Conditioned Medium of Mesenchymal Stromal Cells: A New Class of Therapeutics.

Mesenchymal stromal cell (MSCs) represent a class of biologics with the prospects for employment as immunomodulatory, tissue-protective, and regenerative therapeutics. In parallel with cellular therapy, cell-free therapy based on MSC-secreted bioactive factors is being actively developed. MSCs secrete a variety of protein, peptide, RNA, and lipid mediators which can be concentrated, frozen, or even lyophilized without loss of activity, which gives them a certain advantage over cellular products requiring liquid nitrogen storage and infrastructure to revive frozen cells. This review (i) describes currently conducted clinical trials of cell-free products containing MSC secretome; (ii) summarizes main approaches to the generation and characterization of conditioned media concentrates and extracellular vesicle isolates; (iii) analyzes a variety of preclinical studies where effectiveness of secretome products has been shown; and (iv) summarizes current knowledge about secretome bioactive components obtained by analysis of in vivo models testing the therapeutic potential of the MSC secretome.

Coinhibition of the deubiquitinating enzymes, USP14 and UCHL5, with VLX1570 is lethal to ibrutinib- or bortezomib-resistant Waldenstrom macroglobulinemia tumor cells.

The survival of Waldenstrom macroglobulinemia (WM) tumor cells hinges on aberrant B-cell receptor (BCR) and MYD88 signaling. WM cells upregulate the proteasome function to sustain the BCR-driven growth while maintaining homeostasis. Clinically, two treatment strategies are used to disrupt these complementary yet mutually exclusive WM survival pathways via ibrutinib (targets BTK/MYD88 node) and bortezomib (targets 20 S proteasome). Despite the success of both agents, WM patients eventually become refractory to treatment, highlighting the adaptive plasticity of WM cells and underscoring the need for development of new therapeutics. Here we provide a comprehensive preclinical report on the anti-WM activity of VLX1570, a novel small-molecule inhibitor of the deubiquitinating enzymes (DUBs), ubiquitin-specific protease 14 (USP14) and ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5). Both DUBs reside in the 19 S proteasome cap and their inhibition by VLX1570 results in rapid and tumor-specific apoptosis in bortezomib- or ibrutinib-resistant WM cells. Notably, treatment of WM cells with VLX1570 downregulated BCR-associated elements BTK, MYD88, NFATC, NF-κB and CXCR4, the latter whose dysregulated function is linked to ibrutinib resistance. VLX1570 administered to WM-xenografted mice resulted in decreased tumor burden and prolonged survival (P=0.0008) compared with vehicle-treated mice. Overall, our report demonstrates significant value in targeting USP14/UCHL5 with VLX1570 in drug-resistant WM and carries a high potential for clinical translation.

Chemical characterisation and analysis of the cell wall polysaccharides of duckweed (Lemna minor).

Duckweed is potentially an ideal biofuel feedstock due to its high proportion of cellulose and starch and low lignin content. However, there is little detailed information on the composition and structure of duckweed cell walls relevant to optimising the conversion of duckweed biomass to ethanol and other biorefinery products. This study reports that, for the variety and batch evaluated, carbohydrates constitute 51.2% (w/w) of dry matter while starch accounts for 19.9%. This study, for the first time, analyses duckweed cell wall composition through a detailed sequential extraction. The cell wall is rich in cellulose and also contains 20.3% pectin comprising galacturonan, xylogalacturonan, rhamnogalacturonan; 3.5% hemicellulose comprising xyloglucan and xylan, and 0.03% phenolics. In addition, essential fatty acids (0.6%, α-linolenic and linoleic/linoelaidic acid) and p-coumaric acid (0.015%) respectively are the most abundant fatty acids and phenolics in whole duckweed.

Concurrent ivermectin and Solanum spp. toxicosis in a herd of horses.

