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Supplementary Fig. S5  Growth characteristics of individual treated colonies. a, GR of all colonies used in Fig. 1e. Mean {plus minus} SD in red. b. Pairwise correlations among the three GRs. c. Mean {plus minus} variance of GR2 of U251 glioma colonies according to the number of cells in CS1 or all colonies. Growth rate of colonies with 1 and 2 cells were different from all growth rates (ANOVA, Tukey's multiple comparison post-hoc test). d, Distribution of the number of colonies in each bin for Fig. 1e. e, GR of the indicated cell lines at high and low density. f, CVP and g, hypothesis plot of the glioma cell line A172wt (WT), GFP-tagged alone (GFP) or in the presence of 100 fold excess of untagged cells (GFP 1:100).

File describing in more detail the methods.

Video explaining the use of the DynaFit Desktop app and the R app and some applications.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions.Significance:Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug.

The work presents a theoretical analysis of power generation based on supercritical steam generated by a boiler operating with high-pressure combustion chamber and consuming biomass-water slurry. Steam generated at 30 MPa is injected into turbines and reheated before proceeding to the second turbine stage. The high-pressure flue gas leaving the boiler is used to reheat that steam, and thus, cooled to temperatures below the dewpoints of alkaline species in it, therefore allowing their removal before entering the gas turbines. The exergetic efficiency is chosen as optimization function for the boiler, and the 1st Law efficiency for the whole power generation process. It is shown that a lower efficiency might be expected when compared the predicted by other similar theoretical studies. Such is mainly due the power consumed by compressing the air required by the boiler, the celling temperature of stream injected into the gas turbine, and the amount of energy spent on the vaporization of the water added to form the fuel slurry. A critical analysis of past and current proposal for power generation based on biomass describes how many technical obstacles have been downplayed in previous works.

SUMMARY The two-step approach of applying hydrofluoric acid followed by silane is deemed the gold-standard surface treatment protocol before bonding to glass ceramics. Given hydrofluoric acid is a toxic conditioning agent and with the intention to simplify this step, the dental company Ivoclar Vivadent (Schaan, Lietchtenstein) released a self-etching ceramic primer, Monobond Etch & Prime in 2015, claiming that hydrofluoric acid and silane application would no longer be required prior to luting glass ceramics. Therefore, this clinical case report and retrospective analysis describes the replacement of unsatisfactory anterior veneers due to clinical failures for new feldspathic glass ceramic veneers, using the aforementioned self-etching ceramic primer. After two years, feldspathic glass ceramics presented satisfying clinical performance with absence of debonding, tooth sensitivity, recurrent carious lesions, or marginal infiltration.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions. Significance: Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug.

This retrospective study investigated the outcome of 33 dogs with splenic hemangiosarcoma treated with surgery followed by adjuvant dose-intensified doxorubicin (DOX) with or without low-dose metronomic cyclophosphamide (LDM-C) maintenance therapy. Among the 33 dogs, 18 dogs received LDM-C. Clinical stage was available for all dogs (5 stage I, 18 stage II, and 10 stage III). Nine dogs had macroscopic, and 24 dogs had microscopic disease at the start of DOX treatment. Median progression-free survival (PFS) and overall survival were 125 and 133 days, respectively. Clinical stage and tumor burden (microscopic versus macroscopic) at the start of chemotherapy was prognostic for PFS. No significant difference was observed in PFS or overall survival for the addition of LDM-C after a completed DOX protocol (P = .563 and P = .148, respectively). Based on the results of this retrospective study, the addition of LDM-C therapy as a maintenance regimen following a completed protocol of DOX adjuvant treatment of canine hemangiosarcoma may not improve outcome.

