Induction of human papillomavirus oncogene-specific CD8 T-cell effector responses in the genital mucosa of vaccinated mice.

Cervical cancer, the second leading cause of cancer mortality in women worldwide, results from infection with a subset of human papillomaviruses (HPV), HPV-16 being the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent this cancer in the long term. However, they only target 70-80% of all cervical cancers and cannot control existing HPV infections and associated lesions. Therapeutic vaccines are thus necessary for women who cannot benefit from prophylactic vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines. We report here that mice that received a single subcutaneous (s.c.) vaccination of an adjuvanted long synthetic HPV16 E7(1-98) polypeptide showed induction of 100% tumor protection against s.c. TC-1 tumors and that tumor regression was mainly provided by CD8 T cells. In vivo cytotoxic assay revealed high E7-specific cytolytic T lymphocytes activity in spleen and in genital draining lymph nodes (LN), and E7-specific CD8 T cells could be detected in GM by tetramer staining. More importantly, high-avidity E7-specific INF-gamma secreting CD8 T cells were induced not only in blood, spleen and LN but also in GM of vaccinated mice, thus providing evidence that a parenteral vaccination may be sufficient to provide regression of genital tumors. In addition, there was no correlation between the responses measured in blood with those measured in GM, highlighting the necessity and relevance to determine the immune responses in the mucosa where HPV-tumors reside.

Technological, biopharmaceutical and pharmacokinetic advances: new formulations of application on the skin and oral mucosa

Currently a growing interest to improve the pharmacological therapy exists, not only by the production and the appearance of new drugs, but guaranteeing that the uses of those which already exist, become more effective. In fact, the conventional pharmaceutical formulations of different drugs present a few secondary effects due to oral administration. In order to avoid these undesired side effects, the purpose of current therapeutic is the development and research of formulations as an alternative to others routes of administration. Therefore, in spite of the undoubtedly complete parenteral absorption, the transdermal and transbuccal routes appear to be a rather attractive alternative to provide an efficient absorption. In this chapter a new technological, biopharmaceutical and pharmacokinetic approach of strategies for application on skin and buccal mucosa are reported. In the future new transdermal drug delivery systems will emerge to be more effective, equipped with an improved aesthetic appearance, better adherence and greater diffusion. But to reach these aims, it is necessary previous knowledge of histology and physiology of skin, and factors involved in the penetration of drugs through it.

APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa.

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Electrical colonic stimultation reduces mean transit time in a porcine model

Rapport de synthèse : Introduction : La stimulation électrique représente une nouvelle modalité thérapeutique de divers troubles digestifs. Dans la constipation par exemple, le péristaltisme colique peut être activé par un système électrique alimenté par une batterie. La présente étude a pour but de démontrer l'impact d'une stimulation électrique directe du côlon sur le temps de transit moyen, en utilisant un modèle expérimental chronique porcin. L'effet de la stimulation et du matériel implanté dans la paroi colique est également évalué. Matériel et méthode : Trois paires d'électrodes ont été implantées dans la paroi cæcale de douze porcs anesthésiés. Avant implantation, un temps de transit colique de référence a été déterminé chez chaque animal par utilisation de marqueurs radio-opaques. Cette évaluation a été répétée quatre semaines après implantation, sous stimulation factice, et cinq semaines après implantation, sous stimulation électrique. Des trains séquentiels et aboraux de stimulation (10 V ; 120 Hz ; 1 ms) ont été appliqués quotidiennement durant six jours, en utilisant un stimulateur externe fonctionnant sur batteries. Pour chaque série de marqueurs, une valeur moyenne a été calculée à partir du temps de transit individuel des porcs. Un examen microscopique du cæcum a été systématiquement entrepris après sacrifice des animaux. Résultats : Une réduction du temps de transit moyen a été observée après stimulation électrique (19h ± 13 ; moyenne ± DS), comparativement au temps de référence (34h ± 7 ; p=0.045) et au temps de transit après stimulation factice (36h ± 9 ; p=0.035). L'examen histologique a montré la présence d'une inflammation chronique minime, autour des électrodes. Conclusion : Le temps de transit colique porcin peut être réduit, en conditions expérimentales chroniques, par une stimulation électrique directe et séquentielle de l'intestin. Des lésions tissulaires limitées ont été occasionnées par la stimulation ou le matériel implanté. La stimulation électrique colique représente certainement une approche prometteuse du traitement de certains troubles spécifiques du côlon, avant tout fonctionnels.

