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.

Clinical features of microinvasive stage I oral carcinoma.

BACKGROUND: This study aimed to analyse a case series of microinvasive (tumour thickness <4 mm) stage I oral squamous cell carcinoma (OSCC), with an emphasis on the clinical features of the tumours. METHODS: In total, 32 microinvasive and 67 non-microinvasive stage I lesions, which had been surgically treated, were retrospectively studied and compared. The data analysed included gender, age, risk habits, clinical appearance, lesion site, symptoms, nodal involvement and outcome. RESULTS: The clinical features of microinvasive lesions meant that, more often than not, they resembled premalignant lesions (P = 0.008), and diagnosis was mainly based on accurate clinical examination rather than the presence of symptoms (P = 0.029). During a median follow-up of 4.5 years, one nodal involvement and one cancer-related death were observed in patients with microinvasive lesions. A significantly higher (P = 0.044) level of nodal involvement was observed in the non-microinvasive lesion group. CONCLUSIONS: Stage I OSCC has a favourable prognosis overall, but nodal recurrence is more common in non-microinvasive cancers. As microinvasive lesions tend to present clinically as premalignant lesions, accurate clinical examination is essential if misdiagnosis of early lesions is to be avoided.

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.

Non-surgical periodontal treatment of cyclosporin A-induced gingival overgrowth: immunohistochemical results.

AIM: The present study was planned to analyze the effects of a 12-month non-surgical periodontal treatment on histologic and immunohistochemical features of cyclosporin A (CsA)-induced gingival overgrowth (GO). MATERIALS AND METHODS: Gingival samples were collected from 21 liver transplant subjects exhibiting CsA-induced GO prior to, and 12 months after non-surgical periodontal therapy including oral hygiene instructions, scaling and 2-month recall appointments, and also from 18 healthy control subjects. Gingival biopsy specimens were stained with hematoxylin-eosin and monoclonal antibodies for vimentin, CD3 (T-lymphocytes), CD20 (B-lymphocytes), CD34 (endothelium) and Ki-67 (fibroblasts proliferation rate), using a streptavidin-biotin-peroxidase complex method. RESULTS: Total inflammatory cells, gingival vessels and fibroblast proliferation rate demonstrated significant reduction after non-surgical periodontal treatment (P < 0.0001) in overgrown gingiva, while B- and T-lymphocytes remained nearly unchanged (P = 0.61 and 0.33, respectively). At the 12-month evaluation no significant differences were found when comparing the gingival biopsies from CsA-treated patients and those from healthy controls (P > 0.05). CONCLUSIONS: Control of clinical inflammation by means of non-surgical periodontal treatment results both in lowering of inflammatory infiltrate and in changes in connective tissue composition. Thus, plaque-induced inflammation would seem to modulate the drug-gingival tissue interaction. CLINICAL RELEVANCE: A strict plaque control program play a pivotal role in the management of transplant patients exhibiting cyclosporin A-GO.

Characterizing pearls structures using X-ray phase-contrast and neutron imaging: a pilot study.

Some cultured and natural pearls can be reliably distinguished by visual inspection and by the use of lens and microscope. However, assessing the origin of the pearls could be not straightforward since many different production techniques can now be found in the pearl market, for example in salt or freshwater environments, with or without a rigid nucleus. This wide range of products requires the use of new effective scientific techniques. Indeed, X-ray radiography has been used by gemologists since last century as the only safe and non-destructive way to visually inspect the interior of a pearl, and recently, also X-ray computed micro-tomography was used to better visualize the inner parts of the gems. In this study we analyzed samples of natural and cultured pearls by means of two non-destructive techniques: the X-ray Phase-Contrast Imaging (PCI) and the Neutron Imaging (NI). PCI and NI results will be combined for the first time, to better visualize the pearls internal morphology, thus giving relevant indications on the pearl formation process.

Risk groups of myeloma patients by histologic pattern and proliferative activity.

