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.

Transglutaminase 2 expression is significantly increased in cyclosporine-induced gingival overgrowth.

Cyclosporine A is a potent immunosuppressant used to prevent organ transplant rejection and treat various autoimmune diseases. However, cyclosporine A can also induce gingival overgrowth, which is characterized by increased extracellular matrix due to an altered balance between collagen synthesis and degradation. This study proposed to verify whether trans-glutaminase 2, an enzyme thought to be responsible for the assembly and remodelling of extracellular matrix, plays any role in the pathogenesis of cyclosporine A-induced gingival overgrowth. Cyclosporine A-induced gingival overgrowths were collected from 21 liver transplant patients and case-controlled with 20 non-hyperplastic gingival biopsies from healthy patients who had previous periodontal treatment. In both groups, the presence and tissue distribution of transglutaminase 2 were determined by immunohistochemistry and analyzed in comparison with the tissue morphology and expression of lymphocyte-related antigens (CD3 and CD20) and a vessel-related marker (CD34). Transglutaminase 2 expression showed a significant increase (2.6-fold) in the stromal component of cyclosporine A-treated patients compared with controls (p<0.001), which suggested that transglutaminase 2 had a role in the pathogenesis of the disease. Further studies should investigate the therapeutic effect of anti-transglutaminase 2 drugs (putrescine or 1,4-diamino-butane) in these patients.

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.

Cytology of the oral cavity: a re-evaluation.

Oral exfoliative cytology, while an economical and practical tool for diagnosis of squamous cell carcinoma and potentially malignant lesions, is not extensively used. The results of conventional (n = 89) and liquid-based (n = 411) oral diagnostic cytology cases are reported and compared to histological diagnosis. Cells were collected using either a Cytobrush device for conventional smears or a dermatological curette (AcuDispo) for liquid-based (Thin Prep) cytology. The "curette technique" allowed for the collection of "accidental" tissue fragments, utilized as microbiopsies. The sensitivity was 86.5% in conventional and 94.7% in liquid-based cytology; specificity was 94.3% and 98.9%, respectively; inadequate samples were present in 12.4% and 8.8% of cases, respectively. Although conventional cytology may be useful in oral squamous cell carcinoma and potentially malignant lesions, liquid-based cytology gives better results, enhances both the sensitivity and specificity, and also provides material for further investigations, e.g. DNA ploidy studies, microhistology, etc.

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.

The impact of liquid-based oral cytology on the diagnosis of oral squamous dysplasia and carcinoma.

OBJECTIVE: Even though diagnostic oral exfoliative cytology is a useful, economical and practical tool in the diagnosis of oral dysplasia and carcinoma, it is not yet extensively used. The results of conventional exfoliative and liquid-based diagnostic cytology in oral potentially malignant lesions (PML) are herein reported and compared with the histological diagnosis. METHODS: Either conventional (89) or liquid-based (384) exfoliative cytology was used for the diagnosis of oral dysplasia/carcinoma in 473 subjects and the results were compared with scalpel biopsy histology. Cells were collected using a Cytobrush device for conventional smears and with a dermatological curette for the liquid-based cytology. The 'curette technique' also allowed for the collection of 'accidental' tissue fragments, utilized as microbiopsies. RESULTS: Histological diagnosis was squamous carcinoma in 96 of 473 cases, high-grade dysplasia (oral intraepithelial neoplasia two to three) in 24 and other lesions in 353 cases. The smears in the conventional cytology group were inadequate in 12.4%, with an 85.7% sensitivity and a 95.9% specificity. There were 8.8% of inadequate specimens in the liquid-based cytology group; sensitivity was 95.1% and specificity was 99.0%. CONCLUSIONS: Although conventional cytology is useful when diagnosing oral PML (better sensitivity and predictive positive value if compared with the cervical smear test with similar specificity) and can improve the accuracy of histological diagnosis, liquid-based cytology gives better results, as it not only enhances both sensitivity and specificity, but also provides material for further investigation (AgNORs, DNA, microbiopsies, etc.).

Prognostic relevance of cell proliferation in head and neck tumors.