Electrical colonic stimultation reduces mean transit time in a porcine model

Rapport de synthèse : Introduction : La stimulation électrique représente une nouvelle modalité thérapeutique de divers troubles digestifs. Dans la constipation par exemple, le péristaltisme colique peut être activé par un système électrique alimenté par une batterie. La présente étude a pour but de démontrer l'impact d'une stimulation électrique directe du côlon sur le temps de transit moyen, en utilisant un modèle expérimental chronique porcin. L'effet de la stimulation et du matériel implanté dans la paroi colique est également évalué. Matériel et méthode : Trois paires d'électrodes ont été implantées dans la paroi cæcale de douze porcs anesthésiés. Avant implantation, un temps de transit colique de référence a été déterminé chez chaque animal par utilisation de marqueurs radio-opaques. Cette évaluation a été répétée quatre semaines après implantation, sous stimulation factice, et cinq semaines après implantation, sous stimulation électrique. Des trains séquentiels et aboraux de stimulation (10 V ; 120 Hz ; 1 ms) ont été appliqués quotidiennement durant six jours, en utilisant un stimulateur externe fonctionnant sur batteries. Pour chaque série de marqueurs, une valeur moyenne a été calculée à partir du temps de transit individuel des porcs. Un examen microscopique du cæcum a été systématiquement entrepris après sacrifice des animaux. Résultats : Une réduction du temps de transit moyen a été observée après stimulation électrique (19h ± 13 ; moyenne ± DS), comparativement au temps de référence (34h ± 7 ; p=0.045) et au temps de transit après stimulation factice (36h ± 9 ; p=0.035). L'examen histologique a montré la présence d'une inflammation chronique minime, autour des électrodes. Conclusion : Le temps de transit colique porcin peut être réduit, en conditions expérimentales chroniques, par une stimulation électrique directe et séquentielle de l'intestin. Des lésions tissulaires limitées ont été occasionnées par la stimulation ou le matériel implanté. La stimulation électrique colique représente certainement une approche prometteuse du traitement de certains troubles spécifiques du côlon, avant tout fonctionnels.

Electrical stimulation induces propagated colonic contractions in an experimental model.

BACKGROUND: Direct colonic electrical stimulation may prove to be a treatment option for specific motility disorders such as chronic constipation. The aim of this study was to provoke colonic contractions using electrical stimulation delivered from a battery-operated device. METHODS: Electrodes were inserted into the caecal seromuscular layer of eight anaesthetized pigs. Contractions were induced by a neurostimulator (Medtronic 3625). Caecal motility was measured simultaneously by video image analysis, manometry and a technique assessing colonic transit. RESULTS: Caecal contractions were generated using 8-10 V amplitude, 1000 micros pulse width, 120 Hz frequency for 10-30 s, with an intensity of 7-15 mA. The maximal contraction strength was observed after 20-25 s. Electrical stimulation was followed by a relaxation phase of 1.5-2 min during which contractions propagated orally and aborally over at least 10 cm. Spontaneous and stimulated caecal motility values were significantly different for both intraluminal pressure (mean(s.d.) 332(124) and 463(187) mmHg respectively; P < 0.001, 42 experiments) and movement of contents (1.6(0.9) and 3.9(2.8) mm; P < 0.001, 40 experiments). CONCLUSION: Electrical stimulation modulated caecal motility, and provoked localized and propagated colonic contractions.

p53 Mutation in histologically normal mucosa of the aero-digestive tract is not a marker of increased risk for second primary carcinoma in head and neck cancer patients.

Head and neck cancer patients are at high risk for developing second primary tumors. This is known as field cancerization of the aero-digestive tract. In a previous study, we showed that patients with multiple primary tumors were more likely to have p53 mutations in histologically normal mucosae than patients presenting with an isolated tumor. Based on this observation, we postulated that p53 mutations in normal tissue samples of patients bearing a single primary tumor could have a clinical value as a biomarker for the risk of developing second primary tumors. Thirty-five patients presenting with a single primary tumor were followed-up for a median of 51 months (range 1 month to 10.9 years) after biopsies of histologically normal squamous cell mucosa had been analyzed for p53 mutations with a yeast functional assay at the time of the primary tumor. During this follow-up, recurrences and non-sterilization of the primary tumor, occurrence of lymph node metastases, and of second primary tumors were evaluated. Sixteen (45.7%) patients were found to have p53 mutations in their normal squamous cell mucosa, and 19 (54.3%) patients showed no mutation. No relationship was found between p53 mutations and the occurrence of evaluated events during follow-up. Notably, the rate of second primary tumors was not associated with p53 mutations in the normal squamous mucosa. The correlation between p53 mutations in histologically normal mucosae and the incidence of second primary tumors is generally low. The benefit of analyzing p53 mutations in samples of normal squamous cell mucosa in every patient with a primary tumor of the head and neck is doubtful.

Endoluminal surgical triangulation: overcoming challenges of colonic endoscopic submucosal dissections using a novel flexible endoscopic surgical platform: feasibility study in a porcine model.