BACKGROUND: Representatives from a herd of horses with acute onset of neurologic signs after administration of ivermectin presented for evaluation and treatment. OBJECTIVES: Describe clinical signs of horses intoxicated by ingestion of Solanum sp. and administered ivermectin. ANIMALS: Six of 11 affected unrelated horses presented for evaluation and treatment. The remaining 5 affected horses were treated at the farm. Four additional horses, housed separately, were unaffected. METHODS: Case series is presented. Serum ivermectin concentrations were evaluated in the 6 hospitalized horses. The remnants of the tubes of ivermectin paste were analyzed for ivermectin concentration. The hay fed to the affected horses was analyzed for the presence of toxic plants. RESULTS: Serum ivermectin concentrations were higher than expected, given the dosage of ivermectin administered. The ivermectin concentration remaining in the administration tubes did not exceed specifications. The hay was heavily contaminated by 2 Solanum species. All horses returned to normal neurologic function with supportive care. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses might exhibit signs of ivermectin toxicity after appropriate dosing of the drug if they concurrently consume toxic plants of the Solanum family.

Erythrocyte antioxidant protection of rose hips (Rosa spp.).

Rose hips are popular in health promoting products as the fruits contain high content of bioactive compounds. The aim of this study was to investigate whether health benefits are attributable to ascorbic acid, phenols, or other rose-hip-derived compounds. Freeze-dried powder of rose hips was preextracted with metaphosphoric acid and the sample was then sequentially eluted on a C(18) column. The degree of amelioration of oxidative damage was determined in an erythrocyte in vitro bioassay by comparing the effects of a reducing agent on erythrocytes alone or on erythrocytes pretreated with berry extracts. The maximum protection against oxidative stress, 59.4 ± 4.0% (mean ± standard deviation), was achieved when incubating the cells with the first eluted meta-phosphoric extract. Removal of ascorbic acid from this extract increased the protection against oxidative stress to 67.9 ± 1.9%. The protection from the 20% and 100% methanol extracts was 20.8 ± 8.2% and 5.0 ± 3.2%, respectively. Antioxidant uptake was confirmed by measurement of catechin by HPLC-ESI-MS in the 20% methanol extract. The fact that all sequentially eluted extracts studied contributed to protective effects on the erythrocytes indicates that rose hips contain a promising level of clinically relevant antioxidant protection.

Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems.

Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

Towards the routine use of brain imaging to aid the clinical diagnosis of disorders of consciousness.

Clinical audits have highlighted the many challenges and dilemmas faced by clinicians assessing persons with disorders of consciousness (vegetative state and minimally conscious state). The diagnostic decision-making process is highly subjective, dependent upon the skills of the examiner and invariably dictated by the patients' ability to move or speak. Whilst a considerable amount has been learnt since Jennett and Plum coined the term 'vegetative state', the assessment process remains largely unchanged; conducted at the bedside, using behavioural assessment tools, which are susceptible to environmental and physiological factors. This has created a situation where the rate of misdiagnosis is unacceptably high (up to 43%). In order to address these problems, various functional brain imaging paradigms, which do not rely upon the patient's ability to move or speak, have been proposed as a source of additional information to inform the diagnostic decision making process. Although accumulated evidence from brain imaging, particularly functional magnetic resonance imaging (fMRI), has been encouraging, the empirical evidence is still based on relatively small numbers of patients. It remains unclear whether brain imaging is capable of informing the diagnosis beyond the behavioural assessment and whether brain imaging has any prognostic utility. In this study, we describe the functional brain imaging findings from a group of 41 patients with disorders of consciousness, who undertook a hierarchical speech processing task. We found, contrary to the clinical impression of a specialist team using behavioural assessment tools, that two patients referred to the study with a diagnosis of vegetative state did in fact demonstrate neural correlates of speech comprehension when assessed using functional brain imaging. These fMRI findings were found to have no association with the patient's behavioural presentation at the time of investigation and thus provided additional diagnostic information beyond the traditional clinical assessment. Notably, the utility of brain imaging was further underlined by the finding that the level of auditory processing revealed by functional brain imaging, correlated strongly (rs = 0.81, P < 0.001) with the patient's subsequent behavioural recovery, 6 months after the scan, suggesting that brain imaging may also provide valuable prognostic information. Although further evidence is required before consensus statements can be made regarding the use of brain imaging in clinical decision making for disorders of consciousness, the results from this study clearly highlight the potential of imaging to inform the diagnostic decision-making process for persons with disorders of consciousness.

