An Alternative Bioassay for Synchytrium endobioticum Demonstrates the Expression of Potato Wart Resistance in Aboveground Plant Parts.

The obligate biotrophic chytrid species Synchytrium endobioticum is the causal agent of potato wart disease. Currently, 39 pathotypes have been described based on their interaction with a differential set of potato varieties. Wart resistance and pathotyping is performed using bioassays in which etiolated tuber sprouts are inoculated. Here, we describe an alternative method in which aboveground plant parts are inoculated. Susceptible plants produced typical wart symptoms in developing but not in fully expanded aboveground organs. Colonization of the host by S. endobioticum was verified by screening for resting spores by microscopy and by molecular techniques using TaqMan polymerase chain reaction and RNAseq analysis. When applied to resistant plants, none of these symptoms were detectable. Recognition of S. endobioticum pathotypes by differentially resistant potato varieties was identical in axillary buds and the tuber-based bioassays. This suggests that S. endobioticum resistance genes are expressed in both etiolated "belowground" sprouts and green aboveground organs. RNAseq analysis demonstrated that the symptomatic aboveground materials contain less contaminants compared with resting spores extracted from tuber-based assays. This reduced microbial contamination in the aboveground bioassay could be an important advantage to study this obligate biotrophic plant-pathogen interaction. Because wart resistance is active in both below- and aboveground organs, the aboveground bioassay can potentially speed up screening for S. endobioticum resistance in potato breeding programs because it omits the requirement for tuber formation. In addition, possibilities arise to express S. endobioticum effectors in potato leaves through agroinfiltration, thereby providing additional phenotyping tools for research and breeding. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Combined Effects of Low-Dose Proton Radiation and Simulated Microgravity on the Mouse Retina and the Hematopoietic System.

The purpose of the current study was to characterize the effects of simulated microgravity and radiation-induced changes in retina and retinal vasculature, and to assess the accompanying early changes in immune cells and hematological parameters. To better understand the effects of spaceflight, we used a combination of treatments designed to simulate both the radiation and low-gravity aspects of space conditions. To simulate the broad energy spectrum of a large solar particle event (SPE) and galactic cosmic ray (GCR) radiation, male C57BL/6J mice were exposed to whole-body irradiation using fully modulated beams of 150-MeV protons containing particles of energy from 0 to 150 MeV and a uniform dose-vs.-depth profile. The mice were also hindlimb-unloaded (HLU) by tail suspension. Mice were unloaded for 7 days, exposed to 50 cGy, unloaded for an additional 7 days and then sacrificed for tissue isolation at days 4 and 30 after the combined treatments. Increases in the number of apoptotic cells were observed in the endothelial cells of mice that received radiation alone or with HLU compared to controls at both days 4 and 30 (P < 0.05). Endothelial nitric oxide synthase (eNOS) levels were significantly elevated in the retina after irradiation only or combined with HLU compared to controls at the 30-day time point (P < 0.05). The most robust changes were observed in the combination group, suggesting a synergistic response to radiation and unloading. For hematopoietic parameters, our analysis indicated the main effects for time and radiation at day 4 after treatments (day 11 postirradiation) (P < 0.05), but a smaller influence of HLU for both white blood cell and lymphocyte counts. The group treated with both radiation and HLU showed greater than 50% reduction in lymphocyte counts compared to controls. Radiation-dependent differences were also noted in specific lymphocyte subpopulations (T, B, natural killer cells). This study shows indications of an early effect of low-dose radiation and spaceflight conditions on retina and immune populations.

Acute Effect of Low-Dose Space Radiation on Mouse Retina and Retinal Endothelial Cells.

