CSF Proteomics of Patients with Hydrocephalus and Subarachnoid Haemorrhage.

BACKGROUND: The pathophysiology of brain injury following aneurysmal subarachnoid haemorrhage (SAH) is associated with numerous mediators. The aim of the study is to analyse protein changes after SAH in cerebrospinal fluid (CSF) using mass spectrometry (MS). METHODS: CSF samples were obtained from forty-four control subjects, seven good outcome and ten poor outcome SAH patients. CSF samples were collected at specific time intervals after SAH (days 1, 5 and 10). MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time-of-Flight) and ClinProTools software were utilised for MS, MS/MS (Mass Spectrometry) spectra collection and analysis. Selected masses were identified. The MALDI-TOF profiling experiments allowed for the targeted selection of potential markers in SAH. The study was performed in three steps by comparison of CSF samples: (1) from the control group and SAH patients (both good and poor outcome groups); (2) collected on days 1, 5 and 10 within the groups of poor SAH and good SAH patients, respectively; (3) from poor outcome SAH and good outcome patients at days 1, 5 and 10. RESULTS: 15 new proteins whose CSF level is alternated by SAH presence, SAH treatment outcome and time passed since aneurysm rupture were identified. CONCLUSIONS: We demonstrated new proteins which might play a role in different stages of subarachnoid haemorrhage and could be a new target for further investigation.

Serum free amino acid levels in rheumatoid arthritis according to therapy and physical disability.

BACKGROUND: In presented study the amino acid analysis was performed in serum derived from rheumatoid arthritis patients (RA) according to undertaken therapy and classification of physical disability. The results were compared with previously published data. METHODS: The levels of 31 free amino acids were determined in 50 serum samples derived from RA subjects and 51 controls. The RA patients were divided into two groups according to the therapy (methotrexate/leflunomide, infliximab/adalimumab/etanercept/tocilizumab, prednisolone/NSAID) and classification of physical disability of the patients. Levels of amino acids were measured by LC-MS/MS. The obtained results were subjected to multivariate statistical tests. RESULTS: According to the therapy that was being used, threonine differentiated RA patients treated with methotrexate/leflunomide - infliximab/adalimumab/etanercept/tocilizumab (p = 0.00954) and infliximab/adalimumab/etanercept/tocilizumab - prednisolone/NSAID (p = 0.03109), while tryptophan differentiated RA patients treated with methotrexate/leflunomide - infliximab/adalimumab/etanercept/tocilizumab (p = 0.01723). In the functional classification, arginine differentiated RA samples between class III and IV (p = 0.02332), while glycine differentiated them between class I+II and III of the Steinbrocker functional classification (p = 0.03366). CONCLUSIONS: An analysis of the metabolome profile requires the use of validated bioanalytical methods that are strictly dedicated for this purpose. The obtained results are not accidental (p value less than 0.05), and all of the selected amino acids play an important role in inflammation and immune response. It is suggested that studied amino acids can be considered as a markers for diagnosis of RA and monitoring pharmacotherapy of the disease.

Corrigendum: Amino Acids in Cerebrospinal Fluid of Patients With Aneurysmal Subarachnoid Haemorrhage: An Observational Study.

[This corrects the article on p. 438 in vol. 8, PMID: 28894433.].

Proteomic pattern of cervico-vaginal fluid (CVF) in an ovarian cancer diagnosis - pilot study.

OBJECTIVES: High grade serous ovarian cancer (HGSC) is the most common type of ovarian cancer and is responsible for about 90% of ovarian cancer deaths. The diagnostic tests currently used do not increase the detection rates for ovarian cancer. There is a great necessity to develop new and non-invasive diagnostic tests for ovarian cancer (OC). Cervico-vaginal fluid (CVF) seems to be a potential and valuable source of biomarkers for genital tract diseases including ovarian cancer. The aim of our pilot study was to undertake a preliminary proteomic analysis of CVF derived from ovarian cancer patients and to compare these with results from a control group. MATERIAL AND METHODS: We analysed and compared samples from a group of ovarian cancer patients and a control group of healthy patients. The study used MALDI-TOF coupled with nanoLC and ClinProTools software for MS, MS/MS spectra collection and proteomic analysis. RESULTS: We identified 404 different proteins in the OC group and 417 proteins in the control group. 239 of the proteins were found to be common to both study groups, 165 proteins were unique to the OC subjects, and 178 proteins were unique to the control subjects. We selected three proteins as the OC markers with the greatest potential: cysteine-rich secretory protein 3, fibronectin and Ly6/PLAUR domain-containing protein 3. CONCLUSIONS: The proteins we selected seem to possess great potential as markers for the screening and early detection of OC, especially in non-invasive and low-cost diagnostic tests. However, our findings require more advanced and validated proteomic analysis to confirm the suitability of the selected proteins in everyday medical diagnoses.