When thoughts become action: an fMRI paradigm to study volitional brain activity in non-communicative brain injured patients.

The assessment of voluntary behavior in non-communicative brain injured patients is often challenging due to the existence of profound motor impairment. In the absence of a full understanding of the neural correlates of consciousness, even a normal activation in response to passive sensory stimulation cannot be considered as proof of the presence of awareness in these patients. In contrast, predicted activation in response to the instruction to perform a mental imagery task would provide evidence of voluntary task-dependent brain activity, and hence of consciousness, in non-communicative patients. However, no data yet exist to indicate which imagery instructions would yield reliable single subject activation. The aim of the present study was to establish such a paradigm in healthy volunteers. Two exploratory experiments evaluated the reproducibility of individual brain activation elicited by four distinct mental imagery tasks. The two most robust mental imagery tasks were found to be spatial navigation and motor imagery. In a third experiment, where these two tasks were directly compared, differentiation of each task from one another and from rest periods was assessed blindly using a priori criteria and was correct for every volunteer. The spatial navigation and motor imagery tasks described here permit the identification of volitional brain activation at the single subject level, without a motor response. Volunteer as well as patient data [Owen, A.M., Coleman, M.R., Boly, M., Davis, M.H., Laureys, S., Pickard J.D., 2006. Detecting awareness in the vegetative state. Science 313, 1402] strongly suggest that this paradigm may provide a method for assessing the presence of volitional brain activity, and thus of consciousness, in non-communicative brain-injured patients.

High-throughput, fluorescence-based screening for soluble protein expression.

Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls.

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.

The effect of sucrose application frequency and basal nutrient conditions on the calcium and phosphate content of experimental dental plaque.

A reduced pool of calcium in dental plaque would be expected to increase the ability of plaque fluid to dissolve the underlying enamel when the pH falls during sugar exposure. We have examined the relationship between frequency of sugar application and Ca and P(i) concentrations in artificial mouth plaque microcosm biofilms. Ten plaques were grown simultaneously from a human saliva inoculum using a continuous flow of simulated saliva, DMM, supplemented with either urea or glucose to modulate the resting pH. In addition the plaques received sucrose applications of varying frequency: 12-, 8-, 6-, or 4-hourly, or not at all. After 15 days the plaques were sampled by taking 4 full-thickness specimens of each, and acid-extractable Ca and P(i), and alkali-soluble protein and carbohydrate were determined. Ca and P(i) concentrations were in a range comparable with those in human plaque, except in the DMM + urea plaque receiving no sucrose, when concentrations were higher. Plaque Ca concentration decreased significantly as sucrose application frequency increased. Increasing sucrose application frequency also reduced the protein, i.e. the cell biomass, content of the plaques and, in the case of DMM + urea plaques, increased the water-insoluble hexose content, presumably extracellular polysaccharide. Reduced biomass was partly due to the bulking of plaque with extracellular polysaccharide, but the marked effect of urea on polysaccharide formation is not understood. This study shows that increasing frequency of sugar application alters dental plaque by reducing its mineral protection capacity.

Coffee consumption and serum aminotransferases in middle-aged Japanese men.

We investigated the relation between coffee drinking and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations among 7313 Japanese men receiving a health examination, excluding former alcohol drinkers and men with a history of chronic liver disease. Serum AST > 40 and/or ALT > 40 U/L was defined as liver inflammation. Adjustment was made for alcohol use, smoking, body mass index, serum marker for hepatitis virus infection, and other possible confounders. Adjusted odds ratios of liver inflammation were 1.00 (reference), 0.80, 0.69, and 0.61 for men drinking < 1, 1-2, 3-4, and > or = 5 cups of coffee daily, respectively. Among 6898 men without liver inflammation, serum AST and ALT were inversely associated with coffee consumption, and alcohol-related rise in AST was attenuated with coffee drinking. These findings suggest coffee may have an effect of suppressing the rise of serum aminotransferase, partly by inhibiting the alcohol-related elevation. Studies regarding biological mechanism are warranted.

