Transplantation of mesenchymal stem cells genetically engineered to overexpress interleukin-10 promotes alternative inflammatory response in rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause for long-term disability, yet the treatments available that improve outcomes after TBI limited. Neuroinflammatory responses are key contributors to determining patient outcomes after TBI. Transplantation of mesenchymal stem cells (MSCs), which release trophic and pro-repair cytokines, represents an effective strategy to reduce inflammation after TBI. One such pro-repair cytokine is interleukin-10 (IL-10), which reduces pro-inflammatory markers and trigger alternative inflammatory markers, such as CD163. In this study, we tested the therapeutic effects of MSCs that were engineered to overexpress IL-10 when transplanted into rats following TBI in the medial frontal cortex. METHODS: Thirty-six hours following TBI, rats were transplanted with MSCs and then assessed for 3 weeks on a battery of behavioral tests that measured motor and cognitive abilities. Histological evaluation was then done to measure the activation of the inflammatory response. Additionally, immunomodulatory effects were evaluated by immunohistochemistry and Western blot analyses. RESULTS: A significant improvement in fine motor function was observed in rats that received transplants of MSCs engineered to overexpress IL-10 (MSCs + IL-10) or MSCs alone compared to TBI + vehicle-treated rats. Although tissue spared was unchanged, anti-inflammatory effects were revealed by a reduction in the number of glial fibrillary acidic protein cells and CD86 cells in both TBI + MSCs + IL-10 and TBI + MSC groups compared to TBI + vehicle rats. Microglial activation was significantly increased in the TBI + MSC group when compared to the sham + vehicle group. Western blot data suggested a reduction in tumor necrosis factor-alpha in the TBI + MSCs + IL-10 group compared to TBI + MSC group. Immunomodulatory effects were demonstrated by a shift from classical inflammation expression (CD86) to an alternative inflammation state (CD163) in both treatments with MSCs and MSCs + IL-10. Furthermore, co-labeling of both CD86 and CD163 was detected in the same cells, suggesting a temporal change in macrophage expression. CONCLUSIONS: Overall, our findings suggest that transplantation of MSCs that were engineered to overexpress IL-10 can improve functional outcomes by providing a beneficial perilesion environment. This improvement may be explained by the shifting of macrophage expression to a more pro-repair state, thereby providing a possible new therapy for treating TBI.

Expression of genes associated with inflammation and iron metabolism in 3T3-L1 cells induced with macrophages-conditioned medium, glucose and iron.

Obesity is characterized by a chronic inflammatory process, with an increased volume of total adipose tissue, especially visceral, which secretes pro-inflammatory cytokines such as TNF-α and IL-6. Hepcidin (Hpc), a main iron metabolism regulator, is synthetized by an IL-6 stimuli, among others, in liver and adipose tissue, favoring an association between the inflammatory process and iron metabolism. Still there are questions remain regarding the interaction of these factors. Our aim was to study the effect of a macrophage-conditioned medium (MCM) on adipocyte cells challenged with glucose and/or iron. We studied the mRNA relative abundance of genes related to inflammation in differentiated 3T3-L1 cells challenged with Fe (40 µM), glucose (20 mM) or Fe/glucose (40 µM/20 mM) with or without MCM for 24 h. We also measured the intracellular iron levels under these conditions. Our results showed that when adipocytes were challenged with MCM, glucose and/or Fe, the intracellular iron and mRNA levels of pro-inflammatory cytokines increased. These responses were higher when all the stimuli were combined with MCM from macrophages. Thus, we showed that combined high glucose/high Fe alone or with MCM may contribute to an increase on intracellular iron and inflammatory response in 3T3-L1 differentiated cells, by increased mRNA levels of IL-6, TNF-α, MCP-1, Hpc and reducing adiponectin levels, enhancing the inflammatory processes.

Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.

Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti-programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

Cancer Evolution during Immunotherapy.

Immune checkpoint blockade has revolutionized cancer treatment. In this issue of Cell, insights from a longitudinal multi-omics analysis of the largest yet-reported cohort of melanoma patients reveal how tumor and immunity co-evolve during anti-PD-1 therapy.

The selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen improve ANP levels and decrease nuclear translocation of NF-kB in estrogen-deficient rats.

