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A total of 45 samples of wheat from three different locations in Vojvodina were analyzed for the presence of zearalenone. Analytical methods based on clean-up by solid-phase extraction (SPE) columns and detection by liquid chromatography were used after validation. Limit of detection for ZEA in wheat was 18.6 μg/kg and the limit of quantification was 56.5 μg/kg. Recovery values ranged between 86% and 97%. The occurrence of ZEA in wheat was rather high with 53.3% of positive samples with the average value of 330 μg/kg. Incidences were found from 68 μg/kg to 1079 μg/kg. Contamination levels were above the established maximum limit for unprocessed cereals, other than maize, in as many as seventeen samples. These results were compared to the results of investigation of deoxynivalenol and fumonisin content, established in our previous work on the same samples. The results obtained were also compared to those of the neighboring countries where the relevant data existed and to the data of previous studies in our country. [Projekat Ministarstva nauke Republike Srbije, br. TR31081]

A reliable and easy method has been developed for the determination of aflatoxins B1 and G2 in maize samples. High performance liquid chromatography coupled with FLD (HPLC-FLD) with photochemical derivatization was used. Mycotoxins were extracted from maize using a QuEChERS-based extraction procedure. The optimized analytical conditions were evaluated in terms of recoveries, reproducibility, LOD, LOQ and linearity for aflatoxin B1 and aflatoxin G1 in maize. Extraction, chromatographic and detection conditions were optimized in order to increase sample sensitivity. The linearity was analyzed in the range of 0.4-20 μg/kg and the correlation coefficients (R2) were higher than 0.99 for aflatoxins B1 and G1. Blank samples were spiked at 1.0, 2.0 and 4.0 μg/kg, and the average recovery for aflatoxin G1 was 96.96±1.72% and for aflatoxin B1 it was 86.80±1.24%. RSDs were lower than 25% for both mycotoxins. LOD for both aflatoxins was 0.5 μg/kg and LOQ was 1.0 μg/kg, respectively.

Participation in interlaboratory comparison and proficiency testing schemes is important for laboratories to control the work quality. In this study, a sample of naturally contaminated maize was analyzed for the content of ochratoxin A (OTA) in three laboratories in Serbia. Participating laboratories used enzymatic immunoaffinity method (ELISA) for the determination of OTA and selection of the ELISA kit was free. Between-laboratory precision was acceptable as evidenced by Cochran’s C test. Moreover, z-scores for all three laboratories were z < ± 2, which is considered acceptable. Used OTA confirmation methods were thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC), with fluorescence detector. The results of different methods were comparable. [Projekat Ministarstva nauke Republike Srbije, br. TR 031071 i br. OI 172042]

The subject of this study is the validation of a high-performance liquid chromatography method for the analysis of amino acids in feed. The contents of amino acids were determined in maize, soybean, soybean meal, as well as in their mixtures enriched with different amounts of methionine, threonine and lysine. The method involves the acid hydrolysis of the sample (6 h at 150ºC), automated derivatisation of amino acids with the aid of o-phthaldialdehyde and 9-fluorenylmethyl chloroformate reagents, separation on the ZORBAX Eclipse-AAA column, and detection using a diode-array detector. The method is characterized by high specificity (the difference between the retention times of the feed samples and standard mixtures are below 1.7 %), wide linear range (from 10 to 1000 nmol cm-3, r2 = 0.9999), high accuracy (recovery 93.3-109.4 %), and the precision of the results (RSD below 4.14 % in case of repeatability and below 4.57 % in the case of intermediate precision). The limit of detection and the limit of quantification are in the range 0.004-1.258 μg cm-3 and 0.011-5.272 μg cm-3, respectively. The results demonstrate that the procedure can be used as a method for the determination of the composition of primary amino acids of feed proteins. [Projekat Ministarstva nauke Republike Srbije, br. 31081]

Fusarium head blight (FHB) is present in all growing regions of small grains and causes significant losses in yield and grain quality. In our environmental conditions, dominant species is Fusarium graminearum Group 2. During 2009/10 there was a significant Fusarium infestation on wheat, barley and triticale. The aim of this study was to examine the contents of deoxynivalenol (DON) in cereal samples taken after 2009/10 harvest season. We analyzed 22 NS varieties of small grains from Rimski Šančevi, including 16 varieties of winter wheat, one facultative wheat variety, four varieties of winter barley and one variety of triticale. Analytical methods based on clean-up by solid-phase extraction (SPE) columns and detection by liquid chromatography were used. Fifteen out the 22 analyzed samples were positive for the presence of DON at a mean level of 0.537 mg/kg. The highest concentration was 1.952 mg/kg. These findings were in correlation with percentage of the Fusarium damaged kernels.

