Vascular density analysis in colorectal cancer patients treated with vatalanib (PTK787/ZK222584) in the randomised CONFIRM trials.

BACKGROUND: Pharmacological inhibitors of vascular endothelial growth factor (VEGF) receptors, like vatalanib, have been tested in randomised trials (CONFIRM (Colorectal Oral Novel therapy For the Inhibition of Angiogenesis and Retarding of Metastases) 1 and 2) in colorectal cancer showing activity in a subgroup of patients with high serum LDH expression. In the current study, we assessed the predictive role of vascular density (VD) in patients treated in the above trials. METHODS: Paraffin-embedded materials from 141 patients were analysed with immunohistochemistry for the expression of the CD31 (pan-endothelial cell marker) and of phosphorylated pVEGFR2/KDR on endothelial cells. The VD was correlated with response to therapy and with progression-free (PFS) and overall survival (OS). RESULTS: A significant association of pVEGFR2/KDR+ VD with poor response in the placebo group was noted (response rates (RRs) 15% (3/20) when high VD vs 52% (26/50) when low VD; P=0.006). The RR increased from 15 (3/20) to 50% (11/22) in tumours with high VD when vatalanib was added to chemotherapy (P=0.02). A significantly improved PFS was noted in patients with high pVEGFR2/KDR+ VD when treated with vatalanib (P=0.002). A similar effect was also noted in patients with high CD31+ VD (P=0.07). Overall survival was marginally improved (P=0.07). CONCLUSION: Assessment of the activated vessel density may allow the stratification of patients recruited in randomised trials with VEGFR-targeting anti-angiogenic agents, unmasking their therapeutic potential and enabling their introduction in the clinical practice for the benefit of specific patient subgroups, at the same time reducing the cost of therapy.

Vatalanib for metastatic gastrointestinal stromal tumour (GIST) resistant to imatinib: final results of a phase II study.

BACKGROUND: Vatalanib (PTK787/ZK 222584) inhibits a few tyrosine kinases including KIT, platelet-derived growth factor receptors (PDGFRs) and vascular endothelial growth factor receptors (VEGFRs). We report efficacy and safety results of vatalanib in advanced gastrointestinal stromal tumour (GIST) resistant to imatinib or both imatinib and sunitinib. PATIENTS AND METHODS: Forty-five patients whose metastatic GIST had progressed on imatinib were enrolled. Nineteen (42.2%) patients had received also prior sunitinib. Vatalanib 1250 mg was administered orally daily. RESULTS: Eighteen patients (40.0%; 95% confidence interval (CI), 25.7-54.3%) had clinical benefit including 2 (4.4%) confirmed partial remissions (PR; duration, 9.6 and 39.4 months) and 16 (35.6%) stabilised diseases (SDs; median duration, 12.5 months; range, 6.0-35.6+ months). Twelve (46.2%) out of the 26 patients who had received prior imatinib only achieved either PR or SD compared with 6 (31.6%, all SDs) out of the 19 patients who had received prior imatinib and sunitinib (P=0.324). The median time to progression was 5.8 months (95% CI, 2.9-9.5 months) in the subset without prior sunitinib and 3.2 (95% CI, 2.1-6.0) months among those with prior imatinib and sunitinib (P=0.992). Vatalanib was generally well tolerated. CONCLUSION: Vatalanib is active despite its narrow kinome interaction spectrum in patients diagnosed with imatinib-resistant GIST or with imatinib and sunitinib-resistant GIST.

Ten-year fracture probability identifies women who will benefit from clodronate therapy--additional results from a double-blind, placebo-controlled randomised study.

UNLABELLED: Fracture risk prediction can be enhanced by the concurrent assessment of other clinical risk factors. This study demonstrates that the estimation of an individual's 10-year probability of fracture by the FRAX algorithm identifies patients at high risk of fracture who will respond to bisphosphonate therapy. INTRODUCTION: Treatments for osteoporosis are targeted largely to patients with low bone density (BMD) or a prior fragility fracture. Fracture risk prediction can be enhanced by the concurrent assessment of other clinical risk factors, but it is important to determine whether the risk so identified can be reduced by intervention. We determined the effect of a bisphosphonate on fracture rates when risk was calculated using a new risk algorithm (FRAX). METHODS: Women aged 75 years or more were recruited to a randomised, double-blind controlled trial of 800 mg oral clodronate (Bonefos) daily over 3 years. Baseline clinical risk factors were entered in the FRAX model to compute the 10-year probability of major osteoporotic fractures with or without input of femoral neck BMD. The interaction between fracture probability and treatment efficacy was examined by Poisson regression. RESULTS: In 3,974 women, the interaction between fracture probability and treatment efficacy was significant when probability was assessed without BMD (p = 0.043), but not when BMD was included (p = 0.10). Efficacy was more evident in those deemed at highest risk. For example women lying at the 75th percentile of fracture probability in the absence of BMD (10-year probability 24%) treatment reduced fracture risk by 27% (HR 0.73, 95%CI 0.58-0.92). In those with a fracture probability of 30% (90th percentile), the fracture risk reduction was 38% (HR 0.62, 0.46-0.84). CONCLUSIONS: The estimation of an individual's 10-year probability of fracture by the FRAX algorithm identifies patients at high risk of fracture who will respond to bisphosphonate therapy.