We investigated the histologic pattern and the cell proliferative activity of myeloma cells by the analysis of the nucleolar organizer regions (AgNORs) in bone marrow biopsy specimens from 150 multiple myelomas at diagnosis. The objective was an attempt to define risk groups of myeloma patients. On univariate analysis, the percentage of bone marrow plasma cells (BMPC%), the pattern of infiltration, the degree of plasma cell (PC) atypia, the marrow fibrosis, and the number of AgNOR/PC were correlated with survival time. On multivariate analysis, only AgNOR counts and pattern of infiltration retained independent prognostic significance. At 4-year followup, all patients with BMPC% < or = 20, interstitial pattern of invasion, and well-differentiated (G1) PC plus AgNOR/cell < or = 3.32 were alive, while no patient with BMPC% >50, diffuse pattern of infiltration, and poorly differentiated (G3) PC plus AgNOR/cell >5.15 survived (p < 0.0001). In conclusion, the histologic pattern and proliferative activity of myeloma cells, evaluated by AgNOR counts, are reliable predictors of survival in myeloma. Both parameters can be easily assessed in the same biopsy specimen, are reproducible, and permit identification of a group of patients with favourable outcome at 4-year followup. Thus, bone marrow biopsy should always be included in the diagnostic procedures for myeloma patients.

Biologic differences between noninvasive papillary urothelial neoplasms of low malignant potential and low-grade (grade 1) papillary carcinomas of the bladder.

We investigated the expression of oncogenes p53, c-erbB-2, and bcl-2 and cell proliferative activity in 62 newly diagnosed superficial pTa papillary bladder tumors. Based on the 1998 World Health Organization/International Society of Urological Pathology (WHO/ISUP) and 1999 WHO classifications, 19 were urothelial neoplasias of low malignant potential (LMP) and 43 low-grade (grade 1) papillary carcinomas. All the patients underwent transurethral resection and were followed up to 97 months; 42 had recurrences. Initial biopsies were tested for p53, c-erbB-2, and bcl-2 proteins using DO7, CB11, and bcl-2 124 monoclonal antibodies. Cell proliferation was assessed by MIB-1 mAb and mitotic count. LMP had significantly lower MIB-1 (p = 0.002) and p53 immunopositivity (p = 0.03), mitotic count (p = 0.006), and recurrence rates (p = 0.04) than did grade 1 cases, whereas no difference was observed for c-erbB-2 and bcl-2 expression. The median disease-free survival for LMP was 76 months but only 15 months for grade 1 cases (p = 0.002). Although the cohort is small, the results indicate that the distinction between LMP and low-grade (grade 1) papillary urothelial neoplasias, as proposed by the 1998 WHO/ISUP and 1999 WHO classifications, reflects different biologic activity and clinical behavior; however, a long-term follow-up is advisable also for patients with LMP.

P53 overexpression correlates with proliferative activity and prognosis in carcinomas of the pyriform sinus.

p53 overexpression and proliferative activity were investigated in 28 squamous cell carcinomas of the pyriform sinus of the hypopharynx prior to therapy, using DO1 and MIB-1 monoclonal antibodies in routinely processed biopsies. MIB-1 scores were associated with tumour histological grade (35.4% for grade 3 versus 23.8% for grade 2 cases; p=0.008) and survival (the median of survival was 23 months for cases with MIB-1 scores less than or equal to 33.8% but 11 months only for cases with MIB-1 scores >33.8%; p<0.001). p53 scores were associated with tumour histological grade (56.5% for grade 3 versus 37.1% for grade 2 cases; p=0.02) and survival (median of survival 20 months for cases with p53 scores less than or equal to 56.2% versus 11 months for cases with p53 scores >56.2%; p=0.002). Tumour histological grade was also correlated with prognosis (median of survival 50 months for grade 2 versus 14 months for grade 3 cases; p=0.03). In the multivariate analysis, only MIB-1 (p=0.001) and p53 scores (p=0.003) had an independent prognostic significance. A linear relationship between p53 and MIB-1 scores was observed (r=0.54; p=0.012). With the limitation due to the small number of cases, our findings indicate that p53 overexpression correlates with proliferative activity and survival in squamous cell carcinomas of the pyriform sinus, and suggest the use of p53 and MIB-1 immunostainings in the pretherapeutic assessment of the tumour aggressiveness.

