Objective
To evaluate the expression of type I interferon (IFNα/β)– and type II IFN (IFNγ)–inducible genes in muscle biopsy specimens from patients with juvenile dermatomyositis (DM) and to correlate ...their expression levels with histologic and clinical features.
Methods
Expression levels of IFN‐inducible genes and proinflammatory cytokines were assessed by quantitative polymerase chain reaction in muscle biopsy specimens from patients with juvenile DM (n = 39), patients with Duchenne's muscular dystrophy (DMD), and healthy controls. Muscle biopsy sections were stained and scored for severity of histopathologic features. The charts of patients with juvenile DM were reviewed for clinical features at the time of sampling and long‐term outcomes.
Results
Muscle expression levels of IFNα/β‐inducible genes (type I IFN score), IFNγ, IFNγ‐inducible genes (type II IFN score), and tumor necrosis factor (TNF) were significantly higher in juvenile DM patients not receiving glucocorticoid therapy before muscle biopsy (n = 27) compared to DMD patients (n = 24) (type I IFN score, P < 0.0001; type II IFN score, P < 0.001; TNF, P < 0.05) and healthy controls (n = 4) (type I IFN score, P < 0.01; type II IFN score, P < 0.01; TNF, P < 0.05). Immunofluorescence staining of muscle biopsy sections from untreated juvenile DM patients showed increased immunoreactivity for IFNγ and HLA class II molecules compared to controls. Type I and type II IFN scores were correlated with typical histopathologic features of juvenile DM muscle biopsy samples, such as infiltration of endomysial CD3+ cells (type I IFN score, r = 0.68; type II IFN score, r = 0.63), perimysial CD3+ cells (type I IFN score, r = 0.59; type II IFN score, r = 0.66), CD68+ cells (type II IFN score, r = 0.46), and perifascicular atrophy (type I IFN score, r = 0.61; type II IFN score, r = 0.77). Juvenile DM patients with a high type I IFN score, a high type II IFN score, and high TNF expression levels showed more severe disease activity at biopsy (P < 0.05). In addition, juvenile DM patients with a high type II IFN score at biopsy reached clinically inactive disease significantly later than patients with low type II IFN score (log rank chi‐square value 13.53, P < 0.001).
Conclusion
The increased expression of IFN‐inducible genes in the muscle in juvenile DM patients and their association with histologic and clinical features further support a pathogenic role for both type I and type II IFNs in juvenile DM.
There is growing interest in the complex relationship between the nervous and immune systems and how its alteration can affect homeostasis and result in the development of inflammatory diseases. A ...key mediator in cross-talk between the two systems is nerve growth factor (NGF), which can influence both neuronal cell function and immune cell activity. The up-regulation of NGF described in inflamed tissues of many diseases can regulate innervation and neuronal activity of peripheral neurons, inducing the release of immune-active neuropeptides and neurotransmitters, but can also directly influence innate and adaptive immune responses. Expression of the NGF receptors tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) is dynamically regulated in immune cells, suggesting a varying requirement for NGF depending on their state of differentiation and functional activity. NGF has a variety of effects that can be either pro-inflammatory or anti-inflammatory. This apparent contradiction can be explained by considering NGF as part of an endogenous mechanism that, while activating immune responses, also activates pathways necessary to dampen the inflammatory response and limit tissue damage. Decreases in TrkA expression, such as that recently demonstrated in immune cells of arthritis patients, might prevent the activation by NGF of regulatory feed-back mechanisms, thus contributing to the development and maintenance of chronic inflammation.
The tumor-specific tetrabranched peptide NT4 binds membrane sulfate glycosaminoglycans and receptors belonging to the low density lipoprotein receptor-related protein (LRP) family, like LRP6, which ...are overexpressed in cancer. The binding occurs through a multimeric positively-charged motif of NT4 that interacts with negatively charged motives in both glycosaminoglycans and LRP receptors. LRP6 has an essential function in canonical Wnt signaling, acting together with receptors of the Frizzled family as coreceptor for Wnt ligands. The extracellular domain of LRP6 contains four YWTD β-propellers, which are fundamental for interactions with ligands, such as Wnt and Wnt inhibitors. To investigate the molecular interactions between the NT4 peptide and LRP6 receptor, we synthesized a library of epitope mapping peptides reproducing the YWTD β-propeller 3 and 4 of LRP6. The peptides that showed to bind NT4 represented the portion of LRP6 located on the top face of β-propeller 3 and contained negatively charged residues, including glutamic acid-708 which is known to be involved in Wnt3a interaction. The results pave the way for a possible development of peptide inhibitors of Wnt3a pathway to be used as drugs in oncology.
