Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Oral butyrate for mildly to moderately active Crohn's disease.

BACKGROUND: Butyrate exerts anti-inflammatory effects in experimental colitis and on Crohn's disease lamina propria mononuclear cells in vitro. AIM: To explore the efficacy and safety of oral butyrate in Crohn's disease. METHODS: Thirteen patients with mild-moderate ileocolonic Crohn's disease received 4 g/day butyrate as enteric-coated tablets for 8 weeks. Full colonoscopy and ileoscopy were performed before and after treatment. Endoscopical and histological score, laboratory data, Crohn's disease activity index and mucosal interleukin (IL)-1beta, IL-6, IL-12, interferon-gamma, tumour necrosis factor-alpha and nuclear factor-kappa B (NF-kappaB) were assessed before and after treatment. RESULTS: One patient withdrew from the study, and three patients did not experience clinical improvement. Among the nine patients (69%) who responded to treatment, seven (53%) achieved remission and two had a partial response. Endoscopical and histological score significantly improved after treatment at ileocaecal level (P < 0.05). Leucocyte blood count, erythrocyte sedimentation rate and mucosal levels of NF-kappaB and IL-1beta significantly decreased after treatment (P < 0.05). CONCLUSIONS: Oral butyrate is safe and well tolerated, and may be effective in inducing clinical improvement/remission in Crohn's disease. These data indicate the need for a large investigation to extend the present findings, and suggest that butyrate may exert its action through downregulation of NF-kappaB and IL-1beta.

Immunoglobulin M memory B cell decrease in inflammatory bowel disease.

BACKGROUND & OBJECTIVES: Memory B cells represent 30-60% of the B cell pool and can be subdivided in IgM memory and switched memory. IgM memory B cells differ from switched because they express IgM and their frequency may vary from 20-50% of the total memory pool. Switched memory express IgG, IgA or IgE and lack surface expression of IgM and IgD. Switched memory B cells derive from the germinal centres, whereas IgM memory B cells, which require the spleen for their survival and/or generation, are involved in the immune response to encapsulated bacteria. Since infections are one of the most frequent comorbid conditions in inflammatory bowel disease, we aimed to verify whether IgM memory B cell pool was decreased in Crohn's disease and ulcerative colitis patients. PATIENTS & METHODS: Peripheral blood samples were obtained from 22 Crohn's disease patients, 20 ulcerative colitis patients, 22 healthy controls and 18 splenectomized patients. To analyse peripheral blood lymphocytes, flow cytometry was performed using anti-CD19, anti-CD22, anti-CD27, anti-IgM, anti-IgD and anti-CD38 monoclonal antibodies. RESULTS: Circulating IgM memory B cells were significantly lower in Crohn's disease (median 7.1%, range 1.8-20.7) and ulcerative colitis patients (median 8.1%, range 2.1-18.8) in comparison to control subjects (median 14.0%, range 6.8-31.1). As expected, there was a highly significant difference in the proportion of IgM memory B cells between splenectomized patients (median 2.4%, range 0.9-6.9) and healthy controls. Crohn's disease patients with abscesses showed the lowest frequency of IgM memory B cells. DISCUSSION: Our findings show that peripheral IgM memory B cells are reduced in inflammatory bowel disease patients. Further studies are necessary to answer the question of whether high risk of infection (abscess development) is promoted by the reduction/depletion of IgM memory B-cell pool in inflammatory bowel disease.

Thymomodulin: biological properties and clinical applications.

Thymomodulin (Ellem Industria Farmaceutica s.p.a., Milan, Italy) is a calf thymus acid lysate derivative, composed of several peptides with a molecular weight range of 1-10 kD. Thymomodulin did not exhibit any mutagenic effect. Furthermore, thymomodulin used in animal studies showed no toxicity even when used at high concentrations. Of major significance are the observations in murine and human systems that thymomodulin remains active when administered orally. In vitro and in vivo administered thymomodulin was able to induce the maturation of T-lymphocytes. Additionally, studies in vitro showed that this thymic derivative can enhance the functions of mature T-lymphocytes with cascading effects on B-cell and macrophage functions. Extensive human clinical trials with thymomodulin showed that this agent can improve the clinical symptoms observed with various disease processes, including infections, allergies and malignancies, and can improve immunological functions during ageing.

