Isolated fracture of the talus with asymptomatic contralateral talar fissure in two dogs.

CASE HISTORY: Two adult male dogs were separately presented for acute-onset, severe hind limb lameness isolated to the tarsus. There were no prior orthopaedic concerns and there was no significant trauma associated with the onset of lameness in either case. CLINICAL FINDINGS: Pain and effusion of the affected tarsus were found in both cases. Lameness was not responsive to oral analgesia. Radiography was insufficient to fully determine the extent of the damage in the tarsus; the fracture was visible in one case only. CT imaging demonstrated an isolated, lateral, trochlear ridge talar fracture in both cases and contralateral talar abnormalities of comparable location and direction to the fracture. DIAGNOSIS: Isolated lateral trochlear ridge fracture of the talus without significant trauma or concurrent injury. Abnormalities of talus of the contralateral limb were demonstrated on CT imaging. CLINICAL RELEVANCE: A previously unrecognised pathological process may affect the talus of adult dogs that could predispose them to develop fracture of the lateral talar ridge without significant trauma. Further investigations are required to determine the prevalence and risk of fracture associated with this abnormality.Abbreviations: HIF: Humeral intercondylar fissure.

Association of interleukin-1 gene variations with moderate to severe chronic periodontitis in multiple ethnicities.

BACKGROUND AND OBJECTIVE: Genetic markers associated with disease are often non-functional and generally tag one or more functional "causative" variants in linkage disequilibrium. Markers may not show tight linkage to the causative variants across multiple ethnicities due to evolutionary divergence, and therefore may not be informative across different population groups. Validated markers of disease suggest causative variants exist in the gene and, if the causative variants can be identified, it is reasonable to hypothesize that such variants will be informative across diverse populations. The aim of this study was to test that hypothesis using functional Interleukin-1 (IL-1) gene variations across multiple ethnic populations to replace the non-functional markers originally associated with chronic adult periodontitis in Caucasians. MATERIAL AND METHODS: Adult chronic periodontitis cases and controls from four ethnic groups (Caucasians, African Americans, Hispanics and Asians) were recruited in the USA, Chile and China. Genotypes of IL1B gene single nucleotide polymorphisms (SNPs), including three functional SNPs (rs16944, rs1143623, rs4848306) in the promoter and one intronic SNP (rs1143633), were determined using a single base extension method or TaqMan 5' nuclease assay. Logistic regression and other statistical analyses were used to examine the association between moderate to severe periodontitis and IL1B gene variations, including SNPs, haplotypes and composite genotypes. Genotype patterns associated with disease in the discovery study were then evaluated in independent validation studies. RESULTS: Significant associations were identified in the discovery study, consisting of Caucasians and African Americans, between moderate to severe adult chronic periodontitis and functional variations in the IL1B gene, including a pattern of four IL1B SNPs (OR = 1.87, p < 0.0001). The association between the disease and this IL1B composite genotype pattern was validated in two additional studies consisting of Hispanics (OR = 1.95, p = 0.04) or Asians (OR = 3.27, p = 0.01). A meta-analysis of the three populations supported the association between the IL-1 genotype pattern and moderate to severe periodontitis (OR 1.95; p < 0.001). Our analysis also demonstrated that IL1B gene variations had added value to conventional risk factors in predicting chronic periodontitis. CONCLUSION: This study validated the influence of IL-1 genetic factors on the severity of chronic periodontitis in four different ethnicities.

The effects of long (C20/22) and short (C18) chain omega-3 fatty acids on keel bone fractures, bone biomechanics, behavior, and egg production in free-range laying hens.

Keel fractures in the laying hen are the most critical animal welfare issue facing the egg production industry, particularly with the increased use of extensive systems in response to the 2012 EU directive banning conventional battery cages. The current study is aimed at assessing the effects of 2 omega-3 (n3) enhanced diets on bone health, production endpoints, and behavior in free-range laying hens. Data was collected from 2 experiments over 2 laying cycles, each of which compared a (n3) supplemented diet with a control diet. Experiment 1 employed a diet supplemented with a 60:40 fish oil-linseed mixture (n3:n6 to 1.35) compared with a control diet (n3:n6 to 0.11), whereas the n3 diet in Experiment 2 was supplemented with a 40:60 fish oil-linseed (n3:n6 to 0.77) compared to the control diet (n3:n6 to 0.11). The n3 enhanced diet of Experiment 1 had a higher n3:n6 ratio, and a greater proportion of n3 in the long chain (C20/22) form (0.41 LC:SC) than that of Experiment 2 (0.12 LC:SC). Although dietary treatment was successful in reducing the frequency of fractures by approximately 27% in Experiment 2, data from Experiment 1 indicated the diet actually induced a greater likelihood of fracture (odds ratio: 1.2) and had substantial production detriment. Reduced keel breakage during Experiment 2 could be related to changes in bone health as n3-supplemented birds demonstrated greater load at failure of the keel, and tibiae and humeri that were more flexible. These results support previous findings that n3-supplemented diets can reduce fracture likely by increasing bone strength, and that this can be achieved without detriment to production. However, our findings suggest diets with excessive quantities of n3, or very high levels of C20/22, may experience health and production detriments. Further research is needed to optimize the quantity and type of n3 in terms of bone health and production variables and investigate the potential associated mechanisms.

