Curcumin dietary supplementation ameliorates disease phenotype in an animal model of Huntington's disease.

Huntington's disease (HD) has traditionally been described as a disorder purely of the brain; however, evidence indicates that peripheral abnormalities are also commonly seen. Among others, severe unintended body weight loss represents a prevalent and often debilitating feature of HD pathology, with no therapies available. It correlates with disease progression and significantly affects the quality of life of HD patients. Curcumin, a naturally occurring polyphenol with multiple therapeutic properties, has been validated to exert important beneficial effects under health conditions as well as in different pathological settings, including neurodegenerative and gastrointestinal (GI) disorders. Here, we investigated the potential therapeutic action that curcumin-supplemented diet may exert on central and peripheral dysfunctions in R6/2 mice, a well-characterized HD animal model which recapitulates some features of human pathology. Maintenance of normal motor function, protection from neuropathology and from GI dysfunction and preservation of GI emptying and conserved intestinal contractility, proved the beneficial role of life-long dietary curcumin in HD and corroborated the potential of the compound to be exploited to alleviate very debilitating symptoms associated with the disease.

Clinical and functional outcomes of cad/cam mandibular reconstruction with free fibular flap comparing traditional versus micro-invasive intraoral surgical approaches.

The surgical incision plays a pivotal role in any surgical procedure. A good surgical approach should allow optimal visualization, respect the anatomy and ensure the best aesthetic outcome possible, especially when the lesions involve the face. In this retrospective study, carried out from June 2014 to April 2018, different types of surgical approaches to perform mandibular reconstruction were compared. Twentyone patients who underwent mandibular reconstruction with free fibular flap (FFFs) using CAD-CAM technology and Virtual Surgical Planning (VSP) were included in the study, regardless the condition, the timing of reconstruction (primary vs secondary), the number of fibular segments or the type and size of the mandibular defect. The patients were treated for mandibular defects secondary to benign or low-grade oncological lesions and different non-oncological conditions. However, patients requiring neck dissection were excluded from the study. Patients were divided into two groups according to the type of surgical approach used: 7 patients received a traditional transcervical approach together with an intraoral approach, while 14 patients were operated through an intraoral approach combined with different microinvasive approaches, including the sub-mandibular, the retro-mandibular and the preauricular approaches. Different factors were statistically compared: characteristics of the harvested fibula, surgical timing, days of hospitalization, as well as complication, functional and aesthetic outcomes. According to this study, no statistically significant differences were observed between the two groups in any of the features considered. These results support the hypothesis that the combination of different microinvasive approaches and the traditional approach are superimposable, and they can be safely exchanged when the underlying defects allow it.

Identification of the faecal indicator Escherichia coli in wastewater through the ß-D-glucuronidase activity: comparison between two enumeration methods, membrane filtration with TBX agar, and Colilert®-18.

Escherichia coli (E. coli) is one of the most commonly adopted indicators for the determination of the microbiological quality in water and treated wastewater. Two main types of methods are used for the enumeration of this faecal indicator: membrane filtration (MF) and enzyme substrate tests. For both types, several substrates based on the ß-D-glucuronidase activity have been commercialized. The specificity of this enzyme for E. coli bacteria has generated considerable use of methods that identify the ß-D-glucuronidase activity as a definite indication of the presence of E. coli, without any further confirmation. This approach has been recently questioned for the application to wastewater. The present study compares two methods belonging to the above-mentioned types for the enumeration of E. coli in wastewater: MF with Tryptone Bile X-glucuronide agar and the Colilert®-18 test. Confirmation tests showed low average percentages of false positives and false negatives for both enumeration methods (between 4 and 11%). Moreover, the counting capabilities of these two methods were compared for a set of 70 samples of wastewater having different origins and degrees of treatment. Statistical analysis showed that the Colilert®-18 test allowed on average for a significantly higher recovery of E. coli.

Removal of organics and degradation products from industrial wastewater by a membrane bioreactor integrated with ozone or UV/H2O2 treatment.