Phase II, open-label study of PTK787/ZK222584 for the treatment of metastatic gastrointestinal stromal tumors resistant to imatinib mesylate.

BACKGROUND: We evaluated safety and efficacy of PTK787/ZK222584 (PTK/ZK), a novel tyrosine kinase inhibitor of KIT, platelet-derived growth factor receptors and vascular endothelial cell growth factor receptors (VEGFRs), in patients with imatinib-resistant gastrointestinal stromal tumor (GIST). This is the first study of PTK/ZK in this population. PATIENTS AND METHODS: Patients with metastatic GIST that had progressed after >/= 4-week treatment with imatinib mesylate were eligible. Prior VEGFR-2 inhibitor therapy was not permitted. PTK/ZK 1250 mg orally once-daily was administered to 15 patients (accrued as a two-stage procedure), most of whom (n = 11) had been unsuccessfully treated with imatinib 800 mg daily, until treatment failure. Patients were monitored at 4- to 8-week intervals. RESULTS: All 15 patients enrolled were eligible; two (13%) achieved partial response (PR), eight (53%) had stable disease (SD) >/=3 months, and five (33%) progressed. The clinical benefit rate (PR + SD) was 67% (95% CI 38% to 86%). Median time to progression was 8.5 months (range 0.9-24.8+ months). Three patients had not progressed at the time of analysis, including one PR at 24.8 months and two SDs at 16.6 and 18.6 months on treatment. PTK/ZK was generally well tolerated. CONCLUSION: PTK/ZK 1250 mg p.o. once daily is active and well tolerated in patients with imatinib-resistant GIST.

Screening and selection of lactic acid bacteria strains suitable for ensiling grass.

AIM: Lactic acid bacteria (LAB) strains shown to have broad-spectrum antimicrobial activity were screened for potential as grass silage inoculants. The strains capable of rapidly lowering the pH of the grass matrix and with low proteolytic activity were assessed in laboratory-scale silos in a grass matrix containing natural microbial flora. METHODS AND RESULTS: Screening of nine candidate strains was performed first in a grass extract medium. The four most promising strains were selected on the basis of growth rate in the medium, capacity to reduce pH and ability to limit the formation of ammonia-N. The efficiency of the selected strains was further assessed in a laboratory-scale ensiling experiment. Untreated (no additive) and formic acid served as controls. All tested inoculants improved silage quality compared with untreated. With one exception (Pediococcus parvulus E315) the fermentation losses in the inoculated silages were even lower than in the acid-treated control silage. Pure lactic acid fermentation was obtained in the timothy-meadow fescue silage with all inoculants. The results obtained in the ensiling experiments were consistent with those of the screening procedure, which appeared to predict correctly the potential of LAB as silage inoculants. The strains with a low ammonia production rate in the grass extract medium behaved similarly in the silage. Especially in this respect the strain Lactobacillus plantarum E76 was superior to the other candidates. CONCLUSIONS: The screening method using grass extract proved to be useful in strain selection. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid screening method developed for the LAB strains provides a useful tool for more systematic product development of commercial inoculant preparations. Time consuming and laborious ensiling experiments can be limited only to the most promising strains.

Effects of a dietary organic acid mixture and of dietary fibre levels on ileal and faecal nutrient apparent digestibility, bacterial nitrogen flow, microbial metabolite concentrations and rate of passage in the digestive tract of pigs.