Impact of a mixed chain length omega-3 fatty acid diet on production variables in commercial free-range laying hens.

1. Previous studies found beneficial effects on the prevalence of keel fractures and bone health in laying hens receiving a short chain ω3 fatty acid diet. However, subsequent work using a mixed short and long chain ω3 diet (MCD) found little benefit to bone health and substantial production losses. 2. The current study examined commercial use of a mixed chain length ration in multiple farms and flocks and identified a detrimental effect on productivity including: mortality, number of poor quality eggs, hen daily average, feed consumption, and increased egg weight. 3. Production data were acquired from 11 flocks housed on 5 farms and modelled using MlwiN, a statistical package developed for analysis of hierarchical data. Production responses were individually modelled with duration of exposure to the diet, age of introduction, flock size and farm, included as prediction variables. 4. Analysis indicated birds receiving the MCD manifested dramatic increases in mortality, poor quality eggs, mean egg weight, feed consumption and decreased production in comparison to control flocks receiving a standard ration. Increased exposure time appeared to increase the magnitude of the diet effect. 5. Although no data were collected to explain the potential mechanisms of this diet, the authors discuss several possible scenarios including excessive long chain ω3 content overwhelming eicosanoid regulatory capacity and changes in behaviour patterns.

Broiler carcass quality using head-only electrical stunning in a waterbath.

1. The objective was to assess carcass quality of broilers when they were stunned by immersing their heads in a waterbath with an electric current flowing from one side of it to the other, while a second small current passed through the body to the waterbath to prevent involuntary wing flapping. 2. The prevalence of wing, shoulder and breast fillet haemorrhages and of broken bones in the pectoral region was not greater than that resulting from the normal stunning practice in that plant (63 mA, 610 Hz pDC). 3. These results imply that carcass damage using this technique will be significantly lower than that which will result from the application of higher stunning currents required by the new EU slaughter poultry slaughter regulations.

Identification of neutralising pembrolizumab anti-drug antibodies in patients with melanoma.

Development of anti-drug antibodies (ADAs) can interfere with therapeutic monoclonal antibodies and may lead to drug neutralisation and clinical disease progression. Measurement of circulating drug levels and development of ADAs in the setting of anti-programmed cell death-1 agent pembrolizumab has not been well-studied. Enzyme-linked immunosorbent assays were used to measure pembrolizumab drug level and ADAs in 41 patients with melanoma at baseline, Time-point 1 (3 weeks) and Time-point 2 (21 weeks). Assay results were related to patient demographics and clinical outcome data at 6 months. The median pembrolizumab drug level at 3 weeks was 237 ng/µL and did not correlate with age, sex or body surface area.17/41 patients had an ADA detected at any timepoint, with the highest prevalence at Timepoint 1 (median concentration = 17 ng/µL). The presence of an ADA did not correlate with clinical progression at 6 months. 3/41 (7%) of patients displayed a falling pembrolizumab drug level and rising ADA titre between Timepoint 1 and 2 suggestive of a neutralising ADA. Pembrolizumab drug levels and ADAs can be readily measured. The rates of total and treatment-emergent ADAs may be higher in "real-word" settings than those previously reported. Larger studies are needed to determine effect of neutralising ADAs on long-term clinical outcome.

Organizational behavior of human umbilical vein endothelial cells.

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.

Decoding the locational information in the orb web vibrations of Araneus diadematus and Zygiella x-notata.

