Development of the Nutrition and Functionality Assessment (NFA) among Older Adults in Japan.

OBJECTIVE: Develop and evaluate the feasibility and validity of the Nutrition and Functionality Assessment (NFA) which identifies "target" older adults who could benefit from a personalized program following evaluation of their nutrition status and physical functionality. DESIGN: Cross-sectional study. SETTING: Community and geriatric day-care centers and university in Japan. PARTICIPANTS: 267 older adults aged 65-90. MEASUREMENTS: The "target" individuals were screened based on gait speed (0.6-1.5 m/s). Nutrition (Mini Nutrition Assessment-short form and protein intake), strength (30s chair sit-to-stand and hand-grip strength) and endurance (6-minute walk) were assessed. Physical activity was monitored using a tri-axil accelerometer for a week. Fried frailty phenotype was also assessed. RESULTS: Out of 267 individuals, 185 (69%) had gait speed between 0.6-1.5 m/s, corresponding to our "target" group from which, 184 (95%) completed the nutrition and physical functionality assessments with the physical activity monitoring. The NFA was completed in approximately 30 minutes. No adverse events directly due to the NFA were reported. NFA physical functionality and global scores were significantly related to frailty phenotype but nutrition score was not related to frailty phenotype. CONCLUSION: The study demonstrated that the NFA is a safe and feasible tool to screen target older adults and simultaneously evaluate their nutritional status and physical functionality. Validity of the NFA was partially confirmed by the significant association of the global and physical functionality scores with frailty phenotype. More studies are required to validate and maximize the applicability of the NFA in communities and institutions in Japan and elsewhere.

Oleuropein or rutin consumption decreases the spontaneous development of osteoarthritis in the Hartley guinea pig.

OBJECTIVE: To assess the potential protective effects of three polyphenols oleuropein, rutin and curcumin, on joint ageing and osteoarthritis (OA) development. DESIGN: Sixty 4-week-old Dunkin-Hartley guinea pigs were randomized into four groups and received daily during 31 weeks either standard guinea pig diet (control group) or a standard guinea pig diet enriched with oleuropein (0.025%), rutin (0.5%) or rutin/curcumin (0.5%/0.25%) association. Biomarkers of OA (Coll2-1, Coll2-1NO2, Fib3-1, Fib3-2, ARGS), as well as inflammation prostaglandin E2 (PGE2) were quantified in the serum. Histological assessments of knee cartilage and synovial membrane were performed at week 4 (five young reference guinea pigs) and week 35. RESULTS: At week 35, guinea pigs in the control group spontaneously developed significant cartilage lesions with mild synovial inflammation. The histological scores of cartilage lesions and synovitis were well correlated with the increased level of serum biomarkers. Histologically, all treatments significantly reduced the cartilage degradation score (P < 0.01), but only oleuropein significantly decreased the synovial histological score (P < 0.05) and serum PGE2 levels (P < 0.01) compared to the control group. Coll2-1 was decreased by rutin and the combination of rutin/curcumin, Fib3-1 and Fib3-2 were only decreased by the rutin/curcumin mixture, while Coll2-1NO2 was significantly decreased by all treatments (P < 0.05). CONCLUSION: Oleuropein and rutin ± curcumin significantly slowed down the progression of spontaneous OA lesions in guinea pigs. While no additive effect was seen in the curcumin + rutin group, the differential effects of oleuropein and rutin on inflammatory and cartilage catabolic markers suggest an interesting combination for future studies in OA protection.

Peak bone strength is influenced by calcium intake in growing rats.