Six 34-kg barrows were fitted with a post-valve T-caecum cannula and assigned to six dietary treatments according to a 6 × 5 change-over design to study how a mixture of formic acid, sorbate, and benzoate (0 or 8.4 g/kg feed) influences apparent ileal and faecal digestibility coefficients, bacterial nitrogen (N) flow, microbial metabolite concentrations, and passage rate in pigs fed isoenergetic diets with medium, high, or very high fibre content (neutral-detergent fibre (NDF): 199, 224, and 248 g/kg dry matter, respectively). These barley and soya-bean meal based diets contained 0, 75, and 150 g/kg barley fibre (NDF: 577 g/kg) and 0, 8, and 16 g/kg rapeseed oil, respectively. The dietary organic acid mixture improved the apparent ileal digestibility of 14 of the 17 amino acids analysed (P < 0.05). Increasing levels of dietary fibre linearly decreased the apparent ileal digestibility of six of the 17 amino acids analysed (P < 0.05). Ileal flows of bacterial N and amino acids as assessed on the basis of purine flow were decreased by the dietary organic acid mixture (P < 0.05) but were not affected by dietary fibre level (P>0.05). As assessed on the basis of diaminopimelic acid flow, bacterial N flow was increased by both the dietary organic acid mixture and increased dietary fibre levels (P < 0.05). The dietary organic acid mixture reduced the concentration of lactic acid and increased that of acetic acid in ileal digesta (P < 0.05), while dietary fibre levels had a quadratic effect on concentrations of acetic, propionic, and butyric acid (P < 0.05). The mean retention time of Co (solute marker) and Yb (particle marker) in the large intestine decreased in a linear manner by increasing dietary fibre levels (P < 0.05) but was not affected by the dietary organic acid mixture (P>0.05). The results show that a dietary organic acid mixture has a positive effect on the apparent ileal digestibility of most amino acids irrespective of dietary fibre levels. This could be at least partly related to changes in bacterial N flow in the ileum. However, different bacterial markers showed opposite effects on bacterial N flow, which makes it questionable to use a constant bacterial marker / bacterial N ratio to estimate bacterial N flow. Increasing levels of dietary fibre had negative effects on the apparent ileal amino acid digestibilities and shortened the mean retention time of digesta in the large intestine.

Effects of dietary metabolisable energy, calcium and phosphorus on bone mineralisation, leg weakness and performance of broiler chickens.

1. Performance, gait score (GS), tibial dyschondroplasia (TD), and tibia bone mineralisation and breaking strength were determined in 2880 male and female Ross 208 broilers fed on diets with two different concentrations of dietary metabolisable energy (ME) (11.00 or 12.00 MJ/kg) and 4 different concentrations of available phosphorus (aP) adjusted for dietary ME content (4.0, 4.5, 5.0 or 5.5 g/kg aP in starter and 3.5, 4.0, 4.5 or 5.0 g/kg aP in finisher diets containing 12.00 MJ/kg). 2. Tibia ash, calcium (Ca) and phosphorus (P) contents in broilers given diets with low ME (11.00 MJ/kg) were greater than those of broilers given diets with higher ME (12.00 MJ/kg). Tibia ash, Ca and P contents increased curvilinearly with increasing dietary aP content. The dietary aP level had no effect on GS. 3. Dietary concentration of ME or aP had no effect on tibia breaking strength. 4. Walking ability, as measured by GS, was negatively correlated with the body weight (BW) of tested birds at 23 and 35 d of age, but the dietary ME content or aP level had no significant effect on GS at 35 d of age. 5. The results indicated that bone mineral content had no clear correlation with the walking ability of broilers.

Effects of preservation method and beta-glucanase supplementation on ileal amino acid digestibility and feeding value of barley for poultry.

1. Effects of preservation method (drying or air-tight storage of whole grain and ensiling of rolled high-moisture grain) and beta-glucanase supplementation (Econase) on apparent ileal amino acid digestibilities and metabolisable energy content of barley were evaluated with Ross broiler chickens. In addition, the effect of barley preservation method was assessed using Leghorn cockerels. 2. Birds were given either a semi-purified soyabean meal basal diet or a mixture of the basal diet and barley (50:50 on dry matter basis). Apparent ileal digestibilities (AID) of nutrients were assessed using the slaughter technique. AID of nutrients and nutrient digestibility measured using excreta (AED) were determined using chromium mordanted straw as an indigestible marker. 3. In broilers, AID of amino acids, dry matter and organic matter were lower for dried than air-tight stored barley, particularly for diets based on ensiled barley. In cockerels, barley preservation method had no effect on amino acid AID. The AED of nutrients and nitrogen corrected apparent metabolisable energy content (AMEn) was highest for ensiled barley across both experiments. 4. beta-glucanase supplementation increased nutrient digestibility, phosphorus retention and AMEn content of air-tight stored and dried barley diets in particular but had only negligible effects on ensiled barley. Beta-glucanase improved the AID of amino acids in dried barley but not in air-tight stored or ensiled barley. 5. Amino acid digestibilities were lower in broilers than cockerels and the effect of barley preservation on feeding value of barley was different for broilers and cockerels.