A spider's web is a multifunctional structure that captures prey and provides an information platform that transmits vibrational information. Many physical factors interact to influence web vibration and information content, from vibration source properties and input location, to web physical properties and geometry. The aim of the study was to test whether orb web vibration contains information about the location of the source of vibration. We used finite-element analysis model webs to control and vary major physical factors, investigating webs where spiders use a direct or remote monitoring strategy. When monitoring with eight sensors (legs) at the web centre, a comparison of longitudinal and transverse wave amplitude between the sensors gave sufficient information to determine source direction and distance, respectively. These localization cues were robust to changes in source amplitude, input angle and location, with increased accuracy at lower source amplitudes. When remotely monitoring the web using a single thread connected to the web's hub (a signal thread), we found that locational information was not available when the angle of the source input was unknown. Furthermore, a free sector and a stiff hub were physical mechanisms to aid information transfer, which provides insights for bioinspired fibre networks for sensing technologies.

Prevalence and factors associated with it, of birds dead on arrival at the slaughterhouse and other rejection conditions in broiler chickens.

1. Standardised data on husbandry were recorded for a flock of birds in one house on each of 150 broiler farms in the UK during the 4 d prior to slaughter. 2. For each flock, the incidence of birds found dead on arrival (DoAs) and the Meat Hygiene Service carcase rejection records were recorded at the slaughterhouse. 3. The mean percentage of birds in each flock found DoA was 0.12% (range 0-0.64%) and the mean percentage of Total Carcase Rejects (TCRs) for each flock was 1.23% (range 0.07-5.51%). 4. A general linear model was developed to examine factors associated with flock percentage DoAs. Assuming a linear relationship, all other factors remaining the same, a one percentage point (PP) increase in small/emaciated birds will result in a 0.155 PP increase in DoAs and a 1 PP increase in wheat in diet 4 will result in a 0.003 PP decrease. An increase by one in the total number of vaccines administered will cause a 0.029 PP decrease in DoAs, a 1 g increase in live weight at slaughter will be associated with a 0.000043 PP increase and a 1 PP increase in mortality on farm would be associated with a 0.000044 PP increase. A 1 PP increase in Ross birds decreases DoAs by 0.0004 PPS: there is also a seasonal effect. 5. The model developed for flock percentage TCRs found that a 1 PP increase in wheat in diet 3 will result in a 0.052 PP decrease in TCRs and a 1 PP increase in Ross birds will cause a 0.009 PP decrease. A 1 PP increase in birds culled on farm will be associated with a 0.03 PP increase in TCRs and the diagnosis of disease during the flock cycle increases TCRs by 0.397 PPs. A one day increase in age at slaughter will result in a 0.046 PP increase in TCRs.

Estimating the body temperature of groups of pigs by thermal imaging.

Measurements on 28 pens of pigs containing 384 animals to be slaughtered at a commercial abattoir showed that the mean ear temperatures of the pigs in each pen, measured with a thermal imaging camera, were significantly correlated (r=0.71, P<0.001) with the mean temperature of the blood the pigs lost at exsanguination. In measurements on 220 pigs in 16 of the 28 pens, the mean activity of serum creatine kinase was positively correlated with the mean ear temperature (r=0.55, P<0.05) and the mean concentration of serum cortisol was positively correlated with the mean blood temperature (r=0.50, P<0.05), suggesting that the hotter pigs were suffering from a higher level of stress.

Comparison of the effects of fan-assisted and natural ventilation of vehicles on the welfare of pigs being transported to slaughter.

A total of 417 pigs with an estimated average liveweight of 91 kg were transported to slaughter in three journeys, carried out in late June, each consisting of a three-hour drive followed by a one-hour holding period at the slaughter plant before they were unloaded. The pigs were accommodated on the lower two decks of a three-deck articulated lorry, each deck having five equal-sized pens; half were kept in naturally ventilated pens and half in pens with fan-assisted ventilation. The pigs' body temperature was recorded as the temperature of the blood lost at exsanguination, and as the temperature of the inner surface of the ear measured by thermal imaging. The pigs carried on the upper deck were hotter than those carried on the lower deck, and the pigs carried at the front of the vehicle were hotter than those at the back. There were small differences between the temperatures of the pigs in the different pens, but overall the temperatures of the pigs kept in pens with fan-assisted ventilation were no lower than those of the pigs kept in pens with natural ventilation. The higher temperature of the pigs transported in the front of the vehicle was associated with significantly higher serum concentrations of cortisol and activities of creatine kinase, and a tendency to higher albumin concentrations and osmolality, suggesting that they may have been exposed to more physical and psychological stress, and were possibly slightly more dehydrated, than the pigs in the rearmost pens. The type of ventilation had no effect on the blood composition of the pigs.

Heterokaryon analysis of the genetic control of pigment synthesis in chick embryo melanocytes.

The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, ev and l. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically (One partner in each pair was labeled with 3H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.--Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, l. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-ev combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand the l-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.