In this study we investigated the effect of supplementing the diet of the growing male rat with different levels of calcium (from low to higher than recommended intakes at constant Ca/P ratio), on multiple factors (bone mass, strength, size, geometry, material properties, turnover) influencing bone strength during the bone accrual period. Rats, age 28days were supplemented for 4weeks with high Ca (1.2%), adequate Ca (0.5%) or low Ca level (0.2%). Bone metabolism and structural parameters were measured. No changes in body weight or food intake were observed among the groups. As anticipated, compared to the adequate Ca intake, low-Ca intake had a detrimental impact on bone growth (33.63 vs. 33.68mm), bone strength (-19.7% for failure load), bone architecture (-58% for BV/TV) and peak bone mass accrual (-29% for BMD) due to the hormonal disruption implied in Ca metabolism. In contrast, novel, surprising results were observed in that higher than adequate Ca intake resulted in improved peak bone strength (106 vs. 184N/mm for the stiffness and 61 vs. 89N for the failure load) and bone material properties (467 vs. 514mPa for tissue hardness) but these effects were not accompanied by changes in bone mass, size, microarchitecture or bone turnover. Hormonal factors, IGF-I and bone modeling were also evaluated. Compared to the adequate level of Ca, IGF-I level was significantly lower in the low-Ca intake group and significantly higher in the high-Ca intake group. No detrimental effects of high Ca were observed on bone modeling (assessed by histomorphometry and bone markers), at least in this short-term intervention. In conclusion, the decrease in failure load in the low calcium group can be explained by the change in bone geometry and bone mass parameters. Thus, improvements in mechanical properties can be explained by the improved quality of intrinsic bone tissue as shown by nanoindentation. These results suggest that supplemental Ca may be beneficial for the attainment of peak bone strength and that multiple factors linked to bone mass and strength should be taken into account when setting dietary levels of adequate mineral intake to support optimal peak bone mass acquisition.

Hesperidin inhibits ovariectomized-induced osteopenia and shows differential effects on bone mass and strength in young and adult intact rats.

The main aim of this study was to investigate the bone-sparing effect of hesperidin, one of the main flavonoid present in oranges, in two age groups of ovariectomized female rats, compared with their intact controls. Young (3 mo) and adult (6 mo) female Wistar rats were sham operated (SH) or ovariectomized (OVX) and then pair-fed for 90 days a casein-based diet supplemented or not with 0.5% hesperidin (Hp; n = 10/group). In older rats, Hp intake led to a partial inhibition of OVX-induced bone loss, whereas a complete inhibition was obtained in younger animals. At both ages, while plasma osteocalcin concentrations were unchanged, urinary excretion of deoxypyridinoline was reduced by Hp intake, suggesting that Hp was able to slow down bone resorption. Unexpectedly, in intact young rats, Hp consumption resulted in a significant increase in bone mineral density (BMD). Indeed, 6-mo-old HpSH rats had a similar BMD to 9-mo-old nontreated SH adult rats, suggesting an accelerated bone mass gain in the young rats. In contrast, in intact adult rats, Hp did not further increase BMD but did improve their bone strength. The results of this study show a protective effect of Hp on bone loss in OVX rats of both ages without uterine stimulation and accompanied by a lipid-lowering effect. The unexpected and intriguing findings obtained in intact rats showing improved BMD in young rats and improved femoral load in adult rats merit further investigation. The bone and lipid benefits of hesperidin make it an attractive dietary agent for the management of the health of postmenopausal women.

PIXImus bone densitometer and associated technical measurement issues of skeletal growth in the young rat.

The PIXImus dual-energy X-ray absorptiometer (DXA) is designed to measure body composition, bone mineral content (BMC), area (BA), and density (BMD) in mice and rats. The aims of this study were to longitudinally measure BMC, BA, and BMD in growing rats and to identify potential technical problems associated with the PIXImus. Total femur and lumbar DXA measurements, body weight, and length of initially 3-week-old rats (n = 10) were taken at weeks 5, 9, and 14. BMC and BMD of femoral metaphyseal and diaphyseal regions rich in trabecular and cortical bone, respectively, were obtained. Results showed significant increases in body weight, total femur BMC and BMD, lumbar area, length, BMC, and BMD at each time point. There was a significant positive correlation between body weight and total femur BMD (r = 0.97, P < 0.001) as well as lumbar BMD (r = 0.99, P < 0.001). BMD values for the femoral metaphyseal region and the lumbar spine were also positively correlated (r = 0.96, P < 0.01). Several technical issues (e.g., positioning of animals), difficulties (e.g., in analysis of images), and limitations (e.g., inability to detect underdeveloped calcified bone in growing animals and bone edge detection) of the software pertinent to the PIXImus were evident. In conclusion, despite limitations in the software, the PIXImus is a valuable tool for studying skeletal development of growing rats.

In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids.

