Ingestible light source for intragastric antibacterial phototherapy: a device safety study on a minipig model.

Helicobacter pylori gastric infections are among the most diffused worldwide, suffering from a rising rate of antibiotic resistance. In this context, some of the authors have previously designed an ingestible device in the form of a luminous capsule to perform antibacterial photodynamic inactivation in the stomach. In this study, the light-emitting capsules were tested to verify the safety of use prior to perform clinical efficacy studies. First, laboratory tests measured the capsule temperature while in function and verified its chemical resistance in conditions mimicking the gastric and gut environments. Second, safety tests in a healthy minipig model were designed and completed, to verify both the capsule integrity and the absence of side effects, associated with its illumination and transit throughout the gastrointestinal tract. To this aim, a capsule administration protocol was defined considering a total of 6 animals with n = 2 treated with 8 capsules, n = 2 treated with 16 capsules and n = 2 controls with no capsule administration. Endoscopies were performed in sedated conditions before-after every capsule administration. Biopsies were taken from the corpus and antrum regions, while the gastric cavity temperature was monitored during illumination. The bench tests confirmed a very good chemical resistance and a moderate (about 3 °C) heating of the capsules. The animal trials showed no significant effects on the gastric wall tissues, both visually and histologically, accompanied with overall good animal tolerance to the treatment. The integrity of the administered capsules was verified as well. These encouraging results pose the basis for the definition of successive trials at the clinical level.

Unraveling the metabolism of Mycobacterium caprae using comparative genomics.

In our laboratory, Mycobacterium caprae has poor growth in standard medium (SM) 7H9-OADC supplemented with pyruvate and Tween-80. Our objectives were to identify mutations affecting M. caprae metabolism and use this information to design a culture medium to improve its growth. We selected 77 M. caprae genomes and sequenced M. caprae NLA000201913 used in our experiments. Mutations present in >95% of the strains compared to Mycobacterium tuberculosis H37Rv were analyzed in silico for their deleterious effects on proteins of metabolic pathways. Apart from the known defect in the pyruvate kinase, M. caprae has important lesions in enzymes of the TCA cycle, methylmalonyl cycle, B12 metabolism, and electron-transport chain. We provide evidence of enzymatic redundancy elimination and epistatic mutations, and possible production of toxic metabolites hindering M. caprae growth in vitro. A newly designed SM supplemented with l-glutamate allowed faster growth and increased final microbial mass of M. caprae. However, possible accumulation of metabolic waste-products and/or nutritional limitations halted M. caprae growth prior to a M. tuberculosis-like stationary phase. Our findings suggest that M. caprae relies on GABA and/or glyoxylate shunts for in vitro growth in routine media. The newly developed medium will improve experiments with this bacterium by allowing faster growth in vitro.

Innovative light sources for phototherapy.

The use of light for therapeutic purposes dates back to ancient Egypt, where the sun itself was an innovative source, probably used for the first time to heal skin diseases. Since then, technical innovation and advancement in medical sciences have produced newer and more sophisticated solutions for light-emitting sources and their applications in medicine. Starting from a brief historical introduction, the concept of innovation in light sources is discussed and analysed, first from a technical point of view and then in the light of their fitness to improve existing therapeutic protocols or propose new ones. If it is true that a "pure" technical advancement is a good reason for innovation, only a sub-system of those advancements is innovative for phototherapy. To illustrate this concept, the most representative examples of innovative light sources are presented and discussed, both from a technical point of view and from the perspective of their diffusion and applications in the clinical field.

Nicotinamide-Rich Diet in DBA/2J Mice Preserves Retinal Ganglion Cell Metabolic Function as Assessed by PERG Adaptation to Flicker.

