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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.

Simple Summary Chronic colitis and colon cancer develop for alteration of the mucosa homeostatic regulation, also involving TGF-β1. Dextran sulphate sodium (DSS)-induced colitis and azoxymethane (AOM)-induced colorectal carcinogenesis animal models allow for the investigation of the pathological evolution steps. Since chronic inflammation is a common factor, we aimed to explore in rat models the colon mucosa immunological and structural conditions at one month after the end of the inductions, a transition period between acute effects and established lesions. We found, in comparison to healthy controls, downregulation of inflammatory cytokines (except IL-6) and of TGF-β1. At the same time, the collagen scaffold was significantly remodelled in both groups. We conclude that the pro-inflammatory cytokines, in front of a downregulated TGF-β1, sustained a smouldering inflammation with structural changes preparing the niche of both pathologies (ulcerative colitis with fibrosis; tumour). The collagen scaffold changes pointing to an unnoticed inflammation may be suggested as a possible pre-neoplastic condition marker. Abstract 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-β1, 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-β1, 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-β1 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.

Here we present the polymer conjugates where the core formed by poly(amido amine) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing docetaxel (DTX) attached by a pH-sensitive hydrazone bond. DTX was derivatized with three different keto acids prior to attachment to the polymer carrier to introduce reactive keto groups into the drug. The therapeutic efficacy of such high-molecular-weight star conjugates is based on: (a) the enhanced permeability and retention (EPR) effect facilitating selective accumulation within solid tumors; (b) pH-controlled release of the drug, thus ensuring faster DTX release in the mildly acidic tumor microenvironment. The star DTX conjugate had a remarkably higher maximum tolerated dose in comparison with free DTX when administered as a single i.v. injection (∼160 mg kg−1vs. 40 mg kg−1 of DTX) in C57BL/6 mice. The star DTX conjugate showed significantly higher antitumor activity than free drug in the EL4 T cell lymphoma growing in syngeneic C57BL/6 mice even when given at the same dose (20 mg kg−1 of DTX eq.). Thus, the star DTX conjugates exert a much higher therapeutic activity and yet a lower systemic toxicity than free DTX.

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, α M β 2 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 (>10 7 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.

Aim. Up to now, an appropriate salt intake in renal insufficiency has not been clearly determined. We hypothesize that even a moderate decrease in salt intake may affect functional and morphologic response of the rat remnant kidney after 5/6 nephrectomy.Methods. Subtotal nephrectomy was performed in 77 inbred 12 week-old-female AVN Wistar rats. The two groups of rats were fed either a standard or a low salt diet. Median of salt intake was 14.6 and 10.4 mg/100 g/24 h in the two groups.Results. Ten weeks after ablation, the remnant kidney parenchyma wet weight was 0.66 ± 0.16 g/100 g of body weight and 0.56 ± 0.11 g/100 g of body weight (P<0.01) in rats with a standard and low salt diet, respectively. In these two groups, systolic blood pressure was 151 ± 29 versus 126 ± 21 mmHg (P<0.05), serum creatinine levels were 164 ± 84 versus 106 ± 29 µmol/L (P<0.001), proteinuria was 84 ± 37 versus 83 ± 40 mg/100 g/24 h (N.S.), and the glomerular injury score was 2.06 ± 0.49 versus 1.43 ± 0.62 (P<0.01), respectively.Conclusion. Moderately decreased salt intake slowed down the development of ablation nephropathy in AVN inbred strain of rats.

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 az- oxymethane (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, repre- sents 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 treat- ment. 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 develop- ment 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.

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.

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.

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.The human colostrum samples from 39 healthy mothers were analyzed for autoantibodies by indirect immunofluorescence, dot blots, immunoblots and ELISA.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.The broad spectrum of polyreactive natural autoantibodies present in human colostrum may contribute to proper development of mucosal immune system of the breastfed infant.

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.

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.

