Timing of semen cryopreservation: before or after processing?

Objective: Seminal cryopreservation causes significant damage to the sperm; therefore, different methods of cryopreservation have been studied. The aim of the study was to compare the effects of density gradient processing and washing/centrifugation with seminal plasma removal for cryopreservation in semen parameters. Methods: Seminal samples of 26 normozoospermic patients were divided into 3 parts: with seminal plasma; after washing/centrifugation; and after selection through density gradient. The samples were cryopreserved for at least two weeks. Motility, sperm count, morphology and viability were evaluated before cryopreservation and after thawing. Results: Density gradient processing selected motile and viable sperm with normal morphology in fresh samples (p<0.05). Cryopreservation negatively affected all sperm parameters regardless of the processing performed, and even if the sperm recovery was lower in the density gradient after the thawing, progressive motility, total motility, viability and morphology remained higher (p<0.05). Conclusion: Cryopreservation significantly compromises sperm parameters (motility, morphology, viability). In normozoospermic patients, the density gradients select better quality spermatozoa compared to other processing methods; this benefit was kept after thawing.

Is transumbilical laparoscopic-assisted appendectomy feasible for complicated appendicitis? A single-center experience.

PURPOSE: Transumbilical laparoscopic-assisted surgery (TULS) mixed benefits of laparoscopic and open surgeries. Transumbilical laparoscopic-assisted appendectomy (TULAA) is a well-known procedure, accepted and currently used by pediatric surgeons for treatment of uncomplicated appendicitis (UA). There is no current agreement in its use for the complicated appendiceal infections (CA). We reported our results using TULAA for both UA and CA. METHODS: We retrospectively collected TULAA performed between April 2017 and April 2022. Appendicitis were classified in UA and CA. We analyzed conversion rate, operative time, length of stay, surgical site infections (SSIs) rate, postoperative intra-abdominal abscess and costs. RESULTS: Over 5 years, 316 children underwent TULAA. Conversion rate was 3%. Mean age at surgery was 9.36 years (IQR 2-16). Forty-nine appendicitis were CA. Operative time and hospital stay was higher in CA than in UA group (38.33 vs. 60.73 min, p < 0.00001; 4 vs. 7 days, p < 0.00001). SSIs rate showed no statistically significant difference between two groups. Incidence of postoperative intra-abdominal collections was 11% in CA and 1% in UA. TULAA's cost was 192.07 €. CONCLUSION: In our series, TULAA seems to be safe, feasible and cost-effective for both uncomplicated and complicated appendicitis, with no disadvantage in terms of outcomes compared to what is reported in literature for CLS.

Two-balloon epistaxis catheter to ensure vaginal patency in a complex case of vaginoplasty for vaginal agenesis: a case report.

Congenital vaginal atresia is a rare anomaly of the female genital tract. Many vaginoplasty procedures have been described, but the postoperative risk of vaginal stenosis remains a challenge. We report a case of isolated distal vaginal agenesis in a patient with neurological impairment where the use of an "alternative" dilator was needed. An 11-year-old girl with Down syndrome was admitted to the Emergency Department complaining of pelvic pain. The clinical evaluation showed a hard and painful pelvic mass associated with an imperforate hymen. Abdominal ultrasound and pelvic MRI were suggestive for hematometrocolpos and absence of the lower third segment of the vagina. Vaginoscopy confirmed the diagnosis of congenital vaginal agenesis. The patient then underwent a laparoscopic-assisted vaginoplasty. Considering the difficult management of the postoperative period, an epistaxis catheter was used as a vaginal stent and dilator. The use of an epistaxis catheter to provide adequate vaginal patency after vaginoplasty can be an alternative solution especially in those cases where calibrations with dilators are difficult or not tolerated.

Total Esophagogastric and Cologastric Dissociation in Neurologically Normal Children: Systematic Review.

