Correction of defective CFTR/ENaC function and tightness of cystic fibrosis airway epithelium by amniotic mesenchymal stromal (stem) cells

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with most of the mortality given by the lung disease. Human amniotic mesenchymal stromal (stem) cells (hAMSCs) hold great promise for regenerative medicine in the field of lung disease; however, their potential as therapeutics for CF lung disease has not been fully explored. In the present study, hAMSCs were analysed in co‐cultures on Transwell filters with CF immortalized airway epithelial cells (CFBE41o‐ line) at different ratios to exploit their potency to resume basic defects associated with CF. The results show that F‐actin content was increased in co‐cultures as compared with CF cells and actin was reorganized to form stress fibres. Confocal microscopy studies revealed that co‐cultures had a tendency of increased expression of occludin and ZO‐1 at the intercellular borders, paralleled by a decrease in dextran permeability, suggestive of more organized tight junctions (TJs). Spectrofluorometric analysis of CFTR function demonstrated that hAMSC‐CFBE co‐cultures resumed chloride transport, in line with the appearance of the mature Band C of CFTR protein by Western blotting. Moreover, hAMSC‐CFBE co‐cultures, at a 1:5 ratio, showed a decrease in fluid absorption, as opposed to CFBE cell monolayers that displayed a great rate of fluid resorption from the apical side. Our data show that human amniotic MSCs can be used in co‐culture with CF respiratory epithelial cells to model their engraftment into the airways and have the potential to resume a tight epithelium with partial correction of the CF phenotype.     

Differential expression of TrkB isoforms switches climbing fiber‐Purkinje cell synaptogenesis to selective synapse elimination

Correct neural function depends on precisely organized connectivity, which is refined from broader projections through synaptic/collateral elimination. In the rat, olivocerebellar topography is refined by regression of multiple climbing fiber (CF) innervation of Purkinje cells (PC) during the first two postnatal weeks. The molecules that initiate this regression are not fully understood. We assessed the role of cerebellar neurotrophins by examining tropomycin receptor kinase (Trk) receptor expression in the inferior olive and cerebellum between postnatal days (P)3‐7, when CF‐PC innervation changes from synapse formation to selective synapse elimination, and in a denervation‐reinnervation model when synaptogenesis is delayed. Trks A, B, and C are expressed in olivary neurons; although TrkA was not transported to the cerebellum and TrkC was unchanged during innervation and reinnervation, suggesting that neither receptor is involved in CF‐PC synaptogenesis. In contrast, both total and truncated TrkB (TrkB.T) increased in the olive and cerebellum from P4, whereas full‐length and activated phosphorylated TrkB (phospho‐TrkB) decreased from P4‐5. This reveals less TrkB signaling at the onset of CF regression. This expression pattern was reproduced during CF‐PC reinnervation: in the denervated hemicerebellum phospho‐TrkB decreased as CF terminals degenerated, then increased in parallel with the delayed neosynaptogenesis as new CFs reinnervated the denervated hemicerebellum. In the absence of this signaling, CF reinnervation did not develop. Our data reveal that olivocerebellar TrkB activity parallels CF‐PC synaptic formation and stabilization and is required for neosynaptogenesis. Furthermore, TrkB.T expression rises to reduce TrkB signaling and permit synapse elimination. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Differential expression of TrkB isoforms switches climbing fiber‐Purkinje cell synaptogenesis to selective synapse elimination

Correct neural function depends on precisely organized connectivity, which is refined from broader projections through synaptic/collateral elimination. In the rat, olivocerebellar topography is refined by regression of multiple climbing fiber (CF) innervation of Purkinje cells (PC) during the first two postnatal weeks. The molecules that initiate this regression are not fully understood. We assessed the role of cerebellar neurotrophins by examining tropomycin receptor kinase (Trk) receptor expression in the inferior olive and cerebellum between postnatal days (P)3‐7, when CF‐PC innervation changes from synapse formation to selective synapse elimination, and in a denervation‐reinnervation model when synaptogenesis is delayed. Trks A, B, and C are expressed in olivary neurons; although TrkA was not transported to the cerebellum and TrkC was unchanged during innervation and reinnervation, suggesting that neither receptor is involved in CF‐PC synaptogenesis. In contrast, both total and truncated TrkB (TrkB.T) increased in the olive and cerebellum from P4, whereas full‐length and activated phosphorylated TrkB (phospho‐TrkB) decreased from P4‐5. This reveals less TrkB signaling at the onset of CF regression. This expression pattern was reproduced during CF‐PC reinnervation: in the denervated hemicerebellum phospho‐TrkB decreased as CF terminals degenerated, then increased in parallel with the delayed neosynaptogenesis as new CFs reinnervated the denervated hemicerebellum. In the absence of this signaling, CF reinnervation did not develop. Our data reveal that olivocerebellar TrkB activity parallels CF‐PC synaptic formation and stabilization and is required for neosynaptogenesis. Furthermore, TrkB.T expression rises to reduce TrkB signaling and permit synapse elimination. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