The treatment of a pharmaceutical wastewater resulting from the production of an antibacterial drug (nalidixic acid) was investigated employing a membrane bioreactor (MBR) integrated with either ozonation or UV/H(2)O(2) process. This was achieved by placing chemical oxidation in the recirculation stream of the MBR. A conventional configuration with chemical oxidation as polishing for the MBR effluent was also tested as a reference. The synergistic effect of MBR when integrated with chemical oxidation was assessed by monitoring (i) the main wastewater characteristics, (ii) the concentration of nalidixic acid, (iii) the 48 organics identified in the raw wastewater and (iv) the 55 degradation products identified during wastewater treatment. Results showed that MBR integration with ozonation or UV/H(2)O(2) did not cause relevant drawbacks to both biological and filtration processes, with COD removal rates in the range 85-95%. Nalidixic acid passed undegraded through the MBR and was completely removed in the chemical oxidation step. Although the polishing configuration appeared to give better performances than the integrated system in removing 15 out of 48 secondary organics while similar removals were obtained for 19 other compounds. The benefit of the integrated system was however evident for the removal of the degradation products. Indeed, the integrated system allowed higher removals for 34 out of 55 degradation products while for only 4 compounds the polishing configuration gave better performance. Overall, results showed the effectiveness of the integrated treatment with both ozone and UV/H(2)O(2).

Membrane filtration of municipal wastewater effluents for implementing agricultural reuse in southern Italy.

Membrane filtration was investigated at field scale in order to assess its effectiveness for reusing municipal effluents in agriculture. The study was started on April 2002 and ended on September 2007, as part of a national R&D project (AQUATEC). Preliminary results, which we already reported elsewhere, concerned the first two project years while this paper refers to the subsequent period. Three different crops (processing tomato, fennel and lettuce) were grown in rotation at a test field located in Apulia (Southern Italy) and irrigated with membrane filtered municipal secondary effluents. The quality of the reclaimed water was monitored chemically and microbiologically, and compared with conventional water pumped from a local well. Both water sources were used in parallel for irrigating two plots of the test field. The results showed that the microbiological quality of the treated wastewater was comparable to or even higher than that of the conventional source. Protozoan (oo)cysts were experimentally identified as effective indicators of possible failures of the filtration system. Moreover, long term heavy metals accumulation trends were monitored in soil and crops, showing that despite some lead and copper accumulation in the soil, no measurable increase of these metals was observed in the edible parts of the crops.

[Source apportionment of benzo(a)pyrene in Taranto and carcinogenic risk estimate in general population]. / Individuazione e caratterizzazione delle sorgenti di benzo(a)pirene nel comune di Taranto e stima del rischio cancerogeno associato all'esposizione della popolazione generale.

INTRODUCTION: In 2009 the limit value of benzo(a)pyrene (BaP) in ambient air of 1.0 ng/m3 has been exceeded in the urban district of Taranto near to the industrial area, where a several large plants are located, including an integrated cycle steel plant. OBJECTIVE: To identify emission sources and quantify relative contribution to the PAHs levels; to estimate health impact associated to PAHs exposure in general population. METHODS: Multivariate receptor models have been used. Concentration of PAHs measured in 4 location in Taranto in 2008-2009 have been analyzed. 5 different models estimated profiles of unknown sources and identified significant chemical species. To compute the lung cancer risk the WHO unit risk estimate for BaP (8.7 x 10(5) ng/m3) has been adopted. RESULTS: Models employed identify 3 to 4 emission sources. Estimated profiles have been compared with measured ones. Based on the average annual BaP level measured (1.3 ng/m3), 2 attributable cancer cases in the district Taranto population are estimated to result from a life-time exposure. CONCLUSIONS: Among different emissive sources, the analysis identifies theoretical sources whose profiles, compared with observed data, allow to identify dominant contributions to PAHs pollution and to design corrective actions to reduce environmental and health impact.

TBP-1 protects the human oncosuppressor p14ARF from proteasomal degradation.