Argyrophilic nucleolar organizer region counts and proliferating cell nuclear antigen scores are two reliable indicators of survival in pharyngeal carcinoma.

The proliferative activity of pharyngeal carcinoma has been investigated by means of monoclonal antibody PC10 against proliferating cell nuclear antigen (PCNA/cyclin) and argyrophilic nucleolar organizer region (AgNOR) analysis in formalin-fixed, paraffin-embedded biopsies from 45 primary squamous and undifferentiated carcinomas, prior to therapy. The correlation between AgNOR counts and PCNA(PC10) scores was highly significant (r = 0.73; P < 0.0001) as determined by Pearson's correlation coefficient. Moreover, the univariate Kaplan-Meier survival analysis showed a significant correlation between 3- and 5-year survival rates and the mean AgNOR number per tumour cell (P = 0.0003) or the percentage of PCNA(PC10)-positive cells (P = 0.0001). Our results indicate that both AgNOR counts and PCNA(PC10) scores are reliable markers of the proliferative activity of pharyngeal carcinoma in small, routinely processed biopsies, in which they can allow simultaneous evaluation of the histology and tumour cell kinetics.

G1 heavy chain disease: clinicopathological, ultrastructural and immunochemical study of a new case.

Clinicopathological ultrastructural, and immunohistochemical findings of a new case of IgG1 heavy chain disease are reported in detail. The abnormal protein lacks the VH and CH1 region with sequence starting at 225 residue. The main pathologic feature was plasmacytic tumor of the lymph nodes with B-cell immunoblastic sarcoma patterns. Neoplastic diffusion to other organs was also present. Plasmacytic neoplastic cells have also been found in the bone marrow. The ultrastructure of the noeplastic cells was characterized by more or less abundant plasmacytic-like endoplasmic reticulum with very frequent peculiar whorled configurations. Immunohistochemical methods revealed the abnormal protein production by neoplastic cells in different stages of differentiation. From this case and from the data of the literature it is concluded that the gamma HCD is due to a neoplastic proliferation of lymphoplasmacytic cells whereas the reticulum cells are never involved.

Cell proliferation indices, morphometry and DNA flow cytometry provide objective criteria for distinguishing low and high grade bladder carcinomas.

Argyrophilic nucleolar organizer region (Ag-NOR) analysis, proliferating cell nuclear antigen (PC-NA/PC10) and MIB-1 immunohistochemistry, nuclear morphometry and DNA flow cytometry have been performed on formalin-fixed, paraffin-embedded biopsies from 50 patients with transitional cell carcinoma of the urinary bladder. The mean AgNOR count was 6.01 for the 17 grade 1 (G1), 7.59 for the 21 G2 and 13.33 for the 12 G3 carcinomas (p < 0.001). The mean PCNA score was 15.03% for G1, 24.04% for G2 and 40.01% for G3 cases (p < 0.001). The mean MIB-1 score was 11.31% for G1, 17.09% for G2 and 34.47% for G3 carcinomas (p < 0.001). The mean nuclear area was 35.53 microns2 for G1, 38.65 microns2 for G2 and 83.62 microns2 for G3 cases (p < 0.001). Aneuploidy rates were significantly higher (91.7%) in G3 than in G2 (42.9%, p < 0.01) or G1 cases (47.1%, p < 0.05) but not different for G1 versus G2 cases (p = 0.94). While many overlaps of values were seen between G1 and G2 tumours, no overlaps were found between G3 and G1/G2 tumours. Significant differences of values were also found between pTa and invasive tumours (p < 0.0001 for AgNOR count and PCNA score; p < 0.001 for MIB-1 score and mean nuclear area; p < 0.01 for DNA ploidy); however many overlaps were seen. Our findings indicate that the quantitative parameters obtained with different methods are associated with histological grade of bladder urotheliomas and may improve the grading reproducibility. In addition, the absence of overlaps between G3 and G2/G1 carcinomas supports the tendency to classify bladder urotheliomas in only two categories of malignancy.

