Polymethylmethacrylate dermal fillers: evaluation of the systemic toxicity in rats.

This study evaluated local and systemic reactions after an intravascular injection of polymethylmethacrylate (PMMA) at two concentrations in a murine model. Thirty rats were divided equally into three groups: 2% PMMA, 30% PMMA, and a control group (normal saline only injection). The filler was injected into the ranine vein. The rats were sedated at 7 and 90 days and a clinical evaluation performed. After euthanasia, the right lung, liver, and right kidney were removed, weighed, and microscopically analyzed. The submandibular lymph nodes and tongue were removed and examined microscopically. Serum was subjected to liver and kidney function tests. No groups showed clinical alterations. Microspheres were not observed at any distant organ. Two samples from the 2% PMMA group showed a local inflammatory response at day 7 and another two samples from the 30% PMMA group at day 90. The group injected with 30% PMMA presented higher levels of alanine aminotransferase (P = 0.047) after 90 days when compared with the other groups. The data obtained in this study demonstrate that intravascular injections of PMMA fillers show potential health risks such as chronic inflammation at the implantation site.

Clinical and pathological characteristics of polymethylmethacrylate and hyaluronic acid in the rat tongue.

Adverse effects on the oral mucosa after the use of dermal fillers have been increasingly reported due to their increased use for facial aesthetics. The objective of this study was to evaluate the clinical and histologic effects of two types of product, 10% polymethylmethacrylate and 20mg/ml hyaluronic acid, locally and at long distance, examining initial and late reactions. Each substance was randomly and separately injected in rats' tongues (polymethylmethacrylate, n = 16; hyaluronic acid, n = 18). They were compared with the control group (n = 16) at 3 observation times (7, 60 and 90 days) for clinical analysis, intensity of local inflammatory response (haematoxylin and eosin staining), amount of newly formed blood vessels and macrophages (immunohistochemical assays), density of collagen fibres (picrosirius staining) and systemic migration of the product to the liver and kidney (haematoxylin and eosin staining). The results showed inflammation triggered by the injection of the material, suggesting that both substances cause responses in local tissue, although there was biocompatibility with hyaluronic acid. This research highlights the importance of experimental studies on this subject, since adverse reactions have been observed routinely in dental practice.

Neuroprotective effects of a new skin care formulation following ultraviolet exposure.

BACKGROUND: Chronic ultraviolet (UV) exposure is a major environmental factor involved in extrinsic skin ageing (photo-ageing). Skin nerve fibres are significantly reduced in number following UV irradiation and new skincare compounds with neuroprotective effects are thus highly warranted. OBJECTIVES: We developed a new skincare formulation from a plant extract and evaluated its neuroprotective effects of ex vivo UV irradiation. MATERIALS AND METHODS: The new skincare emulsion was formulated from Echinacea purpurea extract and was enriched with antioxidants (patent no. PROV020110087075). Skin samples were obtained from 20 healthy patients enrolled for plastic surgery and were immediately treated with placebo (SPF 15) or test emulsions. Skin samples were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody, anti-human CD56. Cell damage was quantified by image analysis. RESULTS: UVA and UVB significantly reduced (40-60%) densities of nerve endings in control samples treated with placebo (P < 0.001). Samples treated with test emulsion completely blocked UV-related effects on skin nerve endings. These neuroprotective effects were similarly observed regardless of age or tissue analysed (breast versus abdomen). CONCLUSIONS: Our new skincare formulation obtained from E. purpurea provides important neuroprotective effects of UV irradiation and could be used together with SPFs to prevent chronic deleterious effects of solar exposure.

AIDS-related sinonasal Burkitt lymphoma successfully treated with intensive chemotherapy regimen and high active antiretroviral therapy.