The presence of fumonisin has not been regulated in the legislation of the Republic of Serbia. Therefore, the data on contamination of cereals, especially corn, which is highly susceptible to contamination by this toxin, are not sufficient. This paper presents the results of testing the corn samples collected in the autumn 2009 on the territory of Bačka. Samples were analyzed for the contents of fumonisins and it was determined whether there is a correlation between the moisture content, total number and class of fungi, as well as the content of aflatoxin, ochratoxin and zearalenone. Using enzymatic immunoaffinity method it was discovered that the highest percentage of samples were contaminated with fumonisins, which was probably due to the presence of Fusarium molds as the most abundant ones. The positive samples contained fumonisin in the concentrations from 0.030 to 1.52 mg kg−1. The influence of the climate and moisture content of grain on fungal contamination and mycotoxin production was analyzed in order to investigate the predictability of the presence of mycotoxins.

Deoxynivalenol (DON) is a part of the family of mycotoxins called trichothecenes which are produced by a number of different Fusarium mold species. The presence of DON in 25 wheat and 25 maize samples was examined by Enzyme Linked Immunosorbent Assay (ELISA) and High Performance Liquid Chromatography (HPLC) methods. The presence of DON was detected and determined in 5 (20%) maize and 6 (25%) wheat samples by both of the methods. Correlation between ELISA and HPLC results was established, with the correlation coefficients (r) of 0.9691 and 0.9735 for wheat and maize samples, respectively. The results obtained by ELISA method were significantly higher than those obtained by HPLC method. This fact can be explained by the presence of conjugated or masked mycotoxins in the samples, especially DON-3-glucoside (DON-3-Glc), which could not be determined by HPLC method due to the lack of external standards. Contrary to this, being insufficiently selective towards masked DON, ELISA method measures total DON content of a sample. According to the obtained results, ELISA can be used as a reliable screening method, but the confirmation of positive results must be done by HPLC method.

Reports concerning mycotoxins in horse feed are very rare and are typically restricted to fumonisins. As a non-ruminant monogastric species, horses may be more sensitive to adverse effects of mycotoxins, but the most severe effect of fumonisin B1 (FB1) in equines is that it causes fatal leucoencephalomalacia. In recent years, the European Food Safety Authority (EFSA) has evaluated several mycotoxins as “undesirable substances in animal feed” with the aim of establishing guidance values for the feed industry. In its evaluation of deoxynivalenol (DON), EFSA concluded that this toxin exhibited toxic effects in all species, but that horses were more tolerant towards this toxin than pigs. According to the available data, a systematic survey on mycotoxins in horse feed in Serbia has not been published. Therefore, the aim of this study was to investigate the incidence of mycotoxins in horse feed in Vojvodina. Samples of oats for horse consumption, collected in 2010, were analyzed by enzyme immunoassays (ELISA) for deoxynivalenol contamination. Twelve samples of oats were taken from twelve horse studs, with sport, school and hobby horses.

In the mycological survey of fresh salads of different vegetables, the collected samples were tested for total counts of moulds with special attention paid to the presence of potentially toxigenic species. The survey also included the isolation and the identification of species, as well as the evaluation of mycotoxin biosynthesis ability of potential producers of ochratoxin A (OA) and sterigmatocystin (STC). Mould counts ranged from 10.0 to 4.7x102 cfu g-1. The most common moulds found in fresh salads were Cladosporium (42.89%), Penicillium (25.78%), Aspergillus (14.67%) and Alternaria (6.89%). C. cladosporioides (40.44%), followed by A. niger (10.22%), P. aurantiogriseum (7.55%), A. alternata (6.89%) and Fusarium spp. (3.11%) were the most dominating species. Other species were represented with 2.22% (Eurotium spp.), 1.56% (Botrytis spp.), 0.67% (Phoma spp.), 0.44% (Geotrichum spp., Mucor spp., Phialophora spp.) and 0.22% (Emericella spp., Paecilomyces spp., Trichoderma spp., Xeromyces spp.). Twenty-two of 41 identified mould species were potentially toxigenic, which accounted for 46.18% of the total isolated population. The most frequent were the potential producers of ochratoxin A (17.77%). Potential producers of moniliformin were isolated in 3.11% of samples, while producers of fumonisin and STC were found in 2.67% and 2.44% of samples, respectively. The tested isolates of OA producers did not demonstrate the ability to biosynthetise this mycotoxins, but two out of five isolates of A. versicolor were found to biosynthesise STC in doses of 109.2 ng mL-1 and 56.3 ng mL-1. The obtained results indicate that such products may threaten human health, considering that isolated species were potentially toxigenic, while isolates of A. versicolor also biosynthesised STS.