There is concern that degradation of vision as a result of space flight may compromise both mission goals and long-term quality of life after space travel. The visual disturbances may be due to a combination of intracerebral pressure changes and exposure to ionizing radiation. The retina and the retinal vasculature play important roles in vision, yet have not been studied extensively in relationship to space travel and space radiation. The goal of the current study was to characterize oxidative damage and apoptosis in retinal endothelial cells after whole-body gamma-ray, proton and oxygen (16O) ion radiation exposure at 0.1 to 1 Gy. Six-month-old male C57Bl/6J mice were whole-body irradiated with 600 MeV/n 16O ions (0, 0.1, 0.25, 1 Gy), solar particle event (SPE)-like protons (0, 0.1, 0.25, 0.5 Gy) or 60Co gamma rays (0, 0.1, 0.25, 0.5 Gy). Eyes were isolated for examining endothelial nitric oxide synthase (eNOS) expression and characterization of apoptosis in retina and retinal endothelial cells at two weeks postirradiation. The expression of eNOS was significantly increased in the retina after proton and 16O ion exposure. 16O ions induced over twofold increase in eNOS expression compared to proton exposure at two weeks postirradiation ( P < 0.05). TUNEL assays showed dose-dependent increases in apoptosis in the retina after irradiation. Low doses of 16O ions elicited apoptosis in the mouse retinal endothelial cells with the most robust changes observed after 0.1 Gy irradiation ( P < 0.05) compared to controls. Data also showed that 16O ions induced a higher frequency of apoptosis in retinal endothelial cells compared to protons ( P < 0.05). In summary, our study revealed that exposure to low-dose ionizing radiation induced oxidative damage and apoptosis in the retina. Significant changes in retinal endothelial cells occur at doses as low as 0.1 Gy. There were significant differences in the responses of endothelial cells among the radiation types examined here.

Preclinical research into basic mechanisms of radiation-induced heart disease.

Radiation-induced heart disease (RIHD) is a potentially severe side effect of radiotherapy of thoracic and chest wall tumors if all or part of the heart was included in the radiation field. RIHD presents clinically several years after irradiation and manifestations include accelerated atherosclerosis, pericardial and myocardial fibrosis, conduction abnormalities, and injury to cardiac valves. There is no method to prevent or reverse these injuries when the heart is exposed to ionizing radiation. This paper presents an overview of recent studies that address the role of microvascular injury, endothelial dysfunction, mast cells, and the renin angiotensin system in animal models of cardiac radiation injury. These insights into the basic mechanisms of RIHD may lead to the identification of targets for intervention in this late radiotherapy side effect.

Potential targets for intervention in radiation-induced heart disease.

Radiotherapy of thoracic and chest wall tumors, if all or part of the heart was included in the radiation field, can lead to radiation-induced heart disease (RIHD), a late and potentially severe side effect. RIHD presents clinically several years after irradiation and manifestations include accelerated atherosclerosis, pericardial and myocardial fibrosis, conduction abnormalities, and injury to cardiac valves. The pathogenesis of RIHD is largely unknown, and a treatment is not available. Hence, ongoing pre-clinical studies aim to elucidate molecular and cellular mechanisms of RIHD. Here, an overview of recent pre-clinical studies is given, and based on the results of these studies, potential targets for intervention in RIHD are discussed.

[Women, schizophrenia and oestrogen; neurobiological hypotheses and hormonetherapy studies]. / Vrouwen, schizofrenie en oestrogeen; neurobiologische hypothesen en interventiestudies.

BACKGROUND: Gender differences play a role in the origin and course of schizophrenia. It has been hypothesised that the gonadal hormone, oestrogen, may possibly perform a protective function in the development of certain forms of schizophrenia. AIM: To review neurobiological hypotheses concerning the role of oestrogen in the development and course of schizophrenia. METHOD: The relevant literature was consulted with the help of PubMed, textbooks and bibliographic references; the search terms used were 'oestrogen', 'schizophrenia', 'gender', 'epigenetics', 'psychosis', 'women' and 'brain'. There were no restrictions with regards to the time-period. RESULTS: Neuro-imaging, animal experiments and hormone-therapy studies showed several effects of oestrogen in the field of epigenetics, morphology of the brain, interaction with neurotransmitters and neuroprotection. CONCLUSION: Oestrogen is an important link in a complex of factors that clearly play a role in the varying development of schizophrenia in men and women. So far, however, there is insufficient evidence to support the existence of a specific mechanism that would explain why oestrogen may perform a protective function in schizophrenia.

Histopathology of ventricles, coronary arteries and mast cell accumulation in transverse and longitudinal sections of the rat heart after irradiation.