Total esophagogastric dissociation (TEGD) was first described by Bianchi as a definitive procedure for gastroesophageal reflux disease (GERD) in neurologically impaired children. In the last 20 years, different centers extended the indication to neurologically normal (NN) patients with GERD associated with congenital or acquired esophageal anomalies. The aim of this paper is to analyze the role of TEGD in this cluster of patients. A PubMed and Google Scholar search was conducted. All cases of NN children who underwent TEGD for GERD were collected. Patient characteristics and outcomes were analyzed. Complications were classified according to Clavien-Dindo classification. Forty-eight children were identified. In 56.25%, TEGD was the first anti-reflux procedure, while in 43.75% it was performed after failed fundoplications. Mean follow-up was 5.5 years. Mortality related to surgery was 2.08%. All of the survivors improved their condition, with resolution of GERD and weight gain. In addition, 50% of children weaned off enteral nutrition, with 14.6% having their gastrostomy removed, while 41.67% maintained partial enteral supplementation. Respiratory symptoms almost disappeared in 54.17% of patients. This review suggests that TEGD can also be considered for NN children where conventional methods seem insufficient to control reflux and preserve pulmonary function. Nevertheless, long-term follow-up is still required.

Early Bowel Lengthening Procedures: Bi-Institutional Experience and Review of the Literature.

Early bowel lengthening procedure (EBLP) has been defined as any bowel lengthening procedure performed before six months of age. The purpose of this paper is to compare our experience with literature on this subject to identify common indications. A bi-institutional retrospective analysis was performed. Diagnosis, type of surgery, age at procedure and outcomes were analysed. Eleven EBLP were performed in Manchester and Florence from 2006 to 2021. The median age at surgery was 126 days (102-180), pre-operative median short bowel (SB) length was 28 cm (17-49) with a post-operative median increase of 81%. Furthermore, a PubMed/Embase search was undertaken regarding bowel lengthening procedures performed in the last 40 years. Sixty-one EBLP were identified. The median age was 60 days (1-90). Serial transverse enteroplasty (STEP) was the most frequent procedure used, with a median increased bowel length of 57%. This study confirms that no clear consensus on indication or timing to perform early SB lengthening is reported. According to the gathered data, EBLP should be considered only in cases of actual necessity and performed in a qualified intestinal failure centre.

Endoscopic treatment of ureterocele in children: Results of a single referral tertiary center over a 10 year-period.

INTRODUCTION: The management of UCs remain controversial, especially for UCs with duplex collecting systems that still represent a great challenge in paediatric urology. Several approaches have been used and a shared management is not yet validated. STUDY AIM: Aim of our study is to evaluate the results of the endoscopic treatment of UC comparing ortothopic single-system UC and ectopic duplex-system UC over a 10-year period in a single referral tertiary center. Success was defined as resolution of dilation, lack of urinary infections and preservation of renal function. STUDY DESIGN: We retrospectively reviewed medical records of children with a diagnosis of UC who underwent endoscopic puncture at our division from January 2009 to January 2019. Patients were divided in two groups: Group A composed of patients with ectopic UC associated with renal duplex system and Group B with orthotopic UC in single collecting system. RESULTS: We identified 48 paediatric patients treated with transurethral primary endoscopic incision. Groups result homogeneous for clinical and pathological characteristics. The only statistical significative difference between the two samples was the age at diagnosis (p value with Yates correction = 0.01). DISCUSSION: We considered as a therapeutic success infections control and the elimination of obstruction with preservation of global kidney function. Based on that, our success rate after single (77%) or double (92%) endoscopic treatment is higher than data reported in literature. Differently from previous studies, vescico-ureteral reflux without UTIs was not considered as a failure of the procedure. The present study has some limitations: it is a retrospective and monocentric serie and it lacked a longer follow-up; on the other hand, it has been conducted on a quite large sample size and it is one of the few studies that compares the endoscopic treatment between orthotopic and ectopic UC. CONCLUSION: Our data report primary endoscopic puncture of ureterocele as a simple, effective, and safe procedure also in long-term follow up. This technique avoids the need for additional surgery in the majority of the patients, also in the case of an ectopic UC associated to a duplex system.