Our study examined whether repeated preventive oral administration of live probiotic bacterial strains Escherichia coli O83:K24:H31 (Ec O83), Escherichia coli Nissle 1917 O6:K5:H1 (Ec Nis) and Lactobacillus casei DN 114001 (Lc) can protect mice against dextran sodium sulfate (DSS)-induced colitis. A significant decrease in average symptom score was observed in Ec O83-, Ec Nis- and Lc-pretreated group (p < 0.05). Significant differences in body mass loss between Lc pretreated mice with DSS-induced colitis were found when compared with nontreated mice (p < 0.05). PBS pretreated mice had a significantly shorter colon than Ec O83-, Ec Nis- and Lc-pretreated mice (p < 0.05). Administration of Lc significantly decreased the severity of DSS induced histological marks of inflammation (p < 0.05). A significant difference (p < 0.05) was also found in specific IgA level against given probiotic in enteral fluid between colitic mice and healthy mice pretreated with Ec 083 and Ec Nis.

Organisms live in continuos interaction with their environment; this interaction is of vital importance but at the same time can be life threatening. The largest and most important interface between the organism and its environment is represented by surfaces covered with epithelial cells. Of these surfaces, mucosae comprise in humans approximately 300 m2, and the skin covers approximately 1.8 m2 surface of the human body. Mucosal tissues contain two effector arms of the immune system, innate and adaptive, which operate in synergy. Interaction with commensal bacteria, which outnumber the nucleated cells of our body, occurs physiologically on epithelial surfaces; this interaction could pose the risk of inflammation. The mucosal immune system has developed a complex network of regulatory signalling cascades that is a prerequisite for proper activation but also for a timely inactivation of the pathway. As demonstrated in gnotobiotic animal models of human diseases, impaired regulation of mucosal responses to commensal bacteria plays an important role in the development of several inflammatory and autoimmune diseases.

Commensal microflora (normal microflora, indigenous microbiota) consists of those micro-organisms, which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). The number of bacteria colonising mucosal and skin surfaces exceeds the number of cells forming human body. Commensal bacteria co-evolved with their hosts, however, under specific conditions they are able to overcome protective host responses and exert pathologic effects. Resident bacteria form complex ecosystems, whose diversity is enormous. The most abundant microflora is present in the distal parts of the gut; the majority of the intestinal bacteria are Gram-negative anaerobes. More than 50% of intestinal bacteria cannot be cultured by conventional microbiological techniques. Molecular biological methods help in analysing the structural and functional complexity of the microflora and in identifying its components. Resident microflora contains a number of components able to activate innate and adaptive immunity. Unlimited immune activation in response to signals from commensal bacteria could pose the risk of inflammation; immune responses to mucosal microbiota therefore require a precise regulatory control. The mucosal immune system has developed specialised regulatory, anti-inflammatory mechanisms for eliminating or tolerating non-dangerous, food and airborne antigens and commensal micro-organisms (oral, mucosal tolerance). However, at the same time the mucosal immune system must provide local defense mechanisms against environmental threats (e.g. invading pathogens). This important requirement is fulfilled by several mechanisms of mucosal immunity: strongly developed innate defense mechanisms ensuring appropriate function of the mucosal barrier, existence of unique types of lymphocytes and their products, transport of polymeric immunoglobulins through epithelial cells into secretions (sIgA) and migration and homing of cells originating from the mucosal organised tissues in mucosae and exocrine glands. The important role of commensal bacteria in development of optimally functioning mucosal immune system was demonstrated in germ-free animals (using gnotobiological techniques). Involvement of commensal microflora and its components with strong immunoactivating properties (e.g. LPS, peptidoglycans, superantigens, bacterial DNA, Hsp) in etiopathogenetic mechanism of various complex, multifactorial and multigenic diseases, including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, multiorgan failure, colon cancer has been recently suggested. Animal models of human diseases reared in defined gnotobiotic conditions are helping to elucidate the aetiology of these frequent disorders. An improved understanding of commensal bacteria-host interactions employing germ-free animal models with selective colonisation strategies combined with modern molecular techniques could bring new insights into the mechanisms of mucosal immunity and also into pathogenetic mechanisms of several infectious, inflammatory, autoimmune and neoplastic diseases. Regulation of microflora composition (e.g. by probiotics and prebiotics) offers the possibility to influence the development of mucosal and systemic immunity but it can play a role also in prevention and treatment of some diseases.

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.