High prognostic impact of growth fraction parameters in advanced stage laryngeal squamous cell carcinoma.

One hundred and two cases of laryngeal squamous cell carcinoma, all treated in the same center with total or supraglottic laryngectomy, bilateral neck dissection and postoperative radiotherapy, were investigated with both Ki67 and MIB-1 monoclonal antibodies. The aim was to determine the prognostic impact of growth fraction markers in a homogeneous series of patients. All samples were stained with Ki67 monoclonal antibody on frozen sections, and with MIB-1 monoclonal antibody on paraffin sections, using the ABC immunoperoxidase method. The percentage of positive cells was compared in each case with the overall and disease-free survival, pathologic stage and histologic grading. The values obtained from Ki67 and MIB-1 counts were similar and highly correlated (r=0.90). Two groups of cases with low and high proliferation rate (59 and 43 respectively) were obtained by splitting up the whole series, on the basis of the median value; 84. 6% of the patients with high proliferation relapsed and/or died due to the tumor within two years from diagnosis whereas, at time of writing, 94% of the patients with low proliferation are alive and well (p<0.00001). No relation was found between growth fraction and histologic grading, pathologic stage (pT and pN) and site of the tumor. Only lymph node involvement was correlated with disease-free survival. Our results indicate that Ki67/MIB-1 index represents an independent variable to determine long-term prognosis in laryngeal squamous cell carcinomas. We recommend its use in diagnostic protocols, to distinguish high risk subsets of patients who might benefit from more aggressive treatments.

Bone marrow involvement in Kaposi's disease. Report of a case.

A 71-year-old woman with Kaposi's sarcoma was admitted to the hospital because of severe pancytopenia with negative sternal aspirate. The bone marrow trephine biopsy demonstrated an extensive replacement of the hematopoietic tissue by Kaposi's sarcoma. Skin, soft palate mucosa, spleen, retroperitoneal and axillary lymph nodes were also found to be involved at the post-mortem examination. Therefore the bone marrow biopsy can be used in the staging of Kaposi's sarcoma in presence of pancytopenia and might be able to detect a primary visceral involvement in patients without cutaneous lesions.

Antineoblastic activity of antioxidant vitamins: the role of folic acid in the prevention of cervical dysplasia.

The authors made a study on 90 patients affected by various degrees of uterine cervix dysplasia searching for folic acid plasmatic concentrations. The team members affected by CIN have been compared with a test team consisting of women with normal pap-test and vaginoscopy. The study proved that the average levels of folic acids have significantly decreased in cases of dysplasia compared with the test team. These results allow stating that low folic acid plasmatic concentrations may be associated with cervix neoplasms development.

Danazol in the treatment of endometrial hyperplasia.

The paper reports the results of a study performed in 62 menorrhagic women with endometrial hyperplasia treated with Danazol. The efficacy and tolerability of the above drug was found to be satisfactory.

Bone marrow histopathology of acute nonlymphocytic leukemia following therapy for primary malignancies.

Therapy-related acute nonlymphocytic leukemias occur with increasing frequency owing to modern aggressive antineoplastic therapies. Out of 3,138 bone marrow trephine biopsies, there were 148 cases of acute nonlymphocytic leukemias. Of these, 14 cases occurred 30-156 months following chemotherapy or radiotherapy or both for malignant disease. The male/female ratio was 0.27 (vs. 1.6 of "de novo" leukemias). Primary malignancies (7 Hodgkin's disease, 1 fibrosarcoma and 6 carcinomas) had been treated with chemotherapy+radiotherapy (10 cases), with chemotherapy alone (3 cases) or with radiotherapy alone (1 case) and were apparently cured. All therapy-related leukemias were heralded by a preleukemic cytopenic phase. Response to therapy was poor (mean survival 3.9 months). Bone marrow histopathological findings showed in 13 cases acute myelo- or monoblastic leukemia and in 1 case erythroleukemia. Out of 21 biopsies, there were increased numbers of abnormal megakaryocytes in 10, megaloblastic dyserythropoiesis in 7, and fibrosis in 13 (moderate in 11 cases and severe in 2, with dry tap). Therapy-related acute leukemia appears to be a distinct clinical-pathological entity. Bone marrow trephine biopsy is useful because of the frequency of fibrosis, the possibility of dry tap, and the characteristic histopathological findings that make diagnosis possible also in the preleukemic phase.