The aim of the study was to compare cross sections with longitudinal sections in histopathological examination of the rat heart after irradiation, to find the most optimal method for the detection of cardiac radiation injuries. For this purpose, rats were irradiated locally on the heart with a single dose of 0 or 20 Gy. At different time points after irradiation, hearts were perfused and cut into longitudinal or cross sections. In both sections, several histopathological changes were scored on a graded scale between 0 and 3. Mast cells, which are thought to play a role in tissue remodelling, were counted. After 20 Gy, frequently occurring lesions were most severe in the upper half of the ventricles and the septum. These lesions could only be detected when using longitudinal sections, resulting in a higher total histopathological score than the examination of a single cross section. From 3 months onwards, changes in coronary arteries of irradiated hearts included endothelial cell loss, a loss of smooth muscle cells and fibrosis in media and adventitia. Up to 1 month after irradiation, mast cell densities of the left and right ventricles were decreased after 15 and 20 Gy, compared to time-matched controls, followed by increases from 3 months onwards. In the left ventricle, mast cell densities correlated with myocardial degeneration and fibre loss. The results of this study show that the usage of a single longitudinal section in the histopathological examination of the irradiated rat heart leads to the recognition of more severe injuries, including myocardial degeneration and fibrosis, in ventricular tissue than the usage of a single midventricular cross section. Morphological changes observed in coronary arteries of irradiated hearts might lead to a decreased compliance of the coronary artery wall. Further investigation is needed to determine the role of mast cells in cardiac tissue remodelling after irradiation.

Effects of multiple doses of ionizing radiation on cytokine expression in rat and human cells.

PURPOSE: To determine the effect of daily fractionated irradiation on the expression of growth factors and cytokines in different cardiac and vascular cell types. MATERIALS AND METHODS: Cell cultures of rat cardiac myocytes, fibroblasts, a rat cardiac microvascular endothelial cell line and human artery endothelial cells were irradiated with doses of 2 Gy, given daily during 5 consecutive days. Twenty-four hours after each fraction, gene expression was determined by competitive or semiquantitative polymerase chain reaction. Protein secretion into culture media was determined by enzyme-linked immunoabsorbant assay. RESULTS: Of all investigated mRNA levels, transforming growth factor (TGF)-ss1 and fibroblast growth factor (FGF)-2 were slightly upregulated in the rat cardiac endothelial cell line after irradiation. TGF-ss1 protein secretion by these cells was slightly, but non-significantly, elevated. Interleukin 1ss protein levels in myocyte culture media were decreased in control cultures at days 3 and 4 compared with day 2. No significant changes were observed in expression of FGF-2 in either of the four cell types. Moreover, no changes were observed in gene expression of platelet-derived growth factors A, B and interleukin 8 in the human artery endothelial cells. CONCLUSIONS: Fractionated irradiation leads to minor changes in the expression of specific cytokines in cardiac myocytes, fibroblasts and endothelial cells.

Separation of neonatal rat ventricular myocytes and non-myocytes by centrifugal elutriation.

The preparation of pure cardiac myocyte cultures from neonatal rats is hampered by the presence of non-myocytes, which can proliferate during culturing, thereby causing a progressive decrease in the proportion of myocytes. In order to obtain myocyte cell suspensions of high purity, a method based on centrifugal elutriation was developed. Cardiac cells, isolated from neonatal rat heart ventricles, were subjected to elutriation using flow rates that increased step-wise from 20 to 80 ml/min. The cell fraction obtained at 80 ml/min consisted of 68-90% myocytes. Still, upon culturing, the remaining non-myocytes proliferate, causing the proportion of myocytes to decrease to 60 +/- 2% at day 5. A second elutriation protocol was developed in which myocytes and non-myocytes were separated after a period of co-culturing for 4-5 days. By this approach a fibroblast-rich cell fraction (87 +/- 5%) and a myocyte-rich cell fraction (82 +/- 6%) were obtained. In conclusion, centrifugal elutriation creates the opportunity to separate neonatal rat myocytes from non-myocytes, either freshly isolated or after a period of culturing. Particularly, cell separation after a period of culturing ventricular cells offers an advantage to analyse the experimental effects on myocytes and non-myocytes separately.

Effects of ionizing radiation on gene expression in cultured rat heart cells.