The response of pigs to being loaded or unloaded onto commercial animal transporters using three systems.

Groups of pigs were subjected to three different systems of loading and unloading to and from commercial animal transporters. The systems under trial were: the use of a hydraulic tail-lift, a tail board ramp at an angle of 18 degrees and a modular system. The module was a container 2 m x 2.4 m x 1 m high into which the pigs were loaded. The module could then be lifted on and off the lorry with a fork lift truck. Non-invasive monitoring techniques were used to study the responses of the pigs. A subjective handling score, time taken to load and unload, skin temperature, heart rate and salivary cortisol were recorded. Loading and unloading were subjectively assessed as being easiest and quickest using the modular system, which also appeared to be less physically demanding for the animals, as evidenced by lower heart rate and a reduced maximum heart rate. However, with the modular system, elevated cortisol was found during the loading, unloading and resting periods. This could be interpreted as indicating that the pigs were unable to settle in the module and suffered some small degree of stress, which might become more of a problem during prolonged transport. Of the other two systems, there appeared to be little difference between the hydraulic tail-lift and the ramp.

DNA methylation and differentiation of human keratinocytes.

DNA methylation is a postreplicative modification thought to play a role in gene transcription in eucaryotes. Differences in the amount of 5-methylcytosine as a function of age and differentiation state have been reported. DNA isolated from human skin keratinocytes was analyzed for its 5-methylcytosine content. The 5-methylcytosine in DNA from neonatal and adult human keratinocytes was found to vary as a function of differentiation state. Differentiation of keratinocytes in vitro was promoted using a simple method where keratinocytes were plated directly onto the plastic surface of a culture flask, grown to confluence, and placed on a rocking culture platform that cyclically exposed the cells to air 50% of the time. Terminal differentiation was evident after approximately three weeks in culture. The 5-methylcytosine content of the DNA from differentiated human keratinocytes was 1.4%, whereas that of undifferentiated human keratinocytes was 3.1%. No difference in the 5-methylcytosine content of DNA as a function of the age of the donor was found.

Enalapril in low-renin essential hypertension.

The antihypertensive efficacy of N-[(S)-1-(ethoxycarbonyl)-3-phenyl-propyl]-L-alanyl-L-proline (enalapril maleate) was evaluated in a randomized, double-blind trial in 23 patients with mild low-renin essential hypertension ranging in age from 32 to 70 yr (20 were black and 3 were white). All underwent a 4-wk washout-placebo phase and were then assigned to a dosing schedule of either 10 mg enalapril once daily, 5 mg enalapril twice daily, or placebo twice daily for 12 wk. Conditional on diastolic pressure, the dose was increased at 4-wk intervals to a maximum of 40 mg daily or until control was achieved or the end of the study reached. At the end of the 12-wk titration phase, there was a follow-up period during which measurements were made after discontinuation of the medication. Mean supine diastolic pressure decreased from baseline (98.5 +/- 2.6 mm Hg) during the titration phase (86.3 +/- 4.6 mm Hg) in the group taking enalapril once daily. In three of the eight patients in the once-daily group and five of eight in the twice-daily group, supine diastolic pressures fell below 90 mm Hg. Neither supine nor standing systolic pressure nor standing diastolic pressure decreased significantly from pretreatment levels during enalapril once or twice daily. Heart rates measured after 5 min supine rest were not altered by enalapril. Enalapril induced inhibition of converting enzyme activity at all dose levels and with both dosing schedules. No adverse effect attributable to enalapril occurred during the study. The data indicate that once-daily enalapril is safe and effective treatment for mild low-renin essential hypertension.

Lofexidine and clonidine in moderate essential hypertension.

The efficacy, safety, and tolerability of lofexidine, a centrally acting imidazoline derivative, were compared to that of clonidine in a randomized double-blind trial in 28 patients with moderate essential hypertension. The study consisted of a washout phase, a placebo phase, a drug titration phase (0.2 to 1.6 mg/day, with hydrochlorothiazide added at 0.4 mg daily for supine and erect diastolic blood pressure above 90 mm Hg), and a maintenance phase lasting 3 mo. During the titration phase supine systolic and diastolic pressures fell in lofexidine patients from 143 +/- 4/98 +/- 3 to 122 +/- 3/81 +/- 2 mm Hg and in clonidine patients from 154 +/- 6/101 +/- 2 to 124 +/- 4/81 +/- 2 mm Hg (P less than 0.01), and erect systolic and diastolic pressures fell in lofexidine patients from 143 +/- 3/105 +/- 2 to 116 +/- 3/85 +/- 2 mm Hg and in clonidine patients from 156 +/- 6/104 +/- 2 to 117 +/- 4/82 +/- 2 mm Hg (P less than 0.01). Maximal doses of lofexidine and clonidine in combination with hydrochlorothiazide had equivalent antihypertensive effects, but when the effects of lofexidine and clonidine were compared at each dose level, larger doses of lofexidine patients in either the supine or erect position during the titration phase but heart rate fell in the clonidine patients (P less than 0.05) over the same period. Dry mouth and drowsiness were reported in both groups but were both less frequent and less severe in the lofexidine group than the clonidine group.

