Canine discrimination of ovarian cancer through volatile organic compounds.

Ovarian cancer has a high mortality rate due to its unclear symptomology and the lack of precise early detection tools. If detected in the first stage, over 90% of patients reach remission. As such, developing a reliable method of early detection is crucial in reducing the mortality rate of the disease. One potential method would be to identify specific biomarkers that are unique to ovarian cancer, which could be detected using a blood test. While this can be done using gas chromatography - mass spectrometry (GC-MS), identifying these biomarkers is an enormous task. One way to expedite the process is to utilize trained scent detection canines. In this study, dogs who were previously trained to respond to positive blood samples from ovarian cancer patients were then tested on their ability to recognize samples prepared by micro-preparative gas chromatography (MP-GC) techniques. MP-GC employed a gradient-cooled glass tube connected to the GC outlet to collect GC eluents containing the plasma-derived volatiles in positive blood samples. These post-column fractions were collected at the exit of the GC according to their eluent times (i.e., 0-15 min, 15-25 min and 25-35 min or 0-35 min) and these full or fractional collections were presented to the trained dogs to judge their responses. Dogs' time spent investigating the odor was used as an indication of odor recognition and was significantly longer on the early (0-15 min) and middle (15-25 min) fractions of the ovarian cancer than the late (25-35 min) fraction of plasma odorants or either the negative fractions or distractors odorants. These findings suggest that characteristic odor biomarkers of ovarian cancer for dogs may exist in the relatively small and more volatile compounds. Additionally, variation between dogs suggests that there may be a number of different biomarkers that can be used to identify ovarian cancer.

The bicuspid aortic valve: an integrated phenotypic classification of leaflet morphology and aortic root shape.

OBJECTIVE: To establish a classification of bicuspid aortic valve (BAV) that includes both leaflet morphology and aortic shape. SETTING: Two academic medical centres of the University of Washington, Seattle. PATIENTS: 191 adult patients with BAV. INTERVENTIONS: Review of clinical data and transthoracic echocardiograms. MAIN OUTCOME MEASURES: Assessment of leaflet morphology; valve function; aortic shape and dimensions. RESULTS: We identified three morphologies: type 1, fusion of right and left coronary cusp (n = 152); type 2, right and non-coronary fusion (n = 39); and type 3, left and non-coronary fusion (n = 1). Comparing type 1 and 2 BAV, there were no significant differences in age, height, weight, blood pressure or aortic valve function. Type 1 was more common in men (69 vs 45%). The aortic sinuses were larger in type 1, while type 2 had larger arch dimensions. Myxomatous mitral valves were more common in type 2 BAV (13% vs 2.6%, p<0.05). Three aortic shapes were defined: normal (N), sinus effacement (E), and ascending dilatation (A). Comparing type 1 to type 2 BAV, shape N was more common in type 1 (60% vs 32%), and type A was more common in type 2 (35% vs 54%,); type E was rare (p<0.01 across all groups). CONCLUSION: A comprehensive BAV phenotype includes aortic shape. Type 1 BAV is associated with male gender and normal aortic shape but a larger sinus diameter. Type 2 leaflet morphology is associated with ascending aorta dilatation , larger arch dimensions and higher prevalence of myxomatous mitral valve disease.

Mitral regurgitation.

Mitral regurgitation is a common finding on echocardiography, seen to some degree in over 3/4 of the population. Pathologic mitral regurgitation is a common hospital diagnosis, but the percent of patients with mitral valve disease who ever require surgical correction is very small. There are many etiologies of mitral regurgitation, caused by either pathologic changes to one or more of the components of the mitral valve, including the leaflets, annulus, chordae tendineae, papillary muscles, or by abnormalities of the surrounding left ventricle and/or atrium. Mitral regurgitation can be diagnosed on physical exam or by angiography, but is best diagnosed and quantified using echocardiography. The outcome of mitral regurgitation depends on the acuity of onset of the regurgitation, as well as etiology of the mitral valve disease. Acute mitral regurgitation requires urgent mitral valve surgery. In contrast, most patients with chronic mitral regurgitation will never need corrective surgery. Currently, there is not convincing evidence that medical therapy with vasodilating medications slows the progression of mitral regurgitation. When patients with chronic mitral regurgitation develop symptoms of pathologic changes to the left ventricle, surgical treatment should be offered. Mitral valve repair is the preferred corrective surgery, and only when not possible should mitral valve replacement be performed.