Adhesion molecules (e.g. ICAM-1, CD 54) are known to be upregulated on activated vascular endothelial cells during inflammatory reactions. To study the role of ICAM-1 in intestinal inflammation in vivo, we induced acute experimental colitis in wild-type (C57BL/6) mice and ICAM-1-deficient mice, by feeding the animals with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. In the control strain the immunohistochemical staining showed a very pronounced endothelial upregulation of ICAM-1 after the DSS treatment observed in areas of inflammatory infiltrate, especially in venules or arterioles of the propria and submucosa, and partly in the mesocolon. DSS-fed ICAM-1-deficient mice showed no endothelial enhancement and only faint staining of venules or capillaries approaching that encountered in the control ICAM-1-deficient animals. Our data indicate that ICAM-1 may play a crucial role in the development of acute intestinal inflammation, consistent with our finding that ICAM-1 deficiency can obviate severe forms of experimentally induced colitis in mice.

Allotransplantation (TPL) of the abdominal aortic segments of BN donors was performed in 32 Lewis recipients with or without cyclosporin A (CyA) immunosuppression, and the vascular changes were compared to those of 10 syngeneic grafts (Lewis--Lewis) and to the autologous rat aortae. The vessels were examined 2, 3, 4 and 5 months post TPL by light microscopy, the thickness of intima and media was measured morphometrically and the cell infiltration of adventitia and intima was assessed semiquantitatively. Thirty-six aortae were examined by three-step enzyme immunohistochemistry (proof of selected differentiation, proliferation, cytoskeletal and connective tissue matrix antigens). The adventitia displayed an intense focal and scattered mononuclear cell infiltration; it was more discrete and focal in the intima. This cellularity persisted in the allografts but disappeared from the intima and was reduced in the adventitia of the isografts after four and five months. Disseminated ED1+ activated macrophages were the most prominent population of infiltrates whereas modest numbers of adventitial ED2+ tissue macrophages remained constant throughout the intervals examined. CD4+ cells (focal and scattered) outnumbered (roughly twice) the scattered CD8+ lymphocytes; both these types were rare in the intima. Leukocyte invasion of the media was lacking (except for scarce isolated CD8+ cells in some allografts). In syngeneic grafts the smooth muscle cells (SMC) of media remained intact and the intimal thickening was slight to absent (about 5 microns) four and five months post TPL. On the other hand, the allograft media underwent severe destructive changes (karyolysis, depletion of alpha-SMC actin, focal calcification and general thinning without rupture or aneurysm). The prominent allograft intimal thickening (70-80 microns) was due to the proliferation of longitudinally oriented myointimal cells (alpha-SMC actin, FD2, PCNA and Ki67+) and an increase in matrix substance (strong metachromasia and positivity of chondroitin-sulfate proteoglycan). The deposition of lipids remained discrete, without atheromatous plaques and mural thrombosis. All changes were comparable in CyA-treated and untreated animals. Thus the main lesions of the allografts were (i) persistent mononuclear infiltration chiefly in adventitia, (ii) destruction of medial SMC, and (iii) intimal thickening by proliferation of myointimal cells. At the postTPL intervals examined the proliferation and intimal migration of medial SMC were not apparent and a morphological correlate of significant anti-medial-SMC cytotoxic attack was lacking.

One of the characteristic features of celiac disease is an increase in anti-gliadin antibodies (Abs). Recently we found that some of the monoclonal Abs to gliadin cross-react with molecules on rat enterocytes. One of these cross-reacting molecules was identified as rat calreticulin. This study shows that the levels of serum IgA Abs to gliadin, rat, and human enterocytes; purified enterocyte antigens; and calreticulin in sera from patients with active disease were significantly higher than in patients on a gluten-free diet and healthy controls (P < 0.001). Anti-gliadin Abs were isolated by affinity chromatography from the sera of six active celiac patients. The reactivity of these anti-gliadin Abs was demonstrated to be significantly higher (P < 0.05) with human enterocytes and human calreticulin than with other antigens tested. Furthermore, using isolated patients' anti-gliadin Abs bound to Sepharose 2B, two main proteins of molecular mass 62 and 66 kDa were purified from a lysate of human enterocytes. The 62-kDa enterocyte antigen was identified as human calreticulin. These findings suggest that anti-gliadin Abs may play a pathogenic role in celiac disease by cross-reacting with enterocytes. Calreticulin in enterocytes may be one of the putative targets for autoimmune reactions.