Human and mouse liver microsomes and membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodictyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin, respectively. Microsomal flavonoid metabolism was potently inhibited by the CYP1A2 inhibitors, fluvoxamine and -naphthoflavone. Recombinant CYP1A2 was capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation, but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant CYP1A2, although the reaction rates in general were lower as compared to CYP1A2. CYP2C9 catalyzed the 4'-demethylation of tamarixetin, whereas CYP2D6 did not seem to play any role in the metabolism of the selected flavonoids. The major involvement in flavonoid metabolism of human CYP1A2, which mediates the formation of metabolites with different biochemical properties as compared to the parent compound and furthermore is known to be expressed very differently among individuals, raises the important question of whether individual differences in the CYP enzyme activity might affect the beneficial outcome of dietary flavonoids, rendering some individuals more or less refractory to the health-promoting potential of dietary flavonoids.

Cyclooxygenase-2, malondialdehyde and pyrimidopurinone adducts of deoxyguanosine in human colon cells.

Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.

Comparison of the antioxidant activity of commonly consumed polyphenolic beverages (coffee, cocoa, and tea) prepared per cup serving.

In this study, the in vitro low-density lipoprotein oxidation model was used to assess the relative antioxidant activity of the polyphenolic beverages tea, coffee, and cocoa on a cup-serving basis. The beverages were prepared as 0.7-2.5% soluble coffee and 1.5-3.5% cocoa; teas (green, black, or herbal) were prepared as one tea bag infused over 5 min in 220 mL of hot water. Under these standard cup serving conditions, the antioxidant activity as determined by the lag time was in the range of 292-948 min for coffee, 217-444 min for cocoa, 186-338 min for green tea, 67-277 min for black tea, and 6-78 min for herbal tea. Addition of milk did not alter the antioxidant activity. The influence of coffee bean source and degree of roasting was further investigated. Green coffee beans of Robusta coffee exhibited a 2-fold higher antioxidant activity than Arabica coffee, but after roasting this difference was no longer significant. In conclusion, these commonly consumed beverages have a significant antioxidant activity, the highest being soluble coffee on a cup-serving basis.

Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise.

The present double-blinded, placebo-controlled study investigated whether antioxidant vitamin supplementation was able to modulate the cytokine and lymphocyte responses after strenuous eccentric exercise. Furthermore, muscle enzyme release was examined to see whether antioxidant treatment could reduce muscle damage. Twenty male recreational runners randomly received either antioxidants (500 mg of vitamin C and 400 mg of vitamin E) or placebo for 14 days before and 7 days after a 5% downhill 90-min treadmill run at 75% .VO(2 max). Although the supplemented group differed significantly with regard to plasma vitamin concentration before and after exercise when compared with the placebo group, the two groups showed identical exercise-induced changes in cytokine, muscle enzyme, and lymphocyte subpopulations. The plasma level of interleukin (IL)-6 and IL-1 receptor antagonist increased 20- and 3-fold after exercise. The plasma level of creatine kinase was increased sixfold the day after exercise. The concentrations of CD4+ memory T cells, CD8+ memory and naïve T cells, and natural killer cells increased at the end of exercise. The total lymphocyte concentration was below prevalues in the postexercise period. In conclusion, the present study does not support the idea that exercise-induced inflammatory responses are induced by free oxygen radicals.

Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: mechanisms in rat and human cells.

The coffee-specific diterpenes cafestol and kahweol (C + K) have been reported to be anticarcinogenic in several animal models. Proposed mechanisms involve a co-ordinated modulation of several enzymes responsible for carcinogen detoxification, thus preventing reactive agents interacting with critical target sites. To address the human relevance of the chemoprotective effects of C + K against aflatoxin B(1) (AFB1) genotoxicity observed in rat liver, and to compare the mechanisms of protection involved in both species, animal and human hepatic in vitro test systems were applied. In rat primary hepatocytes, C + K reduced the expression of cytochrome P450 CYP 2C11 and CYP 3A2, the key enzymes responsible for AFB1 activation to the genotoxic metabolite aflatoxin B1-8,9 epoxide (AFBO). In addition, these diterpenes induced significantly GST Yc2, the most efficient rat GST subunit involved in AFBO detoxification. These effects of C + K resulted in a marked dose-dependent inhibition of AFB1-DNA binding in this rat in vitro culture system. Their relevance in humans was addressed using liver epithelial cell lines (THLE) stably transfected to express AFB1 metabolising cytochrome P450s. In these cells, C + K also produced a significant inhibition of AFB1-DNA adducts formation linked with an induction of the human glutathione S-transferase GST-mu. Altogether, these results suggest that C + K may have chemoprotective activity against AFB1 genotoxicity in both rats and humans.

Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies.

PURPOSE: To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies. METHODS: Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays. RESULTS: The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. CONCLUSIONS: The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.

Plasma kinetics in man of epicatechin from black chocolate.

OBJECTIVE: To evaluate the plasma kinetics in man of epicatechin from black chocolate. DESIGN: An intervention study with 8 volunteers. Each served as his own control. Theobromine was used as control marker of the chocolate intake. SETTING: Metabolic Unit, Nestle Research Center, Vers-chez-les-Blanc, Switzerland. SUBJECTS: Eight healthy male volunteers (4 smokers and 4 non-smokers) were enrolled in this study. They abstained from foods rich in polyphenols (coffee, tea, wine, fruit juice, cocoa products) for 24 h prior to the test until its completion. INTERVENTION: Volunteers ate 40 g and 80 g of black chocolate (Nestle Noir) together with bread with a one-week interval. Blood samples were drawn every hour during the first 4h and a last one at 8 h after chocolate consumption. Plasma samples were analysed for epicatechin and theobromine content by HPLC. RESULTS: Plasma concentrations of epicatechin and theobromine increased markedly after chocolate consumption (P = 0.002 and P= 0.001, respectively), reaching a maximum between 2 and 3 h. The maximal concentration and area under the curve of plasma kinetics of both substrates correlated very well with the dose of chocolate. CONCLUSIONS: Epicatechin is absorbed from chocolate and is rapidly eliminated from plasma. Attainable plasma values are 0.7 micromol/l from 80g of black chocolate.

Human corneal epithelial cell functional responses to inflammatory agents and their antagonists.

PURPOSE: To investigate the epithelial nature of primary and SV40 virus-immortalized human corneal epithelial (CEPI) cells and to study a variety of functional responses to some key inflammatory agents (bradykinin [BK], histamine, and platelet-activating factor [PAF]) and their antagonists in these cells. METHODS: Primary CEPI (P-CEPI) and clone 4 of the SV40 virus-immortalized (CEPI-17-CL4) cells were analyzed for their interaction with several monoclonal antibodies selective for various cytokeratins to define their immunocytochemical characteristics and phenotypic traits. Both cell types were tested for their ability to respond to BK, histamine, and PAF and their antagonists, using the production of [3H]inositol phosphates ([3H]IPs) as an index of receptor activation. The ability of BK, PAF, and histamine to stimulate cytokine release and the induction of mRNA for matrix metalloproteinase-1 (MMP-1) were also studied using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction techniques, respectively. RESULTS: P-CEPI and CEPI-17-CL4 cells were both shown to possess the epithelial cell cytokeratins labeled with AE1 and AE3 antibodies. The potencies (EC50s) of BK, histamine, and PAF were similar for stimulating [3H]IPs production in P-CEPI and CEPI-17-CL4 cells: BK = 2.27 to 2.99 nM, PAF = 17.1 to 18.26 nM, and histamine = 1.65 to 5.74 microM (all n = 3 to 6). Both cell types also responded similarly to receptor-selective antagonists for BK, PAF, and histamine (Hoe-140: Ki = 10.1 to 11.9 nM; PCA-4248: Ki = 315 to 421 nM; triprolidine: Ki = 0.8 to 4.76 nM; all n = 5 to 10). Histamine (100 microM) and interleukin-1alpha (IL-1alpha, 10 ng/ml) significantly stimulated IL-6 and granulocyte macrophage colony-stimulating factor release, and histamine, BK, and PAF stimulated the mRNA for MMP-1 in these cells. CONCLUSIONS: These studies have shown that the primary and immortalized human corneal epithelial cells express functional BK (a B2 subtype), histamine (an H1 subtype), and PAF receptors and exhibit very similar immunocytochemical, signal transduction, and pharmacological properties. Therefore, the CEPI-17-CL4 cells (currently at passage 220) appear to provide a useful representative in vitro model system to study the physiological and pathologic aspects of the human corneal epithelium.

Development of in vitro models for cellular and molecular studies in toxicology and chemoprevention.