HACCP (Hazard Analysis and Critical Control Point) is a concept which identifies, evaluates, and controls hazards, significant for food safety. It is applicable to the entire food chain, "from farm to table". Prevention of a food borne mycotoxin contamination of commodities is the most rational and cost-effective method for preventing adverse effects of fungal metabolites on human and animal health. Deoxynivalenol (DON) belongs to the group of mycotoxins produced by certain Fusarium species, which can damage several vital organs, or demonstrate immunotoxic effect, when ingested in small amounts for a longer period of time. Of particular concern is exposure of children to this mycotoxin through cereals, which are believed to lead to reduced weight gain and decreased liver weights. For that reason, we tried to present HACCP concept for preventing wheat contamination with deoxynivalenol. To be able to apply this system, hazards must be identified and risks assessed, and for that purpose a real picture of area (region) in which preventive measures shall be applied, need to be established. According to the results of the study conducted in the laboratory at our department, DON contaminated wheat samples in the region of Vojvodina accounted for 41,6% in 2004 and 2005, whereas amounts of deoxynivalenol ranged from 57 to 1840 μg/kg.

By applying previously established optimal conditions for the determination of deoxynivalenol (DON) by liquid chromatography with DAD detector, in this work, its content was determined in maize samples collected during the past 3 years (2004-2006) from different locations in Vojvodina. Analyzing 103 maize samples in total, the presence of deoxynivalenol was established in 42.7% of the samples. Only 3 samples of maize contained DON in concentrations that exceeded the maximum permitted level (1 μg/g) legislated in most countries.

The aim of this paper was to investigate the potential of zearalenone (ZEN) and type A trichothecenes (T-2 toxin and diacetoxyscirpenole - DAS) for biosynthesis by Fusarium spp. isolated from corn kernels contaminated by mycotoxins. The samples of corn kernels (2004 and 2005 harvest) originating in different regions of Bačka (Vojvodina, Serbia) were tested. Mycotoxicological investigations showed in most cases a significant contamination with deoxynivalenol (DON 200 - 2,460 μg/kg) and ZEA (520 1,680 μg/kg). Isolations and identifications of fusaria established only the presence of species F. verticillioides, after one month storage in freezer conditions, in fusariotoxin positive samples. The control cultures known as ZEA producers - F. graminearum GZ-LES, i.e. T-2 toxin and DAS producer - F. sporotrichioides KF-38/1/R were also tested. In vitro toxicological investigations of isolated fusaria were performed in liquid semisynthetic media (GPK or SPK), and on wet sterilized corn kernels, respectively. Under testing conditions, analyzed F. verticillioide and F. sporotrishioides isolates were not ZEA producers. Contrary to them, F. graminearum GZ-LES pure culture was very good producer of fusariotoxins; it biosynthesized max. 465,900 μg/kg DON, and 4,416 μg/kg ZEA, respectively. Cultivation conditions influenced a great deal of T-2 toxin production under laboratory conditions. In most cases, higher yields were obtained during the cultivation of F. verticillioides in liquid glucose medium (80-240 μg/L). Contrary to the control strain F. sporotrichioides KF-38/1/R that under the same conditions synthesized, besides T-2 toxin (4.000 μg/L) and DAS (240 μg/L), isolates of F. verticillioides from corn grain did not show that ability.