[In vitro development of macrophages from PHA-stimulated blood cultures in Hodgkin's disease and in chronic lymphocytic leukaemia (author's transl)]. / Sulla comparsa di macrofagi in colture di sangue con fitoemagglutinina di soggetti affetti da morbo di Hodgkin e da leucemia linfatica cronica

Blood was cultured from 17 normal subjects, 28 cases of untreated chronic lymphocytic leukaemia (CLL) and 41 cases of Hodgkin's disease. Macrophages were not observed in PHA-stimulated cultures of normal subjects, of CLL or of 18 cases of Hodgkin's disease. The latter had an almost normal rate of lymphocyte blast transformation (70,2 +/- 7,2%).on the other hand, macrophages were numerous in PHA-stimulated cultures of 23 cases of Hodgkin's disease with a low blast transformation (28.4 +/- 13,7%). In Hodgkin's disease the development of macrophages in PHA cultures may be related to the lower blast transformation and cytotoxic activity of lymphocytes. In CLL the dilution of lymphocytes and monocytes by leukaemic B cells may account both for PHA unresponsiveness and low yield of macrophages in culture.

Bone marrow histopathology and prognosis in malignant lymphomas.

Bone marrow trephine biopsies of patients with non-Hodgkin's malignant lymphomas (ML), followed for at least 4 years, were investigated using univariate and multivariate survival analyses to detect which anagraphic data and histomorphologic medullary patterns before therapy were related to the prognosis. In 234 ML (146 low grade, 88 high grade), univariate analysis showed that survival was reduced by bone marrow involvement, absence of reactive lymphoid nodules, and low marrow cellularity. Moreover, in low-grade ML, patients 50 years or older and showing absence of myeloid hyperplasia, excess of hemosiderin and mast cell hyperplasia had significantly lower-survival rates. The prognostic relevance of these parameters did not change when cases without marrow involvement were separately analyzed. Multivariate analysis showed that, besides marrow involvement, age and myeloid hyperplasia had significant prognostic importance in low-grade ML, and lymphoid nodules in high-grade ML. Our data confirm the value of bone marrow histopathology in ML and indicate that the prognosis is related not only to medullary involvement but also to the morphology of the uninvolved marrow.

Angiogenic activity of hyperplastic and neoplastic lymph nodes.

Angiogenic capacity was tested in 14 non-Hodgkin's lymphomas, 7 Hodgkin's lymphomas and 15 cervical lymph nodes nonneoplastic but draining a territory with a laryngeal carcinoma. The objective was to find out whether different groups of lymphomas showed differences in their angiogenic capacity and to compare the ability to induce neovascularization of neoplastic lymphocytes. Frequency and intensity of the angiogenic response were similar for classes of lymphomas different for morphologic and immunologic characteristics. The presence of a carcinoma was sufficient to induce in tributary, nonmetastatic lymph nodes an angiogenic activity comparable to that known to characterize antigenically stimulated lymphocytes.

[Cellular immunity in two familial cases of thyroid carcinoma (author's transl)]. / Aspetti dell'immunità cellulare in due casi di carcinoma tiroideo nella stessa famiglia

Cellular immunity in two brothers with thyroid carcinoma in a family presenting pathological thyroid changes is investigated. In these brothers a low PHA lymphocyte blast-transformation and an elevated number of surface membrane Ig bearing lymphocytes were observed. These data are discussed in relation to the question of immunological defects which frequently are present in carcinoma patients.