Ureteral Reimplantation for Primary Obstructive Megaureter in Pediatric Patients: Is It Time for Robot-Assisted Approach?

Objective: To compare open and robotic approach for treatment of Primary Obstructive Megaureter (POM) in a series of pediatric patients. Materials and Methods: Medical records of all patients who had undergone ureteral reimplantation for POM at our institution, between January 2016 and December 2019, were retrospectively collected and analyzed. Open (Group B) and robotic (Group A) approach were compared. Success was defined as reduction of anteroposterior diameter of pelvis and ureter on postoperative ultrasound scan. Surgical complications were reported according to the Clavien-Dindo Classification. Esthetic results were compared through the Vancouver Scar Scale. Results: Out of 23 patients, 11 belonged to Group A (5M and 6F) and 12 to Group B (9M and 3F). Median age at diagnosis was 38 months in Group A and 46 months in Group B (P = .69). Operative times were comparable (150' Group A and 140' Group B; P = .35), but the hospital stay was significantly shorter in the robotic group (P < .01). Success rate was comparable: 90.9% in Group A and 91.7% in Group B, respectively (P = .95). Incidence of early complications was 9.1% in Group A (grade IIIB) and 8.3% in Group B (grade II) (P = .95). Esthetic evaluation obtained a median score of 1 in Group A and 3 in Group B (P < .01). Conclusions: Our preliminary experience shows that robotic reimplantation can be a safe option in the treatment of POM in pediatric patients with clinical outcomes comparable to the open technique.

Congenital short bowel syndrome: systematic review of a rare condition.

BACKGROUND: Congenital short bowel syndrome (CSBS) is a rare gastrointestinal disorder caused by intrauterine reduction of small bowel length whose etiology is still unknown. Chronic diarrhea, vomiting, and failure to thrive are the most important complications, arising from less absorptive intestinal surface. This review examines clinical features and outcomes of CSBS patients. METHODS: A PubMed and EMBASE research on CSBS was performed. Inclusion criterion was congenital short bowel diagnosis in a range of ages between 33 weeks of gestational age and 15 years old (IQR 38 days). Exclusion criteria were history of atresia of any part of the gastrointestinal tract and extensive surgical bowel resections. Qualitative and quantitative variables were collected and analyzed. Data were expressed in mean and IQR. RESULTS: Sixty-one patients were identified (38 males, 23 females) from 1969 to date. Mean bowel length was 58.24 cm (IQR 37.5). Malrotation of the midgut was seen in 98.4% of cases. Our data showed an interesting trend in improving the survival rate of these patients (from 28.5% before 2008 to 75% in the period after 2008). Sepsis was the most frequent cause of death reported (57.9%). Interestingly, 18 patients were genetically analyzed, finding mutations either in FLNA gene (38.8%) or in CLMP gene (61.1%). CONCLUSIONS: CSBS is a condition that seems to be related to an autosomal recessive (CLMP) or an X linked (FLNA) type of inheritance. Advance in medical management seems to have improved survival of these children in recent years. Further genetic studies can better understand the causes of this disease aiming to create personalized treatment. TYPE OF STUDY: Systematic review. LEVEL OF EVIDENCE: Level IV.

Markers of microbial translocation during pregnancy: differences among HIV+ women of African and European provenance.