Coelomic fluid of Eisenia foetida earthworms is known to exert strong proteolytic, hemolytic, bacteriostatic, and cytolytic properties. Ultrastructural observations revealed that coelomic fluid causes multiple ruptures and defects in the erythrocyte membrane as well as in the membrane of murine peritoneal leukocytes. Incubation of peritoneal cells in coelomic fluid resulted in a disorganization of the macrophage surface microvilli, changes in the organization of cytoplasmic organelles and disruption and degranulation of mast cells. Severe mesothelial damage was observed after intraperitoneal administration of the coelomic fluid.

Background. The side effects of cyclosporine (CsA)-including nephrotoxicity and abnormal differentiation of thymocytes developing in the thymus-can be decreased or even avoided using targeted conjugates of CsA, where both targeting moiety and drug are bound to water-soluble polymeric carrier based on N-(2-hydroxypropyl) methacrylamide (HPMA). Methods. Irradiated, syngeneic bone marrow transplanted-mice (BALB/c and A/Ph) were treated intraperitoneally for 4 weeks with 20 mglkg of free CsA, HPMA-conjugated CsA, or antibody-targeted HPMA-bound CsA. Immunohistology of the thymus was performed together with two-color flow cytometry to detect the effect of different forms of CsA on individual thymocyte subpopulations. Results. We have shown that free CsA strongly abrogated T-cell development. The appearance of mature thymocytes expressing CD3 high is almost completely inhibited (1.8%) after free CsA treatment, whereas these cells are well detectable in controls (22%) and HPMA polymer-bound CsA-treated animals (19%). Immunohistological studies have shown acellular rests of the medulla after free CsA treatment, whereas well-stained medullary thymocytes were detected in controls and after exposure to antibody-targeted HPMA-conjugated CsA. Conclusions. HPMA-conjugates of CsA are generally more specific in their targeting to T lymphocytes. It was found that nonspecific binding of CsA to erythrocytes and plasma lipoproteins is significantly reduced using anti-CD3 targeted, HPMA polymer-bound CsA. In addition, the entry of these macromolecules into the thymus is limited-probably due to the blood-thymus barrier-and HPMA conjugates of CsA, unlike free drug, do not abrogate T-cell development in bone marrow transplanted mice.

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.

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 β-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 β-glucuronidase activity. However, the β-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 β-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.

Structural similarities between external antigen and self components are believed to be one of the possible causes of autoimmunity. This study describes the presence of similar structures shared by gliadin and enterocyte surface molecules recognized by antigliadin mAbs. The reactivity of mAbs to gliadin was followed by ELISA using fixed enterocytes, their brush-border membranes, or purified enterocyte antigen. The specificity of reaction was confirmed by ELISA inhibition studies and by immunohistochemical staining of rat tissue sections using biotin-avidin-peroxidase technique. Immunoprecipitation analysis of 125I-labeled intestinal epithelial cells using antigliadin mAb revealed the presence of two main cross-reactive molecules of 28 and 62 kDa. The 62-kDa and an associated 66-kDa protein were isolated by affinity chromatography. Immunoblotting analysis showed that a 28-kDa protein detected by immunoprecipitation also reacted with IgA of celiac disease patient sera.

BackgroundProbiotic 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 findingsThe 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/significanceOur 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.

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.

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 (O 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.

Summary Commensal bacteria have been shown to modulate the host mucosal immune system. Here, we report that oral treatment of BALB/c mice with components from the commensal, Parabacteroides distasonis, significantly reduces the severity of intestinal inflammation in murine models of acute and chronic colitis induced by dextran sulphate sodium (DSS). The membranous fraction of P. distasonis (mPd) prevented DSS-induced increases in several proinflammatory cytokines, increased mPd-specific serum antibodies and stabilized the intestinal microbial ecology. The anti-colitic effect of oral mPd was not observed in severe combined immunodeficient mice and probably involved induction of specific antibody responses and stabilization of the intestinal microbiota. Our results suggest that specific bacterial components derived from the commensal bacterium, P. distasonis, may be useful in the development of new therapeutic strategies for chronic inflammatory disorders such as inflammatory bowel disease.