Cell proliferative activity has been extensively investigated in head and neck tumors. Ki67/MIB-1 immunostaining, tritiated thymidine or bromodeoxyuridine labeling indices, DNA S-phase fraction, proliferating cell nuclear antigen expression, potential doubling time and analysis of the nucleolar organizer region associated proteins (AgNORs) have shown significant correlation with prognosis in 4806 cases of tumors of the oral cavity, salivary glands, pharynx and larynx. However, this was not observed in 2968 other reported cases. Discrepancies may depend on various factors: the heterogeneity of the series, which include tumors from various anatomic sites and patients treated with different therapy, and the lack of standardization of methods for assessing cell proliferation. Furthermore, none of the methods currently applied can by themselves define the actual proliferative activity, as it depends both on the proportion of cells committed to the cycle (growth fraction) and the speed of the cell cycle. Indeed, the actual proliferative activity of a tumor could well be measured by the equation [PA = Ki67 or MIB-1 scores x AgNORs], as we did in pharyngeal carcinoma. Provided that large and homogeneous series are evaluated by standardized methods, cell proliferative activity can still be regarded as an inexpensive and reliable prognostic factor in head and neck tumors.

[Usefulness of oral exfoliative cytology for the diagnosis of oral squamous dysplasia and carcinoma]. / Utilità della citologia esfoliativa orale per la diagnosi di displasia e carcinoma squamoso orale.

AIM: It is well known that diagnostic oral exfoliative cytology, even if a useful, economical and practical tool in the diagnosis of oral dysplasia and carcinoma, is not yet used so extensively as is cervico-vaginal cytology. METHODS: Exfoliative cytology was used for the diagnosis of oral dysplasia and carcinoma, and the results compared to the histological examination. Cytological smears were taken from 89 patients with oral lesions suspicious for neoplasia (in particular erythro- and leukoplakia and lichen). All patients were also subjected to oral biopsy and histological examination. RESULTS: Out of 89 cases studied, histology showed the presence of an invasive squamous carcinoma in 32, dysplasia in 17, phlogosis in 15 and other types of lesions (2 of which malignant non-epithelial tumours) in 25. The cytological smear was inadequate for diagnosis in 11/89 cases (12.4%). In cytologically adequate and histologically positive cases, cytology confirmed the histological diagnosis of dysplasia and/or carcinoma in 38/45 cases (sensitivity 86.5%, accuracy 89.6%). Moreover, 1 case which was histologically negative at the onset, proved positive at cytology. There were 2 false-positive cytology results (specificity 94.3%, predictive positive value 95.7%). CONCLUSIONS: Despite the small number of cases in the cohort, oral cytology can improve the accuracy of histology, and may be a useful screening tool for the diagnosis of oral neoplasia/dysplasia.

Papnet-assisted cytological diagnosis intensifies the already marked variability among cytological laboratories.

OBJECTIVE: The main objective was to assess the sensitivity, specificity and reliability of PAPNET-assisted diagnosis in comparison with conventional screening. SETTING: Seven Italian and one English University or Research Institutes, and a random sample of an other 20 Italian Laboratories of the Italian National Health Service (INHS) provided the cervical smears. METHODS: During the training phase every center examined in rotation four sets of slides for a total of 300 representative slides. Afterwards, 900 "positive" slides were added to the 3,100 slides which were collected consecutively without any selection or exclusion. The eight main centers were divided into four couples and each couple of centers examined 775 slides with the PAPNET system, "blindly" to the original diagnosis. An expert cytopathologist (M.A.) of the National Institute of Health (NIH) reassessed 40% of the slides with an original negative diagnosis to evaluate the false negative rate. Two expert NIH cytopathologists (M.A., G.M.) re-examined all slides where a disagreement had been observed between the original and one or both of the study diagnoses. The main analyses concerned the following three main categories: WNL and unsatisfactory for evaluation; ASCUS, AGUS and LSIL; HSIL and carcinoma. A special algorithm was devised to define the reference diagnosis for sensitivity and specificity assessment. RESULTS: Laboratories, even belonging to the same couple, classified as "no review" a very different proportion of slides ranging from 35% to 74%. The index of kappa agreement between the members of couples examining the same sets of slides was low or very low, ranging from 0.30 to 0.03. The sensitivity of the review classification was particularly low in some laboratories. Surprisingly, only a small correlation was observed between the sensitivity of the review classification and the proportion of slides classified as "review". The "tentative" diagnosis on PAPNET tiles of the "review" slides was almost as reliable as the microscopic diagnosis. In the overall performance, there were many significant differences among the eight laboratories. The best laboratory had a sensitivity of 95% and a specificity of 96%. At least three laboratories displayed unacceptably low sensitivity and one a very low specificity. CONCLUSION: Altogether these results seem to confirm that there are wide differences among cytological laboratories per se, and that these differences are intensified by the use of an instrument like PAPNET. The huge variation in performance may be explained by differences in basic skills and by different training, but it is difficult to understand exactly what could have been done to reduce it.

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.

Biochemical and histological evaluation of human synovial-like membrane around failed total hip replacement prostheses during in vitro mechanical loading.