PURPOSE: To determine the in vitro effect of ionizing radiation on TGF-beta1, FGF-2, IL-1beta, atrial natriuretic peptide (ANP) and procollagen types I and III gene expression in three different cell types of rat heart. MATERIALS AND METHODS: Primary cell cultures of myocytes and fibroblasts and cultures of a rat heart endothelial cell line (RHEC) were irradiated with single doses of 2.0, 8.5 or 15 Gy. At different time-points after irradiation (4-336 h), gene expression was analysed using a competitive PCR technique. RESULTS: Irradiation of cultured rat heart cells may lead to temporary changes in expression of the genes studied. Analysis of the radiation response of cultured myocytes, cardiac fibroblasts and rat heart endothelial cells reveals different responses with regard to (1) the dose necessary to evoke changes in mRNA expression, (2) the level of and (3) the duration of the 'induced' response. The changes observed were small and between parallel experiments the onset and time-course of the induced gene expression varied between 4 and 48 h. The average expression of TGF-beta1 mRNA between 4 and 48 h was significantly elevated in endothelial cells after a dose of 2.0 Gy, in fibroblasts after a dose of 8.5 Gy and in myocytes after a dose of 15 Gy. Down-regulation of TGF-beta1 mRNA in myocytes was observed after a dose of 8.5 Gy. FGF-2 and procollagen type-I mRNAs were significantly elevated in fibroblasts after a dose of 2.0 Gy. For all three cell types, no effect of dose on the timing or size of the gene expression was observed. CONCLUSIONS: Although irradiation of cultured heart cells influences expression of genes involved in tissue remodelling, the observed differences were too small and too restricted in time and dose to explain the exact role of these cell types in processes leading to radiation-induced cardiac fibrosis.

Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus.

Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a "single-cell" model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Igkappa gene (IGK) rearrangements, followed by IGK deletions and Iglambda gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified. This might be explained by the fact that regulation of the ordered recombination process is not fully strict, particularly when the IGL locus is involved. Unfavorable somatic mutations followed by receptor editing might have contributed to this finding. Eight of these 10 cases indeed contained somatic mutations. In cases with 2 functional Ig light chain rearrangements, both alleles were transcribed, but monospecific Ig expression was still maintained. This suggests that in these cases allelelic exclusion is not regulated at the messenger RNA level but either at the level of translation or protein stability or via preferential pairing of Ig light and Ig heavy chains. Nevertheless, ordered rearrangement processes are the main determinant for monospecific Ig light chain expression.

Detection of prostate cancer: a comparative study of the diagnostic efficacy of sextant transrectal versus sextant transperineal biopsy.

OBJECTIVES: The optimal biopsy strategy for the detection of prostate cancer still needs to be established, since a considerable proportion of clinically significant cancers remains undiagnosed on routine sextant transrectal biopsy. To assess the efficacy of transperineal biopsy to detect prostate cancer, we compared this approach to systematic sextant transrectal biopsy in a simulation experiment. METHODS: Ultrasound-guided sextant transverse (transrectal) biopsy and subsequent sextant longitudinal (transperineal) biopsy were performed on 40 radical prostatectomy specimens of patients with (transrectal) biopsy-detected prostate cancer. Conditions were simulative and may not be completely analogous to clinical settings. Ultrasound-determined prostate volume, biopsy tumor involvement, number of cores with cancer, and tumor volume were determined. Detailed mapping of radical prostatectomy specimens provided insight into the representativeness of the biopsy techniques. RESULTS: Of 40 cancers, 33 (82.5%) were redetected by the transperineal approach; 29 (72.5%) were detected by repeated transrectal biopsies. For both approaches, the tumor volume of the undiagnosed cancers was significantly smaller (P <0.01) and the prostate volume was significantly larger (P <0.01) than in the redetected ones. Between the two approaches, no difference was found for either of the variables determined in the redetected cancers. Prostate maps clarified that transperineal undiagnosed tumors were either small (0.2 cm(3) or less) or notably located at the prostatic base. CONCLUSIONS: The biopsy procedure in which the biopsy needles enter the prostate at the apex for a longitudinal direction may efficiently sample the prostatic peripheral zone. Since the experiment was artificial in design, caution should be observed in extrapolating these results to patient settings.

A genetic polymorphism in connexin 37 as a prognostic marker for atherosclerotic plaque development.

