A period of 10 weeks of increased protein consumption does not alter faecal microbiota or volatile metabolites in healthy older men: a randomised controlled trial.

Diet has a major influence on the composition and metabolic output of the gut microbiome. Higher-protein diets are often recommended for older consumers; however, the effect of high-protein diets on the gut microbiota and faecal volatile organic compounds (VOC) of elderly participants is unknown. The purpose of the study was to establish if the faecal microbiota composition and VOC in older men are different after a diet containing the recommended dietary intake (RDA) of protein compared with a diet containing twice the RDA (2RDA). Healthy males (74⋅2 (sd 3⋅6) years; n 28) were randomised to consume the RDA of protein (0⋅8 g protein/kg body weight per d) or 2RDA, for 10 weeks. Dietary protein was provided via whole foods rather than supplementation or fortification. The diets were matched for dietary fibre from fruit and vegetables. Faecal samples were collected pre- and post-intervention for microbiota profiling by 16S ribosomal RNA amplicon sequencing and VOC analysis by head space/solid-phase microextraction/GC-MS. After correcting for multiple comparisons, no significant differences in the abundance of faecal microbiota or VOC associated with protein fermentation were evident between the RDA and 2RDA diets. Therefore, in the present study, a twofold difference in dietary protein intake did not alter gut microbiota or VOC indicative of altered protein fermentation.

Regulation of Amino Acid Transporters and Sensors in Response to a High protein Diet: A Randomized Controlled Trial in Elderly Men.

BACKGROUND: The mammalian target of rapamycin complex 1 (mTORC1) is fundamental for many cellular processes, yet it is often dysregulated with aging. Increased amino acid (AA) availability is correlated with the expression of AA transporters (AAT) and mTORC1 activity. Although many AA sensors and mediators have been proposed to relay the AA signal to mTORC1, it has not yet been determined if chronic dietary intervention affects the expression of AAT, sensors and mediators and their relationships with mTORC1 activity. OBJECTIVE AND DESIGN: This study investigated whether the consumption of a diet containing either the current recommended daily allowance (RDA) of protein intake (0.8 g/kg/d) or twice the RDA (2RDA) for ten weeks affected the expression of targets associated with AA transport, sensing and mTORC1 regulation in 26 older men (70-81 years). METHOD: Muscle biopsies were collected before and after the intervention under fasting conditions. Diets were controlled by providing fully prepared meals and snacks. Western blot and quantitative polymerase chain reaction were used to measure protein and gene expression respectively. RESULTS: Consumption of 2RDA reduced the protein expression of L-type amino acid transporter 1 (LAT1). However, plasma leucine concentration and basal mTORC1 activity were unaltered. The downregulation of LAT1 did not affect the expression of AA sensors and mediators, including leucyl tRNA synthetase (LRS), cytosolic arginine sensor for mTORC1 (CASTOR1), Sestrin2 and Rag proteins. Instead, total ribosomal protein S6 (RPS6) was upregulated with 2RDA. CONCLUSION: Ten weeks of 2RDA diet did not affect the fasting mTORC1 signaling, but increased total RPS6 might suggest improved muscular translational capacity to maintain muscular mass.

Antiparasitic efficacy of a novel plant-based functional food using an Ascaris suum model in pigs.

Ascaris lumbricoides is the most prevalent soil-transmitted helminth (STH) infection of human beings worldwide. Chemotherapy with synthetic anthelmintics such as albendazole, mebendazole, and pyrantel pamoate is the current method of treatment; however, the emergence of anthelmintic resistance could substantially decrease the efficacy of such treatments and the sustainability of STH control programs. Additionally, benzimidazoles are not recommended for pregnant women or children under age one. A blinded, controlled study was conducted to evaluate the efficacy of two microencapsulated, plant-based essential oil blends, TTN1013 (α-pinene, linalyl acetate, p-cymene, and thymol octanoate) and TTN1014 (α-pinene, linalyl acetate, p-cymene, and thymol acetate) as functional foods against Ascaris suum infection in pigs, an important pathogen that closely resembles human infections with A. lumbricoides. Four groups of 16 female, 21-24 day old, Yorkshire-cross pigs were treated daily with 0.5 or 1.0mg/kg TTN1013, 1.0mg/kg TTN1014, or 1.0mg/kg equivalent of empty capsules, delivered inside a cream-filled sandwich cookie for 14 weeks. Three days after the initiation of daily treatments, pigs were inoculated daily with A. suum eggs for four weeks. Pigs were weighed weekly and fecal egg counts (FEC) were conducted weekly starting five weeks after initial inoculation with A. suum eggs. Fourteen weeks after first infection with eggs, pigs were necropsied and worms were recovered, counted and separated according to sex. TTN1013 administered daily at a dose of 1.0mg/kg yielded a statistically significant reduction in total worm counts (76.8%), female worm counts (75.5%), FEC (68.6%), and worm volume (62.9%) when compared to control group. Reduction of total and female worm numbers and FEC were not significant for TTN1014 or at the 0.5mg/kg dose of TTN1013. All treatments were well-tolerated by all pigs and did not cause any adverse reactions. All pigs remained clinically normal and showed no signs of reduced intestinal health for the duration of treatment. Based on these results, TTN1013 shows promise as a daily supplement to reduce infection burdens of soil transmitted helminths in both pigs and human beings.

