Dynamic role of single-celled fungi in ruminal microbial ecology and activities.

In ruminants, high fermentation capacity is necessary to develop more efficient ruminant production systems. Greater level of production depends on the ability of the microbial ecosystem to convert organic matter into precursors of milk and meat. This has led to increased interest by animal nutritionists, biochemists and microbiologists in evaluating different strategies to manipulate the rumen biota to improve animal performance, production efficiency and animal health. One of such strategies is the use of natural feed additives such as single-celled fungi yeast. The main objectives of using yeasts as natural additives in ruminant diets include; (i) to prevent rumen microflora disorders, (ii) to improve and sustain higher production of milk and meat, (iii) to reduce rumen acidosis and bloat which adversely affect animal health and performance, (iv) to decrease the risk of ruminant-associated human pathogens and (v) to reduce the excretion of nitrogenous-based compounds, carbon dioxide and methane. Yeast, a natural feed additive, has the potential to enhance feed degradation by increasing the concentration of volatile fatty acids during fermentation processes. In addition, microbial growth in the rumen is enhanced in the presence of yeast leading to the delivery of a greater amount of microbial protein to the duodenum and high nitrogen retention. Single-celled fungi yeast has demonstrated its ability to increase fibre digestibility and lower faecal output of organic matter due to improved digestion of organic matter, which subsequently improves animal productivity. Yeast also has the ability to alter the fermentation process in the rumen in a way that reduces methane formation. Furthermore, yeast inclusion in ruminant diets has been reported to decrease toxins absorption such as mycotoxins and promote epithelial cell integrity. This review article provides information on the impact of single-celled fungi yeast as a feed supplement on ruminal microbiota and its function to improve the health and productive longevity of ruminants.

Saccharomyces cerevisiae as a probiotic feed additive to non and pseudo-ruminant feeding: a review.

The production of livestock and poultry faces major challenges to meet the global demand for meat and dairy products and eggs due to a steady increase in the world's population and the ban of antibiotics in animal production. This ban has forced animal nutritionists to seek for natural alternatives to antibiotics. In this context, the yeast Saccharomyces cerevisiae has received considerable attention in the last decade. It has been reported that feed supplementation with live yeast cells improve feed efficiency, enhance feed digestibility, increase animal performance, reduce the number of pathogenic bacteria, improve animal health and reduce the negative environmental impacts of livestock production. The current review sheds light on the effects of the use of live S. cerevisiae cells in the diets of nonruminant and pseudo-ruminant's animals and the mechanisms by which they exert its effects. This review work revealed that the addition of S. cerevisiae in poultry feed causes a phenomenon called competitive exclusion of pathogenic bacteria capable of causing disease adhere to the yeast surface, and so removing a large amount of harmful micro-organisms and allowing the Animal defend more effectively, the production of antimicrobial agents, the balancing the gut microbiota and stimulation of host adaptive immune system and improving gut morphological structure, thus these benefits are reflected on the overall poultry health. In addition, in the presence of live S. cerevisiae cells, the immunity of rabbits was improved due to the high number of white blood cell. In addition, apparent digestibility of acid and neutral detergent fibre was improved in horses and rabbits. Saccharomyces cerevisiae in pig diets augment mucosal immunity by increasing IgM and IgA activity against pathogens, enhance intestinal development and function, adsorb mycotoxins, modulate gut microbiota and reduce postweaning diarrhoea.

Identification of two novel bacterial phosphatase-encoding genes in Pseudomonas putida strain P13.

AIMS: Isolation and identification of genes encoding putative phosphatases from Pseudomonas putida strain P13 DSM 23335. METHODS AND RESULTS: By functional screening of a P. putida P13 genomic library, a number of Pho+ clones were identified. Two genes were identified that encoded proteins exhibiting both phytase and sugar phosphatase activities. The proteins were 249 and 462 amino acids, with molecular masses of 26 and 50 kDa respectively. Sequence alignments revealed no significant similarities to representatives of known phosphatase or phytase gene families. However, the genes were found to have a high similarity to members of the major facilitator superfamily (MFS). Both genes were overexpressed in Escherichia coli and the corresponding partially purified recombinant enzymes were found to have significant phytate-dephosphorylating activity. The protein designated P. putida phytase 1 (Ppp1) displayed the highest activity among potential substrates studied on Na phytate, whereas Ppp2 more likely represents a sugar phosphatase than a phytase. The optimal conditions for phytate dephosphorylation were determined as 60°C and pH 4·5 (Ppp1) or pH 5·0 (Ppp2). CONCLUSIONS: Two novel bacterial phosphatase-encoding genes, named ppp1 and ppp2, were isolated from P. putida P13 DSM 23335 by a functional screening procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphatase-encoding genes are of great importance for industrial applications, particularly in agriculture. The identified phosphatase genes represent a new class of acid phosphatases.