Amino Acids in Cerebrospinal Fluid of Patients with Aneurysmal Subarachnoid Haemorrhage: An Observational Study.

BACKGROUND: The authors are aware of only one article investigating amino acid concentrations in cerebrospinal fluid (CSF) in patients with ruptured intracranial aneurysms, and this was published 31 years ago. Since then, both management of subarachnoid haemorrhage (SAH) and amino acid assay techniques have seen radical alterations, yet the pathophysiology of SAH remains unclear. OBJECTIVE: To analyse the pattern of concentrations of amino acids and related compounds in patients with different outcomes following aneurysmal SAH. METHODS: 49 CSF samples were collected from 23 patients on days 0-3, 5, and 10 post-SAH. Concentrations of 33 amino acids and related compounds were assayed by liquid chromatography tandem mass spectrometry in patients with good [Glasgow Outcome Scale (GOS) 1-3] and poor (GOS 4-5) outcome. RESULTS: Of the 33 compounds assayed, only hydroxyproline and 3-aminoisobutyric acid appeared not to increase significantly following SAH. In poor outcome patients, we found significantly higher concentrations of aspartic acid (p = 0.038), glutamic acid (p = 0.038), and seven other compounds on days 0-3 post-SAH; glutamic acid (p = 0.041) on day 5 post-SAH, and 2-aminoadipic acid (p = 0.033) on day 10 post-SAH. The most significant correlation with GOS at 3 months was found for aminoadipic acid on day 10 post-SAH (cc = -0.81). CONCLUSION: Aneurysmal rupture leads to a generalised increase of amino acids and related compounds in CSF. The patterns differ between good and poor outcome cases. Increased excitatory amino acids are strongly indicative of poor outcome.

The feature selection approach for evaluation of potential rheumatoid arthritis markers using MALDI-TOF datasets.

BACKGROUND: The selection of the most representative mass profiles, in rheumatoid arthritis (RA) serum samples was developed. This allows for selection and identification of potential biomarkers in RA serum samples. METHODS: The RA and controls samples were analyzed using MALDI-TOF. Two different protein elution procedures utilizing ZipTips (E1 and E2) were examined. The statistical evaluation of data was performed using different feature selection (FS) methods in combination with different classifiers, while identification of selected masses was performed using MALDI-TOF-TOF. RESULTS: Utilization of proposed statistical strategy allowed for the selection of different masses according to FS method and elution procedure. Obtained masses were further subjected for targeted identification. The panel of proteins were identified as potential markers. The role of these proteins was discussed in relation to pathomechanism of RA. CONCLUSION: Application of advanced biostatistical analysis of obtained MALDI-TOF datasets, resulted with targeted selection of potential RA biomarkers. Five proteins were identified due the E1 procedure, and six proteins were identified due the E2 procedure, respectively. The panel of identified proteins suggest that presented statistical methodology and proteomic strategy was correct and gave valid results. Obtained results may contribute to development of clinically useful multicomponent diagnostic tool.

STUDY OF OFLOXACIN ENANTIOMERS DISSOLUTION FROM SELECTED SOLID DOSAGE FORMS USING HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS METHOD.