Effects of clodronate on vertebral fracture risk in osteoporosis: a 1-year interim analysis.

The aim of this study was to determine whether clodronate reduced the incidence of vertebral fractures in patients with osteoporosis. We report here the interim analysis after 1 year of a 3-year double-blind placebo-controlled study. The objectives of the interim analysis were to determine whether there was a trend in fracture frequency and to examine the effects of clodronate on bone mineral density (BMD). Patients with densitometrically proven osteoporosis (T-score <-2.5 and <-3 for women and men, respectively) or with at least one prevalent vertebral fracture were recruited to a 3-year double-blind, controlled study. Patients were randomized to three strata, namely women with postmenopausal osteoporosis (stratum I, n = 483), women with secondary osteoporosis (II, n = 110), and men with osteoporosis of any causation (III, n = 84). They received either clodronate 800 mg daily by mouth or an identical placebo, and all patients received a calcium supplement of 500 mg daily. BMD was measured at six monthly intervals, and lateral spine radiographs for vertebral morphometry were obtained at baseline and 1 year. Treatment with clodronate was associated with a significant increase in BMD at the spine of 3.2 +/- 0.3% (p < 0.0001 vs. baseline) compared with a nonsignificant change of 0.5 +/- 0.3% in the placebo group (p < 0.0001 between treatments). At the hip, clodronate was associated with a significant increase in total hip BMD of 1.3 +/- 0.3% (p = 0.018 vs. baseline) compared with a small decrease of 0.4 +/- 0.3% in the placebo group (p = 0.027 for the difference between treatment groups). The mean changes at the spine and hip were similar in all three strata. Incident vertebral fractures were observed in 27 patients at 1 year in the placebo group (9.0%) and in 14 patients receiving clodronate (4.9%) (relative risk 0.54; 95% CI 0.29-1.02; p = 0.07). A trend was observed in all treatment strata. Treatment was well tolerated, with no significant adverse events attributable to clodronate treatment. We conclude that clodronate 800 mg daily is effective in preventing bone loss, and at 1 year, there is a trend consistent with antifracture efficacy in patients with established osteoporosis regardless of causation.

Effect of dietary glycine and benzoate level on benzoate metabolism in mink (Mustela vision), blue fox (Alopex lagopus), and raccoon dog (Nyctereutes procyonoides).

Three 2 x 4 factorial experiments were carried out from August to September with 30 juvenile male mink, 24 raccoon dogs, and 24 blue foxes to investigate the effect of dietary glycine supply (low or high) on the efficiency of these species to excrete hippuric acid with incremental benzoate intake (0, 1, 2, or 4 mmol/kg BW). For mink, two additional treatments with 1 or 2 mmol/kg BW of ethyl benzoate were included. A basal low-glycine diet was formulated to meet the minimum protein requirements of fur animals (30% of ME). This diet was supplemented with 0 or 3 g/kg of glycine, or with 0, 1.0, 2.07, or 4.15 g/kg of sodium benzoate for mink and blue foxes, and with 0 or 4.5 g/kg of glycine and 0, 1.58, 3.17, or 6.34 g/kg of sodium benzoate for raccoon dogs, respectively. Two additional diets with .76 or 1.53 g/kg of ethyl benzoate were made for mink. Fecal and urinary benzoic and hippuric acid excretion were measured for 3 d. The 24-h recovery of [14C]benzoic acid injected intraperitoneally was measured from urine, the liver, and the kidneys. All animals appeared healthy and no clinical signs of benzoate overdose were observed. Dietary benzoate level did not affect ADFI or ADG in any species. Glycine supplementation lowered ADFI in mink. The majority of ingested benzoates were absorbed from the gut (over 95%), except in blue foxes, which excreted 6 to 15% of ingested benzoates in feces with incremental increases in benzoate intake. Urinary free benzoic acid excretion accounted for 10% of the ingested benzoates in blue foxes but less than 5% in mink and raccoon dogs. When benzoate intake was 1 mmol/kg BW, mink, blue foxes, and raccoon dogs excreted 71, 77, and 34% of ingested benzoates as hippuric acid in urine, respectively. With higher benzoate intakes, urinary hippuric acid excretion decreased quadratically with mink to 20%, and linearly with blue foxes and raccoon dogs to 45 and 16%, respectively. The hippuric acid pathway appears to be the principal route of benzoate elimination in the mink and blue fox, whereas, in the raccoon dog, other pathways appear to be more important. In mink, the elimination of ethyl benzoate did not differ from that of sodium benzoate. Because glycine conjugation is the primary route of benzoate elimination, it is recommended that benzoate content in fur animal feeds should not exceed 1 g/kg feed on an as-fed basis.

