Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Regression of subcutaneous B16 melanoma tumors after intratumoral delivery of an IL-15-expressing plasmid followed by in vivo electroporation.

In vivo electroporation has been used to efficiently deliver drugs and 'therapeutic' genes to tumors, including melanoma lesions. This study reports on the effect of intratumoral delivery of an optimized DNA plasmid expressing interleukin-15 (pIL-15) on established murine melanoma tumors. IL-15 has been demonstrated to have a pivotal role in the function of memory CD8+ T cells and natural killer cells, which are critical for tumor immunosurveillance. In this study, C57BL/6 mice were injected with B16.F10 melanoma cells and randomized into different experimental groups: untreated (P-V-E-), treated with pIL-15 (P+) or backbone plasmid (V+), with or without electroporation (E+ or E-). Treatment was performed intratumorally with 50 microg of plasmid on days 0, 4 and 7 and tumor volume/size, tumor regression and long-term survival were measured. At day 100 after initiation of treatment, the percentage of mice surviving with complete tumor regression in the P-V+E+, P+V-E-, P+V-E+ and P-V-E- treatment groups were 0, 12.5, 37.5 and 0%, respectively. These results demonstrate the ability of pIL-15 to mediate B16 melanoma regression, with the effect being significantly enhanced by electroporative delivery. This is the first description of the ability of a naked DNA plasmid expressing IL-15 to alone mediate complete regression of B16 melanoma tumors and underscores the potential clinical use of these plasmids for the treatment of malignant tumors when delivered with in vivo electroporation.

Intestinal colonization of the infant mouse model by attenuated and virulent Vibrio cholerae strains.

BACKGROUND: Intestinal colonization of humans with virulent Vibrio cholerae stimulates substantial, lasting immunity against reinfection. The purpose of this study was to evaluate the colonizing capability of various Vibrio cholerae strains which are promising candidates to oral vaccine. METHODS: Infant mouse model modification was used. In order to standardize the method, several parameters were tested, such as culture medium and optimal time of incubation and appropriate number of cells to be inoculated. The following were tested: Vibrio cholerae strain 81, 413, and 251A, which were obtained at the Molecular Biology Department of the National Center for Scientific Research, Havana, Cuba. Their virulence cassettes which code for the main virulence factors were deleted. RESULTS: Good variance coefficient (VC) was obtained in repeated experiments. The colonizing properties of attenuated Vibrio cholerae strains evaluated by this method correlated well with those observed for parental strains. CONCLUSIONS: Genetically attenuated Vibrio cholera strains have the same intestinal colonization level as their parental strains in the infant mouse model; thus, genetic manipulation does not affect genes that encode for the synthesis of colonization factors.

Evaluation of metal concentrations in mentha herbal teas (Mentha piperita, Mentha pulegium and Mentha species) by inductively coupled plasma spectrometry.

Phytopharmaceuticals are gaining popularity worldwide; however, cases of adverse effects and drug interactions have also increased. One reason is in the high metal content both as ingredients but also as contaminants. Metal monitoring in food, like herbal teas, provides basic information on safety aspects in regulatory processes as well as nutritional values. In the present work, Cd, Pb, K, Na, Ca, Mg, Al, B, Ba, Co, Cr, Cu, Fe, Mn, Zn, Li, Ni, and Mo were determined by inductively coupled plasma spectrometry (ICPS) in 36 samples of Mentha sp. Mint tea bags and loose leaves were randomly obtained from supermarkets, traditional markets, herbal stores, and pharmacies in Tenerife (Canary Islands, Spain). Metal contents varied significantly, dependent on the stores the products were purchased in and on tea packaging (loose leaves versus tea bags). Pb analyses revealed levels (0.65±0.71mg/kg) below legal limits. The maximum permissible limit for Cd, 0.3mg/kg, set by the WHO for medicinal plants, was exceeded by 19.44% of the samples (0.22±0.13mg/kg), but all values were below the limit given in the European Pharmacopoeia for this metal (4mg/kg). We observed high Al (151.24±162.73mg/kg) and Li (5.46±3.94mg/kg) levels. B, Ba, Co, Cr, Cu, Fe, Mn, Ni, Zn, and Mo mean levels were 20.51, 14.15, 0.26, 1.65, 10.65, 406.00, 55.05, 1.72, 33.67, and 0.73mg/kg, respectively. Mean Ca, Mg, K, and Na were detected in concentrations of 10.32, 3.83, 7.23 and 1.17g/kg, respectively. In conclusion, metal exposure through herbal mint teas does not seem to be of health concern, as to most of the studied metals, but regulatory limits for Al contents should be imposed.

[Standardization and evaluation of the modified vibriocidal assay]. / Estandarización y evaluación del ensayo vibriocida modificado.

The vibriocidal antibody test is a reliable and well-documented method to determine bacterial antibodies to Vibrio cholerae 01 antigens. It consists of mixing serum dilutions and a steady quantity of bacteria and supplement to cause cell lysis. Titer is determined by visual observation. In this paper, we implemented a change in the presented method where a pH and glucose indicator was added to the culture medium used to stop the reaction, which allowed a quicker reading by any person who are not very familiar with the carrying out of this test since the colour change in the plaque is quite evident.

[Treatment of esophageal stenosis with Savary-Gilliard balloons in children]. / Tratamiento con las bujias de Savary-Gilliard en las estenosis esofagicas del niño.

30 patients (less than 15 years old) were admitted for esophageal strictures, 16 of them secondary to corrosive injury. All the patients were treated with endoscopic dilation with Savary Gilliard bougie. The dilatation were done with general anesthesia using an Olympus GIF-XP10 endoscope and with fluoroscopic control. In the esophageal stenosis secondary to caustic ingestion endoscopic injection with Betamethasone was also used. The most frequent site of the stenosis was the upper third of the esophagus, and the main type of stenosis was tubular in the secondary to caustic burns and annular in the other group. In the posteaustic group 385 dilatations were performed in 115 sessions. Two perforations and one sepsis were reported in patients with corrosive stenosis. There was no mortality. 43.7% of the patients with corrosive stenosis and 85.7% with stenosis secondary to other causes obtained complete healing. Oesophageal dilatation with Savary-Gilliary bougies represents a safe and reliable method for the treatment of esophageal strictures.

[The excretion pattern and protective capacity in rabbits orally immunized with attenuated strains of Vibrio cholerae O1 biotype El Tor]. / Estudio del patrón de excreción y la capacidad protectora en conejos inmunizados de forma oral con cepas atenuadas de Vibrio cholerae O1 biotipo El Tor.

In order to study the excretion patterns, colonization and protective capacity of live attenuated strains of Vibrio cholerae O1. El Tor, rabbits were immunized in New Zealand with these strains and their corresponding parental strains. 2 doses were administered by the model of oral inoculation in adult rabbits. Rabbits were rotated 2 weeks after the second dose by the model of ligated intestine with highly virulent strains of V. cholerae O1 Ogawa and Inaba serotypes and O139 serogroup. It was proved that the genetically manipulated strains do not effect the excretion patterns when they are compared with their parental strains. It was observed in the challenge a decrease in the levels of colonization of virulent strains of both serotypes, not only among the rabbits immunized with the attenuated strains, but also among those immunized with the parental strains in comparison with control animals immunized with the strain of Escherichia coli K-12, which means that there was certain degree of protection. In the case of the animals challenged with the O139 strain it was demonstrated that the protection is specific for each serogroup, since in this case there was no reduction of the colonization.