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Historic data show that home flea infestations can be managed by treating all animals on the premises with a highly effective flea control product. The use of effective products has also been shown to reduce pruritus and minimize dermatologic lesions in both cats and dogs. Therefore, an in-home study was conducted in West Central Florida USA to evaluate the efficacy of a topically applied selamectin-sarolaner formulation to control fleas in naturally infested cats over a 12-week period. Thirty-seven cats in 21 households were treated once monthly with the selamectin-sarolaner topical solution. In the topical fluralaner treatment (positive control) group, forty-three cats in 20 households were treated once on day 0. A combined total of thirty dogs in both groups were treated once monthly with oral sarolaner. Fleas on cats were counted by flea combing, fleas on dogs were counted using visual area counts and fleas in the indoor premises were assessed using intermittent-light flea traps. Blinded-assessments of feline dermatologic lesions (modified-SCORFAD) were conducted monthly by a boarded-dermatologist and pruritus severity was evaluated by pet owners. Three consecutive monthly treatments of selamectin-sarolaner reduced flea populations on cats by 96.3 % within 7 days and by 100% from week 6 to the end of the 12-week study. The topical application of fluralaner reduced flea populations by 98.1 % within 7 days and efficacy reached 100% by week 12. At the end of the study, fleas were completely eradicated (from cats, dogs and homes) in every home regardless of treatment group. Owner reported cat pruritus was reduced by > 87 % in both treatment groups by week 12. Significant improvements in dermatologic lesion scores (> 81 %) were achieved by both products by the end of the study. Monthly applications of topical selamectin-sarolaner or topical fluralaner to cats living in the heavy flea challenge environment of West Central Florida USA were effective in eradicating flea infestations, reducing pruritus and improving dermatologic lesions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Use global gene expression to characterize differences between high-grade and low-grade clear cell renal cell carcinoma (ccRCC) compared with normal and benign renal tissue., Methods: Tissue samples were collected from patients undergoing surgical resection for ccRCC. Affymetrix gene expression arrays were used to examine global gene expression patterns in high- (n = 16) and low-grade ccRCC (n = 13) as well as in samples from normal kidney (n =14) and benign kidney disease (n = 6). Differential gene expression was determined by analysis of variance with a false discovery rate of 1% and a 2-fold cutoff., Results: Comparing high-grade ccRCC with each of normal and benign kidney resulted in 1,833 and 2,208 differentially expressed genes, respectively. Of these, 930 were differentially expressed in both comparisons. In order to identify genes most related to progression of ccRCC, these differentially expressed genes were filtered to identify genes that showed a pattern of expression with a magnitude of change greater in high-grade ccRCC in the comparison to low-grade ccRCC. This resulted in the identification of genes such as TMEM45A, ceruloplasmin, and E-cadherin that were involved in cell processes of cell differentiation and response to hypoxia. Additionally changes in HIF1α and TNF signaling are highly represented by changes between high- and low-grade ccRCC., Conclusions: Gene expression differences between high-grade and low-grade ccRCC may prove to be valuable biomarkers for advanced ccRCC. In addition, altered signaling between grades of ccRCC may provide important insight into the biology driving the progression of ccRCC and potential targets for therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Gene expression profile classification is a pivotal research domain assisting in the transformation from traditional to personalized medicine. A major challenge associated with gene expression data classification is the small number of samples relative to the large number of genes. To address this problem, researchers have devised various feature selection algorithms to reduce the number of genes. Recent studies have been experimenting with the use of semantic similarity between genes in Gene Ontology (GO) as a method to improve feature selection. While there are few studies that discuss how to use GO for feature selection, there is no simulation study that addresses when to use GO-based feature selection. To investigate this, we developed a novel simulation, which generates binary class datasets, where the differentially expressed genes between two classes have some underlying relationship in GO. This allows us to investigate the effects of various factors such as the relative connectedness of the underlying genes in GO, the mean magnitude of separation between differentially expressed genes denoted by δ, and the number of training samples. Our simulation results suggest that the connectedness in GO of the differentially expressed genes for a biological condition is the primary factor for determining the efficacy of GO-based feature selection. In particular, as the connectedness of differentially expressed genes increases, the classification accuracy improvement increases. To quantify this notion of connectedness, we defined a measure called Biological Condition Annotation Level BCAL(G), where G is a graph of differentially expressed genes. Our main conclusions with respect to GO-based feature selection are the following: (1) it increases classification accuracy when BCAL(G) ≥ 0.696; (2) it decreases classification accuracy when BCAL(G) ≤ 0.389; (3) it provides marginal accuracy improvement when 0.389

