544 research outputs found
An Integrated Analytical Approach for the Characterization of Probiotic Strains in Food Supplements
Research surrounding health benefits from probiotics is becoming popular because of the increasing demand for safer products with protective and therapeutic effects. Proven benefits are species- or genus-specific; however, no certified assays are available for their characterization and quantification at the strain level in the food supplement industry. The objective of this study was to develop a strain-specific Real-time quantitative polymerase chain reaction (RT-qPCR)-based method to be implemented in routine tests for the identification and quantification of Bifidobacterium longum, Bifidobacterium animalis spp. lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus helveticus, starting from a powder mixture of food supplements. The method optimization was carried out in combination with flow cytometry to compare results between the two strategies and implement the analytical workflow with the information also regarding cell viability. These assays were validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) criteria using the plate count enumeration as the gold standard reference. Briefly, probiotic DNAs were extracted from two powder food supplements. Strain-specific primers targeting unique sequence regions of 16S RNA were identified and amplified by RT-qPCR. Primers were tested for specificity, sensitivity, and efficiency. Both RT-qPCR and flow-cytometry methods described in our work for the quantification and identification of Lactobacillus and Bifidobacterium strains were specific, sensitive, and precise, showing better performances with respect to the morphological colony identification. This work demonstrated that RT-qPCR can be implemented in the quality control workflow of commercial probiotic products giving more standardized and effective results regarding species discrimination
Role of glutathionylation in infection and inflammation
Glutathionylation, that is, the formation of mixed disulfides between protein cysteines and
glutathione (GSH) cysteines, is a reversible post-translational modification catalyzed by dierent
cellular oxidoreductases, by which the redox state of the cell modulates protein function. So far, most
studies on the identification of glutathionylated proteins have focused on cellular proteins, including
proteins involved in host response to infection, but there is a growing number of reports showing
that microbial proteins also undergo glutathionylation, with modification of their characteristics and
functions. In the present review, we highlight the signaling role of GSH through glutathionylation,
particularly focusing on microbial (viral and bacterial) glutathionylated proteins (GSSPs) and host
GSSPs involved in the immune/inflammatory response to infection; moreover, we discuss the
biological role of the process in microbial infections and related host responses
Glutathione increase by the n-butanoyl glutathione derivative (GSH-C4) inhibits viral replication and induces a predominant Th1 immune profile in old mice infected with influenza virus
During aging, glutathione (GSH) content declines and the immune system undergoes a
deficiency in the induction of Th1 response. Reduced secretion of Th1 cytokines, which is
associated with GSH depletion, could weaken the host defenses against viral infections.
We first evaluated the concentration of GSH and cysteine in organs of old mice; then, the
effect of the administration of the N-butanoyl GSH derivative (GSH-C4) on the response of
aged mice infected with influenza A PR8/H1N1 virus was studied through the determination
of GSH concentration in organs, lung viral titer, IgA and IgG1/IgG2a production and
Th1/Th2 cytokine profile.
Old mice had lower GSH than young mice in organs. Also the gene expression of
endoplasmic reticulum (ER) stress markers involved in GSH metabolism and folding of
proteins, i.e. Nrf2 and PDI, was reduced. Following infection, GSH content remained low
and neither infection nor GSH-C4 treatment affected Nrf2 expression. In contrast, PDI
expression was upregulated during infection and appeared counterbalanced by GSH-C4.
Moreover, the treatment with GSH-C4 increased GSH content in organs, reduced viral
replication and induced a predominant Th1 response.
In conclusion, GSH-C4 treatment could be used in the elderly to contrast influenza virus
infection by inducing immune response, in particular the Th1 profile
Intracellular redox-modulated pathways as targets for effective approaches in the treatment of viral infection
Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-kB, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-kB, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses
Nerve growth factor is an autocrine survival factor for memory B lymphocytes
AbstractProduction of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD “virgin” B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes
Role of Preoperative Biliary Drainage in Resectable Hilar Cholangiocarcinoma
Background: To review the literature to investigate indications, advantages and complications of the different procedures for biliary drainage
(percutaneous or endoscopic techniques) for resectable hilar cholangiocarcinoma.
Methods: Pubmed and Medline databases were interrogated for articles published between January 1970 and November 2014. After
screening for relevance, 56 articles were selected for the review.
Results: Hilar cholangiocarcinoma is the most common primitive adenocarcinoma of the bile ducts, involving the proximal extra hepatic
biliary system. Prognosis has been strongly related to tumour\u2019s resectability and extended combined hepatic and biliary resections are often
required in order to achieve radical margin and survival benefit. Obstructive jaundice is the most common clinical presentation; preoperative
hyperbilirubinemia is an independent risk factor for increased operative morbidity and mortality.
Conclusion: PBD was shown to be helpful only in those patients having a bilirubin level greater than 3-10 mg/dl; we might conclude that
PBD is probably useless with bilirubin level below 3 mg/dl, advisable between 3-10 mg/dl and mandatory above 10 mg/dl especially in patients
undergoing an extended hepatic resection.
In the initial management of HC, ENBD should be preferred as it is characterized by a less invasive approach and less complications (lower
morbidity) . In case of suboptimal jaundice regression or in presence of complications PTBD might be combined, leaving EBD as last option.
Drain should be limited to FLR. The optimal drain duration is debated: the drain should be left in place the time needed to reduce bilirubin level,
meanly 2-6 weeks
Post-liver Transplantation Pancreatic Pseudocyst without Pancreatitis
Pancreatic pseudocysts rarely complicate acute pancreatitis after liver transplantation. We describe a case of post-transplant pancreatic pseudocyst occurring in the absence of pancreatitis.
A 53-year-old post-hepatitis C cirrhotic man with no history of pancreatic or biliary abnormalities underwent liver transplantation. However, at the time of surgery, a T-tube cholangiogram revealed a mild dilation of the native bile duct and, two months later, a large pancreatic pseudocyst was diagnosed and surgically resected. After two years, biochemical cholestasis developed and a liver biopsy showed biliary damage. ERCP revealed a stenosis of Vater\u2019s papilla, which was successfully treated by sphincterotomy.
Pancreatic pseudocyst may arise as a complication of OLT in the absence of postoperative acute pancreatitis or a history of chronic pancreatitis. In our patient, it is suspected that ampullary dysfunction associated with ischemic pancreatic injury and bacterial infection played a role in generating both the early pancreatic pseudocyst and the late cholestasis
Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress
Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions
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