The chitinase-like protein YKL-40 modulates cystic fibrosis lung disease

The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.     

Identification of nuclear structural protein alterations associated with seminomas

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high‐resolution two‐dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR‐related lipid transfer protein 7 (StARD7). In the non‐seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease. J. Cell. Biochem. 108: 1274–1279, 2009. © 2009 Wiley‐Liss, Inc.     

Cystic fibrosis and calcium oxalate nephrolithiasis.

During the past six years, we have treated eight patients with cystic fibrosis (CF) for nephrolithiasis. In seven patients, the stones were comprised of calcium oxalate. Another six patients had calcium oxalate crystalluria. In our CF population of 140 patients, this represents a cumulative incidence of calcium oxalate nephrolithiasis of 5.7 percent and an additional 4.2 percent incidence of crystalluria. Experience with these patients is reviewed. Pancreatic insufficiency was universally associated with nephrolithiasis or crystalluria. Diabetes and cirrhosis were also common. Predisposing factors and potential mechanisms of stone disease in pancreatic insufficient CF patients are discussed, focusing on the relationship between fat malabsorption in CF to oxalate metabolism.     

HLA‐DRB1 and HLA–DQB1 genes on susceptibility to and protection from allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity pulmonary disease that affects both patients with cystic fibrosis (CF) and those with asthma. HLA‐DRB1 alleles have previously been associated with ABPA–CF susceptibility; however, HLA‐DQB1 allele associations have not been clearly established. The aim of the present study was to investigate HLA class II associations in patients with ABPA–CF and determine their roles in susceptibility or protection. Patients with ABPA–CF, patients with CF without ABPA, patients with asthma without ABPA (AST), and healthy controls were included in this study. DNA was extracted by automatic extractor. HLA‐DRB1 and ‐DQB1 genotyping was performed by the Luminex PCR‐SSOP method (One Lambda, Canoga Park, CA, USA). Allele specific PCR‐SSP was also performed by high‐resolution analysis (One Lambda). Statistical analysis was performed with SSPS and Arlequin software. Both HLA‐DRB1*5:01 and ‐DRB1*11:04 alleles occurred with greater frequency in patients with ABPA–CF than in those with AST and CF and control subjects, corroborating previously published data. On the other hand, analysis of haplotypes revealed that almost all patients with ABPA–CF lacking DRB1*15:01 or DRB1*11:04 carry either DRB1*04, DRB1*11:01, or DRB1*07:01 alleles. In the HLA‐DQB1 region, the HLA‐DQB1*06:02 allele occurred more frequently in patients with ABPA–CF than in those with AST and CF and healthy controls, whereas HLA‐DQB1*02:01 occurred less frequently in patients with ABPA–CF. These data confirm that there is a correlation between HLA‐DRB1*15:01, –DRB1*11:04, DRB1*11:01, –DRB1*04 and –DRB1 * 07:01 alleles and ABPA–CF susceptibility and suggest that HLA‐DQB1*02:01 is an ABPA–CF resistance allele.     

Spectrum of mutations in cystic fibrosis

Cystic fibrosis (CF) is a disorder characterized by elevated sweat electrolytes and thick mucous secretions due to abnormal chloride permeability in epithelial tissues. The gene responsible for this disease, the CF transmembrane conductance regulator (CFTR) was identified by a positional cloning approach 3 years ago. Since that time, over two hundred mutations have been found in CFTR genes from affected individuals. Analysis of these disease-associated mutations has provided new insight into the etiology of this disease and into the mechanisms of epithelial electrolyte secretion.     

DNA marker haplotype association with pancreatic sufficiency in cystic fibrosis.

