA autoantibodies in colostrum of healthy mothers. METHODS: The human colostrum samples from 39 healthy mothers were analyzed for autoantibodies by indirect immunofluorescence, dot blots, immunoblots and ELISA. RESULTS: We found that there is high diversity in reactivities of colostral IgA antibodies to autoantigens among individual samples. Using tissue sections and biochips commonly used for autoimmunity testing, we found that most samples reacted with monkey ovary (79.3%), monkey pancreatic tissue (78.6%), human HEp-2 cells (69%) and monkey adrenal gland (69.0%), fewer samples reacted with monkey liver tissue (47.2%), rat stomach (42.9%), monkey testicular tissue (41.4%), monkey salivary gland (39.3%), rat kidney (32.1%) and monkey cerebellar tissue (17.9%). At the protein level, we detected reactivity of IgA with 21 out of 25 (auto) antigens. The majority of the samples reacted with the pyruvate dehydrogenase complex, E3 ubiquitin ligase, cytosolic liver antigen, promyelocytic leukemia protein and nuclear pore glycoprotein-210. Using ELISA, we found reactivity of colostral IgA antibodies against examined extractable nuclear antigens, double stranded DNA, phospholipids and neutrophil cytoplasm. CONCLUSIONS: The broad spectrum of polyreactive natural autoantibodies present in human colostrum may contribute to proper development of mucosal immune system of the breastfed infant.

Lysate of probiotic Lactobacillus casei DN-114 001 ameliorates colitis by strengthening the gut barrier function and changing the gut microenvironment.

BACKGROUND: Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD. METHODOLOGY/PRINCIPAL FINDINGS: The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4(+)FoxP3(+) regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4(+)FoxP3(+) regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer's patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway. CONCLUSION/SIGNIFICANCE: Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.

Safety and efficacy of the immunosuppressive agent 6-tioguanine in murine model of acute and chronic colitis.

BACKGROUND: Oral thiopurines are effective and widely used in treatment of inflammatory bowel disease (IBD) in humans, although their use is limited due the development of adverse events. Here, we examine the efficacy and toxicity of oral treatment with 6-tioguanine (6-TG) and azathioprine (AZA) in a murine model of IBD. METHODS: We induced acute or chronic colitis in BALB/c mice by one or four cycles of 3% dextran sulphate sodium (DSS), respectively. Mice were treated by daily gavages of various dosages of 6-tioguanine, azathioprine, or by phosphate buffered saline (PBS) starting the first day of DSS or after two cycles of DSS, respectively. We monitored the efficacy and toxicity by measuring the weight change and serum alanine aminotransferase (ALT) activity and by disease severity and histology, at the end of the experiment. Moreover, we measured cytokine production after colon fragment cultivation by enzyme-linked immunoabsorbent assay and numbers of apoptotic cells in the spleen by flow cytometry. RESULTS: 6-TG is effective in the treatment of acute DSS-induced colitis in a dose-dependent manner and 40 µg of 6-TG is significantly more effective in the treatment of acute colitis than both AZA and PBS. This effect is accompanied by decrease of IL-6 and IFN-γ production in colon. We did not observe histological abnormalities in liver samples from control (PBS) or 6-TG treated mice. However, liver samples from most mice treated with AZA showed mild, yet distinct signs of hepatotoxicity. In chronic colitis, all thiopurine derivatives improved colitis, 20 µg of 6-TG per dose was superior. High doses of 6-TG led to significant weight loss at the end of the therapy, but none of the thiopurine derivatives increased levels of serum ALT. Both thiopurine derivatives reduced the proportion of apoptotic T helper cells, but a high production of both IL-6 and TGF-ß was observed only in colon of AZA-treated mice. CONCLUSIONS: Use of 6-TG in the treatment of experimental colitis in mice appears superior to AZA administration and placebo. In contrast to 6-TG, the use of AZA resulted in histological liver abnormalities.

Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 µm), highly porous (Ø 1 µm spaced 3 µm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases.

Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.

Preclinical evaluation of linear HPMA-doxorubicin conjugates with pH-sensitive drug release: efficacy, safety, and immunomodulating activity in murine model.

PURPOSE: In vivo efficacy and safety of HPMA-based copolymers armed with doxorubicin via a spacer containing pH-sensitive linkage that can be prepared within a broad range of attached drug contents (1) was tested in murine tumor models. METHODS: Mice bearing T cell lymphoma EL4 or B cell lymphoma 38C13 were treated with a single dose of the conjugate (15, 25, and 75 mg Dox eq./kg i.v.) in a therapeutic regime. Anti-tumor resistance of the cured animals was proved by a second challenge with a lethal dose of tumor cells without additional treatment. RESULTS: The content of drug bound to the polymer is an important parameter in relation to the conjugate therapeutic efficacy. The best anti-tumor effects were produced by conjugates with 10 - 13 wt% of bound doxorubicin. Free doxorubicin up to 4.6% relative to total drug content had no impact on the treatment efficacy and acute toxicity. The conjugates induced a complete cure of mice and regular treatment-dependent development of specific anti-tumor resistance. No myelosuppression or organ damage was observed. CONCLUSIONS: A well-defined HPMA copolymer-doxorubicin conjugate with pH-sensitive drug release is a good candidate for clinical trials as it has remarkable anti-tumor efficacy and a favorable safety profile.

Colorectal carcinoma: Importance of colonic environment for anti-cancer response and systemic immunity.

The intestinal environment is considered to play an important role both in colorectal tumor development and in the evolution and modulation of mucosal immunity. Studies in animals reared in germ-free (GF, without any intestinal microflora) versus conventional (CV, with regular microflora in bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and anti-cancer immune responses in situ. The lower incidence of colon cancers and better immunological parameters in GF animals versus CV ones after chemically-induced carcinogenesis raises questions about specific characteristics of the immunological networks in each respective condition. Different levels of tolerance/regulatory mechanisms in the GF versus CV animals may influence the development of immune responses not only at the level of mucosal, but also at the systemic, immunity. We hypothesize that GF animals can better recognize and respond to evolving neoplasias in the bowel as a consequence of their less-tolerogenic immunity (i.e., due to their more limited exposure to antigens to become tolerated against at the intestinal level). In this paper, we review the role of bacteria in modulating gut environment and mucosal immunity, their importance in cancer development, and aspects of immune regulation (both at local and systemic level) that can be modified by bacterial microflora. Lastly, the use of GF animals in comparison with conventionally-raised animals is proposed as a suitable and potent model for understanding the inflammatory network and its effect on cancer immunity especially during colorectal cancer development.

Colorectal carcinogenesis in germ-free and conventionally reared rats: different intestinal environments affect the systemic immunity.

Intestinal microbiota are considered to play an important role both in colorectal tumor development and in the modulation of mucosal immunity. Studies on animals reared in germ-free (GF, without intestinal microbiota) versus conventional (CV, with regular microbiota colonization of the bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and the anticancer immune response. The capability of the intestinal environment to modulate anticancer immunity not only at the mucosal but also at the systemic level is still an open question. In our study we found that, following the same protocol of colorectal cancer induction, GF rats developed less and smaller tumors than CV rats. The GF rats that did not develop cancer also presented a better anticancer immune response with an increase in NK, NKT, CTL, B cells and cytotoxicity in peripheral blood. We hypothesize that the lower antigenic challenge and the absence of the 'physiological inflammation', caused by the commensal microbiota in the gut of CV rats, may enhance the capability of the GF rats to develop more efficacious anticancer immune responses. The different levels of tolerance/regulatory mechanisms in GF versus the CV animals may modulate the anticancer response not only at the mucosal but also at the systemic immunity level.