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-β 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.

In the present work, two strategies were elaborated to surface-functionalize implantable polyimide sheets. In the first methodology, cross-linkable vinyl groups were introduced on the polyimide surface using aminopropylmethacrylamide. In the second approach, arc:active succinimidyl ester was introduced on the surface of PI. Using the former approach, the aim is to apply a vinyl functionalized biopolymer coating. In the latter approach, any amine containing biopolymer can be immobilized. The foils developed were characterized in depth using a variety of characterization techniques. including atomic force rnicroscopy, static contact angle measurements, and X-ray photoelectron spectroscopy. The results indicated that both modification strategies were successful. The subcutaneous implantation in mice indicated that both modification strategies resulted in biocompatible materials, inducing only limited cellular infiltration to the surrounding tissue.

The aim of this study was to test the pharmacological effectiveness of antibody-targeted cyclosporin A. Only free drug is currently available; its administration is accompanied by nephrotoxicity, hepatotoxicity and neurotoxicity which severely limits the treatment. Moreover, due to the high nonspecific binding to a number of tissues, the therapeutic doses are considerably higher than required for the actual treatment. We suggest using antibody-targeted CsA conjugate based on a water-soluble synthetic copolymer of N-(2-hydroxypropyl)methacrylamide to overcome these problems. In this construct the antibody to T lymphocyte subsets (anti-CD3 in human and anti-Thy 1.2 in mouse) is responsible for the specific delivery which limits the drug action to the target tissue. Antibody-targeted CsA effectively inhibits lectin- and alloantigen-induced T cell proliferation and considerably reduces CsA nephrotoxicity.

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.

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.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.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.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.

Earthworms, Eisenia foetida, are able to respond to antigenic stimulation by the formation of the antigen-binding molecules by coelomocytes — the effector cells of annelids' defence reactions. The ability to react with gold-labelled antigen was detected in agranular coelomocytes by electron microscopy. Furthermore, flow cytometry analysis used for quantitative evaluation of antigen binding showed significant increase of both antigen-binding cells and the amount of antigen bound per cell after stimulation. The antigen binding was inhibited by preincubation of cells with several similar proteins, although the most potent inhibitor was the immunizing antigen.

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 in biopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis. (J Histochem Cytochem 56:267–274, 2008)

Initial events and effector mechanisms of most inflammatory and autoimmune diseases remain largely unknown. Dysfunction of the innate and adaptive immune systems associated with mucosae (the major interface between the organism and its environment, e.g., microbiota, food) can conceivably cause impairment of mucosal barrier function and development of localized or systemic inflammatory and autoimmune processes. Animal models help in elucidating the etiology and pathogenetic mechanisms of human diseases, such as the inflammatory bowel diseases, Crohn's disease and ulcerative colitis, severe chronic diseases affecting the gut. To study the role of innate immunity and gut microbiota in intestinal inflammation, colitis was induced by dextran sulfate sodium (DSS) in mice with severe combined immunodeficiency (SCID). Conventionally reared (microflora-colonized) SCID mice displayed severe inflammation like that seen in immunocompetent Balb/c mice, whereas only minor changes appeared in the intestinal mucosa of DSS-fed gnotobiotic germ-free SCID mice. The presence of microflora facilitates the inflammation in DSS-induced colitis that develops in immunodeficient SCID mice, that is, in the absence of T and B lymphocytes. Celiac disease, a chronic autoimmune small bowel disorder, afflicts genetically susceptible individuals with wheat gluten intolerance. We showed that, in contrast with any other food proteins, wheat gliadin and its peptic fragments activate mouse macrophages and human monocytes to produce proinflammatory cytokines through the nuclear factor-kappaB signaling pathway. Activation of innate immunity cells by food proteins or components from gut microbiota thus could participate in the impairment of intestinal mucosa and the development of intestinal and/or systemic inflammation.