Le sole termiti del legno secco native dell’Europa appartengono al genere Kalotermes. Fino a poco tempo fa, queste termiti erano considerate appartenenti alla sola specie Kalotermes flavicollis, distribuita in tutto il bacino del Mediterraneo. In due recenti studi, tuttavia, sono state descritte due nuove specie: K. italicus, in Italia, e K. phoenicae, a Cipro e nel Medio Oriente. Inoltre, studi di filogenesi molecolare hanno rivelato diverse linee di K. flavicollis: una in Corsica e Sardegna ed una nella Francia meridionale. Per ampliare le analisi sulla sistematica e la biogeografia di Kalotermes in Europa, sono state analizzate 43 colonie raccolte in 28 diverse località, insieme a campioni ottenuti da studi precedenti. Le analisi di filogenesi molecolare, condotte sul DNA mitocondriale, indicano i) che la distribuzione di K. italicus si estende dall'Italia centrale fino alla Francia meridionale, ii) che la linea Sardo-Corsa si trova anche nell’Italia continentale, e iii) che la linea della francese si estende fino alla penisola iberica. L’analisi dei cronogrammi, inoltre, hanno mostrato che le attuali Kalotermes europee si sono differenziate ~32 milioni di anni fa, con la separazione di K. phoenicae. Le specie K. italicus e K. flavicollis si sono separate ~15 milioni di anni fa, con le linee di K. flavicollis diversificatesi ~6 milioni di anni fa. In confronto alla storia evolutiva di un altro genere di termiti europee, Reticulitermes, è interessante notare come, sebbene si siano originate più di recente, queste ultime presentino una maggiore diversità. Le differenti caratteristiche ecologiche e/o di dinamica delle colonie possono spiegare questa differenza significativa.

Background Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Results Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Conclusions Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of osteosarcoma cells does not appear to be related to EGFR signalling exclusively. Angiogenic responses to radiation and kinase inhibitors are similarly likely to be multifactorial and require further investigation.

The dose-related antinociceptive effects of intravenous (IV) buprenorphine were evaluated in cats. Thermal (TT) and mechanical threshold (MT) devices were used for nociceptive stimulation. After baseline threshold recordings, buprenorphine was administered IV (0.01, 0.02 or 0.04 mg/kg; B1, B2 and B4, respectively) in a randomised, blinded and cross-over study. Data were analysed by ANOVA (P0.05) using 95% confidence intervals (CI). TT increased 15, 30, 45 min and 1 (5.2+/-2.7 degrees C), 2, 3 and 4 h after B1; 15, 30, 45 min and 1 (5.1+/-3.9 degrees C) and 2 h after B2, and 15, 30, 45 min and 1 (5.4+/-3.3 degrees C), 2, 3, 6 and 8 h after B4. MT increased 15 and 45 min after B2 (260+/-171 mmHg), and 30 (209+/-116 mmHg) and 45 min and 1 and 2 h after B4. At 45 min, MT values were significantly higher after B2 compared to B1 (P0.05). With MT, B2 and B4 produced more antinociception and longer duration of action than B1, respectively. No dose response to thermal stimulation was detected.

The aim of this study was to compare the antinociceptive effects of epidural buprenorphine (EB), epidural medetomidine (EM) or epidural buprenorphine-medetomidine (EBM). Eight cats were studied. Thermal thresholds (TT) were measured by increasing the temperature of a probe placed on the thorax. Mechanical thresholds (MT) were measured through inflation of a modified blood pressure bladder to the cat's forelimb. After baseline measurements, EB (0.02 mg/kg), EM (0.01 mg/kg) or half of the doses of each drug (EBM) were administered. Data were analysed using anova (P < 0.05) and 95% confidence interval (CI). TT increased from 30 min to 1 h after EB and at 45 min after EM. MT increased from 45 min to 2 h after EB, from 15 min to 1 h after EM and at 30, 45 min and at 2 h after EBM. MT were significantly lower after EB than EM at 30 min. TT were above the upper 95%CI from 15 min to 24 h after EB, from 15 min to 4 h after EM and from 15 min to 8 h after EBM. MT were above the upper 95%CI from 15 min to 5 h, and at 8, 12 and 24 h after EB, from 15 min to 6 h after EM and from 15 min to 6 h and at 12 and 24 h after EBM. All treatments had similar onset. Overall, EB presented longer period of action than EBM and EM. The same magnitude of analgesia was achieved, but with fewer side effects when EBM was compared with EM.