The therapeutic use of lipoic acid in diabetes: a current perspective.

Lipoic acid and its reduced derivative, dihydrolipoic acid (DHLA) are highly promising antioxidant agents, which are potent attenuators of reactive species-mediated damage in vitro and in animal studies. Lipoic acid is a universal antioxidant, effective in lipophilic and aqueous environments. In contrast to an equivalent endogenous agent, such as oxidised glutathione (GSSG), lipoic acid acts as an antioxidant in its oxidised form. Lipoic acid has been evaluated in diabetic polyneuropathy, a condition which is thought to result in part from oxidant damage caused by long-term hyperglycaemia. Diabetic patients are prone to incur enhanced cellular free radical formation and reduced antioxidant defence. Treatment with lipoic acid has improved nerve conduction velocity during studies in diabetic animals. Trials in diabetic patients have often observed some relief of neuropathic symptoms during treatment with lipoic acid, but consistent objective benefits have been difficult to establish. Lipoic acid is now used in Germany for the treatment of diabetic neuropathy and definitive evidence of efficacy should arise from postmarketing surveillance studies. It is possible that lipoic acid may be more effective as a long-term dietary supplement aimed at the prophylactic protection of diabetics from complications.

A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.

Coffee drinking and serum gamma-glutamyltransferase: an extended study of Self-Defense Officials of Japan.

PURPOSE: To examine the effect of coffee drinking on serum gamma-glutamyltransferase (GGT) level in relation to alcohol drinking, smoking, and degree of obesity in middle-aged Japanese men. METHODS: From 1986 to 1994, a total of 7,637 male officials of the Self-Defense Forces of Japan aged 48-59 years received a preretirement health examination. Coffee drinking was ascertained by a self-administered questionnaire, and serum GGT level was measured. After excluding 1,360 men with a possible pathologic condition influencing liver enzyme levels and 182 former alcohol drinkers, effect of coffee drinking on serum GGT was examined by a multiple linear regression model and analysis of variance adjusting for alcohol drinking, smoking, and body mass index (BMI). RESULTS: The adjusted percentage of difference in serum GGT was -4.3 (95% CI = -5.0; -3.5) per cup of coffee. The inverse coffee-GGT relation was most prominent among men drinking > or = 30 ml of ethanol and smoking > or = 15 cigarettes daily; and positive associations of alcohol and smoking with GGT were attenuated by coffee drinking, more clearly among men with BMI > or = 25.00 kg/m2. Adjusted percentages of difference in serum GGT were -2.6% (p = 0.0003) per cup of brewed coffee, and -5.1% (p = 0.0001) per cup of instant coffee, independently of each other. CONCLUSIONS: The present study suggests that coffee consumption may weaken GGT-induction by alcohol, and possibly by smoking. These effect modifications by coffee may differ according to the degree of obesity.

Synthesis and antimycobacterial activity of some heteroarylcarboxamidrazone derivatives.

A series of pyridine-2-, pyrazine-2- and quinoline-2-carboxamidrazone derivatives was prepared in an automated fashion and tested against Mycobacterium fortuitum in a rapid screen. Seven pyridine-2-carboxamidrazone derivatives were investigated further and four were found to have inhibitory activity with compound 4af being the most active. The structure activity implications are discussed.

Effects of dietary fatty acids on DU145 human prostate cancer cell growth in athymic nude mice.

