Therapeutic drug and immunogenicity monitoring (TDIM) is increasingly proposed to guide therapy with biologics, characterised by high inter-individual variability of their blood levels, to permit ...objective decisions for the management of non-responders and reduce unnecessary interventions with these expensive treatments. However, TDIM has not yet entered clinical practice partly because of uncertainties regarding the accuracy and precision of enzyme-linked immunosorbent assays (ELISA). Here we report the characterisation of a novel surface plasmon resonance (SPR)-based TDIM, applied to the measurement of serum concentrations of infliximab, an antibody against tumour necrosis factor α (anti-TNFα), and anti-infliximab antibodies. SPR has the obvious advantages of directly detecting and measuring serum antibodies in minutes, avoiding the long incubation/separation/washing/detection steps of the methods proposed so far, reducing complexity and variability. Moreover, drug and anti-drug antibodies can be measured simultaneously. This new method was validated for sensitivity and reproducibility, and showed cost-effectiveness over commercial ELISA kits. This method may be applied to other biotherapeutics. These data pave the way for the development of SPR-based point-of-care devices for rapid on-site analysis.
Since many years it has been acknowledged that some bacterial species, among which H. pylori, P. aeruginosa, P. acnes accumulate endogenous photosensitizers (PS) in the form of porphyrins. This makes ...antibacterial photodynamic therapy (PDT) easier to perform due to the possible avoidance of external PS. In this study, we focus on gastric infections associated with the presence of Helicobacter pylori (H. pylori), known to accumulate and release both protoporphyrin IX (PPIX) and coproporphyrins. PDT versus H. pylori can be carried out by modified endoscopes or by new ingestible luminous devices under development.
In both cases of in vitro and in vivo applications, either for therapy (PDT) or diagnosis, scientific literature lacks studies on the possible side-effects of light treatments on the surrounding tissues. To this aim we evaluated in vitro side-effects due to a possible intrinsic photosensitivity of gastric mucosa or to a photosensitization by the PS released from the bacterium itself. Photo-toxicity studies were conducted on the AGS cell line (ATCC® CRL-1739™), commonly used as a model for the stomach mucosa tissue, considering PPIX as the photosensitizing agent. After first evaluations of PPIX dark toxicity, its uptake and accumulation sites, photo-toxicity tests were conducted using a LED light source peaked at 400 nm, by varying both PPIX concentration (50 nM – 2 μM) and light dose in the range 0.6–13 J/cm2, representing different treatment procedures found in literature. The oxidative stress consequent to irradiation was investigated both in terms of ROS production and assessment of the activity of enzymes involved in ROS-related biological mechanisms. A significant phototoxic effect was found only for PPIX concentration > 100 nM for all tested light doses. This indicates that the evaluated photo-treatments do not cause side effects even with the sensitization due to PPIX released by the bacteria.
•Side effects of therapeutic/diagnostic stomach illumination are poorly investigated•Gastric mucosa can be sensitized with porphyrins released by infectant H. pylori•Phototoxicity on gastric cells, photosensitizer PPIX, 400 nm LED source, 0.6–13 J/cm2•Cells respond to oxidative stress induced by light activating enzymes as catalase•A phototoxic effect was found only for high PPIX concentrations for all light doses
Helicobacter pylori (H. pylori) is a Gram-negative pathogen bacterium affecting the mucosa of the stomach and causing severe gastric diseases. H. pylori-related infections are currently treated with ...pharmacological therapies, which are associated with increasing antibiotic resistance and consequent reduction of the efficacy down to 70%-85%. Moreover, drugs have generally side effects that further affect the healthcare system in terms of additional financial and medical efforts. The aim of this study is to present an innovative device for the treatment of H. pylori infection, consisting of an ingestible lighting capsule performing photodynamic therapy by means of light at specific wavelengths. The proposed treatment is minimally invasive and the described system can be considered the first photodynamic swallowable device ever proposed. Preliminary experiments demonstrated that the capsule integrated with LED sources can provide the required lighting power to kill the bacterium with an efficiency up to about 96%.
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.
Graphical abstract
The objective of this study was to investigate the possible synergy between doxycycline and photodynamic therapy against
and to evaluate the possible side effects on adenocarcinoma gastric cells with ...and without protoporphyrin IX.
Three
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.
The photodynamic therapy and doxycycline combination showed an antibacterial synergistic effect with no significant toxicities.
The synergistic treatment could be considered as an interesting therapeutic option.
