Gender-differences of in vitro colonic motility after chemo- and radiotherapy in humans.

Background: The aim of the present in vitro study was to investigate, in different genders, motor responses in surgical colonic specimens from patients with rectal cancer undergoing and not undergoing chemotherapy with capecitabine and radiotherapy. Methods: This in vitro study was conducted from October 2015 to August 2017 at the Experimental Pharmacology Laboratory at the National Institute “S. de Bellis” after collecting samples at the Department of Surgery. Segments of sigmoid colon were obtained from 15 patients (Male (M)/Female (F) = 8/7; control group, CG) operated on for elective colorectal resection for rectal cancer without obstruction and 14 patients (M/F = 7/7; study group, SG) operated on for elective colorectal resection for rectal cancer who also received chemotherapy, based on capecitabine twice daily, and radiotherapy. Isometric tension was measured on colonic circular muscle strips exposed to increasing carbachol or histamine concentrations to obtain concentration-response curves. The motor responses to electrically evoked stimulation were also investigated. Results: In males, carbachol and histamine caused concentration-dependent contractions in the CG and SG. An increased sensitivity and a higher response to carbachol and histamine were observed in SG than CG (P < 0.01). On the contrary, in females, the response to carbachol was not significantly different in CG from the SG and the maximal responses to carbachol were greater in CG than in SG (P < 0.001). The same applied to histamine for half-maximal effective concentrations and maximal response in that they were not significantly different in CG from the SG. Electrically evoked contractions were significantly more pronounced in males, especially in the SG (P < 0.05). Conclusions: This preliminary in vitro study has shown gender differences in motor responses of colonic circular muscle strips in patients who had received chemotherapy with capecitabine and radiotherapy

Rapid Dissemination of SIV Follows Multisite Entry after Rectal Inoculation

Receptive ano-rectal intercourse is a major cause of HIV infection in men having sex with men and in heterosexuals. Current knowledge of the mechanisms of entry and dissemination during HIV rectal transmission is scarce and does not allow the development of preventive strategies. We investigated the early steps of rectal infection in rhesus macaques inoculated with the pathogenic isolate SIVmac251 and necropsied four hours to nine days later. All macaques were positive for SIV. Control macaques inoculated with heat-inactivated virus were consistently negative for SIV. SIV DNA was detected in the rectum as early as four hours post infection by nested PCR for gag in many laser-microdissected samples of lymphoid aggregates and lamina propria but never in follicle-associated epithelium. Scarce SIV antigen positive cells were observed by immunohistofluorescence in the rectum, among intraepithelial and lamina propria cells as well as in clusters in lymphoid aggregates, four hours post infection and onwards. These cells were T cells and non-T cells that were not epithelial cells, CD68+ macrophages, DC-SIGN+ cells or fascin+ dendritic cells. DC-SIGN+ cells carried infectious virus. Detection of Env singly spliced mRNA in the mucosa by nested RT-PCR indicated ongoing viral replication. Strikingly, four hours post infection colic lymph nodes were also infected in all macaques as either SIV DNA or infectious virus was recovered. Rapid SIV entry and dissemination is consistent with trans-epithelial transport. Virions appear to cross the follicle-associated epithelium, and also the digestive epithelium. Viral replication could however be more efficient in lymphoid aggregates. The initial sequence of events differs from both vaginal and oral infections, which implies that prevention strategies for rectal transmission will have to be specific. Microbicides will need to protect both digestive and follicle-associated epithelia. Vaccines will need to induce immunity in lymph nodes as well as in the rectum

Modeling amyotrophic lateral sclerosis in hSOD1G93A transgenic swine

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360\ub0 phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS

Modeling amyotrophic lateral sclerosis in hSOD1 transgenic swine

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS

Amyotrophic lateral sclerosis (ALS) swine models: Production and preliminary characterization