Performance of the plant-based repellent TT-4302 against mosquitoes in the laboratory and field and comparative efficacy to 16 mosquito repellents against Aedes aegypti (Diptera: Culicidae).

Repellent efficacy of the plant-based repellent, TT-4302 (5% geraniol), was compared with 16 other products in laboratory arm-in-cage trials against Aedes aegypti (L). Eight repellents (Badger, BioUD, Burt's bees, California Baby, Cutter Natural, EcoSMART, Herbal Armor, and SkinSmart) exhibited a mean repellency below 90% to Ae. aegypti at 0.5 h after application. Three repellents (Buzz Away Extreme, Cutter Advanced, and OFF! Botanicals lotion) fell below 90% repellency 1.5 h after application. TT-4302 exhibited 94.7% repellency 5 h posttreatment, which was a longer duration than any of the other repellents tested. The positive control, 15% DEET (OFF! Active), was repellent for 3 h before activity dropped below 90%. Additional arm-in-cage trials comparing TT-4302 with 15% DEET were carried out against Anopheles quadrimaculatus Say. At 6 h after treatment, TT-4302 provided 95.2% repellency while DEET exhibited 72.2%. In North Carolina field trials, TT-4302 provided 100% repellency 5 h after application against Aedes albopictus Skuse while DEET provided 77.6% repellency. These results demonstrate that TT-4302 is an efficacious plant-based repellent that provides an extended duration of protection compared with many other commercially available products.

Activity of the plant-based repellent, TT-4302 against the ticks Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis and Rhipicephalus sanguineus (Acari: Ixodidae).

The plant-based repellent TT-4302 (5 % geraniol) was compared to deet (15 %) in laboratory two-choice bioassays against the ticks Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus. At 2.5 and 3.5 h after treatment of filter paper with TT-4302, 100 % repellency was observed for all species at both time points with the exception of I. scapularis at the 3.5 h evaluation where repellency was 95.8 %. Deet was 100 % repellent at both time points for D. variabilis and R. sanguineus and was 100 % repellent at the 2.5 h evaluation for I. scapularis. Repellency of deet to A. americanum was 88.9 and 95.8 % at 2.5 and 3.5 h, respectively which was not significantly different than that of TT-4302. No significant difference against I. scapularis was observed between TT-4302 and deet at 3.5 h after treatment where deet was 87.5 % repellent. A variant of TT-4302, TT-4228 was tested in the laboratory against A. americanum and was compared to deet (15 %) in field trials against wild populations of ticks in North Carolina, USA. In the laboratory, TT-4228 was 94.4 and 87.5 % repellent at 2.5 and 3.5 h after treatment, respectively. In the field where the predominant tick species was A. americanum, significantly fewer ticks were collected from socks worn by human volunteers that were treated with TT-4228 compared to those treated with deet 2.5 or 3.5 h after treatment. Significantly fewer ticks were recovered from socks treated with TT-4228 than their paired untreated controls 2.5 or 3.5 h after treatment and repellencies were 90 and 70 %, respectively. Fewer ticks were collected from deet-treated compared to their paired untreated socks 2.5 h after application; however, no significant difference was found in the number of ticks collected from deet-and untreated socks 3.5 h after treatment.

Effects of high pressure processing on hatching of eggs of the zoonotic rat tapeworm Hymenolepis diminuta.