Patients with cystic fibrosis (CF) generally suffer from chronic obstructive lung disease, pancreatic insufficiency (PI), and a number of other exocrine malfunctions. Approximately 15% of CF patients are, however, pancreatic sufficient. To investigate whether the two clinical subgroups, PI and pancreatic sufficiency (PS), are caused by different CF mutant alleles, we have performed linkage disequilibrium and haplotype association analysis with three DNA markers that are tightly linked to the CF locus. The study showed that the allelic and haplotype distributions for these RFLPs are significantly different between the two groups. The data suggest that most of the CF-PI patients are probably descendants of a single mutational event at the CF locus and that the CF-PS patients resulted from multiple, different mutations. While final interpretation of these data awaits molecular cloning of the CF gene, the information on haplotype association in CF may be useful in genetic counseling and disease prognosis, in identifying the gene itself, and in defining the mutations.     

Terminal glycosylation in cystic fibrosis

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.     

Measurements of birth defect prevalence: Which is most useful as a comparator group for pharmaceutical pregnancy registries?

Pharmaceutical pregnancy registries document birth defects and other complications reported in pregnancies exposed to specific medications or diseases. A baseline estimate of birth defect prevalence is necessary for comparison. To identify potential teratogenic signals, the pregnancy registry must have a comparator that most closely matches the exposed population and data collection methodology, which are characteristics that vary among the multiplicity of birth defect surveillance systems. The system that yields the most accurate prevalence data may be different from that most closely matching the pregnancy registry methods. State public health programs have highly accurate and precise statistics, but their populations are broader than those of a pharmaceutical pregnancy registry. Large collaborative databases may have a more useful covered population, but there are secondary problems related to data precision. Health care databases enroll large numbers of patients and have good information about exposures and health problems, but the data can be difficult to access and lack useful detail. Exposure‐related databases are closer in population definition and collection methods, though the presence of different diseases and exposures can be problematic. Internal comparators are likely to be most useful in formal statistical analysis, but added cost and management burden and may require significantly increased registry enrollment. There is no ideal comparator, and this must be taken into account when planning a single‐exposure or single‐disease pregnancy registry. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Prospects for pluripotent stem cell‐derived cardiomyocytes in cardiac cell therapy and as disease models

The derivation of embryonic stem cells (hESC) from human embryos a decade ago started a new era in perspectives for cell therapy as well as understanding human development and disease. More recently, reprogramming of somatic cells to an embryonic stem cell‐like state (induced pluripotent stem cells, iPS) presented a new milestone in this area, making it possible to derive all cells types from any patients bearing specific genetic mutations. With the development of efficient differentiation protocols we are now able to use the derivatives of pluripotent stem cells to study mechanisms of disease and as human models for drug and toxicology testing. In addition derivatives of pluripotent stem cells are now close to be used in clinical practice although for the heart, specific additional challenges have been identified that preclude short‐term application in cell therapy. Here we review techniques presently used to induce differentiation of pluripotent stem cells into cardiomyocytes and the potential these cells have as disease models and for therapy. J. Cell. Biochem. 107: 592–599, 2009. © 2009 Wiley‐Liss, Inc.     

Overexpression of peptidase inhibitor 16 attenuates angiotensin II–induced cardiac fibrosis via regulating HDAC1 of cardiac fibroblasts

Cardiac hypertrophy and fibrosis are the major causes of heart failure due to non‐ischaemia heart disease. To date, no specific therapy exists for cardiac fibrosis due to the largely unknown mechanisms of disease and lack of applicable therapeutic targets. In this study, we aimed to explore the role and associated mechanism of peptidase inhibitor 16 (PI16) in cardiac fibrosis induced by angiotensin II. In cardiac fibroblasts (CFs), overexpressed PI16 significantly inhibited CF proliferation and the levels of fibrosis‐associated proteins. Further analysis of epigenetic changes in CF revealed that overexpressed PI16 decreases the nuclear level of histone deacetylase 1 (HDAC1) after angiotensin II treatment, resulting in increased histone 3 acetylation in K18 and K27 lysine. However, overexpression of HDAC1 by an adenovirus vector in CFs reversed these changes. Echocardiography showed that PI16 transgenic (Tg) mice have smaller left ventricle mass than wild‐type mice. Histological analysis data showed that PI16 Tg mice demonstrated smaller cardiomyocyte size and less collagen deposition than wild‐type mice. The effects of PI16 on HDAC1 and histone 3 were also confirmed in PI16 Tg mice using immunostaining. Generally, PI16 is a HDAC1 regulator specifically in CFs, and PI16 overexpression prevents cardiac hypertrophy and fibrosis by inhibiting stress‐induced CF activation.     