The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer. The mechanisms that regulate alternative reading frame (ARF) turnover have been obscure for long time, being ARF a relatively stable protein. Recently, it has been described that its degradation depends, at least in part, on the proteasome and that it can be subjected to N-terminal ubiquitination. We have previously reported that ARF protein levels are regulated by TBP-1 (Tat-Binding Protein 1), a multifunctional protein, component of the regulatory subunit of the proteasome, involved in different cellular processes. Here we demonstrate that the stabilization effect exerted by TBP-1 requires an intact N-terminal 39 amino acids in ARF and occurs independently from N-terminal ubiquitination of the protein. Furthermore, we observed that ARF can be degraded in vitro by the 20S proteasome, in the absence of ubiquitination and this effect can be counteracted by TBP-1. These observations seem relevant in the comprehension of the regulation of ARF metabolism as, among the plethora of cellular ARF's interactors already identified, only NPM/B23 and TBP-1 appear to be involved in the control of ARF intracellular levels.

Optimal sludge retention time for a bench scale MBR treating municipal sewage.

Membrane bioreactors allow for higher sludge concentrations and improved degradation efficiencies with respect to conventional activated sludge. However, in the current practice these systems are often operated under sub-optimal conditions, since so far no precise indications have yet been issued on the optimal operating conditions of MBR for municipal wastewater treatment. This paper reports some results of four years of operation of a bench scale membrane bioreactor where steady state conditions were investigated under different sludge retention times. The whole experimental campaign was oriented towards the investigation of optimal process conditions in terms of COD removal and nitrification, biomass activity and growth, and sludge characteristics. The membrane bioreactor treated real municipal sewage, and four different sludge ages were tested (20, 40, 60, and 80 days) and compared with previous data on complete sludge retention. The results showed that the the biology of the system, as assessed by the oxygen uptake rate, is less affected than the sludge physical parameters. In particular, although the growth yield was observed to dramatically drop for SRT higher than 80 days, the biological activity was maintained under all the tested conditions. These considerations suggest that high SRT are convenient in terms of limited excess sludge production without losses of the treatment capacity. Physical characteristics such as the viscosity and the filterability appear to be negatively affected by prolonged sludge retention times, but their values remain within the ranges normally reported for conventional activated sludge.

Membrane bioreactor sludge rheology at different solid retention times.

Rheological characterization is of crucial importance in sludge management both in terms of biomass dewatering and stabilization properties and in terms of design parameters for sludge handling operations. The sludge retention time (SRT) has a significant influence on biomass properties in biological wastewater treatment systems and in particular in membrane bioreactors (MBRs). The aim of this work is to compare the rheological behaviour of the biomass in a MBR operated under different SRTs. A bench-scale MBR was operated for 4 years under the same conditions except for the SRT, which ranged from 20 days to complete sludge retention. The rheological properties were measured over time and the apparent viscosity was correlated with the concentration of solid material when equilibrium conditions were reached and maintained. The three models most commonly adopted for rheological simulations were evaluated and compared in terms of their parameters. Then, steady-state average values of these parameters were related to the equilibrium biomass concentration (MLSS). The models were tested to select the one better fitting the experimental data in terms of mean root square error (MRSE). The relationship between the apparent viscosity and the shear rate, as a function of solid concentration, was determined and is proposed here. Statistical analysis showed that, in general, the Bingham model provided slightly better results than the Ostwald one. However, considering that a strong correlation between the two parameters of the Ostwald model was found for all the SRTs tested, both in the transient growth phases and under steady-state conditions, this model might be used more conveniently. This feature suggests that the latter model is easier to be used for the determination of the sludge apparent viscosity.

Physical characteristics of the sludge in a complete retention membrane bioreactor.

Sludge physical characteristics play an important role in the operation of membrane bioreactors (MBR) due to their influence on filtration and their effects on handling of excess sludge. These systems are designed to maintain high solid concentrations, thus limiting sludge production and the related operational costs of the process. In this study, the sludge from a bench scale MBR operated for about 1 year with complete solid retention was investigated to assess its physical and rheological properties. Concentrations of mixed liquor suspended solids (MLSS) up to 24 gTSSL(-1) affected the diluted sludge volume index (DSVI), the capillary suction time (CST), the specific resistance to filtration (SRF) and the compressibility (s). The MBR sludge displayed similar dewatering properties of conventional waste activated sludge, suggesting that the upgrade of wastewater treatment plants with the MBR technology would not affect the behaviour of the dewatering equipment. The apparent viscosity was expressed as a function of the MLSS and the experimental data were interpreted by comparing different models. Ostwald model was chosen, and two equations for viscosity were proposed. The thixotropy of MBR sludge was also evaluated by measuring the reduced hysteresis area (rHa) and relating this parameter to the characteristics of the sludge. The evaluation of energy consumption for mixing evidenced that, under the tested conditions, the increase of solid concentration from 3 to 30 gTSSL(-1) resulted in a limited increase of energy requirements (25-30%).