Timing of surgery in aortic stenosis.

In adults with valvular stenosis, the importance of prompt aortic valve replacement once symptoms occur is well known. The operative mortality for aortic valve replacement has improved dramatically over the past 4 decades and remains the only effective therapy for severe symptomatic aortic stenosis. Aortic valve replacement in patients with left ventricular dysfunction has a high operative mortality, although those patients who do not undergo surgery at all have an even worse outcome. While issues to consider include the presence or absence of coronary artery disease and expected hemodynamics of the prosthetic valve compared with the native valve, when in doubt, one should err on the side of surgical intervention. Elderly age is not a contraindication to aortic valve replacement for severe symptomatic aortic stenosis, although there is a higher prevalence of comorbid disease and higher operative mortality. Life expectancy is significantly prolonged and quality of life is significantly improved in the elderly who survive surgery. Indications for surgery in asymptomatic patients are controversial. We do not recommend valve replacement in asymptomatic patients at this time due to the known risks of surgery and a prosthetic valve and the lack of evidence for benefit of early surgery. Patients undergoing coronary bypass surgery should be considered for concomitant aortic valve surgery for moderate aortic stenosis that is expected to progress to severe stenosis in less than 5 years.

Effect of culture PO2 on macrophage (RAW 264.7) nitric oxide production.

Macrophages are commonly cultured at a PO2 of 149 Torr, but tissue macrophages in vivo live in an environment of much lower oxygen tension. Despite the many potential mechanisms for changes in oxygen tension to influence nitric oxide (NO) synthesis, there have been few reports investigating the effect of PO2 on macrophage NO production. With the use of a culture chamber designed to rigorously control oxygen tension, we investigated the effects of culture PO2 on macrophage NO production, inducible nitric oxide synthase (iNOS) activity, iNOS protein, and tumor necrosis factor production. NO production and iNOS activity were linearly related in the range of 39.4 to 677 Torr, but not in the range of 1.03 to 39.4 Torr. Therefore, results obtained in vitro for the high oxygen tensions commonly used in cell culture were quantitatively and qualitatively different from results obtained in cells cultured at the lower oxygen tensions that more accurately reflect the in vivo environment. The influence of oxygen tension on NO production has implications for cell culture methodology and for the relationship between microcirculatory dysfunction and inflammatory responses in rodent models of sepsis.

Association of aortic-valve sclerosis with cardiovascular mortality and morbidity in the elderly.

BACKGROUND: Although aortic-valve stenosis is clearly associated with adverse cardiovascular outcomes, it is unclear whether valve sclerosis increases the risk of cardiovascular events. METHODS: We assessed echocardiograms obtained at base line from 5621 men and women 65 years of age or older who were enrolled in a population-based prospective study. On echocardiography, the aortic valve was normal in 70 percent (3919 subjects), sclerotic without outflow obstruction in 29 percent (1610), and stenotic in 2 percent (92). The subjects were followed for a mean of 5.0 years to assess the risk of death from any cause and of death from cardiovascular causes. Cardiovascular morbidity was defined as new episodes of myocardial infarction, angina pectoris, congestive heart failure, or stroke. RESULTS: There was a stepwise increase in deaths from any cause (P for trend, <0.001) and deaths from cardiovascular causes (P for trend, <0.001) with increasing aortic-valve abnormality; the respective rates were 14.9 and 6.1 percent in the group with normal aortic valves, 21.9 and 10.1 percent in the group with aortic sclerosis, and 41.3 and 19.6 percent in the group with aortic stenosis. The relative risk of death from cardiovascular causes among subjects without coronary heart disease at base line was 1.66 (95 percent confidence interval, 1.23 to 2.23) for those with sclerotic valves as compared with those with normal valves, after adjustment for age and sex. The relative risk remained elevated after further adjustment for clinical factors associated with sclerosis (relative risk, 1.52; 95 percent confidence interval, 1.12 to 2.05). The relative risk of myocardial infarction was 1.40 (95 percent confidence interval, 1.07 to 1.83) among subjects with aortic sclerosis, as compared with those with normal aortic valves. CONCLUSIONS: Aortic sclerosis is common in the elderly and is associated with an increase of approximately 50 percent in the risk of death from cardiovascular causes and the risk of myocardial infarction, even in the absence of hemodynamically significant obstruction of left ventricular outflow.