The biochemical role of the synovial-like membrane formed at the interface of eight aseptic failed total hip prosthesis has been investigated during in vitro mechanical loading. The study was carried out on four membranes from cemented prosthesis and four titanium alloy uncemented ones. Intermittent positive pressure leading to 20% deformation of the membrane (100 g/cm(2))was applied to the membrane fragments in cycles (300 cycles/15 min) repeated three times at thirty minutes intervals in which interleukin-6 (IL6), prostaglandin-E2 (PGE2) and interleukin-1beta (IL1beta) levels were quantified both in culture media and in tissue extracts. Histological, morphometrical and immunohistochemical studies were also carried out on the same membranes. Mechanical stress evidenced an increase in the release of the examined cytokines both in cemented and uncemented prosthesis tissues; particularly evident was IL6 trend of increase from cemented prosthesis and IL1beta result from uncemented ones. Histomorphological and immunohistochemical data revealed no differences between membranes obtained from cemented and uncemented prosthesis as to cell proliferation, fibrosis, macrophages lymphocytes B and T population, vessels and nervous fibers. The results indicate that mechanical stress plays a fundamental role in increasing membrane production and release of cytokines known as bone-resorbing agents. Furthermore, the histologic finding of synovial-like membrane with the same histomorphological and immunohistochemical findings but with different biochemical response to mechanical stimulation, suggests that cells involved in the production and release of the considered mediators might have different strain behavior by different development conditions (previous contact with PMMA).

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.

Defective recovery and severe renal damage after acute hemolysis in hemopexin-deficient mice.

Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes.

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.

Histochemical and morphometric observations on the new tissue formed around mammary expanders coated with pyrolytic carbon.

The authors investigated tissue reaction around implanted silicone expanders, focusing on clinical morphological and morphometrical aspects. For use in breast reconstruction in post mastectomy patients, the surface of a medical-grade silicone elastomer was modified, without changing its bulk properties, by the addition of a pyrolytic carbon film. The presence of lipophagy, the number of foreign-body giant cells of histiocytic origin, and the number of MIB-1 positive nuclei (an index of proliferation for the reactive stromal population) were all seen to be influenced by the pyrolytic carbon coating. Indeed, all these parameters were lower in the membrane formed around Carbofilm TM-coated expanders, thus demonstrating the effective protective properties of pyrolytic carbon coating.

Significance of cell proliferation index in assessing histological prognostic categories in Hodgkin's disease. An immunohistochemical study with Ki67 and MIB-1 monoclonal antibodies.

BACKGROUND AND OBJECTIVE: In their review of the Rye histopathological classification of Hodgkin's disease, Bennett and coworkers have proposed that the nodular sclerosis (NS) type should be divided into two diagnostic categories on the basis of their clinical behaviour. In order to evaluate whether the proliferative activity of HD cells might correlate with histology in NS subtypes, we reviewed and re-evaluated cryostat and paraffin-embedded sections from 80 cases sent to our centre from 1986 to 1991. METHODS: In the present study, we investigated the growth cell fraction of 53 cases of Hodgkin's disease with nodular sclerosis by using Ki67 and MIB1 monoclonal antibodies to determine whether proliferative activity is associated with different pathological subtypes and prognostic categories. Eight cases with an interfollicular pattern and 19 with mixed cellularity were also investigated. The results in each group were compared to the others. RESULTS: The values of Ki67 and MIB1 were highly correlated (r = 0.88). In Hodgkin's disease with nodular sclerosis, two groups with significantly different growth fractions were morphologically identified: one with lymphocyte predominance and mixed cellularity subtypes, another composed of cases with variously extensive lymphocyte depletion. The figures were compared with those of interfollicular subtype, which fell into the first group, and of mixed cellularity type, in which the proliferative cell activity was significantly higher than in the second nodular sclerosis group. In all cases, Reed-Sternberg and Hodgkin cells accounted for the majority of the cell growth fraction, although a variable percentage of T-lymphocytes were also Ki67- or MIB1-positive. Taking the median value (15%) of MIB1 positive cells as a cut-off, a significant correlation (p = 0.05) was observed between MIB1 positivity and bulky disease, and a good trend (but not a significant relationship) between MIB1 and overall survival, disease-free survival, staging and the clinical response to therapy. INTERPRETATION AND CONCLUSIONS: Assessment of the growth cell fraction in Hodgkin's disease with different nodular sclerosis patterns provides biological support for the morphological reclassification of their degree of malignancy into two main groups with different impacts on the clinical parameters and a possible relation with the outcome of treatment.