Pharmacology, distribution and development of muscarinic acetylcholine receptor subtypes in the optic tectum of Rana pipiens.

Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5-9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes.These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for excitatory subtypes.

Once weekly administration of nicotine produces long-lasting locomotor sensitization in rats via a nicotinic receptor-mediated mechanism.

RATIONALE: Chronic nicotine administration results in dynamic changes in neuronal function, expressed as behavioral sensitization in animals and addiction in smokers. OBJECTIVES: The present study was undertaken to determine whether once-weekly nicotine injection produces sensitization to the locomotor-activating properties of nicotine as a result of nicotinic receptor activation. METHODS: Once weekly for 6 weeks, rats were administered (s.c.) two saline injections or saline and nicotine (0.35 mg/kg), and locomotor activity was monitored. Rats remained in the home cage for 21 days, and subsequently were injected with the appropriate treatment to determine whether sensitization persisted. Rats were also injected with saline or mecamylamine (1.2 mg/kg) followed by saline or nicotine once weekly for 6 weeks to determine the effect of mecamylamine and whether it inhibited nicotine-induced hyperactivity. A separate group was injected with saline and nicotine once weekly for 4 weeks; on week 5, mecamylamine and nicotine were administered to determine whether mecamylamine inhibited the expression of sensitization. Separate groups were injected with mecamylamine and nicotine once weekly for 5 weeks or 6 weeks; on week 6 or week 9, respectively, saline and nicotine were injected to determine whether mecamylamine inhibited the initiation of sensitization. RESULTS: Sensitization to the locomotor-activating properties of nicotine developed following four nicotine injections across a 28-day period and persisted following 21 days of no drug treatment. Mecamylamine did not alter activity but attenuated both the initiation and expression of sensitization. CONCLUSIONS: Nicotinic receptor activation following once-weekly nicotine administration produces long-lasting behavioral sensitization, suggesting that even infrequent nicotine exposure initiates neuroadaptive processes associated with nicotine addiction.

Prognostic markers in node-negative breast cancer: a prospective study.

BACKGROUND: Despite years of research, it is still unclear which women with node-negative (N-) breast cancer will need adjuvant chemotherapy and which women are being treated unnecessarily. Our goal was to determine which factors best predicted disease free survival (DFS) or cancer-specific overall survival (OS) and, therefore, select the correct patients for treatment. A total of 11 parameters were measured: estrogen receptor (ER), progesterone receptor (PR), age, race, ploidy status, %G0/G1 (% non-DNA synthesis), %S (% S-phase), cathepsin D status, size, stage, and histologic grade. RESULTS: In this prospective study, we followed 556 N- patients diagnosed between 1991 and 1996. The tumors were 56% ER+, 51% PR+, 30% diploid, with a mean %S of 8.9%. The level of cathepsin D ranged from 0.50 to 155 pmol/mg of protein with a mean of 42.9 pmol/mg of protein. There were 87 recurrences (16%) and 72 cancer deaths (13%), with a median follow-up of 7.8 years. Ploidy status (p = 0.01), S-phase activity (p = 0.003), G1 phase activity (p = 0.02) and age (p = 0.01) were able to significantly predict DFS in a univariate manner. All of the measurable factors were significant or borderline significant in predicting OS in a univariate manner except for age, race, and ER status. In multivariate analysis with S-phase included, it was the only remaining factor in DFS and OS; with S-phase excluded, age and ploidy status remained as factors for DFS in stepwise regression, while PR, size, and cathepsin D were the remaining factors that predicted cancer-specific OS. The effect of adjuvant treatment on prognosis was also analyzed. CONCLUSIONS: Both biochemical and clinical parameters have the potential to predict prognosis for N- breast cancer. In this large prospective clinical trial, with a median follow-up of 7.8 years, no individual marker adequately predicted the prognosis for an individual patient. %S activity was the best independent marker, but only 77% of the tumors provided this value. Subset analysis provided improved prognostication, but there were limits to its utility. These data represents a definitive study starting in 1991 and ending in 2002.