Objective—To evaluate the antinociceptive effects of epidurally administered hydromorphone in conscious, healthy cats. Animals—7 healthy adult cats. Procedures—An epidural catheter was implanted in each cat. Thermal threshold (TT) was measured by increasing the temperature of a probe placed on the thorax and monitoring the cat's response. Mechanical threshold (MT) was measured by manually inflating a modified blood-pressure bladder affixed to a thoracic limb and monitoring the response. After the baseline TT and MT values were determined, hydromorphone (0.05 mg/kg) or an equal volume of saline (0.9% NaCl) solution was epidurally injected. The TT and MT were again measured at 15, 30, 45, 60, 120, 180, 240, 300, 360, and 480 minutes after injection. Results—TT and MT did not change significantly from baseline values at any point after saline solution was administered. The MT and TT values were significantly higher than the baseline value at 15 minutes and at 120 and 180 minutes after hydromorphone administration, respectively. The MT and TT values after hydromorphone administration were also significantly different from those obtained at 30 minutes and at 15 minutes and 120 to 300 minutes, respectively, after administration of saline solution. No significant changes in skin temperature were detected after either treatment. Conclusions and Clinical Relevance—Epidural administration of hydromorphone at a dosage of 0.05 mg/kg yielded thermal and some mechanical antinociceptive effects in cats, and no hyperthermia was detected. Additional studies of the antinociceptive effectiveness and duration of epidurally administered hydromorphone in clinical situations are required.

This study evaluated the adverse effects of carprofen in seven healthy cats. Values for CBC, biochemical profiles and platelet aggregation were measured before and at seven days after SID treatment with subcutaneous carprofen: 4 mg/kg (day 1), 2 mg/kg (day 2 and 3) and 1 mg/kg (day 4 and 6) (CG) or 0.35 ml of saline (SG) for six days in a randomized, blinded, cross-over study with a four-week washout period. No treatment was given on day 5. Endoscopy of the GI tract was performed pre-treatment and on day 7 post-treatment. There were no significant changes in hematological profiles, biochemical profiles and endoscopy grading scores within nor between groups, except for lower albumin values at baseline than on day 7 (CG), and globulin and ALP values were higher at baseline than on day 7 in CG and SG. SC administration of carprofen over six days did not cause any adverse effects on gastrointestinal, hematological, or serum biochemical variables.

Avaliou-se os níveis de mercúrio total em amostras de peixes de água doce, procedentes de pesqueiros e pisciculturas de 39 regiões do estado de São Paulo. As espécies de peixes estudadas foram: pacu (Piaractus mesopotamicus), tilápia (Oreochromis niloticus), piauçu (Leoporinum sp), matrinxã (Brycon cephalus), tambaqui (Colossoma macropomum) e carpa (Cyprinus carpis). Foram avaliadas três metodologias de digestão de amostras para a determinação de mercúrio total, variando-se o volume de mistura sulfonítrica utilizada e a concentração do permanganato de potássio, sendo que o uso de 10mL de mistura sulfonítrica e solução de permanganato de potássio a 7% (m/v) na digestão da amostra foi a que forneceu as recuperações mais altas (96%), precisão de 1% e limite de quantificação de 0,3µg/kg. Após a validação do método, foram realizadas as determinações de teor de mercúrio total em 293 amostras de pescado, usando a técnica de espectrometria de emissão com fonte de plasma acoplado em gerador de hidretos. Os níveis médios de mercúrio encontrados variaram entre: tambaqui (0,0003-0,012mg/kg), carpa (0,063mg/kg), matrinxã (0,0003-0,074mg/kg), pacu (0,0003-0,078mg/kg), piauçu (0,0003-0,183mg/kg) e tilápia (0,0003-0,217mg/kg). Os resultados mostraram que nenhuma das amostras procedentes das diferentes regiões e diferentes pesque-pagues, apresentaram níveis de mercúrio total acima do permitido pela legislação brasileira, que é de 0,5mg/kg para pescado não-predador.In this work, the levels of mercury were evaluated in samples of freshwater fish, coming from fishing-grounds and piscicultures of 39 regions of São Paulo State. The species of fish studied were: pacu (Piaractus mesopotamicus), tilápia (Oreochromis niloticus), piauçu (Leoporinum sp), matrinxã (Brycon cephalus), tambaqui (Colossoma macropomum) and carpa (Cyprinus carpis). Three digestion methodologies of sample were evaluated for the mercury determination, with variation of the volume of sulphonitric mixture utilized and the potassium permanganate concentration. After the validation method the determinations of mercury level were recorded in 293 samples of fish, using the technique of emission spectrometry with plasma coupled to an hydrides generator. The medium levels of mercury found were the following: tambaqui (0,0003-0,012mg/kg), carpa (0,063mg/kg), matrinxã (0,0003-0,074mg/kg), pacu (0,0003-0,078mg/kg), piauçu (0,0003-0,183mg/kg) and tilápia (0,0003-0,217mg/kg), and this last one presented the higher contamination rate. The results showed that none of the samples obtained from the different regions and fish-and-pay establishments presented mercury levels above the permitted limits of Brazilian legislation; 0,5mg/kg for non carnivorous fish.