Lactation and metabolic responses to graded abomasal doses of methionine and lysine in cows fed grass silage diets.

Five rumen-cannulated Finnish Ayrshire cows were used in two 5 x 5 Latin square experiments designed to study the lactation and metabolic responses to increasing doses of DL-Met or L-Lys infused into the abomasum. The cows were fed grass silage ensiled with a formic acid additive for ad libitum intake. A supplement with barley and oats was given at a rate of 9 kg/d (Experiment 1) or 7 kg/d (Experiment 2). The experimental treatments were 0, 10, 20, 30, or 40 g of Met/d (Experiment 1) and 0, 15, 30, 45, or 60 g of Lys/d (Experiment 2). The infusion of Met did not significantly affect feed intake or daily milk yield, but increased milk fat content, ECM yield, and C4 to C14 and C18 to C20 fatty acid production in milk. The infusion of Met caused an increase in arterial plasma Met concentration and a decline in branched-chain amino acids (AA). Mammary gland uptake of Met was not related to plasma AA concentration. The infusion of Lys did not affect feed intake, milk yield, or milk composition, except for increases in milk urea and NPN contents. The infusion of Lys increased plasma Lys, BCAA, EAA, and the EAA to TAA ratio. Uptake of plasma BCAA and NEAA by the mammary gland decreased, which suggests that Lys was used as a substrate for milk NEAA synthesis. These data demonstrate that Met is important in the milk fat synthesis, and Lys is important in mammary gland AA metabolism. However, neither Met nor Lys is the first-limiting AA in the milk protein yield of cows fed a grass silage and cereal diet.

The in vitro response to human fibroblast-derived extracellular matrix proteins is restricted by specific HLA class II genes. Relevance for coeliac disease.

Coeliac disease is an immunologic disease of the small intestine which is caused by ingestion of wheat gliadin, the disease-promoting agent. The disease associates strongly with the particular HLA type, HLA-DQA1*0501, DQB1*0201 alleles. Further specific autoantibodies against reticulin and endomysium are found in patients; these autoantibodies appear to be disease specific. An extracellular matrix noncollagenous protein reacts specifically with CD patients' serum immunoglobulin A and is the target of antireticulin antibodies. In this study the immune response to this matrix protein was analyzed in vitro in normal, healthy individuals. Our study shows that the immune response to Fb-CDAP is strictly regulated by the HLA-DR3, DQA1*0501, DQB1*0201 alleles, and that only those cells which were positive for these alleles produced an immune response. On the other hand, half of the cells positive for these HLA alleles were responders. Monoclonal antibodies to DR and DQ inhibited the response in an additive way, showing that both DR and DQ can act as an antigen-presenting structure. The immune response to gliadin has been shown to associate with the same HLA type as CD, but the association is not as strong. Our results show that the immune responses to Fb-CDAP can be generated in vitro in genetically predisposed persons in the absence of CD.

Detection of human papillomavirus DNA by AffiProbe HPV-DNA test kit in cervical scrapes or biopsies-histopathologic correlates.

OBJECTIVE: The aim of this study was to evaluate and compare the efficacy of punch biopsies and cervical scrapes in the detection of human papillomavirus (HPV) DNA from the cervix and compare the results with the histopathologic diagnosis. METHODS: The specimens were collected simultaneously, and HPV DNA was detected using a liquid hybridization test. RESULTS: Biopsies and scrapes were equally efficient, but each detected only two-thirds of all HPV-DNA-positive patients. Thus, the positivity rate increased when both tests were used. Overall, 13% of patients with normal histopathology, 38% of patients with benign atypia, and 66% of patients with squamous intraepithelial lesions (SIL) were HPV-DNA positive. HPV-DNA 16 was found in 54% of HPV-DNA-positive patients with SIL, in 20% of HPV-DNA-positive patients with atypia, and in none of patients with normal histopathology. CONCLUSIONS: The liquid hybridization test used in this study detects HPV DNA equally efficiently from both biopsies and scrapes. The test can be performed in 1 working day. However, the sensitivity of the test is low, and it only detects a limited number of HPV types.