BACKGROUND AND OBJECTIVES: Atherosclerosis is a multifactorial disease, in part characterized by chronic inflammatory changes in the vessel wall and loss of normal physical and biochemical interactions between endothelial cells and smooth muscle cells. Previous studies [Hu J., Cotgreave IA. J Clin Invest; 99: 1-5] have provided molecular links between inflammation and myoendothelial communication via gap junctions, suggesting that these structures may be important in the development of the atherosclerotic vessel phenotype. In order to strengthen this premise, the aim of the present work was to probe for structural polymorphisms in connexin 37, a gap junctional protein uniquely expressed in endothelial cells, and to assess for potential genotypic segregation in individuals displaying atherosclerotic plaque. METHODS AND RESULTS: Computer-based comparisons of Expressed Sequence Tags (ESTs) predicted a polymorphism in the human gap junctional protein connexin 37 (cx37). The C1019-T mutation results in a proline to serine shift at codon 319 (cx37*1-cx37*2). A Restriction Fragment Length Polymorphism (RFLP) assay, involving the insertion of a novel Drd I cleavage site in the proline variant revealed a statistically significant over-representation of the cx37*1 allele in association with atherosclerotic plaque-bearing individuals (Odds-ratio for the homozygote = 2.38, Chi2 = 7.693, P = 0.006), in comparison to individuals lacking plaque, irrespective of a history of hypertension. CONCLUSIONS: These data suggest that the C1019-T polymorphism in cx37 may provide 'single gene marker', which could be useful in assessing atherosclerotic plaque development, particularly in cardiovascular risk groups such as those with borderline hypertension.

Rectal wall contractility in response to an evoked urge to defecate in patients with obstructed defecation.

PURPOSE: The aim of this study was to examine rectal sensory perception and rectal wall contractility in response to an evoked urge to defecate and to identify differences between control subjects and patients with obstructed defecation. METHODS: Twenty control patients (10 men; median age, 47 (range, 17-78) years) and 29 female patients with disabling obstructed defecation (median age, 48 (range, 18-70) years) entered the study. Under radiologic control, an infinitely compliant barostat balloon was inserted over a guide wire into the proximal part of the rectum. Additionally, a latex balloon was introduced into the distal part of the rectum. This latex balloon was inflated until an urge to defecate was experienced. Simultaneously, rectal wall contractility was assessed by measuring the variations in barostat balloon volume. These variations were expressed as percentage changes from baseline volume. RESULTS: By comparing controls and patients with obstructed defecation, a significant difference was found regarding mean distending volume required to elicit an urge to defecate (135 +/- 38 vs. 214 +/- 87 ml of air; P < 0.001, Mann-Whitney U-test). In all controls, the evocation of an urge to defecate induced a pronounced increase in rectal tone, proximal to the distal stimulating balloon. By comparing controls and patients, the increase in rectal tone was found to be significantly higher in control subjects (35 +/- 10 vs. 9 +/- 10 percent; P < 0.001). Twenty-five patients (86 percent) showed no or only minimum (<20 percent) increase in rectal tone during the perception of an urge to defecate. In 14 of these patients, the threshold for this perception was increased. Only four patients (14 percent) showed a relatively normal increase (>20 percent) in rectal tone. However, their threshold for perception was greatly increased. CONCLUSION: The assembly used in this study provides a useful tool for investigation of rectal evacuation. In all of our patients, obstructed defecation was associated with abnormal rectal sensory perception and/or altered rectal wall contractility.

Long-term results of suture rectopexy in patients with fecal incontinence associated with incomplete rectal prolapse.

UNLABELLED: Suture rectopexy is the recommended therapy for complete rectal prolapse that is associated with fecal incontinence. It has been suggested that correction of an incomplete rectal prolapse is also worthwhile for patients with fecal incontinence. PURPOSE: Aims of this study were 1) to evaluate the clinical outcome of suture rectopexy in a consecutive series of patients with incomplete rectal prolapse associated with fecal incontinence, and 2) to compare these results with those obtained from patients with complete rectal prolapse. METHODS: Between 1979 and 1994, suture rectopexy was performed in 13 incontinent patients (3 males; median age, 65 (range, 45-77) years) with incomplete rectal prolapse (Group I) and in 24 incontinent patients (21 females; median age, 71 (range, 24-86) years) with complete rectal prolapse (Group II). RESULTS: After a median follow-up of 67 months, continence was restored in 5 of 13 (38 percent) patients with incomplete rectal prolapse and in 16 of 24 (67 percent) patients with complete rectal prolapse. In both groups, all male patients became continent. CONCLUSIONS: For the majority of incontinent patients with incomplete rectal prolapse, a suture rectopexy is not beneficial. The clinical outcome of this procedure is only good in incontinent patients with complete rectal prolapse. Based on these data, it is questionable whether incomplete rectal prolapse plays a causative role in fecal incontinence.