BACKGROUND: The selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen are used for the treatment of osteoporosis and cancer, respectively, in women. The impairment of both the Atrial Natriuretic Peptide (ANP) cell signaling system and the translocation of nuclear factor-kappa B (NF-kB) to the cell nucleus are associated with detrimental cardiovascular effects and inflammation. The effects of SERMs on these parameters in the cardiac tissue of estrogen-deficient rats has not been reported. METHODS: We investigated the effects of raloxifene and tamoxifen on ANP signaling, p65 NF-kB nuclear translocation, cardiac histology and contractility. Female rats were divided into five groups: control (SHAM), ovariectomized (OVX), OVX-treated 17-ß-estradiol (E), OVX-treated raloxifene (RLX) and OVX-treated tamoxifen (TAM). The treatments started 21days after ovariectomy and continued for 14days. RESULTS: Ovariectomy reduced ANP mRNA in the left atrium (LA), decreased the content of ANP protein in the LA and in plasma, and increased the level of p65 NF-kB nuclear translocation in the left ventricle. Both 17-ß-estradiol and SERMs were able to reverse these alterations, which were induced by the estrogen deficient state. The hemodynamic and cardiac structural parameters analyzed in the present work were not modified by the interventions. CONCLUSIONS: Our study demonstrates, for the first time, the additional benefits of raloxifene and tamoxifen in an estrogen-deficient state. These include the normalization of plasmatic and cardiac ANP levels and cardiac p65 NF-kB translocation. Therefore, these treatments promote cardiovascular protection and may contribute to the prevention of cardiac dysfunction observed long-term in postmenopausal women.

Efficacy and toxicity of treatment with the anti-CTLA-4 antibody ipilimumab in patients with metastatic melanoma after prior anti-PD-1 therapy.

BACKGROUND: Recent phase III clinical trials have established the superiority of the anti-PD-1 antibodies pembrolizumab and nivolumab over the anti-CTLA-4 antibody ipilimumab in the first-line treatment of patients with advanced melanoma. Ipilimumab will be considered for second-line treatment after the failure of anti-PD-1 therapy. METHODS: We retrospectively identified a cohort of 40 patients with metastatic melanoma who received single-agent anti-PD-1 therapy with pembrolizumab or nivolumab and were treated on progression with ipilimumab at a dose of 3 mg kg(-1) for a maximum of four doses. RESULTS: Ten percent of patients achieved an objective response to ipilimumab, and an additional 8% experienced prolonged (>6 months) stable disease. Thirty-five percent of patients developed grade 3-5 immune-related toxicity associated with ipilimumab therapy. The most common high-grade immune-related toxicity was diarrhoea. Three patients (7%) developed grade 3-5 pneumonitis leading to death in one patient. CONCLUSIONS: Ipilimumab therapy can induce responses in patients who fail the anti-PD-1 therapy with response rates comparable to previous reports. There appears to be an increased frequency of high-grade immune-related adverse events including pneumonitis that warrants close surveillance.

Non-HIV-associated Kaposi sarcoma in an immunosuppressed melanoma patient treated with dabrafenib.

WHAT IS KNOWN AND OBJECTIVE: Cutaneous toxicities are commonly seen with BRAF inhibitors, frequently involving painful hyperkeratosis of the feet. We illustrate an unexpected diagnosis of extensive bilateral pedal Kaposi sarcoma masquerading as BRAF inhibitor-related toxicity in a patient treated with dabrafenib for metastatic melanoma. CASE SUMMARY: A HIV-negative, non-diabetic, Italian man with a history of myasthenia gravis and metastatic melanoma presented with enlarging macular/plaque-like rash on his feet preceded by bilateral plantar shooting pains. The rash progressed in the context of acute-on-chronic immunosuppression and was initially thought due to commencement of the BRAF inhibitor (BRAFi) dabrafenib. Histopathological findings from skin biopsies revealed Kaposi sarcoma. The patient was continued on dabrafenib and received superficial radiotherapy to the feet with prompt relief of pain. WHAT IS NEW AND CONCLUSION: This case illustrates the diagnostic pitfalls in patients treated with targeted therapies and highlights the importance of broad differentials for unusual presentations and early biopsy.