The purpose of this study was to examine the effects of diets containing different unsaturated fatty acids (FAs) on DU145 human prostate cancer cell growth in nude mice. In Experiment 1, groups of 25 mice were fed 23% (wt/wt) fat diets containing 18% corn oil (CO)-5% linseed oil (18:2n-6 FA-rich), 18% linseed oil (LO)-5% CO (18:3n-3 FA-rich), or 18% menhaden oil (MO)-5% CO (20:5 and 22:6n-3 FA-rich), and seven days later they were injected subcutaneously with 1 x 10(6) DU145 cells. The diets were continued for six weeks. The growth rates and final weights of tumors from the 18% CO-5% LO and 18% LO-5% CO mice were similar; there was a 30% reduction in tumor growth in the 18% MO-5% CO group (p < 0.001). The tumor phospholipid FA patterns suggested that the inhibitory effect of the high-MO diet was due, at least in part, to a reduction of arachidonic acid available for prostaglandin biosynthesis. In Experiment 2, groups of 25 mice were injected with 5 x 10(5) or 1 x 10(6) DU145 cells directly into the prostate gland and fed a high-fat linoleic acid (n-6 FA)-rich or a low-fat diet for 10 weeks. At necropsy, macroscopic cancers and microscopic intraprostatic tumors were evaluated. When the initial tumor load was 1 x 10(6) cells, all but 7 of the 50 mice had developed large macroscopic tumors; the mean tumor weight in the high-fat group was twice that in the low-fat group (p = 0.047). A stimulatory effect of dietary n-6 FA on DU145 prostate cancer cell growth may require a critical initial tumor cell mass.

Copper supplementation of adult men: effects on blood copper enzyme activities and indicators of cardiovascular disease risk.

In rats, copper deficiency leads to low copper metalloenzyme activity, high serum cholesterol, and cardiovascular lesions. In humans, moderately low copper intake may be common, but the consequences remain largely uncertain. The present study examined the effects of copper supplementation (2 mg/d for 4 weeks in a copper/placebo crossover design) in 20 adult men with moderately high plasma cholesterol. End-point measurements were three copper enzyme activities, erythrocyte superoxide dismutase (SOD), plasma ceruloplasmin (Cp), and plasma diamine oxidase (DAO), and three parameters related to the risk of cardiovascular disease (CVD), plasma cholesterol, plasma lipoprotein (a) [Lp(a)], and lag times for very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) oxidation in vitro. Although copper had no significant effects on any parameter for the entire study group, it did significantly increase two enzyme activities (SOD and DAO), as well as lipoprotein oxidation lag times, in 10 subjects in the lower half of a median split for precopper values. Thus, copper supplementation appeared to influence some types of measurements in subjects beginning with less than median values.

Effect of omega-3 fatty acids on the progression of metastases after the surgical excision of human breast cancer cell solid tumors growing in nude mice.

We showed previously that a diet rich in linoleic acid (LA), an omega-6 fatty acid, stimulates the growth and metastasis of human breast cancer cells in athymic nude mice. In contrast, diets supplemented with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), omega-3 fatty acids, exert suppressive effects. We have now assessed EPA and DHA as adjuvant nutritional therapy in the nude mouse model and compared the responses when the intervention was commenced 1 week before ("neoadjuvant") or immediately after ("postoperative adjuvant") surgical excision of the primary tumor. Female nude mice received a high-fat, 8% LA diet beginning 7 days before 10(6) MDA-MB-435 human breast cancer cells were injected into a thoracic mammary fat pad. As the tumor surface areas approached 0. 7 cm2, the mice were assigned to either continue on the LA-rich diet or to commence one containing 8, 4, or 2% EPA or DHA. Seven days later, the mammary fat pad tumors were excised; the mice still consuming the 8% LA diet were then allocated sequentially to either continue this diet or commence one of the six postexcision omega-3 fatty acid dietary interventions. Eight weeks later, the mice were necropsied and evaluated for local recurrence and lung metastases. Although there were no differences in the incidence of local recurrence between groups, EPA and DHA both inhibited the development of lung metastases. When the dietary interventions were commenced 7 days before surgery, the severity of lung metastasis was reduced by the two omega-3 fatty acids in a dose-dependent manner; at all three levels, the suppressive effects were statistically significant (P < 0.05). Postexcision EPA treatment produced small, statistically insignificant effects, but lung involvement was reduced significantly by feeding DHA at the 2 and 4% levels (P < 0. 05). Overall, these results suggest that omega-3 fatty acids may have a place as adjuvant nutritional therapy in breast cancer and particularly as part of a neoadjuvant regimen.