High hydrostatic pressure processing (HPP) is an effective non-thermal treatment to remove pathogens from a variety of food and food products. It has been extensively examined using prokaryotic organisms but has had limited study on eukaryotic organisms. Treatment using HPP has been shown to be effective in inactivating nematode larvae in food and Ascaris suum eggs. Nothing is known on the efficacy of HPP on tapeworm cysts or eggs. Eggs of important zoonotic tapeworms including Echinococcus and Taenia spp. can potentially contaminate water and food intended for human consumption. The present study examined the efficacy of HPP on the viability of Hymenolepis diminuta eggs. Efficacy of HPP treatment was measured using an egg hatch assay in two experiments. One thousand unhatched H. diminuta eggs in Hanks balanced salt solution were packaged in sealable bags and exposed to 100-600megapascals (MPa; 1MPa=10atm=147psi) for 60s in a commercial HPP unit. Positive (no HPP) and negative (No HPP but frozen/thawed) controls were examined in each experiment. None of the HPP untreated and frozen eggs (negative controls) were able to hatch or exclude trypan blue when placed in the hatching solution in experiment 1 or 2. HPP untreated and nonfrozen eggs (positive controls) hatched and excluded trypan blue; 75% were positive in experiment 1 and 80% were positive in experiment 2. No hatched eggs were observed when they were exposed to 300-600MPa for 60s. Treatment at 400MPa and above caused rupturing of the oncosphere. Results from this study indicate that HPP is a possible method to inactivate tapeworm eggs and that the susceptibility of tapeworm eggs to HPP is similar to or greater than that of nematode eggs or tissue larvae.

A prospective study of change in visual function in patients treated with pegylated interferon alpha for hepatitis C in the UK.

BACKGROUND: Hepatitis C virus and interferon treatment have been associated with retinopathy. Baseline and ongoing assessment by ophthalmologists have therefore been advocated in previous studies. Our experience suggests that the incidence is low, with no or negligible impact of pegylated interferon alpha on actual visual function. This study was conducted to determine whether ophthalmic assessment is necessary in such patients. METHODS: The study was a prospective case series of 52 patients (104 eyes). Visual acuity, contrast sensitivity, colour vision, visual field by perimetry and fundal assessment were measured at baseline and at 3 and 6 months post commencement of interferon alpha treatment. RESULTS: Forty-two men and ten women were followed. No patients reported any subjective visual symptoms. The mean changes in right and left logarithmic minimal angle resolution (LogMAR) visual acuity were negligible between baseline and 6 months (0.05 (SD 0.13) and 0.10 (SD 0.12), respectively). Mean changes in contrast sensitivity and colour vision were also negligible. Of all eyes monitored by serial perimetry for the full follow-up period and deemed to have reliable tests, none developed visual field defects. One patient appeared to develop nasal field defects within 3 months of commencing treatment but failed to attend for repeat testing. No patients developed optic disc changes or permanent fundal changes over the follow-up period. CONCLUSION: In contrast to previous studies in America and south-east Asia, our findings based on a UK cohort suggest that routine ophthalmic screening is not essential for patients with hepatitis C treated with pegylated interferon alpha who have no subjective visual complaints.

Acute retinal necrosis: a national population-based study to assess the incidence, methods of diagnosis, treatment strategies and outcomes in the UK.

AIM: To determine the incidence, methods of diagnosis, treatment strategies and outcomes for acute retinal necrosis (ARN) in the UK. METHODS: A 12-month active case ascertainment study was carried out between March 2001 and March 2002 to record cases of ARN presenting to ophthalmologists via the British Ophthalmological Surveillance Unit (BOSU) reporting system. Questionnaires were sent to the reporting consultants, requesting data on patient characteristics, presentation, clinical findings, investigations and treatment. Diagnosis was made using the American Uveitis Society diagnostic criteria. Further questionnaires were sent at 2 weeks and 6 months to assess outcome and therapies. RESULTS: 74 cases of ARN were reported by 58 consultants between March 2001 and March 2002. Questionnaires were returned for 49 cases (66.2%), of which 18 (36.7%) were excluded. Of the 31 cases included, 22 (71.0%) were male and 9 (29.0%) were female. The age range was 13 to 85 years (mean 54.3 years). 28 cases (90.3%) were unilateral, with 3 patients (9.7%) presenting with bilateral ARN. An aqueous or vitreous biopsy was performed in only 18 patients, with one patient having both. Herpes viral DNA analysis was performed on all 19 biopsies, with identification of the viral DNA in 16; results from 3 biopsies were not documented. Varicella zoster virus (VZV) was the commonest cause identified in 10 patients (56%). Of the 31 subjects, 27 (87.1%) were treated for ARN with systemic antiviral treatment: with intravenous antiviral in 23 cases (85.2%) and oral antiviral in 4 cases (14.8%). 21 of these patients went on to receive oral antiviral maintenance therapy. In addition to antiviral treatment, systemic steroids were given to 16 subjects (51.6%). Surgical intervention for retinal detachment was performed on 5 patients. CONCLUSIONS: During the 12-month study period, 31 cases of ARN met the diagnostic criteria set by the American Uveitis Society. The incidence in the UK based on this study is approximately 1 case per 1.6 to 2.0 million population per year. We have ascertained that the management of ARN throughout the UK is variable, suggesting that national guidelines would be of benefit.

Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers.

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.

Cytomegalovirus Retinitis in infancy.

PURPOSE: To describe the presentation of cytomegalovirus retinitis (CMVR) in a series of infants. METHODS: Immunocompromised infants with either HIV or systemic cytomegalovirus (CMV) were examined for CMVR. Ocular involvement was recorded and monitored by digital imaging. RESULTS: Five infants were detected to have CMVR. All the infants demonstrated changes within the macula. One infant progressed from a fine granular pattern to fulminant CMVR. CONCLUSION: Infants under a year with CMVR have a predilection for the disease to present at the macula, in contrast to the presentation in adults, which tends to involve more peripheral parts of the retina.

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming.

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.

Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases.

Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or stayed in the US exhibited titers over 50, a higher figure than in the group which did not travel out of France or stayed in an other European country. No correlation was found between anti-S. neurona and anti-Neospora sp. antibody titers. Data prompt further study of significance of anti-S. neurona antibodies in clinically healthy or diseased European horses, and identification of putative immunizing parasite(s) and their host(s).

Successful treatment of varicella zoster virus retinitis with aggressive intravitreal and systemic antiviral therapy.

AIMS: To describe the successful treatment of varicella zoster virus retinitis (VZVR) using intravenous cidofovir as part of an aggressive management strategy. CASE REPORTS: Two patients with bilateral VZVR were treated with a combination of intravenous cidofovir and ganciclovir with adjuvant intravitreal foscarnet or ganciclovir. Both patients maintained good vision in the less severely affected eye. Retinal detachment did not occur in either patient. CONCLUSIONS: VZVR should be treated aggressively with a combination of intravenous and intravitreal therapy to improve visual prognosis. Intravenous cidofovir, in the absence of contra-indications, should be considered as part of this aggressive therapeutic approach, especially in patients with AIDS in whom the prognosis is particularly poor.

Rare variants identified in the HNF- 4 alpha beta-cell-specific promoter and alternative exon 1 lack biological significance in maturity onset diabetes of the young and young onset Type II diabetes.

AIMS/HYPOTHESIS: The recently identified alternative promoter (P2) of HNF-4 alpha is the major HNF-4 alpha transcription start site in pancreatic beta cells. The significance of the P2 promoter was shown by the identification of a mutation in the IPF-1 binding site of the alternative promoter which cosegregated with diabetes in a large MODY family. The role of the P2 promoter and the associated alternative exon 1 in both MODY and polygenic Type II (non-insulin-dependent) diabetes mellitus is not known. Linkage to this region in studies of Type II diabetes makes the P2 region a strong candidate for a role in Type II diabetes susceptibility. METHODS: To assess the role of the P2 region we screened MODY, young-onset Type II diabetic subjects, and probands from Type II diabetes families linked to chromosome 20 for variants of the P2 promoter and associated exon of HNF-4 alpha. RESULTS: Two variants were found that were not present in the control subjects. The -79 C/T substitution was present in a MODY family but did not perfectly cosegregate with diabetes. A -276 G/T substitution was identified in two UK young-onset diabetes probands but did not co-segregate with diabetes. Reporter gene studies did not indicate changes in transcriptional activity caused by either the -79 C/T or -276 G/T single nucleotide substitutions. CONCLUSION/INTERPRETATION: We found no evidence to suggest that variation in the P2 proximal promoter region and associated alternative exon 1 of HNF-4 alpha contribute to young onset Type II diabetes susceptibility in Northern Europeans.

A distant upstream promoter of the HNF-4alpha gene connects the transcription factors involved in maturity-onset diabetes of the young.

