An ultrastructural and immunocytochemical analysis of human endothelial cell adhesion on coated vascular grafts

Human adult endothelial cells were enzymatically harvested from adipose tissue. Cell viability was established by Trypan blue exclusion and transmission and scanning electron microscopy. Endothelial cells were identified by immunocytochemical investigation at light microscopy, transmission electron microscopy, and scanning electron microscopy. Isolated cells were positive for actin and vimentin, negative for desmin. Factor VIII RA was mainly expressed at cell surface and occasionally disclosed in the cytoplasm. Reactivity for UEA I and J15 was weak or undetectable. Human endothelial cells were seeded and left to adhere for one hour onto different nonvascular substrates (glass, poly-l-lysine, formvar-carbon, fibronectin, Teflon). Scanning electron microscopy defined surface features, suggesting tenacious cell adhesion on the substrate. Different vascular substrates were tested (preclotted Dacron, albumin Dacron, Hemashield Dacron, Gelseal Dacron, ePTFE, fibronectin-ePTFE). Commercially available coated grafts showed qualitative and quantitative differences in cell adhesion. In particular, Gelseal Dacron provided the best quantitative results, even though a wide variability was observed. In contrast, fibronectin-coated ePTFE gave more reliable results and high spreading efficiency. In the short term, coated grafts do not seem to offer greater advantages than fibronectin-coated ePTFE. However, specific incubation times for each coated graft should be selected and the long-term approach (graft culture) should also be attempted.     

ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

Free insulin-like growth factor-I and cognitive function in older persons living in community.

CONTEXT: Increasing evidences from experimental and human studies suggest that the activity of the growth hormone (GH/insulin-like growth factor-I) axis may contribute to the age-related cognitive decline and poor cognition in late life. OBJECTIVE: The aim of the present study was to evaluate the relationship of total serum free IGF-I and its binding protein-3 with cognitive performance in older persons aged 80 years or older. DESIGN: Data are from baseline evaluation of the ilSIRENTE study (n=353). Cognitive performance was evaluated using five items enclosed in the Minimum Data Set for Home Care assessment form: short-term memory, procedural memory, cognitive skills in daily decision making, verbal expression, comprehension. Free insulin-like growth factor-I (free IGF-I) and IGF-binding protein-3 (IGFBP-3) in blood were measured. Analysis of covariance (ANCOVA) was used to examine the relationship between cognitive impairment and the serum free IGF-I and IGFBP-3 concentrations, after adjustment for potential confounding variables. RESULTS: After adjustment for potential confounders, which included age, gender, education, cerebrovascular disease, ischemic heart disease, congestive heart failure, hypertension, diabetes, depression, Parkinson diseases, thyroid diseases, smoking status, alcohol abuse, body mass index, and number of medications, individuals with verbal expression problems (n=20) and individuals with comprehension problems (n=24) had a significantly lower serum levels of readily dissociable IGF-I than participants without cognitive impairments. The serum IGFBP-3 presented the same behavior of free IGF-I. CONCLUSION: The present study suggests that among old-old subjects living in the community lower levels of total serum free IGF-I and IGFBP-3 are associated with impairment of cognitive performance. This finding suggests that the GH/IGF-I axis may play an important role in the age-related decline of cognitive performance.     

The role of cytogenetics in the classification of soft tissue tumours

 Soft tissue tumours represent a heterogeneous group of mesenchymal lesions, and their classification is the subject of continuous debate. Chromosome analysis, molecular cytogenetics and molecular assays may become increasingly useful in diagnosis, and this review summarises advances in the cytogenetic characterisation and classification of soft tissue tumours. Among the group of fibrous lesions, superficial fibromatosis exhibits trisomy 8. This genomic change is also observed in desmoid fibromatosis in association with trisomy 20. Trisomy 11 is the most frequently observed chromosomal aberration in congenital fibrosarcoma. Dermatofibrosarcoma protuberans and giant cell fibroblastoma share a translocation t(17;22), which supports the concept of the existence of a common differentiation pathway. Adipose tissue tumours is the group in which integration of genetics and pathology has been most fruitful. Ordinary lipomas cytogenetically show an abnormal karyotype in about half the cases. Genomic changes of the 11q13 region are observed in hibernoma. Lipoblastoma exhibits a specific 8q rearrangement in 8q11-q13. Loss of material from the region 16q13-qter and 13q deletions are observed in spindle cell/pleomorphic lipomas. The well-differentiated liposarcoma/atypical lipoma group is characterised karyotypically by the presence of one extra ring and/or extra giant chromosome marker. Myxoid and round cell liposarcoma share the same characteristic chromosome change: t(12;16)(q13;p11) in most cases. In the group of smooth muscle lesions most data are derived from uterine leiomyomas, which can be subclassified cytogenetically into seven different types. Half of all leiomyomas are chromosomally normal; the other half have one of six possible consistent chromosome changes. Alveolar rhabdomyosarcoma is characterised cytogenetically by two variant translocations t(2;13)(q35;q14) and t(1;13)(p36;q14). Among tenosynovial tumours, the localised type of giant cell tumour of tendon sheath exhibits two different karyotypic changes. One involves 1p11 in a translocation with chromosome 2 or with another chromosome. A second type involves 16q24. Synovial sarcoma is characterised cytogenetically by a translocation occurring between chromosome 18 and presumably two adjacent loci on the X chromosome. In neural tumours, abnormalities of chromosome 22 have been reported in benign schwannomas and perineuriomas. Malignant peripheral nerve sheath tumours exist in two main forms: sporadic and associated with the NF-1 syndrome. Karyotypes are very complex, but chromosomes 17q and 22q are very often involved. Clear cell sarcoma is characterised cytogenetically and molecularly by a translocation t(12;22)(q13;q12). The Ewing’s sarcoma/peripheral neuroectodermal tumour category shows a central karyotypic anomaly represented by the translocation t(11;22). The two variants t(21;22) and t(7;22) are found in some cases. Among cartilaginous lesion, the most frequently described anomaly is the t(9;22)(q22;q12) in extraskeletal myxoid chondrosarcoma. Intra-abdominal desmoplastic small round cell tumour is characterised by a t(11;22)(p13;q12). Received: 5 February 1997 / Accepted: 24 February 1997     

Prevalence, clinical characteristics, quality of life, and prognosis of patients with congestive heart failure and isolated left ventricular diastolic dysfunction.