The Laryngeal Mask Airway (LMA) is an accepted adjunct for airway management in emergency patients. There are a number of case reports describing its use in transport medicine for infant to adult patients, including during flight. Although studies of the effect altitude has on air-filled tracheal tubes exists, we were unable to find documentation of the effect of altitude on laryngeal mask airways. Our objective was to assess the effect of altitude on the LMA in both fixed wing and rotary wing models. We performed an in vitro study of the effect of altitude on the LMA cuff. Infant and adult airway trainer mannequins with properly sized and inserted LMA-Classic laryngeal mask airways were monitored for cuff pressure changes while flown at altitudes commonly encountered during air medical transport. Both models demonstrated that LMA cuff pressures may exceed manufacturer recommended levels for safe use even at the relatively low altitudes experienced during rotor wing flight. Properly inserted and inflated laryngeal mask airways at ground level may result in overinflated LMA cuffs when flown to altitudes commonly used for rotor and fixed wing medical transport unless monitored and corrected.

Background: The usual indication for sentinel lymph node biopsy (SLNB) in melanoma is a primary tumour >1mm thickness but under these criteria less than 20% of SLNBs are positive. Of those patients with a negative sentinel node (SN) over 10% will have disease recurrence within 3 years. A more accurate delineation of candidate patients for SLNB and risk profile for negative SN patients is therefore desirable. Melanoma cell adhesion molecule (MCAM) is a predominant cell adhesion molecule of melanomas and its expression has been implicated in tumour progression and metastasis., Aims: To compare MCAM expression in primary and metastatic melanoma and to investigate if MCAM expression in patients meeting the criteria for SLNB correlated with patient outcome., Methods: Tissue arrays of primary (n=78) and metastatic (n=92) melanomas were constructed from archived paraffin embedded tissue and MCAM expression detected by immunohistochemistry. Staining positivity and intensity were assessed by visual scoring and correlated with clinical outcome., Results: In patients meeting the current criteria for SLNB, Cox multivariate analysis showed both MCAM expression positivity and intensity were independently predictive of survival (P=0.007) and development of lymph node disease (P=0.01) in primary melanoma over and above established markers of prognosis, such as Breslow thickness. MCAM-negative patients had a 5-year survival of 92% compared with 40% for MCAM positive., Conclusions: Measurement of MCAM expression represents a potential method to stratify SLNB patients on the basis of risk. This would have considerable benefits in terms of both cost and patient morbidity.

Background: Previous studies from our group had identified c-myc oncoprotein expression as an important prognostic parameter in a series of retrospective studies of primary and metastatic melanoma and other variants of this disease. This study set out to prospectively evaluate the prognostic significance of c-myc positivity in a consecutive series of primary melanomas presenting at Mount Vernon Hospital Regional Plastic Surgery and Burns Centre., Methods: A consecutive series of 117 primary melanomas underwent flow cytometric analysis for c-myc expression at diagnosis. Routine clinical and histological parameters were collected from each patient's clinical records and survival assessed. The mean follow up was 45 months., Results: Kaplan-Meier survival analysis demonstrated that Breslow depth, histogenic subtype, ulceration, age and sex had prognostic significance. Survival analysis revealed high c-myc positivity to be significantly associated with poorer outcome (P<0.043). Each of the main prognostic parameters were assessed for their independent significance using Cox Proportional Hazards; only c-myc retained independent significance (P<0.039)., Conclusions: The strength of this study is that it was performed in a consecutive series of patients followed up in a longitudinal prospective study. c-myc was not the strongest predictor of survival in univariate analysis, but was the only parameter that retained significance in multivariate analysis.