Influence of solid retention time on the rheology of MBR sludge.

The rheological characterization is of crucial importance in sludge management both for biomass dewatering and stabilization purposes and for the definition of design parameters for sludge handling operations. The sludge retention time (SRT) has a significant influence on biomass properties in biological wastewater treatment systems and in particular in membrane bioreactors (MBR). The aim of this work is to compare the rheological behaviour of the biomass in a membrane bioreactor operated under different SRT. A bench scale MBR was operated for four years under the same conditions except for the SRT, that ranged from 20 days to complete sludge retention. The rheological properties were measured over time and the apparent viscosity was correlated with the concentration of solid material under equilibrium conditions. The three models most commonly adopted for rheological simulations were evaluated and compared in terms of their parameters. Steady state average values of these parameters were related to the equilibrium biomass concentration (MLSS). The models were tested to select the one better fitting the experimental data in terms of Mean Root Square Error (MRSE). The relationship between the apparent viscosity and the shear rate, as a function of solid concentration, was determined and proposed.

p14ARF interacts with the focal adhesion kinase and protects cells from anoikis.

The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.

Development of a total colonoscopy rat model with endoscopic submucosal injection of the cecal wall.

BACKGROUND: Experimental models of colorectal tumor require either laparotomy for induction or anastomosis following resection. The long murine cecum avoids the need for an anastomosis, making the cecum the preferred site for induction. This study aimed to evaluate total colonoscopy with submucosal injection of cecal wall (TCWI) in rats in terms of failure rate (FR), complication rate (CR), and reproducibility (R). METHODS: A bolus of bowel prep was given. Anesthesia was injected intraperitoneally. A video fiberscope (5.9 mm outer diameter, 180/90 degrees up/down bending, 100/100 degrees right/left bending, 103 cm working length, 120 degrees view field, and 2.0 mm channel) allowed for irrigation and suction. Saline 1 ml was injected in the cecal wall through a 4-mm-long, 23-gauge needle placed on a 3-mm wire, resulting in a blister. FR was a failure to reach and inject the cecum. Rats were allowed to recover. CR was measured at necropsy. R was assessed by comparing TCWI time, FR, and CR for three investigators. Sample size of 120 (type I error, 0.05; power, 80%) was based on a pilot study. Data are presented as median (range). RESULTS: A total of two of 122 rats (1.6%) died after prep or anesthesia. Bowel prep resulted in 99.1% evacuation of solid feces. A total of 120 male Sprague-Dawley retired breeders weighing 592 g (range, 349-780) underwent TCWI. Scope depth was 28 cm (range, 20-36). Irrigating fluid was 290 ml (range, 100-600). TCWI time was 7 min (range, 4-28). FR was 4%. In three failed cases, the scope reached the ascending colon. CR was 2%. There were two perforations in the ascending colon. All three operators had similar TCWI time (p = 0.673), FR (p > 0.1), and CR (p > 0.1). A total of 98.3% of rats survived to planned sacrifice. At 48-h necropsy, the injection site was macroscopically identified in 118 rats. CONCLUSIONS: A safe and reproducible TCWI rat model has been achieved, which may provide a valuable tool in the future for studies of solid colorectal tumors.

Rheology of sludge in a complete retention membrane bioreactor.

A rheological characterization of the sludge sampled from a complete retention membrane bioreactor was performed in order to correlate the apparent viscosity with the concentration of solids. The three most commonly adopted models were tested to select the one better fitting the experimental data. Ostwald model was choosen, and the relationship between the apparent viscosity and the shear rate was determined for mixed liquor suspended solids (MLSS) concentrations ranging between 9 and 25 g l(-1). Ostwald model parameters k and n were correlated with MLSS concentration, comparing linear, power, and exponential-power (only for k) laws in terms of R2 and Mean Root Square Error (MRSE). Both power and exponential-power functions provided good and comparable correlations for parameter k, while the linear relationship was much less accurate, especially at the highest solid concentrations. The parameter n was better modelled by a power function than by a linear one. Therefore two simulation models were proposed, both based on Ostwald's equation, where the two parameters k and n were expressed as functions of MLSS. Evaluation of energy consumption for mixing showed that the increase of solid concentration from 3 to 30 g l(-1) resulted in a limited increase in energy requirement (25-30 %). In real systems, where Reynolds numbers shift towards the turbulent regime, the increase of energy requirements for increasing solids concentration is even less pronounced. The thixotropy of membrane bioreactor sludge was also evaluated by measuring the rHa (reduced hysteresis area) and relating this parameter to the characteristics of the sludge growing within the reactor.