Os utensílios culinários podem liberar alguns componentes inorgânicos durante a cocção de alimentos. A migração destes pode ser interessante desde que ocorra em quantidades adequadas às necessidades nutricionais do usuário ou não existam implicações toxicológicas. Foi avaliada a migração de metais (Fe, Mg, Mn, Cr, Ni, Ca, Zn, Pb, Cd e Hg) por espectrometria de emissão óptica, utilizando ácidos láctico e acético como simulantes do alimento em panelas de aço inoxidável (304), ferro fundido (GG-10) e pedra-sabão (esteatito), durante 15 cocções seqüenciais. O comportamento da migração durante cinco ciclos de estocagem a 22ºC também foi estudado. Análise de regressão dos dados revelou que, em função do uso, as panelas de inox apresentam redução da migração de metais de forma rápida, enquanto as de pedra-sabão mostram um declínio suave na migração, contrastando com as de ferro, que apresentam aumento na migração de Fe e Mn com o uso contínuo. Em nenhum utensílio foi observada a liberação de metais pesados, como Pb, Cd ou Hg.Cookware may release some inorganic components into foods during cooking. Migration of ions could be of nutritional interest provided they do not pose a toxic hazard or are released in excess of the recommended amounts. In the present study the migration of Fe, Mg, Mn, Cr, Ni, Ca, Zn, Pb, Cd and Hg from stainless steel (304), cast iron (GG-10) and soapstone (steatite) commercial cooking pans was evaluated in 15 sequential operations using lactic and acetic acids as food simulants. The migration patterns were also studied along five cycles of storage at 22ºC. Regression analysis of the data showed that while ions migrated out of the stainless steel pans in a rapidly decreasing fashion, the rate of migration from the stone utensils exhibited a slow and gradual decrease, in contrast with the increasing tendency observed for Fe and Mn in the cast iron pans. In no case was the release of heavy metals such as lead, cadmium or mercury observed.

The purpose of this study was to investigate the association among matrix metalloproteinases (gelatinases A and B, stromelysin-3 (ST3) and matrilysin) mRNAs expressed in primary breast carcinomas and stan-dard prognostic parameters and clinical outcome. mRNA levels were determined by Northern analysis in samples of 81 breast cancer patients (median follow-up, 40 months) and 27 samples of uninvolved adjacent breast tissue. Proteases were expressed by the majority of the tumors and normal breast tissues examined. ST3, gelatinase A and matrilysin mRNAs were more often expressed at high levels in carcinomatous than in normal breast tissues. Differences in the distribution of gelatinase B mRNA were not found. However, paired normal tissues generally produced weaker signals when compared to matched tumor samples. Univariate analysis showed no significant association of gelatinase A and matrilysin mRNAs with the classical prognostic markers (age, menopausal status, stage, size, nodal status, vascular infiltrate, necrosis, steroid receptors, metastasis and survival). Overexpression of ST3 was more frequently found in tumors of post-menopausal women (P < 0.022). Elevated expression of gel B mRNA was associated with the presence of vascular infiltrate (P < 0.026), necrosis (P < 0.039), PR negative tumors (P < 0.014) and inversely corre-lated to the number of survivors (P < 0.021). Multivariate analysis including 68 patients for whom all information was available indicated that neither stromelysin correlated significantly with pathological, clin-ical or biochemical features. High levels of gelatinase A and B mRNAs were inversely associated with the number of survivors. Our findings suggest that measurements of gelatinase A and B mRNAs expression in breast carcinoma may help to identify patients with an agressive form of the disease. ©Lippincott Williams & Wilkins