Macrophage migration inhibitory factor (MIF) is an important proinflammatory cytokine involved in regulation of macrophage function. In addition, MIF may also play a role in murine and human ...reproduction. Although both first trimester trophoblast and decidua express MIF, the regulation and functional significance of this cytokine during human placental development remains unclear. We assessed MIF expression throughout normal human placental development, as well as in in vitro (chorionic villous explants) and in vivo (high altitude placentae) models of human placental hypoxia. Dimethyloxalylglycine (DMOG), which stabilizes hypoxia inducible factor-1 under normoxic conditions, was also used to mimic the effects of hypoxia on MIF expression. Quantitative real-time PCR and Western blot analysis showed high MIF protein and mRNA expression at 7-10 wk and lower levels at 11-12 wk until term. Exposure of villous explants to 3% O(2) resulted in increased MIF expression and secretion relative to standard conditions (20% O(2)). DMOG treatment under 20% O(2) increased MIF expression. In situ hybridization and immunohistochemistry showed elevated MIF expression in low oxygen-induced extravillous trophoblast cells. Finally, a significant increase in MIF transcript was observed in placental tissues from high-altitude pregnancies. Hence, three experimental models of placental hypoxia (early gestation, DMOG treatment, and high altitude) converge in stimulating increased MIF, supporting the conclusion that placental-derived MIF is an oxygen-responsive cytokine highly expressed in physiological in vivo and in in vitro low oxygen conditions.
Introduction In the framework of the growing rate of antibiotic-resistance, new therapeutic solutions are being considered against bacterial infections, among which photodynamic therapy (PDT) is a ...very attractive perspective. In recent years, innovative solutions for endoscopic illumination have been studied, e.g. for catheter infections, pancreas tumours and stomach infections by Helicobacter pylori (Hp). Hp colonizes the human stomach with a worldwide infection prevalence exceeding 50%, besides being a class 1 carcinogen agent (World Health Organization). Currently, Hp infection is treated with pharmacologic therapies, showing high failure rates mainly due to antibiotic resistance. To overcome this limitation, endoscopic PDT devices have been tested. However, they exhibit clear disadvantages, namely great invasivity and adverse effects. Purpose To design and characterize a non-invasive light-emitting device to perform Hp phototherapy: an ingestible “LED antibiotic”, exploiting the presence of endogenous Hp photosensitizers. Materials and methods Merging experimental and simulation methods we obtain the light action spectrum for Hp phototherapy in the gastric environment. We have designed and assembled the pill containing LED sources, a battery and electronic board. Capsule emission parameters are measured by an integrating sphere and in vitro Hp irradiation performed. Results The action spectrum for Hp phototherapy is peaked in the violet and red spectrum regions. Capsule prototypes show emission parameters compatible with in vitro Hp eradication (>99% killing). Conclusion Our device has shown emission spectrum, intensity and duration compatible with an effective phototherapy of Hp , considering also the treatment repeatability (5–10 capsules in 2–3 weeks). Future clinical trials are envisaged. Disclosure GR, GT, BO and FF disclose being also Probiomedica srl ( www.probiomedica.com ).
Recent evidence suggests that interleukin-4 (IL-4) is related to mucosal tolerance by which an injurious immune response is prevented, suppressed or shifted to a non-injurious response. We ...investigated the expression of IL-4 and its splice variant isoform IL-4δ2 in gastric epithelial cells of healthy subjects and gastritis patients infected with Helicobacter pylori (H. pylori) with or without the cag pathogenicity island (cag-PAI). IL-4 and IL-4δ2 mRNAs were evaluated in microdissected gastric epithelium and in AGS cell lines co-cultured with H. pylori B128 or SSI strains. IL-4 mRNA was consistently detected in microdissected gastric epithelial cells from healthy subjects. The IL-4 mRNA expression was low in H. pylori-infected patients, and markedly reduced in cag-PAI-positive ones. IL-4δ2 mRNA was expressed on gastric epithelium of H. pylori-infected patients, but not in healthy subjects. The IL-452 expression was lower in cag-PAI-positive than in cag-PAI-negative H. pylori infected patients. AGS cells also produced IL-4 mRNA upon SSI strain stimulation, whereas IL-4δ2 mRNA expression was detected in AGS co-cultured with either SSI or B128 strains. An inverse correlation was documented between IL-4 and IL-482 mRNA expression by microdissected gastric epithelial cells and the score of gastritis. IL-4, but not IL-452, is expressed by gastric epithelium of healthy subjects, whereas IL-452 and lesser IL-4 mRNA are detectable in the gastric epithelium of H. pylori-infected patients. Data suggest that gastric epithelial cells might regulate the balance between tolerance and immune response by the fine tuning of IL-4 and IL-4δ2 expression.
Interleukin-4 (IL-4) and IL-4delta2 mRNA gastric expression was evaluated in healthy subjects and patients who did not have ulcers but were infected with Helicobacter pylori with or without the cag ...pathogenicity island (cag PAI). IL-4 mRNA was physiologically expressed by gastric epithelium and negatively influenced by H. pylori. Also, nonepithelial cells in the lamina propria of H. pylori-infected patients expressed IL-4 mRNA, whereas IL-4delta2 mRNA was found only in cag PAI-negative patients. Thus, gastric IL-4 takes part in the local immune response to H. pylori.
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