Aims: We report a prospective study examining the prognostic significance of the c-myc oncoprotein, p53 tumour suppressor gene and proliferation rate measurements in malignant melanoma., Methods: Flow cytometry (FCM) was used to measure the expression of c-myc, p53 and proliferation parameters in patients who had received an injection of the thymidine analogue bromodeoxyuridine prior to surgery., Results: Sixty-seven patients had successful FCM measurements of the three parameters. c-myc was detected in 97% of patients with a median cell positivity of 62%. The median p53 positivity was 13%. The median potential doubling time (T(pot)) of the tumours wasf 9.4 days. In univariate analysis, each of the parameters showed an association with survival in metatstatic disease with rapid proliferation (p=0.006) or overexpression of c-myc (p=0.038) related to poor survival whereas increased positivity for p53 predicted better survival (p=0.013)., Conclusions: These data indicate that laser cytometric technology can be used to obtain quantitative data on oncoproteins expression and cell proliferation rates in clinical samples of malignant melanoma.

The c-myc oncogene has been shown to be overexpressed in a number of malignancies, and may play an important role in the pathogenesis of malignant melanoma. Previous prognostic studies have demonstrated c-myc overexpression in a range of cutaneous melanomas, and levels of c-myc oncoprotein expression have been shown to correlate with clinical outcome in both primary and secondary disease. The purpose of this study was to investigate the in vitro manipulation of c-myc expression using antisense oligonucleotides. The human melanoma cell lines A375M, Be11 and WM115 were treated with c-myc antisense oligonucleotides, and the cellular growth was compared with controls. Antisense oligonucleotides reduced the growth rate of all three cell lines, and produced a reduction in c-myc gene expression as measured by flow cytometry. The growth inhibitions in the A375M, Be11 and WM115 cell lines at 72 h were 36.6%, 35.8% and 29.3%, respectively. Each of these was significantly different from control cultures (P<0.01). The c-myc antisense produced a mean 75% reduction in c-myc oncoprotein expression when compared with controls in the A375M cells (P<0.001), a 49% reduction in the Be11 cells (P<0.001) and a 28% reduction in the WM115 cells (P=0.005). This study demonstrates the importance of the c-myc oncogene in controlling melanoma growth. It suggests that blocking the expression of this gene, using an antisense approach, reduces melanoma cell growth, and may potentially provide a novel gene-therapy strategy for the treatment of advanced melanoma., (Copyright 2002 The British Association of Plastic Surgeons. Published by Elsevier Science Ltd. All rights reserved.)

It has been suggested that basal cell carcinomas (BCC) arising in younger patients may be of a more aggressive phenotype than those that present in the older population. The purpose of this study was to investigate this hypothesis by comparing the histological types of BCC in a large cohort of young and old patients.A total of 271 histological specimens and clinical records were evaluated. A consecutive series of 120 patients under 50 years old were compared with a consecutive series of 151 patients over 50 years of age. Tumours were classified according to the accepted definition of aggressive (morphoeic, infiltrative and micronodular) and less aggressive (nodular and superficial) histological growth patterns. Aggressive growth types were found in 35% of patients over 50 and 33% of patients under 50. There was a higher rate of incomplete excision in younger patients but this did not reach significance (16% compared to 9%, P = 0.1). This study found that BCC arising in young patients are not histologically different to those found in the older population. Clinically observed aggressive behaviour of BCC in young patients may result from inadequate surgical excision due to cosmesis and diagnostic doubt., (Copyright 2000 The British Association of Plastic Surgeons.)

This paper reviews the available data for cell production rates of human tissues and tumours, measured in vivo using halogenated pyrimidine labelling and laser cytometry. The technique has now been widely evaluated, and we draw general inferences from the proliferative data over a broad range of tumour and tissue types. Estimates of the S-phase duration, the time taken for DNA synthesis in cycling cells, are consistent over a narrow range with a median value of around 10 hours, notwithstanding the constraints of the experimental and statistical technique, in normal tissues and tumours. This suggests that Ts values may be a species-specific constant. The more easily measured labelled S-phase fraction, or labelling index, shows much greater intra and intertumour variation within any one tumour class. It may thus be a surrogate for time dependent measurements to a first order approximation. The cell production rate, described by the potential doubling time (Tpot), is remarkably rapid in most tumours, a median value of the order of 5 days, and much faster than clinical volume doubling times for most lesions. The rapid cell production rates in normal tissues and tumours highlight the importance of cell loss in the growth and modelling of biological structures. Cell production rate measurements do not adequately describe the biological aggressiveness of tumours. They may be used to refine adjuvant strategies for radiotherapy and chemotherapy in experimental research. Dynamic halogenated pyrimidine labelling has provided unique and valuable insights into the living biology of human tissues and tumours., (Copyright 2000 Harcourt Publishers Ltd.)