Burkitt lymphoma (BL) is the second most common AIDS-related lymphoma. Primary sinonasal BL in HIV patients is extremely rare and treatment data in this subset of patients is almost nonexistent. Recently, a few studies reported promising results treating HIV-associate BL with an intensive chemotherapy regimen. The use of highly active antiretroviral therapy (HAARTHAART) concomitantly with chemotherapy seems to improve patient outcomes, but this topic is still controversial due to potential drug interactions. We report a case of a 29-year old woman diagnosed with AIDS presenting with symptoms of chronic sinusitis. Subsequent investigation by CT scan and endoscopic biopsy discovered a sinonasal BL in an early stage. The patient was treated with intensive chemotherapy and HAARTHAART and achieved a complete remission and long-term immunologic recovery. This case report describes a rare entity whose natural history, treatment and prognosis is infrequently characterized in the medical literature.

Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells.

Plants of Crotalaria genus (Leguminosae) present large amounts of the pyrrolizidine alkaloid monocrotaline (MCT) and cause intoxication to animals and humans. Therefore, we investigated the MCT-induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model. The comet test showed that 24h exposure to 1-500microM MCT and 500microM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42-64% and 53%, respectively. Cells exposed to 100-500microM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1-10microM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1-10microM). Apoptosis was also evidenced in cells treated with 100-500microM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500microM MCT. The data obtained provide support for heterogenic and multipotential effects of MCT on GL-15 cells, either interfering on cell growth and cytoskeletal protein expression, or inducing DNA damage and apoptosis and suggest that the response of glial cells to this alkaloid might be related to the neurological signs observed after Crotalaria intoxication.

Effects of ultraviolet radiation on human cutaneous nerve fibres.

UNLABELLED: Due to an increasing number of skin diseases as a result of exposure to ultraviolet (UV) radiation, it is necessary to evaluate the effectiveness of new skin care formulations with broad-spectrum sunscreens. OBJECTIVES: This study aims to assess the status of nerve fibres in healthy human skin, to quantify effects of UV radiation on nerve endings, and to evaluate neuroprotective effects of new skin care formulations against UV exposure damage. METHODS: Samples were obtained from 34 female patients enrolled for plastic surgery and were immediately treated (10 min) with three emulsions: Cream 1, Cream 2 (placebo) and a sunscreen with sun protection factor 15 (SPF15). Control samples and those treated with the cream emulsions were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody (anti-human CD56/NCAM). Cell damage was assessed by image analysis. RESULTS: Several cellular nervous structures were identified in the skin samples, including free nerve endings. UVA and UVB significantly decreased (40-60%) density of nerve endings in the control samples and those treated with placebo (Cream 2) or SPF15 (all P < 0.001). Cream 1 completely blocked effects of UV radiation on nerve endings (P > 0.05 vs. control). CONCLUSIONS: Quantification of cell damage induced by UV radiation provides useful information for identification of new skin care compounds with neuroprotective properties.

Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures.

Dehydromonocrotaline (DHMC) is the main monocrotaline active cytochrome P450's metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 microM DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 microM, and induced membrane damages at 500 microM. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 microM DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 microM), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/-30%) of the astrocytes exposed to 100-500 microM DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependent effect of DHMC on cultures of rat cortical astrocytes, leading to apoptotic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria.

Exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study.

OBJECTIVE: To assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol. METHODS: (i) Group without lesions. Smears obtained from the lower lip, border of the tongue and floor of the mouth of 31 control individuals (group I), 49 tobacco users (group II) and 27 tobacco/alcohol users (group III) were stained using the Papanicolaou method. The first 100 cells counted on each smear determined the maturation pattern and the keratinization index (KI). Analysis of variance (ANOVA) and the Tukey multiple comparison test were used for statistical analysis, at a 5% significance level. (ii) Group with lesions. Cytopathological and histopathological studies were conducted for 15 patients: eight with leucoplakia without epithelial dysplasia, two with epithelial dysplasia and five with squamous cell carcinoma. RESULTS: (i) Group without lesions. Statistical analysis revealed a smaller number of superficial cells with nuclei in all sites of the group of tobacco/alcohol users (group III) when compared to the control group (group I), and this difference was statistically significant (P<0.005). (ii) Group with lesions. The severity of histopathological findings increased with the increase in the number of cells of the deeper epithelial layers, with a statistically significant difference in the number of intermediate (P=0.013) and parabasal cells (P=0.049), which increased with the severity of the epithelial maturation disorder: leucoplakias with dysplasia had a greater number of intermediate and parabasal cells than leucoplakias without dysplasia; and the number in squamous cell carcinomas was greater than in leucoplakias with dysplasia. CONCLUSION: The maturation pattern of cells in the three anatomic sites showed changes that may be associated with the synergistic effect of tobacco and alcohol. Also, the severity of histopathological findings was associated with the increase in the number of cells in the deeper epithelial layers.