The commercially available coated tablets containing either racemic form of ofloxacin (Tarivid 200 mg, OfloHexal 200 mg and Ofloxacin-Ratiopharm 200 mg) or only levofloxacin S-(-)-isomer (Tavanic 250 mg) were examined. The aim of our study was to establish the kinetics of dissolution rate process of ofloxacin optical isomers (S-(-) and R-(+)-ofloxacin) from solid oral dosage forms using flow-through cell method (USP 4 method). The concentrations of analytes (racemic ofloxacin and its enantiomers) in the samples of tablet extracts as well as in dissolution media (0.1 M/L HCl and phosphate buffer pH 6.8) were determined by validated high performance capillary electrophoresis method. The fraction of the average dose of the individual optical isomers of ofloxacin released from the examined tablets was calculated. In the case of the OfloHexal, Ofloxacin-Ratiopharm and Tavanic it was found to be around 100% for both S-(-) and R-(+)-ofloxacin in 0.1 M/L HCI after 30 min of dissolution test. The fraction of the average dose for the Tarivid tablets was approximately 50% at the same time. A similar results were observed for the Ofloxacin-Ratiopharm and Tavanic tablets examined in phosphate buffer (average fraction about 100% after 30 min), while in the case of Tarivid and OfloHexal the averige fraction of the dose determined in a buffer pH 6.8 was 14% and 44%, respectively. There were not found any differences in the kinetics of dissolution of the S-(-)-ofloxacin and R-(+)-ofloxacin isomers within the same formulation. However, statistically significant differences were found in the dissolution of ofloxacin enantiomers between different preparations.

PRELIMINARY HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS (HPCE) STUDIES OF ENZYMATIC DEGRADATION OF HYALURONIC ACID BY HYALURONIDASE IN THE PRESENCE OF POLYVALENT METAL IONS.

The aim of this study was, at first, to examine the influence of metal ions on digestion process of hyaluronic acid by hyaluronidase (HAse) using high performance capillary electrophoresis (HPCE) method. The influence of copper(H), zinc(Il), manganese(II) ions on enzymatic degradation of HA by hyaluronidase enzyme (HA-se) were investigated. Secondly, the kinetic parameters, V(max), K(m), k(cat), and k (cat),/K(m) were determined to estimate the impact of these metal ions (Me) on digestion process of hyaluronic acid (HA). The two different HA-Me mole ratios were analyzed. The examined data were always compared to the digestion process of pure HA solution by hyaluronidase, to exhibit the differences in the digestion process of pure hyaluronan as well as the hyaluronan in the presence of metal ions. It was observed that all of the investigated metal ions have influenced the hyaluronic acid degradation process. The most important conclusion was a decrease of the kinetic parameters both K,, and V,. In the result, it can be assumed that in all of the studied samples with metal ions addition, the uncompetitive mechanism of enzyme inhibition occurred. The results of this study may give new insight into foregoing knowledge about hyaluronic acid behavior. Due to the fact that our study was carried out only for three different metal ions in two concentrations, it is necessary to continue further research comprising wider range of metal ions and their concentrations.

Activation of Prodrug Treosulfan at pH 7.4 and 37°C Accompanied by Hydrolysis of Its Active Epoxides: Kinetic Studies with Clinical Relevance.

Treosulfan (TREO), originally registered for treatment of ovarian cancer, is currently being investigated for conditioning prior to hematopoietic stem cell transplantation. TREO is a prodrug, which undergoes a pH- and temperature-dependent two-step conversion to active monoepoxide [S,S-EBDM, (2S,3S)-1,2-epoxybutane-3,4-diol-4-methanesulfonate] and diepoxide [S,S-DEB, (2S,3S)-1,2:3,4-diepoxybutane]. In this paper, the kinetics of the nonenzymatic transformation of TREO at pH 7.4 and 37°C were studied for the first time including the effects of the TREO concentration, buffer concentration, ionic strength, and the presence of NaCl. Transformation of TREO was well described by a kinetic model, which included first-order reactions for TREO activation, that is, TREO → S,S-EBDM → S,S-DEB, and pseudo-first-order reactions for the hydrolytic decomposition of S,S-EBDM and S,S-DEB. In contrast to the two-step activation of TREO, the hydrolysis of epoxides was influenced by electrolytes. In phosphate-buffered saline, decomposition of S,S-EBDM and S,S-DEB (mean half-lives 25.7 and 15.4 h) proceeded much slower than their formation (mean half-lives 1.5 and 3.5 h). In conclusion, the kinetics of the nonenzymatic transformation of TREO in the presence of plasma electrolytes cannot contribute to the very low levels of S,S-EBDM and S,S-DEB observed in patient plasma. The results also indicate that elimination of TREO proceeds primarily via conversion to S,S-EBDM.

The pharmacokinetics of the effervescent vs. conventional tramadol/paracetamol fixed-dose combination tablet in patients after total gastric resection.