State of the art of nonthermal and thermal processing for inactivation of micro-organisms.

Despite the constant development of novel thermal and nonthermal technologies, knowledge on the mechanisms of microbial inactivation is still very limited. Technologies such as high pressure, ultraviolet light, pulsed light, ozone, power ultrasound and cold plasma (advanced oxidation processes) have shown promising results for inactivation of micro-organisms. The efficacy of inactivation is greatly enhanced by combination of conventional (thermal) with nonthermal, or nonthermal with another nonthermal technique. The key advantages offered by nonthermal processes in combination with sublethal mild temperature (<60°C) can inactivate micro-organisms synergistically. Microbial cells, when subjected to environmental stress, can be either injured or killed. In some cases, cells are believed to be inactivated, but may only be sublethally injured leading to their recovery or, if the injury is lethal, to cell death. It is of major concern when micro-organisms adapt to stress during processing. If the cells adapt to a certain stress, it is associated with enhanced protection against other subsequent stresses. One of the most striking problems during inactivation of micro-organisms is spores. They are the most resistant form of microbial cells and relatively difficult to inactivate by common inactivation techniques, including heat sterilization, radiation, oxidizing agents and various chemicals. Various novel nonthermal processing technologies, alone or in combination, have shown potential for vegetative cells and spores inactivation. Predictive microbiology can be used to focus on the quantitative description of the microbial behaviour in food products, for a given set of environmental conditions.

A high observed substitution rate in the human mitochondrial DNA control region.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

[The novel intracellular phytase of enterobacteriaceae: isolation and properties].

For the first time phytase enzyme was isolated from Pantoea vagans 3.2 strain and was subjected to investigation. The enzyme was purified about 474-fold to apparent homogeneity from the crude extract of the strain, its primary structure was determined and it was concluded that phytase of Pantoea vagans 3.2 belongs to the family of histidine acid phosphatases. It has a molecular mass of about 46 kDa and Km for the hydrolysis of sodium phytate was 0.28 mM. Some physicochemical properties ofphytase were investigated.

Cost-effectiveness of insulin glargine versus NPH insulin for the treatment of Type 2 diabetes mellitus, modeling the interaction between hypoglycemia and glycemic control in Switzerland.

OBJECTIVE: The objective of this study was to evaluate the short-term and long-term clinical and economic outcomes associated with insulin glargine or NPH insulin in patients with Type 2 diabetes mellitus (T2DM) inadequately controlled with oral anti-diabetic drugs in Switzerland, modeling the interaction between hypoglycemia and glycemic control (HbA1c). METHODS: A validated discrete event simulation model for T2DM was used to predict incidence of short-term complications (symptomatic, nocturnal and severe hypoglycemic events) and long-term complications (microvascular and macrovascular events), life expectancy, quality-adjusted life years (QALYs) and direct medical costs in patients treated with insulin glargine or NPH insulin. The model was populated with published Swiss patient characteristics with T2DM. Baseline risks of hypoglycemic events, utility decrements of diabetes-related long-term complications and the hypoglycemia fear score were derived from the literature. Relative risk reductions of hypoglycemia adjusted for HbA1c using insulin glargine compared with NPH insulin were based on a published negative binomial meta-regression analysis. Costs of severe hypoglycemia, micro- and macrovascular events were analyzed from literature whenever possible otherwise guideline-projected resource-use estimations were valued with Swiss official prices or tariffs in 2006 CHF. Simulations were run with 1,000 patients per cohort over a time horizon of 40 years. Incremental cost effectiveness ratios (ICERs) were presented as cost per QALY and per life year gained (LYG). Future costs and clinical benefits were discounted at 3.5%. Wide-range one-way sensitivity analyses were performed. RESULTS: Insulin glargine was associated with an improvement in quality of life (0.098 QALYs per patient) and additional life expectancy (0.05 life years gained per patient) compared to NPH insulin. Incremental costs of CHF 2,578 resulted in an ICER of CHF 26,271 per QALY and CHF 51,100 per LYG. The cost per QALY was most sensitive to changes in costs, utility decrements and relative risk reductions of hypoglycemia. CONCLUSIONS: This study evaluated, for the first time, the cost effectiveness of insulin glargine versus NPH insulin for the treatment of T2DM considering the interaction between glycemic control and hypoglycemia in Switzerland. The base case and sensitivity analyses demonstrated that insulin glargine proved to be cost-effective with respect to accepted willingness to pay thresholds and therefore represents good value for money.