In the past two decades, the technology of laser cytometry and use of the halogenated thymidine (HP) analogues bromodeoxyuridine and iododeoxyuridine as proliferation labels, have allowed us to quantify the rate of cell turnover in tissues and tumours, in clinical samples as in laboratory models. The principal studies have used injection of bromo- or iododeoxyuridine to measure cell production rates in vivo. Flow cytometry (FCM) has been used to estimate the S phase labelling index (LI) and the S phase duration (Ts) and calculate the cell production rate, represented by the potential doubling time (Tpot). This has allowed calculation of time-dependent indices of proliferation from single biopsies of HP pulse labelled human tissues and tumours. In the first part of this two-part review, we describe the technique and its limitations as a biological assay. The second part summarizes the knowledge gained about cell production rates and the relevance that this information may have to future investigative, prognostic and treatment strategies., (Copyright 2000 Harcourt Publishers Ltd.)

Acral lentiginous melanoma is a particularly aggressive melanocytic lesion but, due to its comparative rarity, biological investigations into the behaviour of this subtype of melanoma are lacking. The activity of the recently described p16 tumour suppressor gene, thought to be the 'familial melanoma gene', was studied in 24 patients with subungual melanoma and 44 patients with plantar melanoma. Lower levels of p16 oncoprotein were demonstrated than that found in other histogenetic types of melanoma. Stratification of patients of all disease stages revealed a poorer survival in patients with low p16 expression (log rank test, chi(2)= 3.9, P = 0. 05). These data suggest that p16 inactivation may play an important role in the development and progression of acral lentiginous melanomas. However, the level of p16 expression was not prognostic since survival analysis on stratification of stage I patients according to p16 level did not reach statistical significance for both survival and disease free interval., (Copyright 2000 The British Association of Plastic Surgeons.)

It has been widely reported that the eyeblink component of the acoustic startle reflex can be modulated by emotionally-toned slide stimuli; pleasant slides reduce eyeblink amplitudes whereas unpleasant slides enhance them. The present study examines the modulation of the acoustic startle reflex by short (2-min) film-clips classified as pleasant, unpleasant or neutral, on the basis of subjective ratings. These film-clips were also evaluated with respect to their test-retest reliability in producing affective states as well as modulating startle reflexes. Overall, results showed significant reduction of startle during pleasant clips and augmentation during unpleasant clips. However, on first showing, one of the two unpleasant clips (a medical demonstration film depicting details of toe surgery) actually inhibited the startle reflex rather than augmented it. This is discussed in terms of the proposition that only stimuli which arouse fear can be guaranteed to augment startle; stimuli that are repulsive may produce perceptual and emotional 'blunting' that reduces startle amplitude.

Acral lentiginous melanoma is a particularly aggressive tumour with a worse prognosis than other varieties of primary cutaneous melanoma. In order to study the biology of this disease, the activity of the c-myc oncogene was studied in tumours from 45 patients using flow cytometry. High levels of oncoprotein were found in all tumours and exceeded that documented in other varieties of cutaneous melanoma. Survival analysis with stratification of patients according to oncogene activity provided a useful prognostic marker with shorter disease free interval (Log-Rank test, chi 2 = 16.7, P < 0.0001) and overall survival (Log-Rank test, chi 2 = 8.9, P = 0.002) in tumours with high oncoprotein levels. Multivariate analysis revealed c-myc oncogene expression to be more accurate in predicting clinical outcome than all existing clinicopathological parameters including the Breslow depth (Cox's proportional hazards model, P = 0.0011). This study provides biological evidence to explain the aggressive behaviour of acral melanoma and supports the application of oncogene measurement as an accurate prognostic marker.