Valvular disease in the elderly.

As the incidence of valvular disease in the elderly is increasing, understanding of its pathogenesis and natural progression as well as surgical approaches and device technologies are improving. Future studies are needed to develop medical interventions that slow or halt the degenerative valvular processes associated with aging. In addition, mechanical approaches with lower operative risks should be explored and the search should continue for a valve substitute that is durable, hemodynamically efficient, easy to implant, and does not require anticoagulation. Hopefully, future intervention trials will include quality of life assessments such as symptoms, functional capacity and perceptions of well being. At present, the degenerative valvular processes must be followed closely by the clinician, and individual management decisions for the elderly based on the type and severity of valve disease, comorbid medical conditions, and the risks and benefits of intervention, along with patient preferences, rather than on the chronologic age of the patient. It is becoming clear that both survival and quality of life outcomes can improve by consideration of surgery at the onset of indications, before further deterioration eliminates the opportunity to provide benefit for the elderly patient with valvular disease.

Longitudinal assessment of valvular heart disease by echocardiography.

Over the past few years, important publications have addressed the issue of longitudinal assessment of valvular heart disease. The American College of Cardiology and the American Heart Association jointly published guidelines on echocardiography. Aortic stenosis continues to be well studied, with papers characterizing the rates of progression of mean and peak gradients, as well as aortic valve area changes. Doppler assessment of the progression of aortic regurgitation has been shown in larger studies than have been performed in the past. Based on an important study in patients with chronic aortic regurgitation, the optimal timing of surgery in asymptomatic mild to moderate aortic regurgitation may be predicted by indices of left ventricular function and left ventricular stress. Finally, mitral stenosis and mitral regurgitation studies evaluated rates of progression and also optimal timing for surgery.

Aortic stenosis. Clinical evaluation and optimal timing of surgery.

Aortic valve disease is common in the elderly with recent data suggesting that aortic sclerosis and stenosis are the end-stage of an active disease process. Aortic atenosis may be diagnosed at symptom onset (angina, heart failure or syncope) but often the diagnosis is suspected in an asymptomatic patient with a systolic murmur. The diagnosis can be confirmed and disease severity evaluated reliably using Doppler echocardiography. Symptomatic severe aortic stenosis is treated with valve replacement, even in the elderly, due to the extremely poor prognosis without relief of outflow obstruction. Management is controversial when there is coexisting moderate aortic stenosis and left ventricular systolic dysfunction.

Production of polyclonal antibodies to feline tumor necrosis factor.

Two 13-amino-acid peptides were synthesized based on the putative feline tumor necrosis factor (FeTNF) sequence. The synthesized peptides were conjugated to keyhole limpet hemocyanin, emulsified in complete Freund's adjuvant, and injected into rabbits. The gene for FeTNF was cloned into the FLAG (International Biotechnologies Inc. [IBI], Kodak, New Haven, Conn.) fusion protein expression vector. The expressed fusion protein was purified by using the M-1 anti-FLAG octapeptide monoclonal antibody (IBI, Kodak). The fusion protein was emulsified in complete Freund's adjuvant and injected into chickens. The immune sera generated to the synthetic peptides and the fusion protein recognized the recombinant FeTNF fusion protein on Western or dot blot assay. The preimmune and immune sera were incubated with naturally occurring FeTNF (supernatants from lipopolysaccharide-stimulated cultured feline peritoneal exudate or peripheral mononuclear cells). The antibody raised to the recombinant FeTNF fusion protein and N-terminal synthetic peptide neutralized bioactivity of native FeTNF and recombinant human TNF. Preimmune sera did not have any neutralizing activity. The polyclonal antibodies were not specific for FeTNF, since both porcine and human recombinant TNF were neutralized by the fusion protein antibodies. The synthetic peptide antibodies recognized recombinant feline and equine TNF on a Western blot.

Prospective study of asymptomatic valvular aortic stenosis. Clinical, echocardiographic, and exercise predictors of outcome.