BACKGROUND: Tramadol/paracetamol is a fixed-dose combination prescribed for the relief of moderate to severe pain. The combination acts synergistically and guarantees the rapid onset of paracetamol and the prolonged analgesic effect of tramadol with good tolerability. These drugs are often used in various formulations in the treatment of patients with postoperative pain, e.g. after stomach resection. Gastrectomy leads to pathophysiological changes within the alimentary tract, which may affect the process of drug absorption. The aim of the research was an analysis of the pharmacokinetics of tramadol/paracetamol from effervescent and conventional tablets in patients after total gastrectomy. METHODS: The research was carried out on patients after gastrectomy with Roux-en-Y reconstruction. The patients received two tramadol/paracetamol fixed-dose combination tablets in a single orally administered dose of 75/650 mg (2 × 37.5/325 mg). The patients were subjected to one of the two study drug group with: I. effervescent tablet (ET) (n = 14; mean [SD] age, 63.4 [10.1] years; weight, 75.5 [15.3]kg; and BMI, 26.0 [4.6]kg/m(2)) and II. conventional tablet (CT) (n = 12; mean [SD] age, 66.8 [7.7] years; weight, 79.8 [17.8]kg; and BMI, 27.4 [5.3]kg/m(2)). Blood samples were collected within 10 h after the drug administration. The plasma concentrations of tramadol and paracetamol were measured with validated HPLC (high-performance liquid chromatography) method with UV detection. RESULTS: The comparison of the paracetamol and tramadol C(max) ratio for the ET group with that of the CT group gave ratios of 1.16 [90% confidence interval (CI) 1.06, 1.27] and 0.86 (90% CI 0.72, 1.02), respectively. The comparison of the paracetamol and tramadol AUC(0-t) ratio for the ET group with that of the CT group showed ratios of 0.99 (90% CI 0.88, 1.10) and 1.00 (90% CI 0.82, 1.22), respectively. The comparison of the difference for the effervescent and conventional formulation gave an estimated decrease in t(max) of 0.5 h for paracetamol and 0.13 h for tramadol. CONCLUSIONS: In view of the changes in the pharmacokinetics of paracetamol and tramadol in the patients after gastric resection for both formulations compared the conventional tablet seems to be more appropriate due to the comparable rate of absorption of both substances, higher concentrations of tramadol and comparable exposure to paracetamol.

Spectroscopic investigations of fluoroquinolones metal ion complexes.

The complex formation reaction, between fluoroquinolones (FQ): ciprofloxacin (CPX), enoxacin (ENX), enrofloxacin (ENRX), lomefloxacin (LOMX), levofloxacin (LEVX), ofloxacin (OFX), norfloxacin (NFX), sparfloxacin (SPRX) and aluminum(III), iron(III), copper(II) and zinc(II) ions were investigated. The spectrophotometic titration method in a wide range of pH was utilized for estimation of complex formation equilibrium. The application of Bjerrum method allowed to estimate the complex equilibrium of analyzed species in the reaction mixture. The overall stability constants (logbeta(pqr)) of fluoroquinolones complexes with metal ions were calculated using HYPRERQUAD program. The most stable complexes FQ were created with iron(III) and aluminum(III) and than copper(II) and zinc(II) ions, respectively. The highest values of calculated logbeta(pqr) were obtained for the Me(FQ)3H3 species and the lowest for the Me(FQ)2OH forms. Furthermore, an additional studies have been performed. The effect of the polyvalent metal ions on the complex structure has been investigated. The IR and 1H, 13C and 19F NMR spectroscopy methods were used for the confirmation of the structure of the FQ-Me complex formations. The most significant shifts of signals of 1H NMR spectra of the fluoroquinolones and their complexes were found for the protons substituted in the positions 2, 5 and 8, whereas the 13C NMR spectra showed up the shifts changes for carbon atoms in positions 2, 3, 3a and 4.

A new method for determination of hyaluronidase activity in biological samples using capillary zone electrophoresis.