Melanomas of the scalp are rare and have a poorer prognosis than melanomas arising at other cutaneous sites. In order to study the biology of this disease, the activity of the c-myc oncogene was studied in tumours from 25 patients with scalp melanoma using the technique of flow cytometry. Survival analysis revealed that stratification of patients according to oncogene activity provided a prognostic marker with shorter overall survival (log rank test, chi 2 = 3.9, P = 0.05) in tumours with high nuclear c-myc oncoprotein positivity. These results support our previous studies of the prognostic value of c-myc expression in melanoma and suggest that estimation of c-myc oncoprotein may be of clinical significance in identifying high risk patients.

This study compared the clinical features, histological subtype, growth fraction (by Ki67 immunohistochemistry) and proliferation pattern of 22 clinically defined horrifying basal cell carcinoma compared to 81 non horrifying lesions. Late presentation was associated with half of the horrifying tumours. The other half developed horrifying tumours despite early intervention. The horrifying tumours exhibited a variety of histological growth patterns. A total of 50% were infiltrative, 23% nodular and 18% micronodular. There was no difference in the growth fraction or proliferation pattern between horrifying and non horrifying tumours of similar growth pattern (P = ns), although infiltrative tumours in either group exhibited a significantly higher growth fraction than nodular tumours (P < 0.01). This suggests that there is no intrinsic biological difference between horrifying and non horrifying tumours to account for their behaviour. We conclude that late presentation, failed or inadequate early management especially of infiltrative tumours (and other subtypes) determines the development of horrifying tumours.

Patients with melanoma who develop nodal metastatic disease represent a group with heterogeneous clinical outcome. Nodal positivity remains the most accurate prognostic marker for regional melanoma although it fails to predict outcome in a significant number of patients. Recent studies have illustrated the prognostic potential of c-myc oncogene expression in melanoma. The aim of this study was to measure c-myc oncoprotein in a series of regional metastatic specimens from 48 patients, and evaluate its use as a marker of clinical outcome. Oncoprotein expression was detected in 46 (96%) of the tumours with a median positivity of 68% (range 0-98%). Survival analysis revealed a significant association between oncoprotein positivity and survival (Long-Rank test, chi 2 = 15.2, P < 0.001). Multivariate analysis of outcome showed c-myc oncoprotein to be an independent prognostic marker more accurate than all other clinicopathological parameters including nodal positivity (chi 2 = 8.34, P = 0.003). Estimation of c-myc oncoprotein is therefore recommended as a powerful prognostic marker for regional metastatic melanoma.

This study investigates the proliferation characteristics of 81 primary basal cell carcinomas (BCC) using detection of the Ki-67 antigen by immunohistochemistry. The tumours were classified into distinct sub-types based on their histological growth pattern and differentiation status. The mean Ki-67 growth fraction was 0.293 and this was found to vary between the different growth patterns, with morpheic, infiltrating and superficial tumours showing the highest levels of proliferation at 0.373, 0.351 and 0.335, respectively; the nodular and micronodular growth patterns were significantly lower at 0.248 and 0.232, respectively. No overall association was seen between proliferation and differentiation status although certain histological growth patterns such as nodular showed a greater propensity to differentiate. Proliferation was related to tumour size, with larger lesions exhibiting higher growth fractions although this may have also been related to tumour subtype as infiltrating and morpheic tumours tended to present with larger tumour diameters. The spatial distribution of proliferating cells by Ki-67 labelling was not related to tumour subtype, differentiation or growth fraction. These studies have shown BCC to possess proliferative characteristics akin to other solid tumours commonly regarded as more rapidly dividing. There was an association between growth fraction and tumour subtype consistent with higher proliferation in the lesions considered to be more aggressive.

Subungual melanoma is a particularly aggressive tumour. However, biological investigations of its behaviour are presently lacking due to its comparative rarity. In order to study the biology of this disease, the activity of the c-myc oncogene was studied in tumours from 24 patients with subungual melanoma using the technique of flow cytometry. High levels of oncoprotein were found in all tumours and exceeded that documented in other varieties of cutaneous melanoma. Survival analysis revealed that stratification of patients according to oncogene activity provided a useful prognostic marker with shorter disease free interval (log rank test, chi 2 = 6.6, P = 0.01) and overall survival (log rank test, chi 2 = 3.6, P = 0.07) in tumours with high oncoprotein levels. This is the first study to investigate oncogene expression in subungual disease and supports its potential application as a prognostic marker.