Alkaloids from Prosopis juliflora leaves induce glial activation, cytotoxicity and stimulate NO production.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

Utilización de dos soportes para la inmovilización de la papaína / Use of two supports for inmobilizing papain

Con la intención de preparar suplementos dietéticos de bajo coste, se inmovilizó papaína en carbón activado (CA) y en alúmina, utilizando suero como fuente de proteínas hidrolizadas. Para determinar el índice de inmovilización se cuantifi caron las enzimas no adsorbidas mediante el método de Lowry. Se analizó el efecto del tiempo de contacto y la temperatura, considerándose 30 min. a 25 ºC como la condición óptima para inmovilizar la papaína en ambos soportes. El CA presentó unos índices de inmovilización muy superiores (entre 95% y 99%) a los de la AL (entre 4% y 13%). Para evaluar la capacidad de reutilización de la papaína se midió la actividad residual de la enzima después de haber sido utilizada hasta 20 veces. Para determinar la actividad de la enzima se cuantifi có el índice de exposición de la fenilalanina mediante espectrofotometría de derivada segunda. En este caso, la AL presentó mejores resultados que el CA, ya que la actividad de la papaína seguía siendo la misma después de haber sido utilizada 15 y 5 veces, respectivamente

Papain was immobilized on activated carbon (AC) and on alumina (AL), with the aim of preparing low cost dietary supplements, using whey as hydrolysed protein source. The quantifi cation of the non-adsorbed enzyme, using Lowry’s method was used to determine the immobilization rate. The effect of the contact time and the temperature was tested, and 30 min at 250C was considered the best condition for immobilizing papain in both supports. AC showed much higher immobilization rates (from 95% to 99%) than AL (from 4% to 13%). The reusability of papain was evaluated by measuring the residual activity of the enzyme after it has been used for up to 20 times. The quantifi cation of exposure rate of phenylalanine by second derivative spectrophotometry was used to determine the enzyme activity. In this case, AL showed better results than AC, since the activity of papain remained unchanged after 15 and 5 times, respectively

Quality of ham pâté containing bovine globin and plasma as fat replacers.

The effect of the incorporation of globin (10%), plasma (10%) and both combined (5% each) as fat replacers on the quality of ham paté was investigated. The chemical composition, the sensorial analysis (color, flavor and consistency) and the instrumental analysis of the texture (hardness, adhesiveness, elasticity, cohesiveness and tackiness) were evaluated. The results showed an increase of moisture and protein contents after the fat replacement, while the fat reduction of 25-35% led to the preparation of light products. No change was observed for the aroma, taste and the consistency of fat replacing products, but an intensification of the cohesivity and a reduction of color, hardness and tackiness were detected in some samples.

Posttransplant diabetes mellitus in renal transplant patients with hepatitis C virus.