Prevalence of isolated left ventricular (LV) diastolic dysfunction has been reported to be as high as one-third of all heart failure (HF) cases, with an increasing prevalence in the elderly population. However, there is a paucity of prospective data about the prevalence and prognosis of isolated LV diastolic dysfunction in an unselected population of patients hospitalized with HF. Therefore, we prospectively evaluated 179 consecutive patients discharged from our hospital with HF to assess the prevalence of systolic versus diastolic LV dysfunction among patients hospitalized with HF and to compare their demographics, clinical features, self-perceived quality of life (QOL), and 6-month readmission rate and mortality. Among them, 133 (59% men, median age 74 years) showed in sinus rhythm and had no significant primary valvular disease. LV diastolic dysfunction was diagnosed on the basis of the European Study Group on Diastolic HF echocardiographic criteria. QOL was assessed at hospital discharge and 6-month follow-up visit using the Minnesota Living with HF questionnaire. Survival of patients with HF was compared with that of age- and sex-matched general population. In all, 29 patients (22%) had isolated LV diastolic dysfunction and 102 (78%) had prevalent LV systolic dysfunction (ie, LV ejection fraction 相似文献   

Significance of cyclin D1 expression in meningiomas: a preliminary study.

Forty-four evaluable patients with intracranial meningiomas were assessed for the expression of the cell-cycle regulator cyclin D1 and of proteins involved in proliferation and apoptosis such as PCNA, MIB-1, p53 and bcl-2. Analyses were carried out by western blot and immunohistochemistry after immediate processing of fresh tumor specimens. By western blot, expression of cyclin D1 significantly correlated with p53 (p=0.02) and with proliferative activity, as assessed by PCNA expression (p=0.0009). By immunohistochemistry, a significant relationship between cyclin D1 and the proliferation marker MIB-1 was confirmed (p=0.05), whereas significance with bcl-2 expression was not found (p=0.01). Moreover, although the association with tumor grade appeared of borderline statistical significance (p=0.07), all the grade II/III meningiomas showed increased expression of cyclin D1 and high proliferative activity. In conclusion, data from this preliminary study seem to suggest a potential value of the combined expression of cyclin D1 and proliferation indicators in defining subgroups of meningiomas with a more aggressive biological behavior.     

Immunohistochemical study of colonic mucosa macrophages in children with Crohn's disease and ulcerative colitis]

In this research the histological characteristics of the macrophages on the colonic mucosa in Crohn's disease and ulcerative colitis were quantified and analysed. Twelve Crohn's disease, 19 ulcerative colitis and 10 specimen of the rectal mucosa, representing the control group according to the followed model, were studied: I period (PI) = pre-treatment, II period (PII) = up two years of evolution and III period (PIII) = more than two years of evolution. The macrophages were identified in a colonic mucosa by the monoclonal CD68 through the immunoperoxidase method. The macrophages quantification was done by chromatic computer images analysis, that express the area (mm2) used by the CD68 positive cells, in percentage. The percentage of the area used by the macrophages was increased in both diseases, in all the studied periods, when compared with the control group, but without statistic significance. The macrophages' distribution inside the control group mucosa was subepithelial, while in the illness group, it reached all the mucosa that was concentrated on the basis of ulcers and all long the fissures. On the Crohn's disease the CD68 positive cells facilited the identification of the microgranulomas, sometimes unnoticed in the hematoxiline-eosine. Although there was no difference between patients and control group in the macrophages area, the difference in the distribution could suggest the macrophages' participation on the injure in both diseases although they do not permit a differential diagnosis because of the variety of the values. The CD68 did not identify the different functional status of the macrophages, but their position in the mucosa suggest that, in terms of fissures and ulcers, their mainly function should be the phagocitosis and in the other cases, they have been the cells that should show the antigens and that recruit the other inflammatory cells.     

Clinical epidemiology and survival of progressive multifocal leukoencephalopathy in the era of highly active antiretroviral therapy: Data from the Italian Registry Investigative Neuro AIDS (IRINA)

Human immunodeficiency virus (HIV)-associated progressive multifocal leukoencephalopathy (PML) remains a relevant clinical problem even in the era of highly active antiretroviral therapy (HAART). Aims of the study were to analyze clinical and treatment-related features and the survival probability of PML patients observed within the Italian Registry Investigative Neuro AIDS (IRINA) during a 29-month period of HAART. Intravenous drug use, the presence of focal signs, and the involvement of white matter at neuroradiology increased the risk of having PML. A reduced probability of PML was observed when meningeal signs were reported. Patients starting HAART at PML diagnosis and previously naïve for antiretrovirals showed significantly higher 1-year probability of survival (.58), compared to those continuing HAART (.24), or never receiving HAART (.00). Higher CD4 cell count were associated with a higher survival probability (.45). At multivariate analysis, a younger age, higher CD4, starting HAART at PML diagnosis, the absence of previous acquired immunodeficiency syndrome (AIDS)-defining events, and the absence of a severe neurologic impairment were all associated with a reduced hazard of death. The use of cidofovir showed a trend towards a reduced risk of death.