"Food allergy in children: an attempt to improve the effects of the elimination diet with an immunomodulating agent (thymomodulin). A double-blind clinical trial".

During 90 days of elimination diet nineteen children with food allergy manifesting atopic dermatitis were treated with either 120 mg/day of thymomodulin (10 subjects) or placebo (9 subjects) in a double blind design. After this period an improvement in skin lesions was observed in both groups. Subsequently a food challenge was performed for two weeks: in the group treated with thymomodulin skin lesions did not modify while they worsened in the placebo group and the comparison was statistically significant (p less than 0.01). Before the beginning of the trial laboratory assessments evidenced an increase in total and specific IgE serum levels, which decreased by the end of the study only in the group receiving the thymic derivative (p less than 0.05).

Thymomodulin increases the depressed production of superoxide anion by alveolar macrophages in patients with chronic bronchitis.

Thymomodulin is an immunomodulating agent which is derived from calf thymus by partial acid lysis. It promotes T-cell maturation, enhances antibody synthesis and improves the phagocytic response of neutrophils. Clinical trials have revealed the effectiveness of this thymic derivative in the prevention of recurrent respiratory infections (RRI) in children and in adults; 11 patients (8 males and 3 females; age range 18-76 years) with chronic bronchitis dominated by recurrent respiratory infections were studied. They were treated orally for 6 months during the winter season with 120 mg/day of thymomodulin. All the subjects were asked to keep a diary recording the intensity of their symptoms, the number of working days lost (days of illness) and the use of antibiotic and/or mucolytic drugs. At the beginning and at the end of the trial each patient was subjected to a control with a flexible fibreoptic bronchoscope with bronchoalveolar lavage to evaluate the phagocytic response of alveolar macrophages. At the end of therapy a significant improvement of the clinical status, evaluated by the above-mentioned parameters, of the bronchial mucosa aspect and an increase in alveolar macrophage superoxide production was noticed (from 0.1 +/- 0.09 and 0.8 +/- 0.5 nmol to 1.6 +/- 0.8 and 4.1 +/- 2.2 nmol with PMA or zymosan particles respectively; p less than 0.001). During thymomodulin treatment no side-effects were recorded.

The combined administration of thymomodulin and interleukin 2 reverses T-cell unresponsiveness in mice infected with Mycobacterium bovis-BCG.

Mice infected intravenously with high doses of M. bovis, strain BCG, showed a marked impairment of delayed-type hypersensitivity to PPD evaluated in in vivo skin tests and in vitro as splenocyte blast transformation. However, this state of unresponsiveness could be partially reversed after 1 month of infection by the intraperitoneal injection of a calf thymus acid lysate (thymomodulin). Furthermore, BCG-infected mice treated in vivo simultaneously with both thymomodulin and interleukin 2 immediately developed positive skin reactions and blast transformation to PPD and did not become anergic in the course of infection.

The effect of a calf thymus acid lysate on bone marrow cell growth in vitro.

Granulocyte-macrophage colony forming units (CFU-GM) were studied in cultures of bone marrow from 16 apparently healthy normal controls, 9 patients with the myelodysplastic syndrome, 5 patients with myeloproliferative disease and 2 with myeloma. Supernatants from non-stimulated 72 hr cultures of nonadherent mononuclear blood cells ("lymphocytes") stimulated the forming of an average of 38.4 colonies per 100,000 cells from normal marrow. The addition of GIBCO's commercial conditioned medium or of a medium produced by lymphocytes stimulated with different concentrations (5, 10 and 20 mcg/ml) of an acid lysate of thymus (thymomoduline), increased growth to 65.2 - 55.4 colonies (p less than 0.001 to 0.05). Similarly, a significant increase (p less than 0.05) was found in the number of clusters and colonies formed in cultures of marrow from patients with the myelodysplastic syndrome. In contrast, no growth was found when the thymus acid lysate was added directly to the bone marrow cultures, suggesting that the lysate induces the production of colony stimulating activity by lymphocytes, but does not contain it. Similarly no significant increase was found as regards the initially high number of colonies from the five patients with myeloproliferative disease, or as regards the initially low number in the two myeloma patients.