INTRODUCTION: Microbial translocation (MT) markers are indicators of HIV-related immune activation, but reference values are mostly derived from European or North American populations and could be substantially different in populations living in developing countries. Here we evaluate possible differences in MT markers levels in HIV+ pregnant women of different geographical provenance. METHODOLOGY: This study is nested within an observational study of pregnant women with HIV in Italy. Women were dichotomized on the basis of provenance in two groups of European (n = 14) and African (n = 26) origin. Soluble CD14, lipopolysaccharide-binding protein (LBP) and intestinal-fatty acid binding protein (I-FABP) were measured in plasma samples collected between the first and second trimester of pregnancy. RESULTS: Demographic and viroimmunological characteristics were similar between groups, although European women were more commonly smokers and HCV-coinfected. Irrespective of origin, LBP plasma levels were positively correlated with I-FABP (r = 0.467, p = 0.004) and sCD14 levels (r = 0.312 p = 0.060). Significantly higher levels of sCD14 (1885 vs. 1208 ng/mL, p = 0.005) LBP (28.5 vs. 25.3 µg/mL, p = 0.050) and I-FABP (573.4 vs. 358.2 pg/mL, p = 0.002) were observed in European compared with African women. A multivariable linear regression analysis, adjusted for smoking and HCV coinfection confirmed the association between sCD14 levels and women provenance (p = 0.03). CONCLUSIONS: Our observations indicate significant differences in soluble markers among women of different provenance. In the design and analysis of studies evaluating MT markers, population-specific reference values should be considered.

Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection.

Hepatitis C virus infection (HCV) represents a unique model to characterize, from early to late stages of infection, the T cell differentiation process leading to exhaustion of human CD8+ T cells. Here we show that in early HCV infection, exhaustion-committed virus-specific CD8+ T cells display a marked upregulation of transcription associated with impaired glycolytic and mitochondrial functions, that are linked to enhanced ataxia-telangiectasia mutated (ATM) and p53 signaling. After evolution to chronic infection, exhaustion of HCV-specific T cell responses is instead characterized by a broad gene downregulation associated with a wide metabolic and anti-viral function impairment, which can be rescued by histone methyltransferase inhibitors. These results have implications not only for treatment of HCV-positive patients not responding to last-generation antivirals, but also for other chronic pathologies associated with T cell dysfunction, including cancer.

Effects of dexamethasone on the Li-pilocarpine model of epilepsy: protection against hippocampal inflammation and astrogliosis.

BACKGROUND: Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy and is accompanied, in one third of cases, by resistance to antiepileptic drugs (AED). Most AED target neuronal activity modulated by ionic channels, and the steroid sensitivity of these channels has supported the use of corticosteroids as adjunctives to AED. Assuming the importance of astrocytes in neuronal activity, we investigated inflammatory and astroglial markers in the hippocampus, a key structure affected in TLE and in the Li-pilocarpine model of epilepsy. METHODS: Initially, hippocampal slices were obtained from sham rats and rats subjected to the Li-pilocarpine model of epilepsy, at 1, 14, and 56 days after status epilepticus (SE), which correspond to the acute, silent, and chronic phases. Dexamethasone was added to the incubation medium to evaluate the secretion of S100B, an astrocyte-derived protein widely used as a marker of brain injury. In the second set of experiments, we evaluated the in vivo effect of dexamethasone, administrated at 2 days after SE, on hippocampal inflammatory (COX-1/2, PGE2, and cytokines) and astroglial parameters: GFAP, S100B, glutamine synthetase (GS) and water (AQP-4), and K+ (Kir 4.1) channels. RESULTS: Basal S100B secretion and S100B secretion in high-K+ medium did not differ at 1, 14, and 56 days for the hippocampal slices from epileptic rats, in contrast to sham animal slices, where high-K+ medium decreased S100B secretion. Dexamethasone addition to the incubation medium per se induced a decrease in S100B secretion in sham and epileptic rats (1 and 56 days after SE induction). Following in vivo dexamethasone administration, inflammatory improvements were observed, astrogliosis was prevented (based on GFAP and S100B content), and astroglial dysfunction was partially abrogated (based on Kir 4.1 protein and GSH content). The GS decrease was not prevented by dexamethasone, and AQP-4 was not altered in this epileptic model. CONCLUSIONS: Changes in astroglial parameters emphasize the importance of these cells for understanding alterations and mechanisms of epileptic disorders in this model. In vivo dexamethasone administration prevented most of the parameters analyzed, reinforcing the importance of anti-inflammatory steroid therapy in the Li-pilocarpine model and possibly in other epileptic conditions in which neuroinflammation is present.