Pathophysiological aspects and clinical outcome of intra-anal application of isosorbide dinitrate in patients with chronic anal fissure.

BACKGROUND: Relaxation of the internal anal sphincter can be achieved by local application of exogenous nitric oxide donors. AIM: To evaluate the influence of topical application of isosorbide dinitrate (ISDN) on anal pressure, anodermal blood flow, and fissure healing. PATIENTS: Thirty four consecutive patients (male/female: 18/16; mean age (SEM): 39 (10)) with a chronic anal fissure were studied. METHODS: All patients were treated for at least six weeks or a maximum period of 12 weeks. Before treatment and at three and six weeks 22 patients underwent conventional anal manometry and laser Doppler flowmetry of the anoderm. RESULTS: Within 10 days the fissure related pain was resolved in all patients. At six, nine, and 12 weeks the anal fissure was completely healed in 14, 22, and 30 patients respectively. At three and six weeks manometry was performed at least one hour after the last application of ISDN. These recordings showed a reduction of the maximum resting anal pressure (mean (SD), pretreatment 111 (26) mm Hg; three weeks 86 (19); six weeks 96 (27), p < 0.001). Simultaneous recordings of anodermal blood flow showed a significant increase of flow (pretreatment 0.53 (0.17); three weeks 0.80 (0.16); six weeks 0.76 (0.31), p < 0.005). The mean (SEM) duration of follow up after successful outcome was 11 (5) months. Within this period fissure relapsed in two of 30 patients (7%), eight and 10 weeks after treatment had been stopped. CONCLUSIONS: Local application of ISDN reduces anal pressure and improves anodermal blood flow. This dual effect results in a fissure healing rate of 88% at 12 weeks. This new and simple treatment modality seems to be an attractive alternative for the current available surgical procedures.

Anal fissure: new concepts in pathogenesis and treatment.

The posterior commissure of the anal canal is less well perfused than the other segments of the anoderm. There is growing evidence that the increased activity of the internal anal sphincter, which is found in almost all patients with a chronic anal fissure, further decreases the anodermal blood supply, especially at the posterior midline. Reduction of anal pressure, either by anal dilatation or by lateral internal sphincterotomy, is the most important step in the treatment of chronic anal fissure. However, both procedures frequently result in permanent sphincter defects and subsequent continence disturbances. Recently, nitric oxide (NO) has been identified as the chemical messenger mediating relaxation of the internal anal sphincter. It has been shown that local application of exogenous NO donors such as nitroglycerin and isosorbide-di-nitrate reduces anal pressure and improves anodermal blood flow. This dual effect results in fissure healing in more than 80% of patients.

[Intra-anal application of isosorbide dinitrate in chronic anal fissure]. / Intra-anale applicatie van isosorbidedinitraat bij chronische fissura ani.

OBJECTIVE: To evaluate the effect of intra-anal application of isosorbide dinitrate on the healing rate of chronic anal fissure. DESIGN: Prospective, descriptive. SETTING: Outpatient clinic of the department of Surgery, University Hospital Dijkzigt, Rotterdam. METHOD: Sixteen patients with chronic (more than three months' duration) anal fissure were treated by intra-anal application of isosorbide dinitrate ointment every 3 hours, except during the night. The maximal duration of therapy was 12 weeks. Every three weeks the following aspects were investigated: clinical symptoms, side-effects and fissure healing. RESULTS: All patients experienced mild and transient headache shortly after the beginning of the treatment. At three weeks the fissure-related pain was resolved in all patients. At 6, 9 and 12 weeks the fissure was completely healed in 9, 11 and 15 patients respectively. CONCLUSION: The majority of chronic anal fissures can be treated effectively by local application of isosorbide dinitrate. This new and simple treatment modality appears to be an attractive alternative to the currently available surgical procedures.