BACKGROUND: Only limited data on the rate of hemodynamic progression and predictors of outcome in asymptomatic patients with valvular aortic stenosis (AS) are available. METHODS AND RESULTS: In 123 adults (mean age, 63 +/- 16 years) with asymptomatic AS, annual clinical, echocardiographic, and exercise data were obtained prospectively (mean follow-up of 2.5 +/- 1.4 years). Aortic jet velocity increased by 0.32 +/- 0.34 m/s per year and mean gradient by 7 +/- 7 mm Hg per year; valve area decreased by 0.12 +/- 0.19 cm2 per year. Kaplan-Meier event-free survival, with end points defined as death (n = 8) or aortic valve surgery (n = 48), was 93 +/- 5% at 1 year, 62 +/- 8% at 3 years, and 26 +/- 10% at 5 years. Univariate predictors of outcome included baseline jet velocity, mean gradient, valve area, and the rate of increase in jet velocity (all P < or = .001) but not age, sex, or cause of AS. Those with an end point had a smaller exercise increase in valve area, blood pressure, and cardiac output and a greater exercise decrease in stroke volume. Multivariate predictors of outcome were jet velocity at baseline (P < .0001), the rate of change in jet velocity (P < .0001), and functional status score (P = .002). The likelihood of remaining alive without valve replacement at 2 years was only 21 +/- 18% for a jet velocity at entry > 4.0 m/s, compared with 66 +/- 13% for a velocity of 3.0 to 4.0 m/s and 84 +/- 16% for a jet velocity < 3.0 m/s (P < .0001). CONCLUSIONS: In adults with asymptomatic AS, the rate of hemodynamic progression and clinical outcome are predicted by jet velocity, the rate of change in jet velocity, and functional status.

Endotoxemia and tumor necrosis factor activity in dogs with naturally occurring parvoviral enteritis.

A prospective, nonrandomized study was performed to evaluate the role of endotoxin and tumor necrosis factor (TNF) in dogs with parvoviral enteritis. Seventeen dogs with naturally occurring parvoviral enteritis were enrolled in the study. Plasma samples were obtained for quantification of endotoxin and TNF on presentation and at 3 and 6 hours after therapy with either fluids prior to antibiotics, or fluids concurrently with antibiotics. All dogs received standard supportive therapy. Fourteen of 17 dogs had endotoxin in their plasma during the study period; 7 of 17 dogs had measurable TNF. No endotoxin or TNF was detectable in plasma from normal puppies. An increase in TNF activity was predictive of mortality (P = .041). There was a trend for increasing endotoxin activity to predict mortality (P = .0769). Animals that received antibiotics with fluids were significantly older than those that received fluids prior to antibiotics, and there was a trend for animals that received antibiotics with fluids to have a decrease in endotoxin activity after treatment (P = .054). Endotoxin and activation of the cytokine cascade are integral to the pathophysiology of parvoviral enteritis. Measures to limit endotoxemia and the systemic inflammatory response may improve survival.

Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study.

OBJECTIVES: The aim of this study was to determine the prevalence of aortic sclerosis and stenosis in the elderly and to identify clinical factors associated with degenerative aortic valve disease. BACKGROUND: Several lines of evidence suggest that degenerative aortic valve disease is not an inevitable consequence of aging and may be associated with specific clinical factors. METHODS: In 5,201 subjects > or = 65 years of age enrolled in the Cardiovascular Health Study, the relation between aortic sclerosis or stenosis identified on echocardiography and clinical risk factors for atherosclerosis was evaluated by using stepwise logistic regression analysis. RESULTS: Aortic valve sclerosis was present in 26% and aortic valve stenosis in 2% of the entire study cohort; in subjects > or = 75 years of age, sclerosis was present in 37% and stenosis in 2.6%. Independent clinical factors associated with degenerative aortic valve disease included age (twofold increased risk for each 10-year increase in age), male gender (twofold excess risk), present smoking (35% increase in risk) and a history of hypertension (20% increase in risk). Other significant factors included height and high lipoprotein(a) and low density lipoprotein cholesterol levels. CONCLUSIONS: Clinical factors associated with aortic sclerosis and stenosis can be identified and are similar to risk factors for atherosclerosis.

Aortic root complications in Marfan's syndrome: identification of a lower risk group.