Tandem repeats constitute a consistent fraction of eukaryotic genomes and are given by monomers or gene clusters repeated in a head-to-tail direction. Main examples are represented by microsatellites, minisatellites, satellites, rDNA genes, and protein-coding genes. Tandem repeats dynamics is mainly due to nonreciprocal genomic turnover mechanisms, leading to copy number variation. They differently impact on genome evolution and some of them appear clearly linked to human diseases and are applied for DNA fingerprinting.

The effects of ketoconazole, a synthetic imidazole derivate, were evaluated in 42 women affected by acne (17 cases) and/or hirsutism (36 cases) treated with 400 mg/day for 3-6 months. Androstenedione, total and free testosterone, 5 alpha dihydrotestosterone and dehydroepiandrosterone levels progressively dropped during treatment while 17 alpha hydroxyprogesterone, estradiol, ACTH, cortisol, LH and FSH levels increased. Dehydroepiandrosterone sulfate decreased only towards the end of treatment, while estrone, sex hormone binding globulin, and PRL remained unchanged. Daily mean +/- SD rate of hair growth, measured by a special image analysis processor, decreased within 3 months of therapy from 0.258 +/- 0.058 to 0.184 +/- 0.039 mm/day (P less than 0.02) and mean +/- SD hair diameter from 0.123 +/- 0.015 to 0.110 +/- 0.013 mm (P less than 0.05) together with decreasing hormone levels. The therapeutic effects of ketoconazole on hirsutism was evident at 6 months in only 14 subjects, while no significant change in hirsutism score was recorded in 22 women who failed to complete the therapy. Acne improved in all cases. Several side effects and complications arose during treatment, such as headache, nausea, loss of scalp hair, hepatitis, and biochemical changes. Even though ketoconazole improves hyperandrogenism, only selected patients are eligible for treatment as scrupulous monitoring is required.

Vitamin A and its metabolic forms, like all-trans retinoic acid (ATRA), are used with promising results in the treatment of many tumors. Two major problems in the clinical use of retinoids are that the doses needed for successful treatment are often toxic, leading to "hypervitaminosis A syndrome" and that patients often develop drug resistance. In order to find compounds that can overcome these problems, many new derivatives of retinoids have been synthesized and tested. Here we present a study on the effect of a new derivative of retinoic acid, IIF (pat. WIPO W0 00/17143), on growth and differentiation of two colon carcinomna cell lines, CaCo-2 and HT-29, with different degrees of tumorigenicity, the second one being more undifferentiated. The effect of IIF was compared with that of ATRA, whose antitumoral action on colon cancer cells and other tumoral cells is widely described in the literature. Besides exerting a strong antiproliferative effect, even higher than that of ATRA, IIF induced cellular differentiation, as demonstrated by the appearance of morphological (domes and microvilli formation) and biochemical (alkaline phosphatase induction) markers. Therefore, these findings indicate the new retinoid IIF as a possible candidate in the treatment of colon cancer.