Response to MAPK pathway inhibitors in BRAF V600M-mutated metastatic melanoma.

WHAT IS KNOWN AND OBJECTIVE: The management of metastatic melanoma has changed significantly in the past decade with the development of immunotherapies and targeted molecular therapies. Trials of targeted therapies have focused mainly on patients with the most common BRAF V600 mutations, namely V600E/K substitutions, with very little information available on the benefit of targeted therapies on less commonly occurring mutations such as V600R/D and M. CASE SUMMARY: We present a 54-year-old man with metastatic melanoma harbouring a rare BRAF V600M mutation, who experienced clinical and radiological response to combined therapy with the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. WHAT IS NEW AND CONCLUSION: As our understanding of these therapies evolves and an increasing number of patients have mutational testing performed, there is a clear imperative--as highlighted by this case--to test for rarer mutations and facilitate their inclusion both in everyday practice and in future clinical trials.

Triterpenes from the Protium heptaphyllum resin - chemical composition and cytotoxicity

Protium heptaphyllum (Aubl) Marchand, Burseraceae, is popularly used as an analgesic and anti-inflammatory agent. However, the cellular mechanism of action remains unknown. This study aims to evaluate the chemical composition of P. heptaphyllum resin and cytotoxicity on a breast cancer cell line (MCF-7). The chemical composition of the resin was determined by Gas Chromatography coupled to a Mass Spectrometer. The cytotoxicity was evaluated using an MTT assay. Annexin V-FITC, caspase-3, Angiotensin Converting Enzyme activity and Tumor Necrosis Factor alpha (TNF- α) assays were performed to evaluate apoptosis and inflammatory events. The resin consisted of triterpenes, such as α- and β-amyrin. Cytotoxicity was only observed in fractions enriched with α- and β-amyrin. The resin and fractions elicited antiproliferative activity, increased activity of caspase-3 and ACE, and a decrease in the TNF-α level. Altogether, the resin and fractions enriched with α- and β-amyrin promoted cytotoxicity and apoptosis.

Effect of calcium, tannic acid, phytic acid and pectin over iron uptake in an in vitro Caco-2 cell model.

Calcium, phytic acid, polyphenols and fiber are major inhibitors of iron absorption and they could be found in excess in some diets, thereby altering or modifying the iron nutrition status. The purpose of this study is to evaluate the effect of calcium, tannic acid, phytic acid, and pectin over iron uptake, using an in vitro model of epithelial cells (Caco-2 cell line). Caco-2 cells were incubated with iron (10-30 µM) with or without CaCl2 (500 and 1,000 µM) for 24 h. Then, cells were challenged with phytic acid (50-150 µM); pectin (50-150 nM) or tannic acid (100-500 µM) for another 24 h. Finally, (55)Fe (10 µM) uptake was determined. Iron dialyzability was studied using an in vitro digestion method. Iron uptake in cells pre-incubated with 20 and 30 µM Fe was inhibited by CaCl2 (500 µM). Iron uptake decreased in cells cultured with tannic acid (300 µM) and CaCl2 (500-1,000 µM) (two-way ANOVA, p = 0.002). Phytic acid also decreased iron uptake mainly when cells were treated with CaCl2 (1,000 µM) (two-way ANOVA; p < 0.05). Pectin slightly decreased iron uptake (p = NS). Iron dialyzability decreased when iron was mixed with CaCl2 and phytic or tannic acid (T test p < 0.0001, for both) but not when mixed with pectin. Phytic acid combined with calcium is a strong iron uptake inhibitor. Pectin slightly decreased iron uptake with or without calcium. Tannic acid showed an unexpected behavior, inducing an increase on iron uptake, despite its low Fe dialyzability.

Morphometry to identify subtypes of leukocytes.

INTRODUCTION: Recent studies in image cytometry evaluated the replacement of specific markers by morphological parameters. The aim of this study was to develop and evaluate a method to identify subtypes of leukocytes using morphometric data of the nuclei. METHOD: The analyzed images were generated with a laser scanning cytometer. Two free programs were used for image analysis and statistical evaluation: Cellprofiler and Tanagra respectively. A sample of leukocytes with 200 sets of images (DAPI, CD45 and CD14) was analyzed. Using feature selection, the 20 best parameters were chosen to conduct cross-validation. RESULTS: The morphometric data identified the subpopulations of the analyzed leukocytes with a sensitivity and specificity of 0.95 per sample. CONCLUSION: The present study is the first that identifies subpopulations of leukocytes by nuclear morphology.

