Congenital heart block and immune mediated sensorineural hearing loss: possible cross reactivity of immune response.

Immune-mediated sensorineural hearing loss may complicate systemic autoimmune diseases. We have previously reported the presence of antibodies directed against inner ear antigens in patients with Cogan syndrome, a disease characterized by sudden hearing loss and interstitial keratitis. Such autoantibodies cross-react with an epitope of SSA/Ro60 protein. Anti-Ro/SSA antibodies in pregnant women cross the placenta and reach the fetal tissues inducing an immune-mediated damage of the cardiac conduction system. We wanted to evaluate whether mothers with anti-Ro/SSA antibodies who gave birth to children with congenital heart block have antibodies directed against inner ear antigens and whether these antibodies are connected with the presence of immune-mediated sensorineural hearing loss. We did not find anti-inner ear antibodies in the majority of the mothers. On the contrary a 13-year-old boy with congenital heart block and sensorineural hearing loss was positive for the presence of anti-inner ear antigens antibodies. Moreover his serum was positive for the presence of anti-Ro60 peptide antibodies but did not recognize the entire protein Ro60 (TROVE2), a behaviour similar to that of sera from patients with Cogan syndrome. In conclusion the data obtained so far show that anti-inner ear antibodies do not recognize the entire protein TROVE2 and do not support the hypothesis that such antibodies may be involved in the pathogenesis of congenital heart block.

Infections and autoimmunity: role of human cytomegalovirus in autoimmune endothelial cell damage.

Molecular mimicry between infectious agents and normal human host cell proteins represents one of the possible mechanisms responsible for autoimmunity. Among infectious agents, human cytomegalovirus (HCMV) is an ideal candidate for involvement in autoimmune disorders because of its lifelong persistence through periods of reactivation and latency and because of the extensive manipulation of innate and adaptive immunity. HCMV has been implicated in the pathogenesis of vascular damage in systemic sclerosis (SSc) and atherosclerosis. Based on our data, which demonstrate a cause-and-effect relationship between HCMV and endothelial cell aggression in SSc and atherosclerosis, we propose that immune responses to particular HCMV proteins may result in autoaggression through a mechanism of molecular mimicry of normally expressed endothelial cell surface molecules.

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.

In chronic idiopathic urticaria autoantibodies against Fc epsilonRII/CD23 induce histamine release via eosinophil activation.

BACKGROUND: Chronic idiopathic urticaria is a common skin disorder characterized by recurrent, transitory, itchy weals for more than 6 weeks. An autoimmune origin has been suggested based on the findings of auto-antibodies (Abs) directed against either the alpha subunit of the high-affinity IgE receptor or the IgE molecule in nearly half of the patients. OBJECTIVE: To identify other autoantigen targets in patients with chronic idiopathic urticaria. METHODS: We used pooled IgG derived from 133 patients with chronic idiopathic urticaria to screen a random peptide library to identify disease-relevant autoantigen peptides. Among the identified peptides, one was recognized by the vast majority of patients' sera. Abs against this peptide were affinity purified from the patients' sera and assayed for their ability to induce histamine release from basophils. RESULTS: We identified a peptide that showed similarity with the low-affinity IgE receptor (Fc epsilonRII/CD23) expressed on lymphomonocytes and eosinophils. Anti-peptide IgG Abs purified from the patients' sera bound cell surface CD23 and were able to induce histamine release from basophils. This effect appeared to be mediated by the release of major basic protein from eosinophils upon engagement of CD23. The same effects were obtained with the sera from mice immunized with the CD23 peptide. CONCLUSION: Our results indicate that patients with chronic idiopathic urticaria have Abs against CD23 and that eosinophils, which infiltrate the skin of these patients, play a crucial role in maintaining the disease through the release of major basic protein upon engagement of the low-affinity IgE receptor by such auto-Abs.

Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells.

Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin-NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.

Glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders.

Our objective was to investigate the presence of a B and T cell immune response directed against the glycine-rich cell wall protein (GRP) in patients with different autoimmune disorders and with food allergy. GRP is an ubiquitous food protein that has high homology with cytokeratins and other self proteins [Epstein-Barr virus nuclear antigen-1 (EBNA-I), heterogeneous nuclear ribonucleoprotein, fibrillar collagen] which are common targets in autoimmune disorders. A peptide (GGYGDGGAHGGGYGG) derived from GRP was used to screen human sera in direct and competitive ELISA assay. Anti-GRP-specific IgG were analyzed for their ability to cross-react with autoantigens. The intracellular cytokine profiles of the peptide-specific T cell clones obtained from representative patients have been studied. BALB/c mice were immunized with the peptide coupled to the carrier protein keyhole limpet hemocyanin (KLH). Serum IgG antibodies directed against the GRP peptide were detected in several autoimmune disorders and in food allergic patients, and were able to cross-react with autoantigens including keratin, collagen and EBNA-I. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide and were of the T(h)0 phenotype. Eight of the 10 BALB/c mice immunized with the peptide coupled to KLH developed an autoimmune response. Our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals. They also indicate that the antigen spreading of a particular sequence among apparently divergent proteins may participate to initiate or amplify an immune response.

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.

Androgen metabolism and inhibition of interleukin-1 synthesis in primary cultured human synovial macrophages.

The presence of androgen receptors on synovial macrophages in human normal and rheumatoid synovial tissues has been described previously. It is now reported that primary cultured human macrophages obtained from normal and rheumatoid synovia express functional androgen receptors. We have investigated the capacity of cultured macrophages to metabolize androgens and have found that these cells were capable of metabolizing testosterone to the bioactive metabolite dihydrotestosterone. Therefore, macrophages contain the key enzymes of steroidogenesis, in particular the 5alpha-treductase. Furthermore, interleukin-1beta production by primary cultured rheumatoid macrophages was analysed, following exposure to physiological concentrations of testosterone (10(-8) M). A significant decrease of IL-1beta levels in conditioned media after 24 h (p < 0.05) was observed. It is concluded that androgens may act directly on human macrophages and may interfere with some of their functions via receptor-dependent mechanisms.

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.