Objective: To compare the antitumoral effects of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) with those of 5-OH,11-O-hydrophenanthrene (IIF), a new derivative of retinoic acid. Materials and Methods: The effect of retinoids was tested on cell line HL-60. Cell differentiation and apoptosis were evaluated by morphological and biochemical analysis as bcl-2 protein and by DNA fragmentation assay. The ability to activate retinoic acid receptors (RAR) and/or retinoid X receptors (RXR) and to modulate gene expression was determined by transactivation assay. Results: With cell line HL-60, the antiproliferative effect of IIF was stronger than that of ATRA and 9-cis RA. Following retinoid treatment, cells appeared to differentiate and apoptotic cells were observed. The appearance of DNA laddering and a decrease in the amount of bcl-2 protein confirmed apoptosis. IIF transcriptionally activated RXR-γ more than RAR-α. Conclusion: The findings indicate that IIF transcriptionally activates RXR-γ preferentially, induces apoptosis and has a more antiproliferative activity than ATRA and 9-cis RA on cell line HL-60.

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

In the parthenogenetic Bacillus atticus atticus, widespread over most of the Mediterranean basin, a highly repeated satellite DNA (BaB300) has been detected and analysed. BgI II, Taq I and Alu I restriction enzymes cut 316 bp long repeating units from the BaB300 family. These are non-coding, AT rich and well conserved in the three isolated populations studied. By in situ hybridization, the satellite has been located in the centromeric heterochromatin of a subset of medium- and small-sized chromosomes, including the Xs. Related sequences have been found in the bisexual B. grandii grandii and in its specific hybrid B. whitei (=B. rossius/grandii grandii), while they are absent from B. rossius redtenbacheri. These data support the genetic affinity relationships within the genus Bacillus inferred from morphological, allozymic and karyological data. They also encourage further comparisons among Bacillus hybrids, in order to trace their parental genomes in the hybrid nuclei, and within the B. atticus complex (i.e. additional samples of B. a. atticus and diploid/triploid cytotypes of B. a. carius) to study repetitive DNA turnover in unisexual organisms.

We have shown by in vitro experiments, using immunofluorescence techniques, that precipitated immune complexes of IgM antibodies and ovalbumin (ICIgM) are able to bind to rabbit blood polymorphonuclear leucocytes (PMN), as well as immune complexes of IgG antibodies (ICIgG). This binding capacity for both classes of immune complexes is exhibited by more than 80% of the PMN cell population and is independent of Ca2+ in the medium. For ICIgG the binding to PMN can be completely inhibited by preincubation of the cells with soluble IgG used at physiological concentrations (competition for the Fc gamma receptors) while for ICIgM there is no such inhibition by fluid-phase IgM. After binding to the leucocytes there was a striking difference in the fate of ICIgM and ICIgG: whereas the ICIgG was readily phagocytosed (endocytosed), the ICIgM remained mostly on the cell surface, being only poorly endocytosed after 1 hr incubation at 37 degrees. This was demonstrated by a quantitative fluorimetric method developed to assay phagocytosis of immune complexes, and was confirmed by a qualitative fluorescence quenching technique. These results may have implications for understanding the fate of these classes of immune complexes formed in circulation or deposited in tissues, and the participation of PMN in inflammatory reactions and tissue injury in immune complex diseases.

Phagocytosis mediated by IgM and C3 receptors was studied by in vitro experiments with peritoneal macrophages from mice treated with repeated i.p. injections of glycogen. The immune complexes were sheep red cells sensitized with mouse IgM antibody or antibody and complement. Macrophages from the inflammatory exudate induced by glycogen were able to phagocytize immune complexes of red cells and IgM antibodies in contrast to normal macrophages, which can only bind to these complexes. Evidence was also found for phagocytosis mediated by interaction with C3 receptors in the macrophages from the glycogen-treated animals. The implications of these findings in the mechanism of phagocytosis and the possible relevance of the observed changes in the function of macrophage receptors for the afferent and efferent branches of immune response are discussed.

(1986). Taxonomy and phylogenetic inference. Bollettino di zoologia: Vol. 53, No. sup001, pp. 53-60.