Cost-effectiveness and cost-utility of insulin glargine compared with NPH insulin based on a 10-year simulation of long-term complications with the Diabetes Mellitus Model in patients with type 2 diabetes in Switzerland.

OBJECTIVE: The objective of this study was to evaluate the cost-effectiveness of insulin glargine compared with NPH insulin in patients with type 2 diabetes and in whom OAD (oral anti-diabetics) had failed in Switzerland. METHODS: Long-term diabetes outcomes were simulated with the Diabetes Mellitus Model (DMM) over a period of 10 years. The incidences of long-term complications (micro- and macrovascular events) were simulated for 10,000 patients over 10 years for six different scenarios. The scenarios were based on HbA1c reductions observed in clinical trials. For insulin glargine, HbA1c reductions of 0.96% (pessimistic case) and 1.24% (optimistic case) were simulated for three different HbA1c baseline values (10, 9 and 8%). For NPH insulin the HbA1c reduction was assumed to be 0.84%. A cost model and a utility model were developed in order to use the cumulated incidences of the simulations for the calculation of cost and QALYs (quality-adjusted life years). The unit costs of micro- and macrovascular events were assessed on the basis of published literature and guideline-projected resource-use estimations for Switzerland. Disutility values of diabetes-related long-term complications were derived from the literature. Total direct medical costs or QALYs were assessed by a combination of cumulated incidences of each event up to 10 years with the corresponding unit cost per event (in addition to the acquisition cost) or with disutility values per event, respectively. Events, total cost, and QALYs were discounted at 3%. In scenarios where no savings could be shown for insulin glargine, incremental cost-effectiveness ratios were calculated as the incremental cost per event prevented and the cost per QALY gained. RESULTS: Cost comparison demonstrated that insulin glargine is the dominant strategy for the optimistic case scenario starting at a baseline HbA1c value of 10% as savings in the management of complications exceeded the difference in acquisition costs after 8 years of treatment. Optimistic case scenarios for baseline HbA1c values of 9 and 8% achieved costs per QALY gained amounting to CHF 2,853 and CHF 5,711 and costs per event prevented amounting to CHF 2,054 and CHF 4,899, respectively. Pessimistic case scenarios for baseline HbA1c values of 10, 9 and 8% resulted in costs per QALY gained amounting to CHF 40,441, CHF 45,701 and CHF 49,468 and costs per event prevented amounting to CHF 27,742, CHF 32,451 and CHF 41,620, respectively. CONCLUSIONS: This study investigated the long-term health-economic implications of treating type 2 diabetes patients, in whom OAD had failed, with insulin glargine versus NPH insulin in Switzerland. The 10-year simulations demonstrated that the deltaHbA1c reductions of 0.4 and 0.12% achieved with insulin glargine led to a reduction of long-term complications, mortality and associated costs as well as to an improved quality of life. Insulin glargine proved to be cost-effective and represents good to excellent value for money compared to NPH insulin.

Effects of gamma radiation on maize samples contaminated with Fusarium verticillioides.

The efficacy of gamma-irradiation as a method of decontamination of maize containing Fusarium verticillioides under controlled conditions of relative humidity (RH) (97.5%) and water activity has been studied. Maize grains inoculated with a spore suspension of F. verticillioides were irradiated to 2, 5, and 10 kGy. Thereafter, the irradiated and control samples were analyzed for the presence of fumonisins, their viable cells were counted, and their morphology was investigated by electronic microscopy. It was found possible to decrease the risk of exposure to fumonisins by irradiating maize to 5 or 10 kGy. However, at the dose of 2 kGy, the survived fungi (36%) can produce more fumonisins than the fungi in the control unirradiated samples under the same conditions.

Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer.

Hypoxia has long been recognized as detrimental to the successful treatment of malignant tumors with ionizing radiation. Because hypoxia-inducible factor (HIF)-1alpha plays an essential role in oxygen homeostasis in vitro, we explored the predictive potential of this factor in a cohort of 98 patients with squamous cell cancer of the oropharynx, who were treated by curative radiation therapy. Ninety-four % of the primary tumors showed overexpression of HIF-1alpha, relative to the surrounding tissue, as determined by immunohistochemistry. The degree of HIF-1alpha immunoreactivity correlated inversely with both the rate of complete remission of the primary tumor (odds ratio, 0.33; P = 0.03) and lymph node metastases (odds ratio, 0.34; P = 0.02) as well as with local failure-free survival (risk ratio, 2.15; P = 0.006), disease-free survival (risk ratio, 2.01; P = 0.008), and overall survival (risk ratio, 2.17; P = 0.002). The multivariate analysis revealed the predictive power of HIF-1alpha to be independent of other covariables. We conclude that HIF-1alpha is overexpressed in the vast majority of patients with squamous cell cancer of the oropharynx and that the degree of expression has predictive and prognostic significance in individuals undergoing curative radiation therapy.

First isolated locoregional recurrence following mastectomy for breast cancer: results of a phase III multicenter study comparing systemic treatment with observation after excision and radiation. Swiss Group for Clinical Cancer Research.

PURPOSE: We performed a randomized phase III multicenter study to compare systemic treatment versus no treatment after complete excision and radiotherapy for isolated first locoregional recurrence in patients with breast cancer. PATIENTS AND METHODS: One hundred sixty-seven good-risk patients with an estrogen receptor (ER+) positive recurrence or, in case of unknown receptor status, a disease-free interval (DFI) of greater than 12 months and < or = three recurrent tumor nodules each < or = 3 cm in diameter were entered onto the study. They were randomized to observation subsequent to local treatment or to receive tamoxifen (TAM) until disease progression. Seventy-nine percent of the patients were postmenopausal. RESULTS: The median observation period for the entire study population was 6.3 years. The median disease-free survival (DFS) duration was 26 months for observation and 82 months for TAM patients (P = .007). This was mainly due to the reduction of further local recurrences, whereas the occurrence of early distant metastases was delayed. A multivariate analysis identified DFI and treatment with TAM as significant prognostic factors for DFS. The 5-year overall survival (OS) rates were 76% and 74%, respectively (P = .77). DFI was also a prognostic factor for OS. CONCLUSION: Systemic therapy with TAM after isolated locoregional recurrence of breast cancer significantly increased 5-year DFS rates from 36% to 59% compared with observation alone and prolonged median DFS by more than 4.5 years in patients with ER+ tumors or in the case of unknown ER status with a DFI of greater than 12 months and minimal tumor burden. Treatment with TAM currently has no significant impact on OS, but the median survival duration of the study population has not yet been reached.

Accelerated radiotherapy, carbogen, and nicotinamide in glioblastoma multiforme: report of European Organization for Research and Treatment of Cancer trial 22933.