Maturity-onset diabetes of the young (MODY) is a monogenic, autosomal dominant subtype of early-onset diabetes mellitus due to defective insulin secretion by the pancreatic beta-cell in humans. Five different genes have been identified including those encoding the tissue-specific transcription factors expressed in pancreatic beta-cells, i.e. HNF-4alpha (MODY1), HNF-1alpha (MODY3), IPF-1 (also known as PDX-1, MODY4) and HNF-1beta (MODY5). Analyzing the transcription of the HNF-4alpha gene, we now identify an alternative promoter, P2, which is 46 kb 5' to the previously identified P1 promoter of the human gene. Based on RT-PCR this distant upstream P2 promoter represents the major transcription site in pancreatic beta-cells, but is also used in hepatic cells. Transfection assays with various deletions and mutants of the P2 promoter reveal functional binding sites for HNF-1alpha, HNF-1beta and IPF-1, the other transcription factors known to encode MODY genes. We demonstrate the significance of this alternative promoter in a large MODY family where a mutated IPF-1 binding site in the P2 promoter of the HNF-4alpha gene co-segregates with diabetes (LOD score 3.25). These data suggest a regulatory network of the four MODY transcription factors interconnected at the distant upstream P2 promoter of the HNF-4alpha gene.

CMVR diagnoses and progression of CD4 cell counts and HIV viral load measurements in HIV patients on HAART.

AIM: To assess the impact of highly active antiretroviral therapy (HAART) on the prevalence and progression of CMV retinitis (CMVR) among AIDS patients with baseline CD4 cell counts <100 cells x 10(6)/l. METHODS: A longitudinal cohort study of 1292 patients. CD4 cell counts and HIV viral load measurements were obtained before commencing therapy, at 3 months, 1 year, 2 years, and at last follow up. The CMVR prevalence rate was measured for the subgroup with baseline CD4 cell counts <100 cells x 10(6)/l. CMVR adverse event (AE) rates per 100 person days at risk were calculated for the subgroup with CMVR and baseline CD4 cell counts <100 cells x 10(6)/l. RESULTS: 1292 patients were started on HAART. 8% of patients had CD4 counts <50 cells x 10(6)/l and 40% had detectable HIV viral load at last follow up. The prevalence of CMVR for the subgroup with baseline CD4 <100 cells x 10(6)/l was 10%. For those with baseline CD4 <100 cells x 10(6)/l, the mean CMVR AE rate was greatest during the first 6 months of follow up after HAART commencement (p <0.003). The mean AE rate per 100 person days at risk was 0.36 (95% CI 0.167 to 0.551) before starting HAART, and 0.14 (95% CI 0.085 to 0.199) after starting HAART (p = 0.03). CONCLUSIONS: HAART significantly prolongs the disease-free intervals in patients with pre-existing disease but recurrences persist within the first 6 months of starting therapy. AE were absent beyond 18 months of follow up in all patients including those with persistently low CD4 counts and detectable HIV viral load indicating clinical immunorestoration. New methods for monitoring the response to therapy are needed to identify those at risk.

Chronic cocaine-mediated changes in non-human primate nucleus accumbens gene expression.

Chronic cocaine use elicits changes in the pattern of gene expression within reinforcement-related, dopaminergic regions. cDNA hybridization arrays were used to illuminate cocaine-regulated genes in the nucleus accumbens (NAcc) of non-human primates (Macaca fascicularis; cynomolgus macaque), treated daily with escalating doses of cocaine over one year. Changes seen in mRNA levels by hybridization array analysis were confirmed at the level of protein (via specific immunoblots). Significantly up-regulated genes included: protein kinase A alpha catalytic subunit (PKA(calpha)); cell adhesion tyrosine kinase beta (PYK2); mitogen activated protein kinase kinase 1 (MEK1); and beta-catenin. While some of these changes exist in previously described cocaine-responsive models, others are novel to any model of cocaine use. All of these adaptive responses coexist within a signaling scheme that could account for known inductions of genes(e.g. fos and jun proteins, and cyclic AMP response element binding protein) previously shown to be relevant to cocaine's behavioral actions. The complete data set from this experiment has been posted to the newly created Drug and Alcohol Abuse Array Data Consortium (http://www.arraydata.org) for mining by the general research community.

Specific affinity depletion of cell adhesion molecules and growth factors from serum.

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.