A calf thymus acid lysate improves clinical symptoms and T-cell defects in the early stages of HIV infection: second report.

Thymomodulin is a calf thymus acid lysate capable of inducing T lymphocyte maturation. Fifteen patients with HIV infection at different stages according to the Walter Reed classification were treated with 60 mg/day of thymomodulin syrup for more than 50 days. Two WR6B subjects had clinical and immunological parameters unchanged and died, while the patient suffering from Kaposi's sarcoma presented an evident clinical and laboratory improvement with remission of the neoplasia. The other 12 patients ranging from WR2 to WR5B showed an improvement of clinical symptoms after thymomodulin therapy accompanied by the normalization of CD4/CD8 ratio (P less than 0.001). This helpers/suppressors increase was due to a significant increase of CD4 cells (P less than 0.01) and also to a decrease of the CD8 lymphocytes (P less than 0.05). Thymomodulin administration did not cause an enhancement of the urinary levels of neopterin, a marker of T-cell activation.

[Perennial allergic rhinitis. Prophylaxis of acute episodes using thymomodulin]. / Rinopatia allergica perenne. Profilassi degli episodi acuti mediante l'impiego di timomodulina.

The therapeutical effectiveness of a calf thymic derivative (thymomodulin), orally administrable, in patients suffering from perennial allergic rhinitis is described. The use of thymomodulin for 4 months at the dosage of 120 mg daily in 20 patients significantly reduced (p less than 0.001), if compared with the previous year, the monthly mean frequency of allergic episodes. Also objective symptoms improved (p less than 0.05). The clinical course was associated with the normalization of IgE plasmatic levels (p less than 0.001) and consequently the mean of RAST and PRICK tests scores decreased (p less than 0.001). Eosinophil count was significantly reduced (p less than 0.001), while IgG serum levels were increased (p less than 0.02).

[Prevention of recurrent respiratory infections in adults]. / Prevenzione delle infezioni respiratorie ricorrenti dell'adulto.

The authors compared the results obtained by using antibiotic therapy, vaccine, thymomodulin (a calf thymus acid lysate) and association of vaccine-thymomodulin in order to prevent acute infectious episodes in a group of 85 patients suffering with recurrent respiratory infections. The use of thymomodulin, alone and in association with vaccine, at the dose of 120 mg/die for 20 days/month during the period of observation (4 months), determined a higher reduction, (p less than 0.001) if compared with the other treatments, of the number and the duration of infectious episodes and moreover of the antibiotics' intake. Also the respiratory symptoms, and in particular the fits of coughing, showed an improvement. The pulmonary function indices and the laboratory parameters were unchanged in all groups studied.

Update and future perspectives of a thymic biological response modifier (Thymomodulin).

Thymomodulin (Ellem Industria Farmaceutica spa, Milan, Italy) is a calf thymus acid lysate with immunomodulating activities. It is composed of several peptides with a molecular weight range of 1-10kD. Extensive studies in animal systems showed that Thymomodulin exhibited no, or very little toxicity even when used at high doses. Studies done in vitro and in vivo demonstrated that Thymomodulin is a biologically active compound which regulates the maturation of human and murine pre T lymphocytes, as well as modulate the functions of apparently mature human and animal B and T lymphocytes. It was observed that Thymomodulin can promote myelopoiesis as demonstrated by an increase of granulocyte-macrophage colonies in agar. Although additional studies to examine its target cell lineage are required, it appears that Thymomodulin exhibits specificity toward T cells. Therefore, enhancement of other cell lineage functions by Thymomodulin may be indirect, and mainly due to its effect on T cells. Of major importance is to note that Thymomodulin is prepared in a manner which allows it to maintain its biological activity when administered orally.