Tumor development is modulated by the interplay between the transformed cells and the host, and produces changes in the immune system. We followed the cancer progression and the variation of immune parameters in a rat in vivo model of induced colorectal carcinoma. Retrospective data collected from different experiments illustrated the dynamics of the tumor development, and of the immune cells (NK, NKT, T, CD4+, CTL, B and gammadeltaTCR+ cells), cytotoxicity, and CD4/CD8 ratio, at the third, sixth and eighth month of carcinogenesis. The chemically-induced carcinogenesis involved the complete large bowel, with progressive generation of multiple tumors during the complete considered period. Reduction in number and function of cytotoxic and regulatory cells of the innate immunity were crucial for cancer progression.

Glyco-coat changes on cancer cells due to aberrant glycosylation are potential targets for immune recognition through lectin-like receptors on immune cells. These cells include natural killer (NK), CD8+ and CD4+ lymphocytes, all reported to have, together with cytokines, important functions in antitumor immunity. The aim of this study was to evaluate a possible role of synthetic monodisperse multivalent neo-glycoconjugates, namely glycodendrimers, as a new approach to anticancer immune modulation through carbohydrate-mediated immune recognition. Octavalent polyamidoamine dendrimers functionalized with N-acetyl-glucosamine residues (PAMAM-GlcNAc8), with in vitro high affinity for the recombinant lymphocyte receptor NKR-P1A, were employed. To follow the fate of the compound, a fluorescent marker was conjugated to the tetra-branched semi-component of the dendrimer. Tumor development and immunity were evaluated in C57BL/6 mice. Animals were inoculated with B16F10 melanoma cells and underwent different protocols of PAMAM-GlcNAc8 administration. Advantages on survival and reduction of tumor growth were obtained in dose-dependent manner, by IP route. Increase of CD69+ cells in the spleen and their appearance inside the tumors, early progressive release of IL-1beta, a later production of INFgamma and IL-2 concomitant to an increment of CD4+ cells were observed. Cytotoxicity assays, performed ex vivo, showed an enhanced NK cell activity proportioned to the percentage of activated NK cells. Our data suggest that well-defined multivalent neo-glycoconjugates can stimulate an antitumor immune response engaging both innate and acquired immunity.

Cell adhesion molecules and their ligands are essential for regulating lymphocyte recirculation and leucocyte emigration into an inflamed or injured tissue. Vascular endothelial selectins as mediators of leucocyte rolling and intercellular cell adhesion molecule-1 (ICAM-1) have been found to be up-regulated on activated endothelium during acute allograft rejection. This study was designed to investigate whether ICAM-1 or selectin-ligand deficiency, or a combination of both, affected graft survival during acute cardiac allograft rejection. To this goal, we performed cardiac transplantation using mice deficient in genes for ICAM-1 or α(1,3)fucosyltransferase Fuc-TVII, representing a model for general absence of selectin-ligand expression, and a newly developed strain with a double mutation in Fuc-TVII and ICAM-1 alleles. Transplantation of a heart from ICAM-1 −/− or Fuc-TVII/ICAM-1 double-mutated mice into allogeneic recipients resulted in limited (2–2.5 days) but nevertheless significant prolongation of the graft survival (P

Chronic rejection is the major cause of late kidney allograft failure. We evaluated the efficacy of LF 08-299 (LF), an analogue of 15-deoxyspergualin, in a rat aortic allograft model of chronic rejection. BN aortic allografts were transplanted to Lew recipients. LF was administered at a dose of 6 mg/kg and 2.5 mg/kg on days 0-20 and 6 mg/kg on days 60-90. CyA was used at a dose of 5 mg/kg on days 0-20. Untreated isografts and allografts were used as controls. Histological changes and immunohistochemistry were monitored sequentially at 8, 12, 16 and 20 weeks. There were no differences in intimal proliferation between LF-treated allografts and untreated or CyA-treated controls. Only a tendency in adventitial infiltration reduction was seen in LF-treated animals. We found a significantly less pronounced reduction in media diameter in LF-treated animals. We concluded that LF 08-0299 is only able to reverse reduction in media thickness in aortic allografts, but not intimal proliferation in this model of chronic rejection.