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates.

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

Zone-specific inducibility of cytochrome P450 2B1/2 is retained in isolated perivenous hepatocytes.

The expression and induction of the cytochrome P450 2B1/2 isoenzyme is heterogeneous, exhibiting a regional pattern in the intact liver and a varied response to phenobarbital in isolated cultured hepatocytes. We report that P450 2B1/2 immunostaining of hepatocytes isolated from the perivenous liver region and cultured in the presence of phenobarbital is much stronger than that of cells identically treated but isolated from the periportal region. P450 2B1 mRNA, quantified by a sensitive and specific RNAase protection assay, is also preferentially induced in perivenous hepatocytes, demonstrating that the difference in induced expression is at the pretranslational level. Our results suggest that perivenous and periportal hepatocytes are differentially imprinted to retain regiospecific factors governing their inducibility after isolation.

A rapid and quantitative solution hybridization method for detection of HBV DNA in serum.

A sensitive and convenient solution hybridization technique was adapted for the semiquantitative detection of hepatitis B virus DNA in serum. The assay utilizes 35S-isotope as label and biotin-avidin interaction for collection of the hybrids onto microtitre plate wells. Results are obtained as numerical values, which allow quantification. 10(6) molecules of HBV DNA/ml serum could be detected by a 3-h hybridization followed by a 2-h collection reaction. By analyzing 500 microliters of serum, 30 (88%) of 34 patient sera positive for HBeAg were also positive for HBV DNA and 17 HBsAg-positive sera, 16 of which were anti-HBe-positive, were DNA-negative. The amount of HBV DNA varied from 5 x 10(6) to 3 x 10(9) molecules/ml. The solution hybridization method which was developed allows fast and accurate quantification of HBV DNA in serum providing an estimate of the virus titre.

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.

A rapid solution hybridization method for detection of human papillomaviruses.

Nucleic acid hybridization methods in routine diagnosis of micro-organisms have been limited by the tedious assay procedures. We have previously described the sandwich hybridization method which allows convenient testing of biological specimens. In this paper we describe the adaptation of the solution hydridization method into the microtitre plate format using 35S-isotope as label. Using 3-hour hybridization followed by 2-hour collection of the hybrids a sensitivity of 5 x 10(5) target DNA molecules was achieved. The method was applied for identification of human papillomaviruses in crude gynaecological specimens. A simple 1-day assay protocol was achieved with high HPV type specificity. The specificity was confirmed by testing a variety of unrelated micro-organisms, none of which gave a positive signal in the test. Results, obtained as numerical values, were easy to interpret; positive and negative samples gave clearly distinguishable signals.

Use of AffiProbe HPV test kit for detection of human papillomavirus DNA in genital scrapes.

The presence of human papillomavirus (HPV) DNA in cervical and vaginal scrapes was analyzed by the AffiProbe HPV test kit (Orion Corp., Orion Pharmaceutica, Helsinki, Finland), which is a 1-day solution hybridization test for HPV type 6/11, 16, or 18. The AffiProbe test was compared with a commercially available dot blot test (ViraPap and ViraType tests; Life Technologies Inc., Gaithersburg, Md.). The study group consisted of 178 patients seen in a gynecological outpatient clinic. Altogether, 64 specimens (36 cervical and 28 vaginal scrapes) from 49 patients were positive by the AffiProbe test. Concurrently collected cervical scrapes from 174 patients were available for the reference test, which yielded 27 positive results for HPV type 6/11 or 16/18 and 25 positive results for HPV type 31/33/35. Agreement as to the presence of HPV type 6/11, 16, or 18 by the two tests was reached in 85% of the specimens. Eleven cervical specimens were positive by the AffiProbe test only, and nine cervical specimens were positive by the ViraType test only. Independent evidence obtained by the polymerase chain reaction, repeat examination, or the concurrent presence of HPV DNA in vaginal or vulval epithelium supported the AffiProbe and the ViraType test results for 6 of the 11 and 6 of the 9 specimens with discrepant results, respectively. Thus, the DNA tests had similar sensitivities for HPV type 6/11, 16, and 18 DNAs, but the results were obtained within 1 day by the AffiProbe test, whereas results for the ViraPap and ViraType analyses required from 4 days to 2 weeks.