Hyperammonemia compromises glutamate metabolism and reduces BDNF in the rat hippocampus.

Ammonia is putatively the major toxin associated with hepatic encephalopathy (HE), a neuropsychiatric manifestation that results in cognitive impairment, poor concentration and psychomotor alterations. The hippocampus, a brain region involved in cognitive impairment and depressive behavior, has been studied less than neocortical regions. Herein, we investigated hippocampal astrocyte parameters in a hyperammonemic model without hepatic lesion and in acute hippocampal slices exposed to ammonia. We also measured hippocampal BDNF, a neurotrophin commonly related to synaptic plasticity and cognitive deficit, and peripheral S100B protein, used as a marker for brain damage. Hyperammonemia directly impaired astrocyte function, inducing a decrease in glutamate uptake and in the activity of glutamine synthetase, in turn altering the glutamine-glutamate cycle, glutamatergic neurotransmission and ammonia detoxification itself. Hippocampal BDNF was reduced in hyperammonemic rats via a mechanism that may involve astrocyte production, since the same effect was observed in astrocyte cultures exposed to ammonia. Ammonia induced a significant increase in S100B secretion in cultured astrocytes; however, no significant changes were observed in the serum or in cerebrospinal fluid. Data demonstrating hippocampal vulnerability to ammonia toxicity, particularly due to reduced glutamate uptake activity and BDNF content, contribute to our understanding of the neuropsychiatric alterations in HE.

Efficacy and safety profile of boceprevir- or telaprevir-based triple therapy or dual peginterferon alfa-2a or alfa-2b plus ribavirin therapy in chronic hepatitis C: the real-world PegBase observational study.

BACKGROUND: The aim of the study was to determine the efficacy and safety of triple therapy with a first-generation protease inhibitor (PI; boceprevir, telaprevir) plus peginterferon alfa-2a or -2b plus ribavirin, and dual therapy (peginterferon alfa-2a or -2b plus ribavirin) in patients with chronic hepatitis C (CHC) in routine clinical practice. METHODS: PegBase was an international, prospective, observational study in which 4441 patients with CHC were enrolled in 27 countries. This analysis focuses on results in 4100 treatment-naïve and previously treated patients treated with PI-based triple therapy or dual therapy, according to the discretion of the investigator and local standards of practice. The primary efficacy outcome was sustained virological response after 12-week follow up (SVR12). RESULTS: SVR12 rates in treatment-naïve genotype (G) 1 patients were 56.6% and 62.9% for recipients of boceprevir plus peginterferon alfa-2a/ribavirin and boceprevir plus peginterferon alfa-2b/ribavirin, respectively, and 65.3% and 58.6% for recipients of telaprevir plus peginterferon alfa-2a/ribavirin and telaprevir plus peginterferon alfa-2b/ribavirin, respectively. In previously treated patients assigned to these four regimens, SVR12 rates were 43.6%, 48.3%, 60.3% and 56.1%, respectively. Among treatment-naïve patients assigned to peginterferon alfa-2a/ribavirin and peginterferon alfa-2b/ribavirin, respectively, SVR12 rates were 49.2% and 41.9% in G1 patients, 75.7% and 83.3% in G2 patients, 65.9% and 65.9% in G3 patients, and 49.7%, and 51.1% in G4 patients. The safety and tolerability of dual and triple therapy were consistent with previous reports. CONCLUSION: The efficacy and safety of first-generation PI-based triple-therapy and dual-therapy regimens in this real-world cohort were broadly comparable to those of previous studies.

Lipopolysaccharide modulates astrocytic S100B secretion: a study in cerebrospinal fluid and astrocyte cultures from rats.