This study compares the ultrastructure of three syngeneic and three allogeneic grafts of rat abdominal aorta (Lewis to Lewis and BN to Lewis, respectively); the tissue was sampled three months after transplantation (TPL). The endothelial plate was preserved and mononuclear cell adherence was absent. In syngeneic grafts the intima and media remained close to normal with well-preserved smooth muscle cells (SMC). The thickened allograft neointima consisted of elongated spindle cells and rich intercellular matrix. The cells were typical SMC without apparent signs of dystrophy or degeneration. On the other hand, most SMC of the media showed complete disruption and disorganization of membrane and organelles suggestive of accomplished necrosis. However, the framework of elastic lamellae was preserved, without apparent ruptures or lytic changes. Intraintimal migration of medial SMC was not recorded while some cytoplasmic strips were seen to extend across the outer elastic lamella (possible rudimentary outgrowth of SMC?). Lymphocytes and histiomonocytic cells (macrophages) were found in the adventitia but not in the destroyed media. Thus electron microscopy elucidated the histological picture of “anuclear allograft media” and confirmed the predominance of SMC in the thickened neointima. However, signs of the mediointimal SMC invasion were not apparent three months post TPL.

Coelomic fluid of earthworms contains a 42 kDa protein designated CCF-1 (coelomic cytolytic factor 1), which accounts for approximately 40% of cytolytic activity of the entire coelomic fluid. CCF-1 was documented to be present on cells of the mesenchymal lining of the coelomic cavity as well as on free coelomocytes. Both cellular and humoral levels of CCF-1 were significantly increased after parenteral injection of endotoxin. Moreover, CCF-1 seems to be involved in cell mediated cytotoxicity, because cytotoxic activity is blocked in the presence of anti-CCF-1 monoclonal antibody (mAb).

We have recently reported that thymic B lymphocytes (TBL) are the first B-cell subpopulation undergoing isotype switching to IgG and IgA during embryonic life. The aim of this study is to analyze the influence of antigenic stimulation on TBL location and activity using a germ-free (GF) newborn pig model, in which maternal antibodies and antigens do not affect B-cell development. Immunohistological analysis showed that TBL were disseminated mainly in the thymic medulla. There were no differences in the distribution of TBL, both in GF newborn piglets before and after colonization withEscherichia coliand in older conventionally reared (CONV) piglets. The number of immunoglobulin (Ig)-secreting cells measured by the ELISPOT method was not influenced by microflora and food antigens. IgM-positive cells secreting IgM and CD45RC-positive cells spontaneously producing IgM, IgG, and IgA were detected in newborn thymus.Our findings suggest that TBL differentiation and Ig switching to IgG and IgA-secreting cells is not influenced by external antigens and that the thymic microenviroment plays an important role in this process.

This study compares the toxic effects of native cyclosporin A (CyA) with those of targeted CyA that is conjugated with the anti-rat-thymocyte antibody of rabbit originvia the N-(2-hydroxypropyl)methacrylamide (HPMA) carrier bearing digestible, reactive oligopeptide side chains. Ten toxic doses of native CyA (50 mg/kg i.p.) given to young adult rats in the course of 14 d produced a severe renal lesion—diffuse microvacuolization of the proximal tubules in the deep cortex, and hypergranulation of juxtaglomerular regions. Severe atrophy of the thymic medulla was documented by morphometry. In the cortex the epithelial reticular (but not deep interdigitating) cells showed ultrastructural signs of severe degeneration and lysis. The immature CD4+8+ double-positive cortical lymphocytes were preserved whereas, the single-positive medullary thymocytes were greatly depleted; there was also a restriction of MHC class II antigen expression in the medulla. The number of medullary B cells was increased. The cytokeratin net was focally shrunken in the cortex and almost negative in the medulla, with loss of Hassall's corpuscles. After ten corresponding doses of antibody-targeted conjugated CyA no damage to the renal tubules and arterioles appeared and the antiGBM or immune-complex deposition was absent. The thymus had a normal medulla with numerous mature thymocytes and the cortical epithelial reticulum remained well preserved. Thus, the main toxic effects of CyA could be eliminated by targeting. The T-cell-targeted drug was tested for preserved immunosuppressive properties and non-toxic character of HPMA copolymer carrier.

A method for in vitro cultivation of earthworm gut wall tissue explants is described. Tissue explants are able to respond to protein antigen by secretion of antigen-binding protein into the culture medium. Large irregular cells and round-shaped cells are released from tissue explants during the cultivation. This method represents an advantageous tool for in vitro studies providing the possibility of parallel assays from the same donor.