Abnormal regulation of ion channels in cystic fibrosis epithelia.

Cystic fibrosis (CF), the most common lethal genetic disease in Caucasians, is characterized by defective electrolyte transport in several epithelia. In sweat duct, pancreatic, intestinal, and airway epithelia, abnormalities in transepithelial ion transport may account for the manifestations of the disease. A Cl- impermeable apical cell membrane is a common feature in these CF epithelia. The rate of transepithelial Cl- transport is controlled in part by hormonally regulated apical membrane Cl- channels; in CF epithelia, Cl- channels are present but their regulation is defective. Most regulation studies have focused on an outwardly rectifying Cl- channel, although other channels may be involved in Cl- secretion. Phosphorylation of Cl- channels or associated regulatory proteins by cAMP-dependent protein kinase or by protein kinase C (at a low internal [Ca2+]) in excised patches of membrane activates Cl- channels in normal cells but not in CF cells. Phosphorylation with protein kinase C at a high internal [Ca2+] in excised patches of membrane inactivates the channel; such inactivation is normal in CF cells. Cl- channels can also be activated by other maneuvers including an increase in the cytosolic [Ca2+], sustained membrane depolarization, an increase in temperature, proteolysis, and changes in osmolarity; the response to such maneuvers is not defective in CF. In addition to the Cl- channel abnormalities, Na+ absorption is increased in CF epithelia. It is not certain whether the increased rate of Na+ absorption results from an increase in the number of cation channels or an alteration of their kinetics. The relation of these ion channel abnormalities to the CF gene product is unknown, but an understanding of the function of the protein product and its defective function in CF should yield important new insights into the pathogenesis and potential therapy of this disease.     

Greenhouse Gas Emissions Quantification and Reduction Efforts in a Rural Municipality

The present article aims to determine the current carbon footprint (CF) of Zernez, a Swiss mountain village, and to identify reduction potentials of greenhouse gas (GHG) emissions. For this purpose, material and energy flows were assessed mainly based on detailed household surveys, interviews, and energy bills, but also by means of other information sources, for example, national statistics, traffic censuses, and literature values. To set up the GHG balance, special attention was paid to the consistent definition of system boundaries by adopting two fundamentally different perspectives: purely geographical accounting (PGA) and the consumption‐based footprint (CBF) method. Each of these two perspectives total approximately 10 tonnes of carbon dioxide equivalents per capita per year. The PGA revealed that 70% of the direct emissions in Zernez are caused by agricultural activities, whereas no consumption area dominated the consumption‐induced CF. For the identification of targeted measures, both perspectives were considered in a complementary manner. The building stock and its underlying energy supply system showed a GHG reduction potential of 80%. The building sector was thus detected as a reasonable first step for the municipality to adopt GHG mitigation strategies. In the case of Zernez, building‐stock‐related measures are predicted to decrease the current CF by 13% (CBF) and 17% (PGA), respectively.     

Expression of the cystic fibrosis gene in human development.

The specialised epithelia lining the respiratory tract, pancreatic ducts, male genital ducts and sweat gland ducts are defective in the severe inherited disease, cystic fibrosis (CF). We have looked at the expression of the CF gene in human fetal tissues to throw light on the development of function in specialised ductal epithelia and to determine the age of onset of the CF disease process. The CF gene is already seen to be transcribed in mid-trimester fetal lung, pancreas and male genital ducts. Hence, by this developmental stage, and before they are fully differentiated, these epithelia have the capability to perform important transport functions. Epithelial cell cultures derived from fetal pancreas and male genital ducts maintain expression of the CF gene in vitro and so form good models for analysing CF gene function and differentiation of these specialised epithelia.     