During the year 2006, climatic conditions were favourable for the appearance of head blight in the majority of localities in which wheat was grown in our country. In the locality of Apatin, in certain plots, the amount of detected infection was up to 25 infected heads per m2. During the harvest, heads with distinct disease symptoms and sporulation of Fusarium graminearum fungi were gathered. Grains from the parts of heads with manifested disease symptoms were separated into separate samples, together with the grains above and below the infested head part. Apart from ocular evaluation, the percentage of grain infestation by Fusarium genus fungi was determined in all three sample categories, using wet chamber method. Deoxynivalenol (DON) was determined in the samples after extraction, using acetonitrile-water (84:16 v/v) solution. Quantitative amount of DON was determined using liquid chromatography with DAD detector at 220 nm. The content of DON in the samples was as follows: grains with manifested disease symptoms 353,4 ppm (μg/g), grains above the infested head part 0,225 ppm (μg/g), grains below the infested part 0,125 ppm (μg/g). The content of zearalenone in the samples was determined using thin layer chromatography method. This toxic agent was determined only in the samples taken from the head part in which disease symptoms were clearly manifested in the amount of 2,1 ppm (μg/g).

The determination of deoxynivalenol (DON) in corn by liquid chromatography with DAD detection was optimized. The separations were achieved on a Hypersil ODS column (100 x 4.6 mm; particle size 5 μm) by isocratic elution (0.6 cm3/min), a mobile phase consisting of acetonitrile–water in the ratio of 16:84. UV Detection was performed at 220 nm. Linear calibration curves were constructed in the concentration range of 0.72 – 12.00 ng/μl (equivalent to 0.29 – 4.8 μg/g com). The detection limit measured as the signal-to-noise ratio (3:1) was 0.16 ng/μl for DON (equivalent to 0.06 μg/g corn). The efficiencies of two clean-up principles for crude corn extract were compared solid-phase and immunochemical extraction. The efficiency of solid-phase extraction was found to be higher, with a value of 92.7 % when MycoSep 225 columns were used, while its value was 97.6 % when self-made activated charcoal–alumina–Celite–cationic columns were used. In contrast, the efficiency of the immunochemical columns (IMA) was only 73.8%. It was also found that the self-made columns could be used at least three times in a row in that way differing from the MycoSep 225 columns, which could not be reused either with or without regeneration, as well as from the IMA columns, which had a regeneration efficiency of 53.6 %.

By applying previously established optimal conditions for determination of deoxynivalenol by liquid chromatography with DAD detector, its content was determined in this work in corn samples artificially inoculated with Fusarium graminearum. The samples presented material of different genetic origin, and are frequently used as parent components of corn hybrids in the moderate belt. The obtained values for deoxynivalenol content were not higher than the maximum permitted level (1 g/g), regardless of the fact that the fungus was artificially inoculated. This is probably a consequence of the absence of humid weather, as well as extremely high temperatures during the sampling period (year 2003).

Ochratoxin A is cancerogenic, teratogenetic, immunotoxic and nephrotoxic The mentioned order stresses the importance of this toxin concerning its harm to human health. The harmful effects of ochratoxin A include the effects at molecular level, such as DNA fragmentation, protein synthesis inhibition gluconeogenesis, lipid peroxidation, disorder of oxydative phosphorization in mitochondria, inhibition of blood coagulation and apoptosis. The presence of ochratoxin A in a great number of food samples, both of plant and animal origin, is the obvious risk to human health, which is confirmed by the high incidence of this toxin in samples of human serum and milk. It could be stated, with certainty, that the above - mentioned facts are the reason for which the EU has paid great attention to this mycotoxin in recent years. This paper deals with the results of the analysis of the animal feed component samples for the period 2000-2003 concerning the ochratoxin A content. The analysed feed components were taken from the farms with significant health problems of animals (not monitoring). The samples were analysed by chromatography on a thin layer and with a limited detection method for ochratoxin A of 40 ppb. The analysis was carried out on 108 maize samples, 11 barley samples, 21 wheat samples, 42 sunflower pellets samples and 47 soybean pellets samples (Table 1). The samples of sunflower pellets were contaminated in the greatest percentage, which indicates the inadequate storage of this feed component.

Ensiling is a preservation of moist forage crops based on an anaerobic solid-state fermentation. Different types of mini-silos have been used for laboratory examination of fermentation dynamics in silage. Suitability of these mini-silos is dependent on preventing air from getting into silage, as the presence of air can lead to undesirable microbiological reactions. In this study, whole crop maize was ensiled, with and without bacterial inoculants, in a simple system consisting of polypropylene (PP) containers with special water valve, which prevented air from entering the container while fermentation gases could freely pass out. The containers were opened on the 15th and 50th day and the samples were taken for determination of total mould and yeast count and total bacteria count. After 50 days, the silage was in good condition and there were no significant weight losses. The silage examination system proposed in this study is simple, economical and prevents air penetration into the silage.