The aim of this study was to assess the possible association between posttransplant diabetes mellitus (DM) and hepatitis C virus (HCV) infection in renal transplant recipients. This study included 124 patients who underwent renal transplantation between 1997 and 2002. Inclusion criteria were patients who were not diabetic prior to transplantation and posttransplant follow-up longer than 6 months. DM was defined as fasting blood glucose levels higher than 126 mg/dL on at least two occasions. HCV infection was detected using second- or third-generation ELISA methods and/or polymerase chain reactions for HCV-RNA. Twenty-five HCV positive (HCV+) patients were compared with 25 consecutive HCV negative (HCV-) transplant patients. Demographic and clinical data of the groups were compared. Posttransplantation DM was observed in 24% of the HCV+ patients. There were no statistical differences in age, gender, race, family history of DM, follow-up, or body mass index between the two groups. There was a higher prevalence of posttransplantation DM in HCV+ patients, but the difference did not reach statistical significance (24% vs 12%, P = NS). Alternatively, comparing patients of the two groups (n = 50) who did versus not develop DM, the incidence of posttransplantation DM was higher among HCV+ patients, but the difference did not reach statistical significance (66.6% vs 46.3%, P = NS). In conclusion, there was no association between HCV infection and the development of posttransplantation DM in this cohort of renal transplant recipients. However, there was a trend that suggested an association.

Transcontinental communication and quantitative digital histopathology via the Internet; with special reference to prostate neoplasia.

OBJECTIVE: To describe practical experiences in the sharing of very large digital data bases of histopathological imagery via the Internet, by investigators working in Europe, North America, and South America. MATERIALS: Experiences derived from medium power (sampling density 2.4 pixels/microm) and high power (6 pixels/microm) imagery of prostatic tissues, skin shave biopsies, breast lesions, endometrial sections, and colonic lesions. Most of the data included in this paper were from prostate. In particular, 1168 histological images of normal prostate, high grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) were recorded, archived in an image format developed at the Optical Sciences Center (OSC), University of Arizona, and transmitted to Ancona, Italy, as JPEG (joint photographic experts group) files. Images were downloaded for review using the Internet application FTP (file transfer protocol). The images were then sent from Ancona to other laboratories for additional histopathological review and quantitative analyses. They were viewed using Adobe Photoshop, Paint Shop Pro, and Imaging for Windows. For karyometric analysis full resolution imagery was used, whereas histometric analyses were carried out on JPEG imagery also. RESULTS: The three applications of the telecommunication system were remote histopathological assessment, remote data acquisition, and selection of material. Typical data volumes for each project ranged from 120 megabytes to one gigabyte, and transmission times were usually less than one hour. There were only negligible transmission errors, and no problem in efficient communication, although real time communication was an exception, because of the time zone differences. As far as the remote histopathological assessment of the prostate was concerned, agreement between the pathologist's electronic diagnosis and the diagnostic label applied to the images by the recording scientist was present in 96.6% of instances. When these images were forwarded to two pathologists, the level of concordance with the reviewing pathologist who originally downloaded the files from Tucson was as high as 97.2% and 98.0%. Initial results of studies made by researchers belonging to our group but located in others laboratories showed the feasibility of making quantitative analysis on the same images. CONCLUSIONS: These experiences show that diagnostic teleconsultation and quantitative image analyses via the Internet are not only feasible, but practical, and allow a close collaboration between researchers widely separated by geographical distance and analytical resources.

Quantitative study of ductal breast cancer progression: nuclear signatures for evaluation of progression grade.

The evaluation of progressive morphological changes, with 93 morphometric parameters in tissue lesions representative of ductal breast cancer progression, has been performed in order to define in great detail the profile of chromatin texture (nuclear signature) changes. A gradual, distinctive increase in nuclear signature alterations from hyperplasia to infiltrating carcinoma has been found. The nuclear signatures' analysis of microinfiltrating foci in comedo DCIS showed sharp differences compared with those of comedo DCIS they derived from: these foci consist of cells with smaller and also more homogeneous nuclei. Opposite to the prominent heterogeneity of those of comedo DCIS: they appear to express a reduced clonality in the new, more progressed, cell population. Digital analysis of chromatin patterns seems to be useful, beyond mere extraction of individual features of value, in getting objective data for individual grading and prognosis of breast cancer.

Measurement of chemopreventive efficacy in skin biopsies.

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.

Progression curves for endometrial lesions.