BACKGROUND: Inflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calcium-binding protein secreted by astrocytes, has properties of a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B. METHODS: In this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide. RESULTS: Our data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 µg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein. CONCLUSIONS: Together, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

Genetically engineered resistance to Plum pox virus infection in herbaceous and stone fruit hosts.

Plum pox virus (PPV), a Potyvirus, is the causal agent of sharka, the most detrimental viral disease affecting stone fruit trees. This review focuses on research carried out to obtain PPV- resistant transgenic plants and on how biotechnological strategies evolved in light of the scientific advances made during the last several years. Successful RNA silencing strategies that confer high level of resistance to strains of PPV have been developed and tested under laboratory and greenhouse conditions. Moreover, field tests showed that transgene-mediated RNA silencing was effective in protecting plum plants against aphid-mediated PPV infection. The new emerging biotechnological approaches for conferring PPV resistance are discussed.

Silencing of Plum pox virus 5'UTR/P1 sequence confers resistance to a wide range of PPV strains.

An effective disease-control strategy should protect the host from the major economically important and geographically widespread variants of a pathogen. Plum pox virus (PPV) is the causal agent of sharka, the most devastating viral disease of Prunus species. We have shown previously that the hairpin RNA expression driven by h-UTR/P1, h-P1/HCPro, h-HCPro and h-HCPro/P3 constructs, derived from the PPV-M ISPaVe44 isolate, confers resistance to the homologous virus in Nicotiana benthamiana plants. Since the production of transgenic stone fruits and their evaluation for PPV resistance would take several years, the ISPaVe44-resistant plant lines were used to evaluate which construct would be the best candidate to be transferred to Prunus elite cultivars. To do that, nine PPV isolates of the D, M, Rec, EA and C strains originally collected from five Prunus species in different geographical areas, were typed by sequencing and used to challenge the transgenic N. benthamiana lines; 464 out of 464 virus-inoculated plants of lines h-UTR/P1, h-HCPro and h-HCPro/P3 showed complete and long-lasting resistance to the seven PPV isolates of D, M and Rec strains. Moreover, the h-UTR/P1 plants were also fully resistant to PPV-C and -EA isolates. Our data suggest that the h-UTR/P1 construct is of particular practical interest to obtain stone fruit plants resistant to the sharka disease.

Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response.

BACKGROUND & AIMS: Radiofrequency thermal ablation (RFA) is a minimally invasive technique used as standard local therapy of hepatocellular carcinoma and second-line treatment for metastatic liver tumors. Studies in preclinical models and in patients have shown that thermal destruction of tumor tissue can enhance anti-tumor cellular responses, but our knowledge of its impact on natural killer (NK) cells is still very limited. METHODS: Thirty-seven patients undergoing RFA for hepatocellular carcinoma were studied for peripheral blood lymphocytes counts followed by phenotypic and functional characterization of NK-cell population. RESULTS: Peripheral blood lymphocytes kinetics revealed an increased frequency and absolute number of NK cells expressing higher levels of activatory along with reduced levels of inhibitory NK receptors, and increased functional NK-cell activity. A prevalent expansion of the CD3(-)CD56(dim) NK subset was observed compared to the CD3(-)CD56(bright) counterpart. Interferon-gamma production, anti-K562 cell cytotoxicity, and antibody-dependent cell cytotoxicity, appeared consistently increased in terms of both absolute activity and killing efficiency at 4 weeks after RFA, as compared to baseline. Interestingly, when recurrence-free survival was assessed in 2 groups of patients separated according to higher vs lower enhancement of cytotoxicity and/or interferon-gamma production, a significant difference was observed, thus suggesting a potential predictive role of NK functional assays on efficacy of RFA. CONCLUSIONS: RFA can lead to stimulation of NK cells with a more differentiated and proactivatory phenotypic profile with general increase of functional activities. This observation may be relevant for development of adjuvant immunotherapeutic strategies aimed at enhancing NK-cell responses against primary and metastatic liver tumors.

Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.

We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.