Lysosomal enzyme release by rabbit polymorphonuclear leukocytes induced by immune complexes of IgM or IgG antibodies and ovalbumin was studied by in vitro experiments. It was shown that IgM immune complexes in the form of precipitates were able to stimulate the release of the lysosomal enzymes beta-glucuronidase and alkaline and acid phosphatases, in contrast to heat-aggregated IgM, which had only a small effect. This stimulation was dependent on the presence of Ca2+ in the extracellular medium; Mg2+ showed a slight effect as a substitute for Ca2+. IgG precipitated immune complexes prepared from different regions of the precipitation curve were tested. It was shown that immune precipitates from the equivalence region were the most effective; those at antibody excess have a smaller but comparable capacity, whereas the precipitated immune complexes at antigen excess were the least effective. Soluble immune complexes of IgG with an excess of antigen concentration in the range of 2.5 to 5 times that of equivalence were also able to stimulate the enzyme release, but that capacity decreased rapidly with the increase in antigen concentration. The enzyme release induced by IgM immune complexes was not inhibited by competition with free immunoglobulin in the medium, either IgM or IgG in approximate physiologic concentrations; contrarily, with IgG immune complexes, free monomeric IgG (but not IgM) can completely block the process. The possible implications of these findings in the immune complex diseases are discussed.

An open randomised crossover study (20 patients) was performed to compare the efficacy in stable effort angina of atenolol 100mg once a day, and pindolol 5mg thrice daily.

Sheep red cells (E) sensitized with IgG antibody (EA) or with antibody and complement (EAC) interact in vitro with mouse peritoneal macrophage monolayers. The role of IgG and of C3 in the attachment and ingestion of the erythrocytes was examined by means of quantitative technique utilizing 51Cr-labeled E. Controlled osmotic lysis permitted the separate measurement of the radioactivity associated with bound or with ingested E. IgG-125I was used to estimate the number of IgG molecules bound per E as a function of the IgG concentration. Control experiments showed that iodination did not influence the extent of binding of IgG to E and that the binding of IgG prepared from immune serum could be essentially ascribed to its anti-E antibody content. Only between 103 and 104 rabbit anti-E IgG molecules per erythrocyte were needed for detectable attachment and ingestion of EA (a maximum number of 6 x 105 IgG antibody molecules could be accomodated on one erythrocyte). Evidence was obtained that C3 is primarily involved in particle attachment, whereas only IgG is able to markedly promote the ingestion of particles attached to macrophages: (a) Addition of complement to the EA substantially increased the binding to the macrophages, whereas ingestion was increased to a smaller extent. Both binding and ingestion of EAC were markedly inhibited by papain fragments of IgG obtained from a rabbit antiserum to mouse C3. (b) Low doses (2 µg/ml) of papain fragments of IgG from a rabbit antiserum to mouse IgG markedly reduced the ingestion of EAC, whereas attachment of EAC to macrophages was inhibited to a much smaller degree. The possible relevance of these findings for the in vivo fate of particulate immune complexes as they interact with macrophages is discussed. It is suggested that in the primary immune response, when the complexes are predominantly in the form of EA (IgM) or EA (IgM) C3, they would tend to remain on the surface of the macrophages and thus be in a position to stimulate immunocompetent cells. In the secondary response, when EA (IgG) or EA (IgG) C3 predominate, the complexes would tend to be more rapidly interiorized and degraded by the mononuclear phagocytes

1. The effect of benzylation of tryptophan residues of immunoglobulin G (IgG) on the interaction with macrophage receptors was studied by measuring the competition between free IgG and immune complexes (sheep red cells covered with IgG antibodies). 2. The modification of approximately three tryptophan side chains greatly decreased the capacity of IgG for binding to the phagocytic cell. Under conditions in which normal IgG inhibited the binding of immune complexes to macrophages by 49 +/- 5% and the urea-treated IgG control inhibited it by 40 +/- 11%, IgG modified by 2-hydroxy-5-nitrobenzyl bromide in the presence of urea inhibited the binding by 7.5 +/- 5%. 3. These results indicate that tryptophan residues are located at or near the site of IgG that binds to the macrophage receptor and that they are important for the interaction.

A molecular mechanism for B and T lymphocyte collaboration is proposed. Two sets of polivalent receptors, each one on a cell type, would interact with different epitopes of the antigen, resulting in a cross-linking of the receptors as the initial step in B lymphocyte activation. The mechanism can explain many known featrues of humoral immune response and accords with experimental findings on the blast transformation of B lymphocytes.