This study was undertaken to assess the value of flow cytometric measurements of total p53 protein content and proliferation indices derived from in vivo halogenated pyrimidine labelling. Two series of colorectal cancer specimens were studied for which clinical outcome data were recorded. A series of 84 archival, ethanol-fixed, bromodeoxyuridine (BrdUrd) labelled colorectal tumours were analysed by flow cytometry for their total and cell cycle phase p53 protein content using the pAb1801 monoclonal antibody. A second series of 33 freshly obtained tumours was used for assay evaluation and for comparison with the archival material. In the archival series (n=84), the median p53-pAb1801 LI was 81.9% (range: 11.1-99.8%). In only three tumours could significant amounts of p53 protein not be detected. The median phase specific p53-pAb1801 LI in G0/G1 was 71.6%, in S was 95.5%, and in G2/M was 98.5%. In the series of fresh tumours (n=33), the median p53-pAb1801 labelling index (LI) was 94.6% (range: 17.9-99.9%). Only two tumours failed to express significant amounts of p53 protein. There was no significant difference in the generally high levels of p53 protein content between the fresh and archival series. Life-table analysis of the patients in the archival series failed to demonstrate a statistical difference in life expectancy in relation to Dukes' stage when tumours were stratified by the median total p53 labelling index. In this study, p53 content and proliferative indices measured by flow cytometry do not have independent predictive value over Dukes' grading in determining the outcome of colorectal cancer. Flow cytometry is confirmed as a practical tool for multi-parametric and cell cycle analysis of oncoprotein expression in human tumour biopsies.

Programmed cell death (apoptosis) is now recognized as an important factor in tumour growth. Bcl-2 is an oncogene which promotes tumour progression by specifically inhibiting programmed cell death. Bcl-2 oncoprotein was measured using flow cytometry in 42 surgically excised regional lymph node metastases from patients with a median follow-up of 45 months. Fifteen patients in the study were found to have bcl-2 expression which was associated with significantly shorter survival (log-rank test, P<0.002). In addition, multivariate analysis confirmed the predictive value of bcl-2 independent of other established prognostic markers (chi(2)=7.02, P<0.01). Oncogenic control of programmed cell death is therefore important in melanoma progression and bcl-2 measurement provides a useful marker of prognosis for regional lymph node metastases.

Melanoma produces specific tumour antigens which are capable of eliciting an immune response. However, this tumour evades the immune system, in part, by downregulation of class I HLA antigens on the cell surface, which are required for T cell recognition. It has been suggested that the oncogene c-myc may have a role in effecting this change in vitro, however, the relationship between oncoprotein level and tumour antigenicity has not been established in human tumours. This study measured c-myc oncoprotein in 94 melanoma specimens (46 primary tumours and 48 regional metastases) using flow cytometry and evaluated class I HLA expression with immunohistochemistry. C-myc expression was found in 91 tumours (96%) with higher expression in metastases than primary melanomas (P<0.005). Class I HLA expression was found to show great variation although metastases showed less antigenicity than primary tumours (P<0.01). Analysis of the relationship between these two parameters revealed a highly significant correlation in both primary (P<0.01) and metastatic disease (P<0.01), with high oncoprotein being associated with down regulation of cell surface antigens. Knowledge of the control of tumour antigenicity is likely to provide an objective platform for the development of new strategies for immunotherapy.

Forty-six tumors from patients with oropharyngeal carcinoma were analysed by flow cytometry after injection of bromodeoxyuridine (Budr) for the labelling index, the duration of S phase and the potential doubling time (Tpot). The results show large variations in Tpot (from 2.6 to 16.7 days) among these tumors from the same site and with the same histology. The variations in Tpot were not significantly related to TNM status and differentiation grade. However, aneuploid tumors had statistically significant shorter Tpot. The predictive value of Tpot regarding the response to radiotherapy is currently under investigation.