Background: In the last few years, Klebsiella pneumoniae strains producing K. pneumoniae carbapenemase (KPC) enzymes have emerged as important multidrug-resistant pathogens in hospitalized patients. This report describes KPC-producing isolates collected through the Croatian antimicrobial resistance surveillance program in the early stage of their dissemination in Croatia., Materials and Methods: Forty-eight KPC-producing K. pneumoniae isolates, collected during a period from February 2011 to August 2013, were analyzed in this study. Antimicrobial susceptibility profiles were determined using disk diffusion and E-test. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. Identification of β-lactamase genes and associated antibiotic resistance mechanisms was performed by polymerase chain reaction and positive products were sequenced. Localization of bla KPC was investigated by S1 PFGE and Southern hybridization., Results: Of 40 participating centers in Croatia, KPC isolates were recorded in 9 of them. They all had multidrug-resistant phenotype, but showed varying levels of resistance to carbapenems. All isolates displayed ST258, and PFGE showed that all but one were closely related. All isolates harbored bla KPC-2 . Isolate with a unique PFGE pattern produced TEM-1, while others produced TEM-116. All isolates harbored bla SHV-11 , but were negative for bla CTX-M and bla AmpC genes. All isolates contain one KPC-harboring plasmid, ranging in size from ∼60 to ∼210 kb, characterized as FIIs and IncR., Conclusion: This report describes that the early stage of KPC-producing K. pneumoniae dissemination in Croatia is associated with a prolific PFGE type belonging to ST258. So far, the spread of an outbreak strain is limited to the northwest region of the country.

Generation of reactive oxygen species (ROS) is one of the earliest responses of plant cells to various biotic and abiotic stresses. ROS are capable of inducing cellular damage by oxidation of proteins, inactivation of enzymes, alterations in the gene expression, and decomposition of biomembranes. On the other hand, they also have a signaling role and changes in production of ROS can act as signals that change the transcription of genes that favor the acclimation of plants to abiotic stresses. Among the ROS, it is believed that H₂O₂ causes the largest changes in the levels of gene expression in plants. A wide range of plant responses has been found to be triggered by H₂O₂ such as acclimation to drought, photooxidative stress, and induction of senescence. Our knowledge on signaling roles of singlet oxygen (¹O₂) has been limited by its short lifetime, but recent experiments with a flu mutant demonstrated that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. In this review we summarize the latest progress on the signaling roles of ROS during senescence and abiotic stresses and we give a short overview of the methods that can be used for their assessment.