Flickering light increases metabolic demand in the inner retina. Flicker may exacerbate defective mitochondrial function in glaucoma, which will be reflected in the pattern electroretinogram (PERG), a sensitive test of retinal ganglion cell (RGC) function. We tested whether flicker altered the PERG of DBA/2J (D2) glaucomatous mice and whether vitamin B3-rich diet contributed to the flicker effect. D2 mice fed with either standard chow (control, n = 10) or chow/water enriched with nicotinamide (NAM, 2000 mg/kg per day) (treated, n = 10) were monitored from 3 to 12 months. The PERG was recorded with superimposed flicker (F-PERG) at either 101 Hz (baseline) or 11 Hz (test), and baseline-test amplitude difference (adaptation) evaluated. At endpoint, flat-mounted retinas were immunostained (RBPMS and mito-tracker). F-PERG adaptation was 41% in 3-month-old D2 and decreased with age more in control D2 than in NAM-fed D2 (GEE, p < 0.01). At the endpoint, F-PERG adaptation was 0% in control D2 and 17.5% in NAM-fed D2, together with higher RGC density (2.4×), larger RGC soma size (2×), and greater intensity of mitochondrial staining (3.75×). F-PERG adaptation may provide a non-invasive tool to assess RGC autoregulation in response to increased metabolic demand and test the effect of dietary/pharmacological treatments on optic nerve disorders.

Synergistic effect of photodynamic therapy at 400 nm and doxycycline against Helicobacter pylori.

Aim: The objective of this study was to investigate the possible synergy between doxycycline and photodynamic therapy against Helicobacter pylori and to evaluate the possible side effects on adenocarcinoma gastric cells with and without protoporphyrin IX. Materials & methods: Three H. pylori strains (ATCC 700392, 43504 and 49503) were grown on solid medium either with, or without, doxycycline at subinhibitory concentrations, and irradiated for 10, 20 and 30 minutes with a 400 nm-peaked light source. The phototoxicity tests on AGS cells were evaluated by MTT assay. Results: The photodynamic therapy and doxycycline combination showed an antibacterial synergistic effect with no significant toxicities. Conclusion: The synergistic treatment could be considered as an interesting therapeutic option.

Two-stage Turnbull-Cutait pull-through coloanal anastomosis versus coloanal anastomosis with protective loop ileostomy for low rectal cancer. Protocol for a randomized controlled trial (Turnbull-BCN).

PURPOSE: The aim of this study was to determine whether patients that underwent ultra-low rectal resection for cancer can benefit from the recently reintroduced two-stage Turnbull-Cutait abdominoperineal pull-through procedure. METHODS: Patients with low rectal tumors undergoing radical sphincter-sparing resection are eligible for inclusion in a randomized multicenter study. Whether two-stage Turnbull-Cutait coloanal anastomosis provides significant benefits over hand-sewn coloanal anastomosis and associated lateral ileostomy in terms of postoperative morbidity is the primary endpoint. In addition, the study aims to assess secondary endpoints such as quality of life, fecal incontinence, and locoregional recurrence of the neoplasm. Patients with adenocarcinoma of the lower rectum diagnosed by rigid proctoscopy, with histological confirmation of malignancy, and who are candidates of rectal removal and coloanal anastomosis will be included in a randomized controlled and multicenter trial. Postoperative morbidity is defined as complications that occur within 30 days of the data of the second surgical procedure of the last patient included in the trial. Patients will be followed for a minimum period of 3 years. CONCLUSIONS: The two-stage Turnbull-Cutait coloanal anastomosis may constitute an effective surgical alternative in the current approach to the treatment of low rectal cancer without the need of a temporary loop colostomy, preventing the wide range of complications related to stoma surgery. TRIAL REGISTRATION: This trial is registered at clinicaltrials.gov (trial number: NCT01766661). This trial is registered in January 10, 2013.

Critical role of bevacizumab scheduling in combination with pre-surgical chemo-radiotherapy in MRI-defined high-risk locally advanced rectal cancer: Results of the BRANCH trial.