Many natural dietary phytochemicals found compounds found in fruits, vegetables, spices and tea have been shown in recent years to be protective against cancer in various animal models. In the light of the potential impact of these compounds on human health it is important to elucidate the mechanisms involved. We therefore developed and characterized relevant in vitro models using immortalized human epithelial cell lines derived from target tissues in carcinogenesis, such as lung, liver and colon. Assays were established, allowing the evaluation of the cytotoxic and genotoxic effects of various procarcinogens, including nitrosamines, mycotoxins and heterocyclic amines on these metabolically-competent human epithelial cell lines. These cellular models appeared to be a useful tool to study the capacity of certain food components to block the initiation stage of carcinogenesis. The ability of carnosol and carnosic acid from rosemary as well as the synthetic dithiolethione, oltipraz, to block the formation of DNA adducts, and their effects on the expression of phase I and phase II enzymes was investigated. We have observed that both rosemary extracts and oltipraz inhibited benzo(a)pyrene- or aflatoxin B1-induced DNA adduct formation by strongly inhibiting CYP450 activities and inducing the expression of glutathione S-transferase. These results in human cell models give some insight into the different mechanisms involved in the chemopreventive action of both natural and synthetic compounds in relation to phase I and phase II enzymes.

Mechanisms involved in the chemoprotective effects of rosemary extract studied in human liver and bronchial cells.

Natural polyphenols found in rosemary have not only potent antioxidant activities but also anticarcinogenic properties. We have studied some of the molecular mechanisms involved in their chemopreventive action using in vitro human liver and bronchial cell models. Rosemary extract, or its active components, carnosol or carnosic acid are potent inhibitors of DNA adduct formation induced by benzo(a)pyrene or aflatoxin B1. At least two mechanisms are involved in the anticarcinogenic action of rosemary extract: (i) inhibition of the metabolic activation of procarcinogens catalysed by the phase I cytochrome P450 enzymes; (ii) induction of the detoxification pathway catalysed by the phase II enzymes such as glutathione S-transferase.

A comparison of energy expenditure by a high level trans-femoral amputee using the Intelligent Prosthesis and conventionally damped prosthetic limbs.

Comparisons were made between the Intelligent Prosthesis (IP), Mauch and pneumatic swing phase control damping systems on the same prosthesis worn by a high level trans-femoral amputee. Speeds self selected by corridor walking (4.4-5.5 kmh-1) proved not to be sustainable for treadmill walking. Comfortable speeds were attained when the subject walked on a treadmill at 2.0, 2.6 and 3.2 kmh-1 in two tests for each prosthesis type. Oxygen uptake (VO2), cadence and heart rate were measured over 5 minute walks interspersed with rest periods. Spearman's correlation was used to test for differences between prosthesis types at each speed. At the two slower speeds no significant difference was found, but at the higher speed of 3.2 kmh-1, the IP was associated with a significantly lower VO2 (p < 0.05). A two way analysis of variance with replication (ANOVA) demonstrated a significant difference between VO2 for different limb types (p = 0.015). A square law function was fitted to the mean VO2 for each prosthesis type by the method of least squares regression. ANOVA demonstrated a significant difference between velocity coefficients for the different prosthesis types (p < 0.05). Cadence was almost constant during the period of each walk, varying by 1 step min-1 at most. However the test-retest differences in cadence were considerable. It is concluded that there was little difference in energy expenditure between prosthesis types at slower speeds, but at higher speeds (==> 3.2km h-1) the IP gave a lower oxygen uptake by about 10%.

Functional promoters in the genome of human papillomavirus type 6b.

Viral mRNAs from lesions containing human papillomavirus type 6 (HPV-6) have previously been mapped on the viral DNA but relatively little is known about the control of mRNA production, or whether the mapped RNA termini correspond to promoters. By analysis of run-off transcripts synthesized in vitro, primer extension and measurements of promoter activity in fragments of the viral DNA introduced into cells, we have identified three promoters in the early region of the HPV-6b genome. These are: (i) at the end of the long control region upstream of the E6 open reading frame; (ii) upstream of E7 and (iii) upstream of E1. The promoter upstream of E1 was the most active. These results contrast with results of similar assays with HPV-18, in which the strongest promoter was that controlling expression of the transforming genes E6 and E7. In addition, a novel promoter was detected close to E5a, upstream of the late genes.