PURPOSE: A three-step phase I/II trial associating accelerated radiotherapy with carbogen (step 1, ARCO), with nicotinamide (step 2, ARN), or with both (step 3, ARCON) was conducted, the aim of which was to overcome the effects of proliferation and hypoxia as potential causes of tumor radioresistance in glioblastoma multiforme. PATIENTS AND METHODS: Radiotherapy consisted of 60 Gy delivered over 4 weeks in 1.5-Gy fractions twice daily, 5 days a week. Carbogen breathing was started 5 minutes before each fraction and continued until the end of each treatment session. Nicotinamide was given daily as a single oral dose of 85 mg/kg. RESULTS: A total of 115 patients with a median age of 55 years were registered. Of 107 eligible patients, 23 were registered in step 1, 28 in step 2, and 56 in step 3. The planned treatment was administered without any interruption in 72% of patients (86% in ARCO but 68% in ARN and ARCON). The incidence and severity of acute skin and mucous membrane toxicity were higher in patients who received nicotinamide (ie, the ARN and ARCON groups). Grade 1 to 2 gastrointestinal toxicity was observed in 44% of patients in the ARN group and 32% of patients in the ARCON group, but only in 8% of patients in the ARCO group. Eight percent of evaluated patients presented with abnormal liver test results at treatment completion. The dose of corticosteroids had to be increased in 44% of patients. Late neurologic side effects were similar in all treatment steps and were observed mostly in patients with disease progression. Median survival times for patients treated with ARCO, ARN, and ARCON were 10.1, 9.7, and 11.1 months, respectively. CONCLUSION: Feasibility of ARCO treatment was good but that of ARN and ARCON was only fair. This probably reflected the higher acute toxicity rate, particularly gastrointestinal, for patients receiving nicotinamide. The dose of corticosteroids had to be increased frequently during treatment, suggesting a higher than expected acute neurologic toxicity. Overall survival was similar in the three treatment steps and not different when compared with results of other series that used radiotherapy alone.

Fluorouracil plus racemic leucovorin versus fluorouracil combined with the pure l-isomer of leucovorin for the treatment of advanced colorectal cancer: a randomized phase III study.

PURPOSE: To compare the efficacy and toxicity of fluorouracil (FU) and racemic leucovorin (d,l-LV) versus FU combined with the l-isomer of leucovorin (l-LV) in the treatment of advanced colorectal cancer. PATIENTS AND METHODS: A total of 248 patients with advanced measurable colorectal cancer previously unexposed to chemotherapy were randomly assigned to treatment with either FU (400 mg/m2/d by intravenous [I.V.] infusion for 2 hours) and racemic LV (100 mg/m2/d by I.V. bolus injection) given for 5 consecutive days, or the combination of FU and the pure l-isomer of LV using the same dose schedule. In both treatment arms, courses were administered every 28 days if toxicity allowed for a total of 6 months, unless evidence of tumor progression was documented earlier. RESULTS: There were no significant differences between the FU/racemic LV and the FU/l-LV arm in the overall response rate (25% v 32%), duration of response (7.2 v 8.0 months), median time to progression or death (6.25 v 8.0 months), or median overall survival time (14.5 v 15.0 months). Except for minor myeloid toxic effects associated with FU/l-LV, there was also no significant difference in terms of adverse reactions. Gastrointestinal symptoms, specifically mucasitis and diarrhea, were less frequent and less severe in both treatment arms compared with other trials with FU/racemic LV reported in the literature, which might be because of the prolonged administration of FU used in both arms. CONCLUSION: The combination of FU/l-LV produced response rates, response durations, and survival times similar to those with FU/d,l-LV. Biochemical modulation of FU by either pure l-LV or racemic LV thus appears to result in equivalent clinical efficacy.

A multi-criteria approach to Great Barrier Reef catchment (Queensland, Australia) diffuse-source pollution problem.

This paper presents a multi-criteria based tool for assessing the relative impact of diffuse-source pollution to the Great Barrier Reef (GBR) from the river basins draining into the GBR lagoon. The assessment integrates biophysical and ecological data of water quality and pollutant concentrations with socio-economic information pertaining to non-point source pollution and (potential) pollutant impact. The tool generates scores for each river basin against four criteria, thus profiling the basins and enabling prioritization of management alternatives between and within basins. The results support policy development for pollution control through community participation, scientific data integration and expert knowledge contributed by people from across the catchment. The results specifically provided support for the Reef Water Quality Protection Plan, released in October 2003. The aim of the plan is to provide a framework for reducing discharge of sediment, nutrient and other diffuse-source loads and (potential) impact of that discharge and for prioritising management actions both between and within river basins.

No predictive value of the micronucleus assay for patients with severe acute reaction of normal tissue after radiotherapy.