Hepatic and adipocyte cells respond differentially to iron overload, hypoxic and inflammatory challenge.

Adipose tissue secretes numerous pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α that can lead to insulin resistance (IR). In the liver, both IL-6 and TNF-α induce IR by inhibiting phosphorylation or ubiquitination of IRS1. In IR development, Fe is a risk factor in type-2 diabetes development. We studied the expression of genes related to inflammation, hypoxia, and mitochondrial function in hepatic (HepG2) and adipose (3T3-L1) cells. HepG2 and 3T3-L1 cells were incubated with 20 µM Fe, 40 µM Fe, or 40 µM Fe/20 mM glucose for 7 days and then challenged with 20 ng/ml IL-6 and/or 100 µM CoCl(2) for 20 h. We measured intracellular Fe levels and the relative expression of hepcidin, NF-κB, IL-6, TNF-α, hypoxia inducible factor 1α (HIF-1α), and mitofusin 2 (Mfn-2) mRNA using qRT-PCR. The intracellular Fe concentration in HepG2 cells did not change with 20 or 40 µM Fe. However, levels were decreased with Fe/glucose and IL-6 and/or CoCl(2). 3T3-L1 cells showed an increase in intracellular Fe with high Fe plus either IL-6 or CoCl(2). HepG2 cells incubated with 40 µM Fe alone or Fe/glucose and challenged with IL-6 and/or CoCl(2) showed increased IL-6, NF-κB, and TNF-α mRNA expression and decreased mRNA expression of Mfn-2 in all experimental conditions. 3T3-L1 cells incubated with 40 µM Fe alone or Fe/glucose and challenged with IL-6 showed increased NF-κB mRNA expression and decreased Mfn-2 expression in all experimental conditions. Thus, high Fe, inflammation, and hypoxia trigger the expression of genes related to inflammation and Fe metabolism in HepG2 cells, in 3T3-L1 cells the same stimuli increased NF-kB and hepcidin expression.

Ferritin levels and hepcidin mRNA expression in peripheral mononuclear cells from anemic type 2 diabetic patients.

This study aims to measure iron nutrition parameters and to determine the presence of anemia in obese type 2 diabetic patients and to analyze the mRNA relative abundance of genes related to inflammation, immune system, iron metabolism, and mitochondrial activity. Obese type 2 diabetic (OBDM, n=30) and healthy subjects (Cn, n=30) were studied. Biochemical, anthropometric, and iron nutrition parameters were determined. Peripheral mononuclear cells from type 2 diabetic and control group were challenged with high concentrations of iron (Fe) and glucose and total mRNA was isolated. The frequency of anemia among diabetic patients was 4/30. OBDM patients with or without anemia had higher levels of ferritin and high-sensitivity C-reactive protein than the Cn group. mRNA relative abundance of nuclear factor kappa-light-chain-enhancer of activated B cells was elevated in OBDM with anemia, and mRNA expression of interleukin-6 and toll-like receptor (TLR) 2 was increased in OBDM group in basal high Fe and high glucose concentrations. The expression of tumor necrosis factor alpha and TLR-4 was increased in OBDM with anemia in all experimental conditions. Hepcidin mRNA expression was increased in OBDM with anemia even in basal Fe concentration, and mitofusin 2 was decreased in all experimental conditions. This study shows that obese type 2 diabetic patients have iron distribution disorders associated to their proinflammatory state, and anemic subjects have a marked elevation of hepcidin mRNA expression.

Thermal stability of the hydrogen-bonded water network in the hydration shell of islet amyloid polypeptide.