The antimicrobial peptide SET-M33 is under study for the development of a new antibiotic against major Gram-negative pathogens. Here we report the toxicological evaluation of SET-M33 administered ...intravenously to rats and dogs. Dose range finding experiments determined the doses to use in toxicokinetic evaluation, clinical biochemistry analysis, necroscopy and in neurological and respiratory measurements. Clinical laboratory investigations in dogs and rats showed a dose-related increase in creatinine and urea levels, indicating that the kidneys are the target organ. This was also confirmed by necroscopy studies of animal tissues, where signs of degeneration and regeneration were found in kidney when SET-M33 was administered at the highest doses in the two animal species. Neurological toxicity measurements by the Irwin method and respiratory function evaluation in rats did not reveal any toxic effect even at the highest dose. Finally, repeated administration of SET-M33 by short infusion in dogs revealed a no-observed-adverse-effect-level of 0.5 mg/kg/day.
The increasing frequency of multi-resistant Gram-positive and Gram-negative bacteria and a long-term decreasing trend in the development of new antimicrobial molecules prompts research for new ...anti-infective agents with new modes of action. Antimicrobial peptides (AMPs) are considered an interesting class of antibacterial molecules. Many new AMPs have been discovered and some are being evaluated for the development of new antibacterial therapeutics. Since the development of new antibacterial drugs has been neglected for decades, we are now faced with extreme medical need combined with a lack of technical experimental progress in setting up efficient models of antibacterial activity in animals. Here we review experiments with AMPs in animal models of sepsis, pneumonia and skin infection caused by bacteria. Animal models of infection have been of enormous predictive value in antibacterial drug discovery, both for elucidating AMP efficacy in the treatment of experimentally induced infection and for comparing the effectiveness of two or more antibiotics.
SET-M33 is a synthetic peptide that is being developed as a new antibiotic against major Gram-negative bacteria. Here we report two in vivo studies to assess the toxicity and efficacy of the peptide ...in a murine model of pulmonary inflammation. First, we present the toxicity study in which SET-M33 was administered to CD-1 mice by snout inhalation exposure for 1 h/day for 7 days at doses of 5 and 20 mg/kg/day. The results showed adverse clinical signs and effects on body weight at the higher dose, as well as some treatment-related histopathology findings (lungs and bronchi, nose/turbinates, larynx and tracheal bifurcation). On this basis, the no observable adverse effect level (NOAEL) was considered to be 5 mg/kg/day. We then report an efficacy study of the peptide in an endotoxin (LPS)-induced pulmonary inflammation model. Intratracheal administration of SET-M33 at 0.5, 2 and 5 mg/kg significantly inhibited BAL neutrophil cell counts after an LPS challenge. A significant reduction in pro-inflammatory cytokines, KC, MIP-1α, IP-10, MCP-1 and TNF-α was also recorded after SET-M33 administration.
The increasing frequency of multidrug-resistant bacteria and a recent slowing in the development of new antimicrobial agents place mankind in a state of emergency with regard to the threat of new ...bacterial infections. Antibacterial peptides (AMPs) are considered an important class of molecules to develop against bacteria. AMPs have been known for many years but very few have yet been extensively used in clinical practice, mainly because of their general toxicity and manufacturing cost. Now, thanks to new technologies for screening and development, interest in these molecules has grown. Many new AMPs have been discovered and some are under evaluation for the development of new antibacterial therapeutics. Here we review the major AMPs currently used in clinical practice and others in the phase of preclinical and clinical development.
The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. ...Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.
The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up ...molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.
Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron ...density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells.
In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model.
NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.
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