Activated sludge diffusion for odour removal --effects of H2S on the biomass.

The removal of odours from wastewater treatment plants through diffusion of odour-containing air volumes into the aerated basins was investigated in a bench scale experimental campaign which lasted more than 200 days. Hydrogen sulphide was selected as a model odorous compound and its removal efficiencies were experimentally evaluated along with its effects on the biomass and on the main biochemical processes. Two bench scale sequencing batch reactors were fed in parallel on real primary sewage and monitored for chemical oxygen demand removal, nitrification and denitrification. The balance of H2S was also monitored after adding to one of them a Na2S liquid solution of 17 mgS l(reactor)(-1) d(-1), corresponding to a gas-phase concentration of 240 mgS (Nm3)(-1). Results showed an average sulphide removal of 94% in the reactor supplied with Na2S. Moreover, microbial composition did not show relevant variations after the addition of sulphide, and the good features of activated sludge flocs were maintained also in terms of sludge settleability. No relevant effects of sulphide were detected on carbon and nitrogen metabolism and chemical oxygen demand removal, nitrification and denitrification efficiencies were always above 75%, 95%, and 50% respectively, and comparable across the two reactors.

Model for the genetic evolution of human solid tumors.

A conceptual model is proposed for the genetic evolution of many human solid tumors that is based on the observations that cancer cells may spontaneously double their chromosome number; that cells with excessive chromosome numbers may be cytogenetically unstable, both losing chromosomes randomly during subsequent cell divisions, and often developing structural abnormalities in the chromosomes that are retained; and that some structural chromosome abnormalities may activate growth-promoting genes. The sequence of tetraploidization with chromosome loss can occur repeatedly in a given tumor. The available evidence supporting the model is reviewed. A computer simulation system that embodies these concepts is described and the model is used to generate distributions of chromosome number/cell under various simulated conditions and in a variety of simulated biological settings. A simulation of the time course of changes in chromosome number per cell that accompany the spontaneous neoplastic transformation of mouse fibroblasts in vitro is described. The best fit to the data was obtained when provision was made for the activation of at least two growth-promoting genes. The conditions for generating discrete aneuploid peaks in cytogenetic and flow cytometric studies were explored; our modeling studies suggest that the activation of a growth promoting gene is required in order to produce a discrete aneuploid peak. Our modeling studies suggest that the overrepresentation of individual oncogene-bearing chromosomes in aneuploid cell lines may require the activation of gene dose-dependent growth-promoting genes and is not likely to occur in cell lines in which at least two copies of each normal chromosome are required for cell survival. Overall, the results obtained using the model are consistent with a wide variety of flow cytometric and cytogenetic studies in human solid tumors.

Karyotypic evolution of a human undifferentiated large cell carcinoma of the lung in tissue culture.

Serial cytogenetic studies were performed on a cell line derived from a pleural effusion from a patient with undifferentiated large cell carcinoma of the lung. The initial sample had a broad range of chromosome numbers per cell, with a hypodiploid/pseudodiploid stem line and a hypotetraploid sideline. A sequence consisting of a doubling of chromosome number per cell followed by chromosome loss was observed repeatedly during 40 culture passages. The presence of metaphase spreads showing evidence of endoreduplication suggested this as a likely mechanism for the doubling of chromosome number per cell. Eleven marker chromosomes were observed in the cells of the primary sample; these markers persisted through all subsequent passages. Chromosomes 1, 2, 6, 7, 8, 11, and 16 were consistently overrepresented; each of these chromosomes was involved in marker formation. Chromosomes 4, 5, 9, 10, 19, 21, and 22 were consistently underrepresented. Every chromosome, either in its normal form and/or as part of a marker, was represented on the average by at least one copy per diploid cell. Eighteen new marker chromosomes were observed during the course of cell cultivation; one of these evolved into a clonal marker over the course of six cell passages. Of the new marker chromosomes that were formed during the observation period, the majority were found in hypotetraploid cells.