BACKGROUND: We have previously shown that an intensified preoperative regimen including oxaliplatin plus raltitrexed and 5-fluorouracil/folinic acid (OXATOM/FUFA) during preoperative pelvic radiotherapy produced promising results in locally advanced rectal cancer (LARC). Preclinical evidence suggests that the scheduling of bevacizumab may be crucial to optimize its combination with chemo-radiotherapy. PATIENTS AND METHODS: This non-randomized, non-comparative, phase II study was conducted in MRI-defined high-risk LARC. Patients received three biweekly cycles of OXATOM/FUFA during RT. Bevacizumab was given 2 weeks before the start of chemo-radiotherapy, and on the same day of chemotherapy for 3 cycles (concomitant-schedule A) or 4 days prior to the first and second cycle of chemotherapy (sequential-schedule B). Primary end point was pathological complete tumor regression (TRG1) rate. RESULTS: The accrual for the concomitant-schedule was early terminated because the number of TRG1 (2 out of 16 patients) was statistically inconsistent with the hypothesis of activity (30%) to be tested. Conversely, the endpoint was reached with the sequential-schedule and the final TRG1 rate among 46 enrolled patients was 50% (95% CI 35%-65%). Neutropenia was the most common grade ≥ 3 toxicity with both schedules, but it was less pronounced with the sequential than concomitant-schedule (30% vs. 44%). Postoperative complications occurred in 8/15 (53%) and 13/46 (28%) patients in schedule A and B, respectively. At 5 year follow-up the probability of PFS and OS was 80% (95%CI, 66%-89%) and 85% (95%CI, 69%-93%), respectively, for the sequential-schedule. CONCLUSIONS: These results highlights the relevance of bevacizumab scheduling to optimize its combination with preoperative chemo-radiotherapy in the management of LARC.

In vitro assessment of antibody-conjugated gold nanorods for systemic injections.

BACKGROUND: The interest for gold nanorods in biomedical optics is driven by their intense absorbance of near infrared light, their biocompatibility and their potential to reach tumors after systemic administration. Examples of applications include the photoacoustic imaging and the photothermal ablation of cancer. In spite of great current efforts, the selective delivery of gold nanorods to tumors through the bloodstream remains a formidable challenge. Their bio-conjugation with targeting units, and in particular with antibodies, is perceived as a hopeful solution, but the complexity of living organisms complicates the identification of possible obstacles along the way to tumors. RESULTS: Here, we present a new model of gold nanorods conjugated with anti-cancer antigen 125 (CA125) antibodies, which exhibit high specificity for ovarian cancer cells. We implement a battery of tests in vitro, in order to simulate major nuisances and predict the feasibility of these particles for intravenous injections. We show that parameters like the competition of free CA125 in the bloodstream, which could saturate the probe before arriving at the tumors, the matrix effect and the interference with erythrocytes and phagocytes are uncritical. CONCLUSIONS: Although some deterioration is detectable, anti-CA125-conjugated gold nanorods retain their functional features after interaction with blood tissue and so represent a powerful candidate to hit ovarian cancer cells.

Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis.

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

Long-term functional assessment of antegrade colonic enema for combined incontinence and constipation using a modified Marsh and Kiff technique.

PURPOSE: Constipation and fecal incontinence can severely affect quality of life for patients, particularly when simultaneously present. Malone antegrade colonic enema enables periodic colonic emptying, thus preventing uncontrolled passage of feces and constipation. METHODS: Eleven patients with fecal incontinence and severe constipation or perineal colostomy after Miles' operation underwent a modified Marsh and Kiff ileostomy for antegrade colonic enema. Before and after surgery, the patients were fully evaluated for gastrointestinal functions, including gallbladder and stomach emptying time, H(2)-breath test, colonic transit time, dynamic defecography, and anorectal manometry. The severity of incontinence and constipation was scored preoperatively and postoperatively by using the American Medical System score and Cleveland Clinic Constipation scale, respectively, whereas the quality of life was measured by the Gastrointestinal Quality of Life Index. The surgical technique involved division of the terminal ileum 10 to 15 cm from the ileocecal valve, anastomosis and intussusception of the ileum with the cecum, narrowing of the ileal conduit with a linear stapler, and a small, introflexed ileostomy with an advanced skin flap. RESULTS: During the postoperative period, the mean American Medical System score decreased significantly from 77 to 11 (P<0.01) and the mean Cleveland Clinic Constipation score from 23 to 8.5 (P<0.01) with a significant improvement of quality of life. Antegrade colonic enema did not affect gallbladder, gastric, or orocecal transit time, which remained comparable with baseline. Colonic scintigraphy showed that antegrade colonic enema was efficient to clean the whole colon and rectum, leaving only 24 (range, 6-40) percent of the initial radioactivity after 30 minutes. Ileal manometry confirmed the presence of a high-pressure zone, preventing accidental reflux. CONCLUSIONS: Modified Marsh and Kiff technique is a safe and effective surgical option to treat patients with combined fecal incontinence and severe constipation and those with perineal colostomy after Miles. It should be recommended as a last option before colostomy.