Lidocaine (L) in still considered the drug of choice in the treatment of life threatening ventricular arrhythmias in the setting of acute myocardial infarction (A.M.I.): this is mainly due to its proved efficacy and high therapeutic index. Since however L has well defined electrophysiologic properties (class 1 B) and does not seem to be effective in all of these patients we compared its antiarrhythmic efficacy with the one of amiodarone (A), an antiarrhythmic agent provided of electrophysiologic properties quite different from L (class 4) and usually well tolerated. Twenty-five consecutive patients with A.M.I. without haemodynamic consequences, were randomly assigned to L (bolus of 1 mg/kg followed by an infusion of 10 mg/min for 20 minutes and thereafter of 1,5 mg/min) or A (bolus of 5 mg/kg and an eventual repeat dose of 150 mg followed by an infusion of 1,8 g/24 h) The baseline arrhythmia was classified as Lown class 2 ore more in all the patients. The ventricular arrhythmias were completely relieved in 47% of the patients assigned to L and in 60% of those treated with A (p = N.S.); a minor efficacy was found in 40% of the patients of both groups; in two cases ventricular fibrillation occurred after the acute infusion of L. The antiarrhythmic effectiveness kept fit with both drugs over a 24 hours period. A prolongation of QTc interval was found to occur both after the bolus and 24 hours of treatment with A. In addition A provoked a slight decrease of systolic blood pressure. No important side effects were observed with both drugs regimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium oxalate (CaOx) stone formation depends on both oversaturation with CaOx and the level of inhibitory activity that retards the crystallization of the salt. Robertson et al. (1) showed that naturally occurring glycosaminoglycans (GAG) are potent inhibitors of the growth and aggregation of CaOx crystals in vitro. Since then the effects of specific urinary GAG on CaOx crystallization have been evaluated by numerous investigators (2–5). Many attempts have been made to detect a decreased excretion of GAG by CaOx stone-forming individuals, and a decrease has indeed been reported (1,6). Other investigators using a different technique failed to detect such a difference between normal and stone-forming subjects (7, 8).

The effect of complement components incorporated into precipitated immune complexes (IC) of IgG or of IgM on their capacity for stimulating lysosomal enzyme release from rabbit polymorphonuclear leucocytes was studied in vitro. We have found that: (i) complement causes an amplification in the stimulatory capacity of both classes of IC, the effect being dependent on the concentration of the IC, and higher for the IgM class; (ii) dose-effect experiments of competition by fluid-phase immunoglobulins have shown that IgG (in physiological or smaller concentrations) can inhibit greatly the stimulation by this class of immune complex; this inhibition can be prevented, however, by the presence of complement in the IC (a situation expected to occur in vivo); for IgM immune complexes there was no such competitive inhibition, so complement would not be necessary; (iii) the relevant complement factors must be located in the range C1-C3. These results help us to understand the importance of complement (besides the well-known generation of chemotactic factors) in the mechanisms of tissue injury produced by neutrophils in immune complex diseases.

Transposable elements are widely distributed within genomes where they may significantly impact their evolution and cell functions. Short interspersed elements (SINEs) are non-autonomous, fast-evolving elements, but some of them carry a highly conserved domain (HCD), whose sequence remained substantially unchanged throughout the metazoan evolution. SINEs carrying the HCD called V are absent in amniote genomes, but V-like sequences were found within the miniature inverted-repeat transposable element (MITE) MER6 in Homo sapiens. In the present work, the genomic distribution and evolution of MER6 are investigated, in order to reconstruct the origin of human V domain and to envisage its possible functional role. The analysis of 85 tetrapod genomes revealed that MER6 and its variant MER6A are found in primates, while only the MER6A variant was found in bats and eulipotyphlans. These MITEs appeared no longer active, in line with literature data on mammalian DNA transposons. Moreover, they appeared to have originated from a Mariner element found in turtles and from a V-SINE from bony fishes. MER6 insertions were found within genes and conserved in mRNAs: in line with previous hypothesis on functional role of HCDs, the MER6 V domain may be important for cell function also in mammals.