OBJECTIVES: The purpose of this study was to examine clinical and echocardiographic predictors of outcome in a cohort of patients with Marfan's syndrome. BACKGROUND: Serial echocardiographic measurements of aortic root dimensions are an important clinical method for monitoring patients with Marfan's syndrome. However, there are few data on the prognostic importance of echocardiographic variables for risk stratification and timing of aortic root replacement. METHODS: In 89 consecutive patients with Marfan's syndrome (age range 1-54 years) clinical and serial echocardiographic data (n = 62) were evaluated as potential predictors of outcome (mean (range) follow up 4 (< 1-16) years). Aortic sinus diameter and an aortic ratio normalised for age and body surface area were examined using Kaplan-Meier life table and Cox regression analysis, with the end point defined as death or surgery for ascending aortic dissection and events defined as an end point or surgery for ascending aortic aneurysm. RESULTS: Overall actuarial survival at two and five years was 96% and 92% and event free survival was 85% and 76%, respectively. There were five deaths due to aortic dissection, four patients survived surgery for ascending dissection, and nine underwent root replacement with a composite graft for ascending aneurysm. Those with aortic events were older (35 (12) v 25 (13) years, P = 0.007) and had greater initial aortic root dimensions (47 (14) v 33 (8) mm, P < 0.0001) and ratios (1.6 (0.5) v 1.3 (0.2), P < 0.0001). In the 62 patients with serial echocardiographic follow up, the rate of aortic root dilatation was more rapid in those with events (15 (17) v 0 (3)%/year, P < 0.0001). Utilising a Cox proportional hazards model, the groups with an initial aortic ratio > or = 1.3 or an annual change in aortic ratio > or = 5% had a relative risk of an aortic complication of 2.7 and 4.1, respectively (95% confidence limits 1.5 to 4.8 and 1.8 to 9.3). Only one of 31 patients with an initial aortic ratio of < 1.3 and a rate of change of < 5% had an event (five year event free survival 97%). CONCLUSIONS: A low risk subgroup of patients with Marfan's syndrome can be identified as those with an aortic ratio < 1.3 and an annual change in aortic ratio of < 5%. These findings are helpful in optimising echocardiographic monitoring and risk stratification of patients with Marfan's syndrome.

Tumor necrosis factor production in cats in response to lipopolysaccharide: an in vivo and in vitro study.

Supernatants from feline peritoneal exudate cells (PECs) exposed to lipopolysaccharde (LPS) produced significantly (P < 0.05) more tumor necrosis factor (TNF) activity than supernatants from cells exposed to media. An in vitro LPS response was obtained following incubation of whole blood with 10 micrograms ml-1 LPS for 2 h. Intravenous infusion of LPS (750 micrograms kg-1 rapidly increased plasma TNF activity to a maximum at 60 min after initiation of LPS infusion. By 180 min, TNF activity returned to baseline. Cats produce TNF in response to LPS in a manner similar to other species. Measurement of TNF activity in plasma or in LPS-stimulated whole blood are methods to further characterize the inflammatory response in feline diseases.

Expression of recombinant feline tumor necrosis factor is toxic to Escherichia coli.

The tumor necrosis factor (TNF) genes from cats, horses, and pigs have all been cloned into the pFLAG-1 fusion protein expression vector (International Biotechnologies, Inc., Kodak, New Haven, Conn.). Growth curves for Escherichia coli containing the pFLAG-1 vector alone and the pFLAG-1 vector containing the TNF gene from each species were determined by visible light spectrophotometry (at 600 nm). Porcine TNF, equine TNF, and feline TNF cultures had slower doubling rates than cultures containing the pFLAG-1 vector alone. Cultures of cells transformed with feline TNF reached peak densities at 3 to 4 h and then decreased to near initial densities prior to the recovery of growth. The induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) arrested the growth of fresh feline TNF cultures for 6 h, which was followed by complete recovery. This inhibition occurred in two strains of E. coli (LL308 and JM101). Induced feline TNF cultures expressed the TNF-FLAG fusion protein for the first 6.5 h. Uninduced cultures expressed low levels of fusion protein. The feline TNF-pFLAG-1 vector was purified from cells expressing fusion protein and from cells with recovered growth curves. Sequencing the vector demonstrated the complete feline TNF gene and tac promoter in cells expressing the fusion protein and a deletional mutation of the tac promoter site in recovered cells. In contrast to equine and porcine TNF, the expression of recombinant feline TNF is toxic to E. coli. Alterations in protein folding and the prevention of secretion of the feline protein may explain the toxic effect.