Antiflagellin antibodies recognize the autoantigens Toll-Like Receptor 5 and Pals 1-associated tight junction protein and induce monocytes activation and increased intestinal permeability in Crohn's disease.

BACKGROUND AND OBJECTIVES: Bacterial flagellin is considered an important antigen in Crohn's disease (CD) as it activates innate immunity through Toll-Like Receptor 5 (TLR5) engagement and induces an elevated adaptive immune response. Little is known about the presence of an autoimmune process in CD. We aimed to identify pathogenically relevant autoantigen targets in CD. METHODS: We screened a random peptide library with pooled sera of patients with active CD. Transepithelial flux of [3H] mannitol in T84 human intestinal epithelial cell line was used to study the epithelial barrier function. Monocyte activation was evaluated by surface expression of activation markers and by production of pro-inflammatory cytokines. Gene modulation of T84 cells exposed to antipeptide antibodies was analysed by gene array. RESULTS: We identified a peptide that shares homology with Salmonella typhimurium flagellin and with self-antigens such as TLR5 and cell junction protein, Pals 1-associated tight junction protein. The affinity-purified antipeptide antibodies recognized the self-antigens and induced increased intestinal epithelial cell permeability. Moreover, the antibodies induced monocyte activation upon binding TLR5. Finally, in cultured intestinal cells (T84) the purified antibodies induced the modulation of clusters of proinflammatory genes similar to the one induced by the engagement of TLR5 by its natural ligand flagellin. CONCLUSIONS: Antibodies directed against an immunodominant peptide of flagellin recognize self-antigens and are functionally active suggesting the presence of an autoimmune process that can both facilitate loss of tolerance to intestinal microflora by increasing cell permeability and amplify the innate immunity involvement through a novel mechanism of TLR5 activation.

Granulocyte-macrophage colony-stimulating factor activity in cerebrospinal fluid.

OBJECTIVES: The purpose of this study was to analyse the presence of the granulocyte-macrophage colony-stimulating factor (GM-CSF) in human cerebrospinal fluid (SF) of patients affected by multiple sclerosis (MS) in comparison with non-inflammatory neurological diseases. MATERIAL AND METHODS: All SFs were collected from 59 patients for diagnostic purpose. The presence of GM-CSF was revealed by measuring its activity and by immunoassay. The data obtained were statistically evaluated. RESULTS: We found that GM-CSF is constitutively present in human SF; this presence was confirmed by its stimulating activity of colony-forming-unit granulocyte-macrophage (CFU-GM) production. No significant changes of the GM-CSF concentration in the SFs were observed among different neurological disorders (degenerative or vascular) and MS. CONCLUSION: Our data suggest that GM-CSF is a constitutive component of human SF, relatively uninfluenced by the different morbid conditions of the nervous system.

Mitoxantrone in elderly patients with advanced breast cancer: pharmacokinetics, marrow and peripheral hematopoietic progenitor cells.

The aims of this study were to evaluate the pharmacokinetics, tolerability and hematopoietic toxicity of mitoxantrone in elderly women. Thirteen patients with advanced breast cancer, median age of 73 years, received escalating doses of mitoxantrone 8, 10, 12 and 14 mg/m2 on day 1, q 21. There was a linear relationship between the mitoxantrone dose administered and the mitoxantrone exposure (AUC) in plasma (r = 0.856, pc0.001). After 4 courses of treatment, a significant decrease in bone marrow cellularity (p = 0.0067), and HPC content (BFU-E p = 0.0077) was observed. A remarkable, though not statistically significant decrease in circulating HPCs was observed after 4 courses and was still present 8-12 months after the termination of treatment. Therapy with mitoxantrone in elderly women was well tolerated at the dose of 12 mg/m2 for four courses. The significant hematological toxicity observed in marrow cellularity and HPC content warrant further studies.

Increased bronchoalveolar granulocytes and granulocyte/macrophage colony-stimulating factor during exacerbations of chronic bronchitis.

Although inflammatory changes are found throughout the airways of patients with chronic bronchitis, the mechanisms of the pathogenesis of chronic bronchitis are still unclear. The aim of this study was to investigate airways inflammation in patients with and without an exacerbation of bronchitis. Thirteen chronic bronchitic patients and nine normal subjects were studied. Eight of the patients were studied under baseline conditions (B), and five during an exacerbation of bronchitis (E). Bronchoscopy and bronchoalveolar lavage (BAL) with cytological analysis were performed, and the levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) were determined in sera and in BAL supernatants by a solid phase enzyme immunoassay. Compared with patients under baseline conditions, chronic bronchitic patients with an exacerbation had increased numbers of BAL neutrophils (10+/-3 and 83+/-18x10(3) cells x mL(-1), respectively; p<0.0001) and of BAL eosinophils (1.9+/-0.5 and 6.7/-1.9x10(3) cells x mL(-1), respectively; p=0.014). Patients with chronic bronchitis, as a whole, had significantly increased levels of BAL GM-CSF compared to control subjects (36+/-5 and 19+/-4 pg x mL(-1), respectively; p=0.035), and similar levels of serum GM-CSF. Serum levels of GM-CSF were markedly increased in chronic bronchitic patients with an exacerbation, as compared with patients under baseline conditions (1.4+/-0.4 and 13+/-1 pg x mL(-1), respectively; p <0.0001). BAL levels of GM-CSF were also increased in chronic bronchitic patients with an exacerbation (25+/-5 and 54+/-8 pg x mL(-1), respectively; p=0.009). During exacerbations of chronic bronchitis there are changes in the cell populations in bronchoalveolar lavage of patients consistent with a recruitment of polymorphonuclear leucocytes in the airway lumen. The increased levels of granulocyte/macrophage colony-stimulating factor might suggest a role for this cytokine in the inflammatory processes of chronic bronchitis.