In approximately 5% of cancer patients undergoing radiotherapy, this treatment has to be interrupted because of an acute reaction of normal tissues. To test the possibility of predicting this type of reaction, the micronucleus assay was used to determine radiosensitivities of peripheral blood lymphocytes of 15 patients with severe acute reaction of normal tissue, 15 patients without this reaction and 15 healthy donors. Whole-blood cultures were irradiated with X-rays (4 Gy, 1.08 Gy/min) and treated with cytochalasin B. The micronuclei scores observed in irradiated cells were corrected for the scores in unirradiated cells. Intra-individual and interindividual variations in micronuclei scores were analysed in samples from healthy donors, and highly significant interindividual differences were found (P < 0.001). Scores of cells not irradiated in vitro were higher for cancer patients before radiotherapy than for healthy donors (P < 0.001), and those for cancer patients after radiotherapy were higher than for patients before radiotherapy (P < 0.001). Average micronuclei scores induced by in vitro irradiation were significantly higher in samples from cancer patients compared with those from healthy donors (P < 0.01). Moreover, all subgroups of cancer patients included individuals with very high levels of micronuclei after in vitro irradiation. There was, however, no relationship between the micronuclei scores and the occurrence of severe acute reactions in normal tissues.

Conformal radiotherapy for unresectable retroperitoneal soft tissue sarcoma.

Local tumor control remains a continuing challenge in the treatment of retroperitoneal soft tissue sarcoma. Though complete resection by means of wide excision or excisional biopsy can be performed in a minority of patients only, aggressive surgical approach remains the treatment of choice. Unresectable sarcoma can rarely be controlled by conventionally applied radiotherapy--only a few percent of patients survive. A superior dose distribution of external radiation is demanded in order to spare healthy tissue. The presumably greatest advantage will occur when radiotherapy is used preoperatively. The possible clinical gain of superior dose distribution is demonstrated by results of the dynamic, 3-D conformal pion radiotherapy at PSI. Between April 1983 and June 1988 a total of 21 patients were treated with high doses (greater than or equal to 30 Gy) for unresectable retroperitoneal soft tissue sarcoma. The follow-up time is 13-74 months, median 24. Fifteen patients were treated with 20 fx, and 19 patients were treated with fraction sizes of 150 or 165 cGy. Except for one patient with thrombocytopenia after chemotherapy, no treatment interruption was necessary. Five patients developed late reactions, caused also by surgery and chemotherapy: two intestinal obstructions, one liver abscess, one leg edema, and one superficial skin necrosis. Nine patients had laparotomy after pion irradiation, five for resection of the previous unresectable tumor; 3/5 sarcoma were completely resected. Morbidity rate after post-pion laparotomy did not increase. Three patients had local tumor progression, 1/3 inside the treatment volume. The actuarial five-year local tumor control rate of these unresectable retroperitoneal sarcoma is 60%, the actuarial five-year survival rate is 33%. Out of the 21 patients, 15 are alive, two have died from local progression, one from peritoneal progression, and three from metastases.

Intratumoral microvessel density predicts local treatment failure of radically irradiated squamous cell cancer of the oropharynx.

PURPOSE: To determine the predictive value of intratumoral microvessel density (IMD), and of the expression of p53, vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) for the radiocurability of patients with squamous cell cancer of the oropharynx. MATERIALS AND METHODS: 139 patients with squamous cell cancer of the oropharynx were radically irradiated (median dose, 74 Gy) between 1991 and 1997. Biopsies from 100 patients were processed for immunohistochemistry. IMD was determined in hot spot areas of tissue stained with anti-CD31 at a magnification of x200. Staining for p53 was considered positive if more than 10% of the cell nuclei overexpressed the protein. Immunostaining of VEGF and TSP-1 was assessed semiquantitatively. RESULTS: Increasing IMD (range, 54-282) was strongly correlated with incomplete remission of both the primary tumors (p = 0.01) and lymph node metastases (p = 0.02). Moreover, multivariate Cox regression analysis revealed local failure-free survival to decline with increasing IMD (IMD continuous: risk ratio = 1.01 per increase of 1 microvessel, p = 0. 0001; IMD categorical: 130: risk ratio = 13.01). Neither the expression of p53, nor that of VEGF or TSP-1 was associated with the treatment outcome or IMD, but VEGF and TSP-1 expression were positively correlated (p = 0.02). CONCLUSION: IMD represents a powerful and independent predictive factor for local treatment failure in radically irradiated patients with squamous cell cancer of the oropharynx.