OBJECTIVE: To derive a numeric measure for the progression of endometrial lesions as a baseline study for an eventual assessment of chemopreventive intervention efficacy. STUDY DESIGN: Tissue sections from normal endometrium at the proliferative and secretory phase, simple hyperplasia, atypical hyperplasia from cases free of concomitant adenocarcinoma and adenocarcinoma of the endometrium were recorded at high spatial resolution. Six cases from each diagnostic category were chosen as "typical," and 60 epithelial nuclei were randomly selected for measurement for each case. Discriminant analyses were carried out to derive a direction of progressive change in feature space and to correct the progression curve for the presence of cells not expressing progressive change among the random sample of nuclei. RESULTS: A well-conditioned progression curve was derived based on the mean discriminant function scores for each diagnostic category and the mean nuclear abnormality of the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. The lesion signatures showed a clear trend toward extension into the range of higher nuclear abnormalities with increasing progression. There was an indication that abnormal endometrial lesions may comprise cases with distinctly different degrees of nuclear abnormality. CONCLUSION: A numeric assessment of lesion progression for endometrial lesions, based on karyometric measurements, is possible. The data suggest that additional analysis may provide further characterizing information for individual lesions.

Karyometry in Barrett's esophagus.

OBJECTIVE: To derive a progression curve for lesions in Barrett's esophagus based on karyometric features. STUDY DESIGN: High-resolution imagery of 900 nuclei from normal gastric tissue, Barrett's metaplasia, Barrett's high grade dysplasia and adenocarcinoma of the esophagus was recorded. Karyometric features were computed, and nuclear signatures and lesion signatures for these lesions were derived. A progression curve was defined. RESULTS: Esophageal lesions were distinctly different from the normal gastric fundus tissue, with nuclei from Barrett's metaplasia deviating from normal almost as much as nuclei from high grade dysplasia and adenocarcinoma. There was considerable case-to-case variability and overlap between lesions histologically assigned to different diagnostic categories. CONCLUSION: The karyometric data suggest that Barrett's metaplasia is a more developed lesion than previously assumed.

Carcinomatous meningitis as a clinical manifestation of pancreatic carcinoma.

Carcinomatous meningitis (CM) is clinically less common than brain metastasis or spinal cord compression, having dire consequences for both the quality of life and the overall survival of patients with solid tumors. It occurs in about 5% of all adult cancer patients, but autopsies may double this number. If leukemia and lymphoma are excluded, most cases are due to breast cancer, lung cancer and melanoma. In this report, we describe a 49-year-old male patient with metastatic pancreatic adenocarcinoma who developed carcinomatous meningitis. To our knowledge, this is only the second case of carcinomatous meningitis secondary to a pancreatic carcinoma described so far.

Data representation and reduction for chromatin texture in nuclei from premalignant prostatic, esophageal, and colonic lesions.

BACKGROUND: To identify nuclei and lesions with great specificity, a large set of karyometric features is arranged in the form of a linear profile, called a nuclear signature. The karyometric feature values are normalized as z-values. Their ordering along the profile axis is arbitrary but consistent. The profile of the nuclear signature is distinctive; it can be characterized by a new set of variables called contour features. A number of data reduction methods are introduced and their performance is compared with that of the karyometric features in the classification of prostatic, colonic, and esophageal lesions. METHODS: Contour characteristics were reduced to descriptive statistics of the set of z-values in the nuclear signature and to sequence information. The contour features derived were (1) relative frequencies of occurrence of z-values and of their differences and (2) co-occurrence statistics, run lengths of z-values, and statistics of higher-order dependencies. Performance was evaluated by comparing classification scores of diagnostic groups. RESULTS: Rates for correct classification by karyometric features alone and contour features alone indicate equivalent performance. Classification by a combined set of features led to an increase in correct classification. CONCLUSIONS: Image analysis and subsequent data reduction of nuclear signatures of contour features is a novel method, providing quantitative information that may lead to an effective identification of nuclei and lesions.