The aim of the study was to develop a new capillary zone electrophoresis (CZE) method for determination of enzymatic activity of hyaluronidase. The method permits monitoring of the process of hyaluronic acid digestion by hyaluronidase. Studies were performed using CZE instrument equipped with capillary of 64.5 cm total length, 56 cm effective length and internal diameter 75 µm. Separation was performed in the phosphate buffer (pH 8.10) in the electric field of 20 kV, λ = 220 nm. The procedure was based on mixing a known quantity of hyaluronic acid and an aliquot of hyaluronidase solution, followed by obtaining CZE profiles after a known period of incubation (0.5 h). The activity of hyaluronidase was calculated using multiple regression analysis in which sizes of the peaks of the main degradation products were used. The newly developed method was fully validated and it is appropriate to evaluate the activity of hyaluronidase originating from different sources with high precision and accuracy. t-Tests showed that there were no significant differences between results obtained using turbidimetric, viscosimetric and the new CZE method. The developed method is characterized by a short duration of analysis, low volume of analyzed sample, small amount of buffers used and low cost of analysis.

New CZE-DAD method for honeybee venom analysis and standardization of the product.

The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 µm ID, and 360 µm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 µA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002-2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A(2) from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A(2), and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product.

Comparison of the pharmacokinetics of paracetamol from two generic products in patients after total gastric resection.

Gastrectomy leads to pathophysiological changes within the alimentary tract, which may affect drug absorption and pharmacokinetic parameters (PK). The need to apply orally administered analgesics in this group of patients is often related with alternative application of currently available generic products. Thus, from the clinical point of view it is necessary to evaluate the PK of these drugs to confirm their equivalence. The aim of the study was therefore an analysis of the pharmacokinetics of paracetamol from two generic products in patients after total gastric resection. The research was carried out on two groups of patients after gastrectomy with Roux-en-Y reconstruction (n = 30; mean [SD] age, 63.0 [11.5] years; weight, 67.6 [13.7] kg; and height, 166.4 [9.1] cm). The patients received paracetamol in a single orally administered dose of 1,000 mg. Blood samples were collected within 6 h of drug administration. The concentration of paracetamol and paracetamol glucuronide in the blood plasma was marked by means of a validated high-pressure liquid chromatography with ultraviolet detection (HPLC-UV). The main PK for paracetamol in group 1 (n = 17) and 2 (n = 13) were as follows: C(max), 9.46 (3.66) and 12.79 (5.32) µg/ml, respectively (p = 0.0517); AUMC(0-t), 77.64 (30.37) and 51.01 (15.76) µg h(2)/ml (p = 0.0046); AUC(0-inf), 41.61 (23.52) and 30.28 (9.74) µg h/ml (p = 0.0862); t(max), 1.68 (0.63) and 0.50 (0.25) h (p < 0001). The obtained C(max) and AUC values in patients after gastrectomy were reduced in comparison with healthy subjects. Total gastrectomy therefore affected the pharmacokinetics of paracetamol administered in tablets. In our patients, we also observed significant differences between the PK of paracetamol and two generic preparations. These two drugs can thus be used interchangeably, but with caution.

Analysis of the factors that significantly influence the stability of fluoroquinolone-metal complexes.

The aim of the present study was to evaluate factors contributing to the differences between the overall stability constants (logbeta(pqr)) of the fluoroquinolone-metal ion complexes. The experiments were performed using potentiometric titration method in wide pH range. The overall stability constants (logbeta(pqr)) were determined using the Hyperquad program. Complexation equilibria of eight different fluoroquinolones with six divalent and trivalent metal ions were investigated in this study. The authors employed a multifactorial ANOVA analysis, fixed effect model to describe the influence of particular variables affecting the stability of the analyzed complex species. Four different variables were set at different levels labeled. The ligand number (LF) was the first factor. LF determined the number of fluorochinolone molecules in the complex structure, and could take the values 1, 2 or 3. The second factor (Me) was connected with the type of the metal ion bonded in the complex. Since six different metal cations were studied, the Me factor was described with six levels. The number of hydrogen or hydroxide groups substituted into the complex molecule was the third variable (HR) with many levels labeled: q, a, s, d, f and g. The last factor FQ described the type of the fluorochinolone used for complex formations. All variables analyzed here were statistically significant (p value lower than 0.01), which indicates that all of them strongly affect the logbeta(pqr) value. Binary interactions (LF-Me, LF-FQ, Me-HR and Me-FQ) between variables were also stated, which suggests that the effects of these variables were higher than we could calculate based on the effect of each variable alone. The ANOVA analysis has shown that the following factors Me, LF and HR were the most important for the stability of the fluoroquinolone-metal ion complexes. It was also found that according to the FQ factor (type of ligand molecule) all analyzed fluoroquinolones formed stable complexes with metals. It was proved that the application of ANOVA for the entire complexation profile of analyzed fluoroquinolones with polyvalent metal ions was a valid technique for detecting the statistically significant differences in the complexation profiles. Such information may be very useful for better understanding and interpretation of differences in bioavailability of fluoroquinolones and their interactions with antacids and other multimineral drugs.