BACKGROUND: Colonic endoscopic submucosal dissection (ESD) is challenging as a result of the limited ability of conventional endoscopic instruments to achieve traction and exposure. The aim of this study was to evaluate the feasibility of colonic ESD in a porcine model using a novel endoscopic surgical platform, the Anubiscope (Karl Storz, Tüttlingen, Germany), equipped with two working channels for surgical instruments with four degrees of freedom offering surgical triangulation. METHODS: Nine ESDs were performed by a surgeon without any ESD experience in three swine, at 25, 15, and 10 cm above the anal verge with the Anubiscope. Sixteen ESDs were performed by an experienced endoscopist in five swine using conventional endoscopic instruments. Major ESD steps included the following for both groups: scoring the area, submucosal injection of glycerol, precut, and submucosal dissection. Outcomes measured were as follows: dissection time and speed, specimen size, en bloc dissection, and complications. RESULTS: No perforations occurred in the Anubis group, while there were eight perforations (50 %) in the conventional group (p = 0.02). Complete and en bloc dissections were achieved in all cases in the Anubis group. Mean dissection time for completed cases was statistically significantly shorter in the Anubis group (32.3 ± 16.1 vs. 55.87 ± 7.66 min; p = 0.0019). Mean specimen size was higher in the conventional group (1321 ± 230 vs. 927.77 ± 229.96 mm(2); p = 0.003), but mean dissection speed was similar (35.95 ± 18.93 vs. 23.98 ± 5.02 mm(2)/min in the Anubis and conventional groups, respectively; p = 0.1). CONCLUSIONS: Colonic ESDs were feasible in pig models with the Anubiscope. This surgical endoscopic platform is promising for endoluminal surgical procedures such as ESD, as it is user-friendly, effective, and safe.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

TLR5 signaling stimulates the innate production of IL-17 and IL-22 by CD3(neg)CD127+ immune cells in spleen and mucosa.

In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3(neg)CD127(+) immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3(neg)CD127(+) cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.

Buccal mucosa graft for laryngotracheal reconstruction in severe laryngeal stenosis.

OBJECTIVE: An operative technique is described as a salvage treatment for severe subglottic and supraglottic laryngeal stenosis. In addition to expansion of the laryngeal framework with an anterior cartilage graft, as used in a classical laryngotracheal reconstruction, the scar tissue obliterating the airway lumen is excised and a mucosal graft is placed to reconstruct the inner lining of the airway. The graft is harvested from buccal mucosa. METHODS: The operative technique is outlined. Three cases, 2 paediatric and one adult, with complete or near complete laryngeal stenosis are presented where this operative technique was employed. In all patients several surgeries had been performed previously which were unsuccessful. RESULTS: In all 3 patients a patent airway was achieved with decannulation of the tracheostomy in the 2 paediatric patients. CONCLUSIONS: In patients with severe subglottic or supraglottic airway stenosis where other surgeries have failed, excision of endoluminal scar tissue and placement of a buccal mucosal graft, in addition to conventional laryngotracheal reconstruction, is a promising technique. In revision cases of subglottic stenosis cricotracheal resection might not be an option because of scarring from previous surgeries. This operation is an alternative, which allows an increase in the airway lumen by excising the scar tissue then re-lining the exposed internal lumen. The buccal mucosa reduces granulation formation and re-stenosis.

Colonic perianastomotic carcinogenesis in an experimental model

Background To examine the effect of anastomosis on experimental carcinogenesis in the colon of rats. Methods Forty-three 10-week-old male and female Sprague-Dawley rats were operated on by performing an end-to-side ileorectostomy. Group A:16 rats received no treatment. Group B: 27 rats received 18 subcutaneous injections weekly at a dose of 21 mg/kg wt of 1–2 dimethylhydrazine (DMH), from the eighth day after the intervention. Animals were sacrificed between 25–27 weeks. The number of tumours, their localization, size and microscopic characteristics were recorded. A paired chi-squared analysis was performed comparing tumoral induction in the perianastomotic zone with the rest of colon with faeces. Results No tumours appeared in the dimethylhydrazine-free group. The percentage tumoral area was greater in the perianastomotic zone compared to tumours which had developed in the rest of colon with faeces (p = 0.014). Conclusion We found a cocarcinogenic effect due to the creation of an anastomosis, when using an experimental model of colonic carcinogenesis induced by DMH in rats.

Human cytomegalovirus induced pseudotumor of upper gastrointestinal tract mucosa: Effects of long-term chronic disease?

Human cytomegalovirus-induced lesions resembling malignancies have been described in the gastrointestinal tract and include ulcerated or exophytic large masses. The aim of this study was to review the cases registered in the databases of two academic hospitals and formulate a hypothesis concerning the pathogenic mechanisms responsible for cytomegalovirus-induced pseudotumor development. All the diagnoses of human cytomegalovirus infections of the upper gastrointestinal tract recorded from 1991 to 2013 were reviewed. Cases of mucosal alterations misdiagnosed endoscopically as malignancies were selected. Large ulcers occurring in the stomach (three cases) and an irregular exophytic mass at the gastro-jejunal anastomosis were misdiagnosed endoscopically as malignancies (4 cases out of 53). Histologically, all lesions reflected hyperplastic mucosal changes with a prevalence of epithelial and stroma infected cells, without signs of cell atypia. The hypothesis presented is that the development of human cytomegalovirus-induced pseudotumors may be the morphological expression of chronic mucosa damage underlying long-term infection.