Objective: We determined and compared acute effects of different alcoholic beverages on oxygen-induced increase in oxidative stress plasma marker and arterial stiffness in healthy humans., Methods: Ten males randomly consumed one of four tested beverages: red wine (RW), vodka, beer (0.32 g ethanol/kg body wt) and water as control. Every beverage was consumed once, a week apart, in a cross-over design. The volunteers breathed 100% normobaric O(2) between 60th and 90th min of 3h study protocol. Plasma lipid peroxides (LOOH) and uric acid (UA) concentration, blood alcohol concentration (BAC) and arterial stiffness (indicated by augmentation index, AIx) were measured before and 30, 60, 90, 120 and 180 min after beverage consumption., Results: Intake of all alcoholic beverages caused a similar increase of BAC. The oxygen-induced elevation in AIx was similarly reduced in all three groups relative to the control (3.4 ± 1.3%, 5.4 ± 2.2% and 0.2 ± 1.6% vs. 13.7 ± 2.6% for red wine, vodka, beer and control, respectively, 60 min after intake). Exposure to oxygen resulted in increased plasma LOOH in all groups. However, in RW group this increase was lowest (1.1 ± 0.5) in comparison to the vodka (2.1 ± 0.5), beer (1.6±0.3) and control (2.5 ± 0.4μM/L H(2)O(2)). 60 min after intake of RW and beer plasma UA significantly increased (34 ± 4 and 15 ± 3) in contrast to vodka and control (-6 ± 2 and -8 ± 2μmol/L)., Conclusion: All three alcoholic beverages provided similar protection against oxygen-induced increase in arterial stiffness, probably due to central vasodilatatory effect of alcohol itself, but only RW provided protection against oxygen-induced oxidative stress., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: More severe vertebral fractures have more personal impact. In the European Prospective Osteoporosis Study, more severe vertebral collapse was predictable from prior fracture characteristics. Subjects with bi-concave or crush fractures at baseline had a 2-fold increase in incident fracture size and thus increased risk of a disabling future fracture., Introduction: According to Euler's buckling theory, loss of horizontal trabeculae in vertebrae increases the risk of fracture and suggests that the extent of vertebral collapse will be increased in proportion. We tested the hypothesis that the characteristics of a baseline deformity would influence the size of a subsequent deformity., Methods: In 207 subjects participating in the European Prospective Osteoporosis Study who suffered an incident spine fracture in a previously normal vertebra, we estimated loss of volume (fracture size) from plane film images of all vertebral bodies that were classified as having a new fracture. The sum of the three vertebral heights (anterior, mid-body, and posterior) obtained at follow-up was subtracted from the sum of the same measures at baseline. Each of the summed height loss for vertebrae with a McCloskey-Kanis deformity on the second film was expressed as a percentage., Results and Conclusions: In univariate models, the numbers of baseline deformities and the clinical category of the most severe baseline deformity were each significantly associated with the size of the most severe incident fracture and with the cumulated sum of all vertebral height losses. In multivariate modeling, age and the clinical category of the baseline deformity (crush > bi-concave > uni-concave > wedge) were the strongest determinants of both more severe and cumulative height loss. Baseline biconcave and crush fractures were associated at follow-up with new fractures that were approximately twice as large as those seen with other types of deformity or who previously had undeformed spines. In conclusion, the characteristics of a baseline vertebral deformity determines statistically the magnitude of vertebral body volume lost when a subsequent fracture occurs. Because severity of fracture and number of fractures are determinants of impact, the results should improve prediction of the future personal impact of osteoporosis once a baseline prevalent deformity has been identified.

There is important geographic variation in the occurrence of the major osteoporotic fractures across Europe. The aim of this study was to determine whether between-center variation in limb fracture rates across Europe could be explained by variation in the incidence of falls. Men and women, aged 50-79 years, were recruited from population-based registers in 30 European centers. Subjects were followed by postal questionnaire to ascertain the occurrence of incident fractures, and were also asked about the occurrence and number of recent falls. Self-reported fractures were confirmed, where possible, by review of the radiographs, medical record, or subject interview. The age- and gender-adjusted incidence of falls was calculated by center using Poisson regression. Poisson regression was also used to assess the extent to which between-center differences in the incidence of limb fractures could be explained by differences in the age- and gender-adjusted incidence of falls at those centers. In all, 6302 men (mean age 63.9 years) and 6761 women (mean age 63.1 years) completed at least one questionnaire concerning fractures and falls. During a median follow-up time of 3 years, 3647 falls were reported by men and 4783 by women. After adjusting for age and gender, there was evidence of significant between-center differences in the occurrence of falls. There was also between-center variation in the occurrence of upper limb, lower limb, and distal forearm fractures. Variation in the age- and gender-adjusted center-specific fall rates explained 24%, 14%, and 6% of the between-center variation in incidence of distal forearm and upper and lower limb fractures, respectively. Given the constraints inherent in such an analysis, in men and women aged 50-79 years, variation in fall rates could explain a significant proportion of the between-center variation in the incidence of limb fracture across Europe., (Copyright 2002 by Elsevier Science Inc.)