Human T lymphocyte cell line (Mo) and its subclone (J) produce colony stimulating activity on normal and malignant T cell precursors.

Conditioned medium from a T-lymphoblastic cell line (Mo) is known to produce factors promoting CFU-GM, BFU-E and CFU-MK. In our study we investigated the potential CSA of conditioned media obtained from Mo and its subclone J on normal and malignant lymphoid progenitors of both T and B lineage. Both cell lines release factors inducing a significant increase in number and size of T-lymphoid colonies when compared to standard source of factors (PHA-LCM). On the contrary, they presented a low CSA on B cell precursors confirming the difficulties in identifying a source of growth and differentiation factors for human B cell ontogeny. This study contributes to the knowledege of biological properties of these tumor cell lines, suggesting the possibility to employ Mo- and J-derived supernatants in vitro for improving growth potential of normal and malignant T cell progenitors.

Release of peripheral blood progenitor cells during standard dose cyclophosphamide, epidoxorubicin, 5-fluorouracil regimen plus granulocyte colony stimulating factor for breast cancer therapy.

BACKGROUND: High dose chemotherapy with the support of peripheral blood progenitor cells (PBPC) is increasingly used in the treatment of solid tumors. Although the best method of PBPC mobilization is still under investigation, it should be optimized for different tumor types to obtain antitumor effect and mobilizing activity. The authors report these results in terms of the number of PBPC released and the time of maximum mobilization induced by standard dose cyclophosphamide, epidoxorubicin, 5-fluorouracil (CEF) (cyclophosphamide 600 mg/m2, epidoxorubicin 60 mg/m2, 5-fluorouracil 600 mg/m2) plus granulocyte colony stimulating factor (G-CSF) in patients with breast cancer. METHODS: Peripheral blood progenitor cells were studied by clonogenic assay of granulocyte macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming unit (CFU-Meg) and erythrocyte burst-forming unit (BFU-E) and by flow cytometric analysis of CD34+ cells in 12 patients with early breast cancer throughout three cycles of CEF chemotherapy plus G-CSF. RESULTS: Colony assays and CD34+ cell determination were performed on 111 and 151 blood samples, respectively. The peak of CFU-GM and CD34+ cells occurred consistently at day 11 throughout all three cycles. At day 11 of the first cycle, the median peak values were 2223 CFU-GM/mL and 256 CD34+ cells/microL. A progressive decrease in peak value from the first to the third cycle was observed. CONCLUSIONS: Standard dose CEF chemotherapy plus G-CSF is a disease specific regimen allowing PBPC mobilization without any relevant toxicity. Maximum mobilization was recorded at day 11 of the first cycle.

Serum levels of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in treated patients with chronic myelogenous leukemia in chronic phase.

BACKGROUND: Despite the fact that granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are increasingly used in clinical practice, little is known of their endogenous production, especially in myeloproliferative disorders such as chronic myelogenous leukemia (CML). METHODS: In order to define serum levels of GM-CSF and G-CSF in subjects affected by CML, the sera of 17 patients with CML in chronic phase treated either with hydroxyurea or interferon-alpha were tested by specific enzyme immunoassays. Fifteen age- and sex-matched healthy volunteers were used as normal controls. RESULTS: Eight out of the 17 patients (44%) with CML showed detectable (> 3 pg/mL) serum levels of GM-CSF (range 3.9-55 pg/mL). Detectable levels (> 50 pg/mL) of G-CSF were observed in 9 of these patients (52%) (range 150-2,830 pg/mL). On the contrary, among the normal controls only one had detectable GM-CSF concentrations, and none had detectable G-CSF concentrations. The highest concentrations of both GM-CSF and G-CSF were seen in patients with the highest white blood cell counts, although a linear correlation between the levels of these growth factors and the number of circulating leukocytes was not demonstrated. CONCLUSIONS: Our data indicate that significant amounts of both endogenous GM-CSF and G-CSF are detectable in the serum of a substantial percentage of patients with CML in chronic phase. The pathophysiological meaning of this finding remains to be determined.