[Scanning electron microscopy study of changes induced by thymomodulin on the morphology of mononuclear elements of peripheral blood from healthy subjects and patients with neoplasms]. / Studio al S.E.M. delle modificazioni indotte dalla timomodulina sulla morfologia degli elementi mononucleati del sangue periferico di soggetti sani e di pazienti con neoplasia.

The authors studied with scanning electron microscopy the surface of peripheral mononucleated blood cells of healthy subjects and neoplastic patients before and after incubation with thymomodulin, a calf thymus derivative with immunomyelomodulating activity. In the healthy subjects before incubation with thymomodulin a wide number of lymphocytes with villous surface was observed, while monocytes appeared as bigger cells with a central portion of cytoplasm with an adhesion plate. Already after an hour of incubation with the thymic derivative an increase of rough lymphocytes and the appearance of cytoplasmic bridges between lymphocytes and monocytes were observed. Afterwards thymomodulin induced in lymphocytes the forming of cytoplasmic appendages named "uropods" and "pseudopods" which are expression of lymphocytic activation. In cancer patients at base line "monstrous" cells, considered as suffering lymphocytes, were seen and they became morphologically normal after incubation with the thymic derivative.

A double-blind clinical trial for the evaluation of the therapeutical effectiveness of a calf thymus derivative (Thymomodulin) in children with recurrent respiratory infections.

The effectiveness of an orally administrable thymic derivative (Thymomodulin) for the treatment of the recurrent respiratory infections (RRI) in children has been studied in a double-blind clinical trial with historical comparison. In the Thymomodulin treated group a significant decrease of the monthly frequency of RRI has been observed in comparison with the previous year (P less than 0.05) and with the placebo treated group (P less than 0.002). The evaluation of the laboratory data at the beginning of the study didn't show in the two groups typical alterations of the common hematological and immunological parameters, but at the end of the trial a statistically significant increase of the levels of salivary IgA has been noticed only in the Thymomodulin treated group (P less than 0.02).

Demonstration of lysozyme in primary ovarian tumors using the immunoperoxidase technique.

The presence of lysozyme in ovarian tumors has been detected for the first time using the immunoperoxidase technique. A study of the distribution of this enzyme in 50 primary ovarian neoplasms revealed its presence exclusively in mucinous and mixed (serous-mucinous) cystadenomas and cystadenocarcinomas. The secretion of lysozyme seems to be correlated to the production of strongly acidic mucins.

Orcein-positive material in hepatocytes in viral hepatitis and other liver diseases.

Liver biopsy samples from 110 patients with various liver diseases were stained by orcein according to the method of Shikata et al. Orcein-positive hepatocellular material was observed in only the 31.7% of HBsAg seropositive cases. A positive orcein reaction was frequently found in protracted and chronic viral hepatitis and occasionally in other liver diseases, such as alcoholic and cholestatic hepatitis, as well as in cryptogenetic cirrhosis and in liver metastases. The results obtained suggest a more cautious evaluation of the diagnostic and prognostic significance of orcein-positive hepatocellular material.

Histochemical study of orcein-positive hepatocellular material in paraffin sections of liver biopsy samples in the course of various liver diseases.

A histochemical study was carried out on orcein-positive hepatocellular material in 111 cases, 27 of which were stained positively by orcein. Orcein-positive material was very frequently found in protracted viral hepatitis and in chronic active hepatitis, as well as in other liver diseases with or without cholestasis; it was absent in liver cirrhosis. In all the cases considered the orcein-positive material was mainly formed of proteins, rich in amino acids with sulfhydryl and disulphide radicals, tryptophan and, to a lesser extent, of NH2 alpha amino acids. The orcein-positive substance was apparently carbohydrate-free. The presence of copper was also confirmed.