Flavonoid profiling among wild type and related GM wheat varieties.

Pleiotropic effects are one of the main concerns regarding genetically modified organisms (GMOs). This includes unintended side effects of the transgene or its genome insertion site on the regulation of other endogenous genes, which could potentially cause the accumulation of different secondary metabolites that may have not only an impact on diet as repeatedly worried by the public but also on the environment. Regarding amount and possible environmental effects, flavonoids represent the most prominent group of secondary metabolites in wheat. Many flavonoids function as signalling or defence molecules. We used a robust and reproducible analytical method to compare the flavonoid content of genetically modified (GM) wheat (Triticum aestivum L., Gramineae) expressing genes that confer increased fungal resistance with their non-GM siblings. The transgenes provide either a broad-spectrum fungal defence (chitinase/glucanase from barley) or bunt-specific resistance by a viral gene (KP4). Significant differences in flavonoid composition were found between different wheat varieties whereas different lines of GM wheat with increased antifungal resistance showed only minor differences in their flavonoid composition relative to their non-GM siblings. In a field test, no significant differences were detectable between infected and non-infected wheat of the same variety regardless of the presence of the transgene. Our results are in agreement with the hypothesis that the transgenes we used to increase wheat defence to fungal pathogens do not interfere with the flavonoid biosynthesis pathway. More significantly, the genetic background resulting from conventional breeding has a direct impact on the biological composition of flavonoids, and thus possibly on the environment.

Exploration and Swiss field-testing of a viral gene for specific quantitative resistance against smuts and bunts in wheat.

The viral gene for the killer protein 4 (KP4) has been explored for its antifungal effect in genetically modified wheat to defeat specifically the seed-transmitted smut and bunt diseases. In vitro both important seed-transmitted diseases of wheat, loose smut (Ustilago tritici) and stinking smut (Tilletia caries), are susceptible to KP4, whereas all other organisms tested so far proved to be not susceptible to KP4. For studies in planta we used stinking smut as a model fungus. In greenhouse experiments, two KP4-transgenic wheat lines showed up to 30% lower symptom development as compared to the nontransgenic control. As the last step in the proof of concept, field-testing has shown for the first time increased fungal resistance of a transgene in wheat. Due to its specificity against smuts and bunts, KP4 presents a very low risk to humans and the environment. Field-testing in Switzerland is regulated by a strong law, which for research is acceptable if legally and scientifically correctly applied.

Increased field resistance to Tilletia caries provided by a specific antifungal virus gene in genetically engineered wheat.

The field performance of a viral gene in two Swiss wheat (Triticum aestivum) varieties showed 10% increased fungal resistance against Tilletia caries (stinking smut). To the best of our knowledge, this is the first report of improved resistance against any fungus in the field achieved by genetic engineering in wheat. The genetically modified wheat lines previously showed a c. 30% decrease in symptoms of T. caries in the glasshouse (Clausen, M., Kräuter, R., Schachermayr, G., Potrykus, I. and Sautter, C. (2000) Antifungal activity of a virally encoded gene in transgenic wheat. Nat. Biotechnol. 18, 446-449), depending on the fungal strain inoculated. A glasshouse experiment run in parallel to the field test, and using the same collection of T. caries, gave the same results. In a dose-response experiment with isolated fungal strains, in which the infection pressure was varied via the spore concentration, the transgene behaved as a quantitative resistance gene and shifted the S-shaped dose-response curve towards higher resistance. The transgene was shown to be highly specific for fungi of the order Ustilaginales. Tests of the transgene using cell cultures of eukaryotes, including hamster and human, showed no significant side-effects with respect to biosafety. Endogenous pathogen-related genes were also activated on fungal infection in the presence of the kp4 transgene.