Implementing Carbon‐Footprint‐Based Calculation Tools in Municipal Greenhouse Gas Inventories

Policy makers and practitioners working on local climate action continuously strive for improved greenhouse gas (GHG) inventories to support their mitigation strategies. The provision of such inventories, with the sufficient detail and quality needed, has proven to be challenging. This article describes the application of a carbon‐footprint–based (CF‐based) calculation tool to Norwegian municipalities. The aim is to improve the local GHG inventory, with a particular focus on the emissions resulting from the provision of services by local governments. Through selective CF analysis, we aim to illustrate the knowledge gained from implementing these tools in local climate action plans and to further contrast CF‐based calculations with more traditional production‐based accounting. Municipalities report they have revised their understanding of the most important GHG mitigation strategies after implementing this tool.     

A comprehensive review of cystic fibrosis in Africa and Asia

Cystic fibrosis (CF) was earlier thought to be a disease prevalent in the West among Caucasians. However, quite a number of recent studies have uncovered CF cases outside of this region, and reported hundreds of unique and novel variant forms of CFTR. Here, we discuss the evidence of CF in parts of the world earlier considered to be rare; Africa, and Asia. This review also highlighted the CFTR mutation variations and new mutations discovered in these regions. This discovery implies that the CF data from these regions were earlier underestimated. The inadequate awareness of the disease in these regions might have contributed towards the poor diagnostic facilities, under-diagnosis or/and under-reporting, and the lack of CF associated health policies. Overall, these regions have a high rate of infant, childhood and early adulthood mortality due to CF. Therefore, there is a need for a thorough investigation of CF prevalence and to identify unique and novel variant mutations within these regions in order to formulate intervention plans, create awareness, develop mutation specific screening kits and therapies to keep CF mortality at bay.     

Biomarkers for prostate cancer

The detection of prostate cancer using a blood test has by many standards changed the face of the disease. Despite this tremendous success, there are limitations attributed to the use of prostate specific antigen (PSA) as a means to screen and detect prostate cancer. PSA, as its name implies, is not specific for prostate cancer and as such is often found elevated in other prostatic diseases/symptoms associated with the aging male. Clearly, more specific marker(s) that could identify which individuals actually have prostate cancer and differentiate them from those without the disease would be of tremendous value. The search for more accurate and clinically useful biomarkers of prostate cancer has been extensive. This has focused on individual markers, as well as groups of markers. Included among these are PSA isoforms, pathological indicators and stains, nucleic acids and others. This article highlights the discovery of PSA as a first blood‐based biomarker for prostate cancer detection, as well as other molecular biomarkers and their potential application in detection of the disease. J. Cell. Biochem. 108: 3–9, 2009. © 2009 Wiley‐Liss, Inc.     

Different mechanisms of Trichoderma virens‐mediated resistance in tomato against Fusarium wilt involve the jasmonic and salicylic acid pathways

In the present study, we investigated the role of Trichoderma virens (TriV_JSB100) spores or cell‐free culture filtrate in the regulation of growth and activation of the defence responses of tomato (Solanum lycopersicum) plants against Fusarium oxysporum f. sp. lycopersici by the development of a biocontrol–plant–pathogen interaction system. Two‐week‐old tomato seedlings primed with TriV_JSB100 spores cultured on barley grains (BGS) or with cell‐free culture filtrate (CF) were inoculated with Fusarium pathogen under glasshouse conditions; this resulted in significantly lower disease incidence in tomato Oogata‐Fukuju plants treated with BGS than in those treated with CF. To dissect the pathways associated with this response, jasmonic acid (JA) and salicylic acid (SA) signalling in BGS‐ and CF‐induced resistance was evaluated using JA‐ and SA‐impaired tomato lines. We observed that JA‐deficient mutant def1 plants were susceptible to Fusarium pathogen when they were treated with BGS. However, wild‐type (WT) BGS‐treated tomato plants showed a higher JA level and significantly lower disease incidence. SA‐deficient mutant NahG plants treated with CF were also found to be susceptible to Fusarium pathogen and displayed low SA levels, whereas WT CF‐treated tomato plants exhibited moderately lower disease levels and substantially higher SA levels. Expression of the JA‐responsive defensin gene PDF1 was induced in WT tomato plants treated with BGS, whereas the SA‐inducible pathogenesis‐related protein 1 acidic (PR1a) gene was up‐regulated in WT tomato plants treated with CF. These results suggest that TriV_JSB100 BGS and CF differentially induce JA and SA signalling cascades for the elicitation of Fusarium oxysporum resistance in tomato.