Vertebral fracture is one of the major adverse clinical consequences of osteoporosis; however, there are few data concerning the incidence of vertebral fracture in population samples of men and women. The aim of this study was to determine the incidence of vertebral fracture in European men and women. A total of 14,011 men and women aged 50 years and over were recruited from population-based registers in 29 European centers and had an interviewer-administered questionnaire and lateral spinal radiographs performed. The response rate for participation in the study was approximately 50%. Repeat spinal radiographs were performed a mean of 3.8 years following the baseline film. All films were evaluated morphometrically. The definition of a morphometric fracture was a vertebra in which there was evidence of a 20% (+4 mm) or more reduction in anterior, middle, or posterior vertebral height between films--plus the additional requirement that a vertebra satisfy criteria for a prevalent deformity (using the McCloskey-Kanis method) in the follow-up film. There were 3174 men, mean age 63.1 years, and 3,614 women, mean age 62.2 years, with paired duplicate spinal radiographs (48% of those originally recruited to the baseline survey). The age standardized incidence of morphometric fracture was 10.7/1,000 person years (pyr) in women and 5.7/1,000 pyr in men. The age-standardized incidence of vertebral fracture as assessed qualitatively by the radiologist was broadly similar-12.1/1,000 pyr and 6.8/1,000 pyr, respectively. The incidence increased markedly with age in both men and women. There was some evidence of geographic variation in fracture occurrence; rates were higher in Sweden than elsewhere in Europe. This is the first large population-based study to ascertain the incidence of vertebral fracture in men and women over 50 years of age across Europe. The data confirm the frequent occurrence of the disorder in men as well as in women and the rise in incidence with age.

This paper reports 2 cases of primary hypertrophic osteoarthopathy (PHO) which evolved into secondary hypertrophic osteoarthopathy (SHO) under the influence of bronchogenic carcinoma. The patients had a clinical picture of primary hypertrophic osteoarthropathy but without any signs of disease activity until in the last several months when a malignant bronchopulmonary condition developed. This activated all the symptoms: joint swelling; enlarged fingers and distal forearms and legs; moist palms and soles; unpleasant odour of perspiration; and deeper folds of the forehead and nasolabial furrow. A bone scan showed increased accumulation of the radioisotope in specific regions of the skeleton. To our knowledge, no similar cases have been described in the literature.

Objective: To assess the safety and effectiveness of leflunomide versus placebo in patients with active rheumatoid arthritis (RA) treated for 6 months., Methods: Four hundred two patients were randomly assigned to receive placebo or leflunomide at 5 mg, 10 mg, or 25 mg daily. A washout period of 6-12 weeks from prior second-line therapy was required., Results: Statistically significant improvement in primary and secondary outcome measures, as well as by responder analyses, occurred in the 10-mg and 25-mg dosage groups compared to placebo. Twenty-one patients (7.0%) in the active treatment groups withdrew due to adverse events (AEs). The incidence of AEs was higher with leflunomide than with placebo. Gastrointestinal symptoms, weight loss, allergic reactions, skin rash, and reversible alopecia were more common in the 10-mg and 25-mg dosage groups. The incidence of infections was similar between the treatment and placebo groups; no opportunistic infections were seen. Transient elevations in liver function studies were noted in a small number of patients., Conclusion: Leflunomide is effective in daily doses of 10 mg and 25 mg in patients with active RA. Improved efficacy at the 25-mg dose was associated with a higher incidence of AEs. Randomized, placebo-controlled trials using daily doses of 10 mg and 20 mg are under way in the US and Europe to confirm these positive results.

Pachydermoperiostosis (PDP) is a disease characterized by the presence of pachydermia, periostosis and finger clubbing. Evidence that the skin and soft tissues are involved in the disease prompted the in vitro investigation of the behaviour of fibroblasts obtained from cutaneous biopsies of involved and apparently uninvolved PDP skin. PDP fibroblasts from affected skin demonstrated an abnormal proliferation, very rapid and tumultuous when compared to the growth of fibroblasts derived from apparently uninvolved skin and fibroblasts from the skin of healthy subjects. This characteristic was confirmed by the rate of thymidine incorporation, which was increased in PDP-affected fibroblasts (1152 dpm) compared to apparently non-PDP involved fibroblasts (273 dpm) and controls (262 dpm). Ultracentrifuged and non-centrifuged conditioned medium (CM) of fibroblasts affected or apparently not affected with PDP were used to evaluate the effect on the proliferation of healthy skin fibroblasts, compared to the effect of CM derived from healthy fibroblasts and from healthy fibroblasts incubated with 10% and 1% foetal calf serum. The CM of non-centrifuged PDP fibroblasts resulted in a statistically significant stimulation of fibroblast growth when compared to that expressed by ultracentrifuged PDP CM, healthy fibroblast CM and 10% stimulated CM. These data show that PDP fibroblasts maintain in vitro the capacity to proliferate at a higher rate than healthy fibroblasts and that in the CM residual cells and/or their debris may be present, inducing the abnormal growth of healthy fibroblasts. This evidence suggests that fibroblasts in PDP may play a role in the development of the disease.