Bacillus megaterium SF185 induces stress pathways and affects the cell cycle distribution of human intestinal epithelial cells.

The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.

Dose-dependent fetal complications of warfarin in pregnant women with mechanical heart valves.

OBJECTIVES: The purpose of this study was to assess the incidence of warfarin fetal complications and whether they are dose-dependent. BACKGROUND: Gravid patients with mechanical heart valves require long-term anticoagulant therapy. Controversy exists concerning the appropriate treatment of these patients. METHODS: Forty-three women on warfarin carrying out 58 pregnancies were studied. For each patient with full-term pregnancy a caesarian section was scheduled for the 38th week during brief warfarin discontinuation. Maternal and fetal complications were evaluated. Fetal complications were divided according to the warfarin dosage < or = 5 mg and > 5 mg necessary to keep an international normalized ratio (INR) of 2.5 to 3.5, and analyzed subsequently. RESULTS: A total of 58 pregnancies were observed: 31 healthy babies (30 full term, 1 premature) and 27 fetal complications (22 spontaneous abortions, 2 warfarin embryopathies, 1 stillbirth, 1 ventricular septal defect, 1 growth retardation) were recorded. Two maternal valve thromboses occurred. No fetal or maternal bleeding was observed during caesarian sections or premature vaginal delivery. Patients whose warfarin doses during pregnancy were > 5 mg had 22 fetal complications, whereas those taking a dose < or = 5 mg had only five fetal complications (p = 0.0001). For an increase of the warfarin dose there was a substantially increased probability of fetal complications (p < 0.0001; p < 0.7316). CONCLUSIONS: There is a close dependency between warfarin dosage and fetal complications. Patients on warfarin anticoagulation may be delivered by planned caesarian section at the 38th week while briefly interrupting anticoagulation.

Correlations among p53, Her-2/neu, and ras overexpression and aneuploidy by multiparameter flow cytometry in human breast cancer: evidence for a common phenotypic evolutionary pattern in infiltrating ductal carcinomas.

Human solid tumors develop multiple genetic abnormalities that accumulate progressively in individual cells during the course of tumor evolution. We sought to determine whether there are specific sequences of occurrence of these progressive evolutionary changes in human breast cancers by performing correlated cell-by-cell measurements of cell DNA content, p53 protein, Her-2/neu protein, and ras protein by multiparameter flow cytometry in 56 primary tumor samples obtained at surgery. In addition, p53 allelic loss and Her-2/neu gene amplification were determined by fluorescence in situ hybridization in cells from the same samples. We reasoned that if there is a specific order in which genetic changes occur, the same early changes would be found consistently in the cells with the fewest abnormalities. We reasoned further that late-developing abnormalities would not occur alone in individual cells but would almost always be found together with the early changes inherited by the same cells. By these criteria, abnormalities involving p53 generally occurred early in the course of development of invasive breast cancers, whereas ras protein overexpression was found to be a late-occurring phenomenon. Within individual tumors, cellular p53 overexpression was often observed alone in individual cells, whereas ras protein overexpression was rarely observed in the absence of p53 overexpression and/or Her-2/neu overexpression in the same cells. Furthermore, the intracellular level of each abnormally expressed protein was found to increase progressively as new abnormalities were acquired. Infiltrating ductal carcinomas exhibited characteristic phenotypic patterns in which p53 allelic loss and/or p53 protein overexpression, Her-2/neu amplification and/or overexpression, aneuploidy, and ras overexpression accumulated within individual cells. However, this pattern was not a prominent feature of lobular breast cancers. All six lobular breast cancers studied were diploid. p53 allelic loss and/or early p53 overexpression, and late ras cooverexpression in the same cells were less common in lobular breast cancers than in infiltrating ductal carcinomas. Although Her-21neu overexpression was a common finding in lobular breast cancers, Her-2/neu amplification was not observed in these tumors.