Introduction. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. As employment of transgenic SOD1 rodent models in ALS research didn’t result in an improvement of patient prognosis, another model, more closely related to human species, is strongly demanded by the scientific community. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic blastocysts and swine carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces patients phenotype progression. Materials and Methods. SCNT blastocysts on fifth day of development were transplanted by midventral laparatomy to the synchronized sows uterus. A cesarean delivery was performed at the 114th day of gestation. In order to achieve a preliminary characterization of our swine model, tissue banking was performed on stillborn piglets and on animals that died soon after birth. Immunocytochemistry on ear biopsy fibroblasts and western blot on homogenized snap-shot frozen tissues (rabbit policlonal antibody 07–403 Millipore, concentration 1:200 and 1:1000 respectively) were performed. To assess SOD1G93A deposition pattern Immunohistochemistry (rabbit policlonal antibody GTX 100659; 1:250) and Immunofluorescence (GTX 100659; 1:250 and NeuN MAB377; 1:1000) were employed on FFPE tissues. Genomic SOD1G93A swine DNA digested by SalI+BglII (10 U/μg DNA) was hybridized with SOD-DIG probes (20 ng/ml) to assess transgene integrations number by Southern Blot. Results and Conclusions. The transfer of 638 embryos to eight recipient sows resulted in four pregnancies and in the birth of 16 vital and 12 stillborn piglets (mean blastocyst development to term efficacy, 8.78%). Five animals developed normally while the remaining piglets died due to events commonly reported in commercial herds. The transgenic protein expression was confirmed by both immunocytochemistry and western blot. Furthermore Southern blot revealed a transgene integration number ranging from 1 to about 6 copies. IHC demonstrated granular mutant protein aggregates in both perikarya and neurites of neurons (nucleus labeled with NeuN in immunofluorescence experiment) in brain (from area hypothalamica lateralis to the third ventricle) and in spinal cord neurites. Despite these encouraging results, further molecular and pathological investigations are required since data have been obtained in stillborn or extremely young animals. A detailed phenotypical characterization, adapting to pig currently employed human diagnostic devices, is in progress on adult living swin

Generation of pre-implantation pig SCNT embryos harboring the amyotrophic lateral sclerosis related hSOD1G93A gene

Introduction. ALS is a fatal neurodegenerative disease that occurs in two forms: sporadic and familial, the latter linked to mutations in the SOD1 gene. Pathogenic hypotheses mainly rely on transgenic rodent models however doubts have been raised about their suitability to faithfully reproduce the human disease. Therefore, a more suitable model is strongly demanded to provide a better tool to study the disease and to facilitate the preclinical findings extrapolation. Swine model plays an emerging role in biomedical research as its anatomical, physiological and biochemical features are more closely related to human species. On this basis, our group produced, by Genetic Engineering and SCNT, transgenic swine blastocysts carrying the hSOD1G93A mutation, which is the most frequently studied in rodents, since it reproduces the ALS patients phenotype progression. Material and Methods. Using the Multisite Gateway System (Invitrogen) we obtained the \u201cEntryClone\u201d pENTRL1L2- hSODG93A, containing the cDNA coding the hSOD1G93A gene whose open reading frame was confirmed by sequencing, and the \u201cDestinationVector\u201d pMGOrfA5\ua23\u2019MARpuro5171. The exchange reaction between the \u201cDestinationVector\u201d and the \u201cEntryClone\u201d was mediated by LR Clonase-Invitrogen, and was used to transform chemically competent E.coli cells (OneShotMatch1-Invitrogen). The resulting \u201cExpressionVector\u201d pMG5\ua23\u2019MARPuro-hSODG93A was purified and analyzed. The same construct was successfully used to develop an ubiquitous EGFP expression vector that maintains high expression level through the next generation of pigs. This vector carries the pCAGGS hybrid promoter (CMV-IE enhancer + chicken \u3b2-actin promoter) inserted between two insulators (5\u2032MAR of chicken lysozyme gene) to prevent positional or copy number silencing effects. 5 \ub5g of linearized vector were used to transfect 1 7 106 Pig Adult Fibroblast (PAF) cultured in DMEM/ M199[1:1] + 10%FCS+1% \u3b2-FGF by Nucleofector (Amaxa Biosystem). Transgene expression was detected by ICC using an anti-hSOD1 antibody ([1:200]07\u2013403 Millipore) to select PAF colonies to be employed as donor cells. The endogenous expression level was evaluated in Huvec cells. SCNT-embryos were reconstructed following a zona-free method, as described previously. Transgenic hSOD1G93A-embryos were finally grown in vitro to assess morphology, development and survival. Results. PAFs showed a transgene expression level ranging from lower (about 0.5 times) to higher (about 3\u20134 times) than the endogenous one. Thirteen hSOD1G93A-PAF colonies carrying the complete range of expression levels were employed in eight SCNT experiments. A total of 1,630 oocytes were enucleated and 1430 SCNT-embryos were obtained. The average viable embryos percentage was 39.16% of the total manipulated oocytes. These results, consistent with those obtained from similar experiments conducted with other transgenes, have ruled out the hSOD1G93A lethality in the pre-implantation embryonic phases, regardless of its expression level. Conclusion. On the basis of these results the hSOD1G93Ablastocysts can be generated by SCNT. The next step will be to implant them in recipient sows to generate offspring. Taking into account the similarity in size and physiology of neuromuscolar system, a swine ALS model may represent a more suitable model in reproducing the human disease than current transgenic rodents