The effect of temperature on the connectivity of hydrogen bonds in the hydration shells of the islet amyloid polypeptides (IAPPs) is studied by means of computer simulations. The hydrogen-bonded network of hydration water homogeneously envelopes a peptide at low temperature and breaks into an ensemble of small clusters upon heating. This thermal break occurs via a percolation transition, which is not found to be sensitive to the chemical modifications of IAPP (IAPP with and without a disulfide bridge, human and rat IAPP). The radius of gyration of IAPP starts to increase when the hydration water network breaks upon heating. The fluctuations of the number of intra-peptide hydrogen bonds show negative correlation with the fraction of molecules in the largest cluster of hydration water. The thermal stability of the network of hydration water is enhanced upon increasing number of intra-peptide hydrogen bonds, which makes the peptide surface more hydrophobic. The thermal stabilities of the hydrogen-bonded water networks in the hydration shells of IAPPs and of several other biomolecules are found to be rather similar: the network breaks between 300 and 330 K, i.e., in the temperature interval where the biological activity of living organisms is maximal.

Volumetric properties of human islet amyloid polypeptide in liquid water.

The volumetric properties of human islet amyloid polypeptide (hIAPP) in water were studied in a wide temperature range by computer simulations. The intrinsic density rho(p) and the intrinsic thermal expansion coefficient alpha(p) of hIAPP were evaluated by taking into account the difference between the volumetric properties of hydration and bulk water. The density of hydration water rho(h) was found to decrease almost linearly with temperature upon heating and its thermal expansion coefficient was found to be notably higher than that of bulk water. The peptide surface exposed to water is more hydrophobic and its rho(h) is smaller in conformation with a larger number of intrapeptide hydrogen bonds. The two hIAPP peptides studied (with and without disulfide bridge) show negative alpha(p), which is close to zero at 250 K and decreases to approximately -1.5 x 10(-3) K(-1) upon heating to 450 K. The analysis of various structural properties of peptides shows a correlation between the intrinsic peptide volumes and the number of intrapeptide hydrogen bonds. The obtained negative values of alpha(p) can be attributed to the shrinkage of the inner voids of the peptides upon heating.

On the mechanical properties of bovine serum albumin (BSA) adhesives.

Biological adhesives, natural and synthetic, are of current active interest. These adhesives offer significant advantages over traditional sealant techniques, in particular, they are easier to use, and can play an integral part in the healing mechanism of tissue. Thus, biological adhesives can play a major role in medical applications if they possess adequate mechanical behavior and stability over time. In this work, we report on the method of preparation of bovine serum albumin (BSA) into a biological adhesive. We present quantitative measurements that show the effect of BSA concentration and cross-linker content on the bonding strength of BSA adhesive to wood. A comparison is then made with synthetic poly(glycidyl methacrylate) (PGMA) adhesive, and a commercial cyanoacrylate glue, which was used as a control adhesive. In addition, BSA samples were prepared and characterized for their water content, tensile strength, and elasticity. We show that on dry surface, BSA adhesive exhibits a high bonding strength that is comparable with non-biological commercial cyanoacrylate glues, and synthetic PGMA adhesive. Tensile testing on wet wood showed a slight increase in the bonding strength of BSA adhesive, a considerable decrease in the bonding strength of cyanoacrylate glue, and negligible adhesion of PGMA. Tests performed on BSA samples demonstrate that initial BSA concentration and final water content have a significant effect on the stress-strain behavior of the samples.

Design, development and characterization of cyst phantom for ultrasound elastography applications.

Polyacrylamide based tissue mimicking phantom with embedded cysts is designed and analyzed for better understanding of cyst elastograms. Cysts filled with different fluids of varying density and bulk moduli are embedded in the phantom. Characterization is done based on parameters measured from the ultrasound B mode and elastogram of the cystic lesions. Such a phantom can serve as tool for better understanding of the elastographic appearance of cysts. Thus simple and complex cysts can be easily distinguished. It can also be used to teach a complex procedure like ultrasound guided fine needle aspiration.

Role for dopamine-3 receptor in the hyperphagia of an unanticipated high-fat meal in rats.

Behavioral studies have indicated that midbrain dopamine projections arising in the ventral tegmental area and substantia nigra play a central role in integrating violations of expectancy in reward-related paradigms. The present study was designed to assess violations of dietary expectation and the role the dopamine-3 receptor plays in integrating reward-related food intake in violations of expectancy. Two groups of rats were conditioned to a meal-feeding schedule (3 h of access to food per day) in which they received either standard rodent chow or a preferable, high-fat diet. Animals either received the diet they had access to during the training period (no contrast) or the opposite diet (negative and positive contrast). As predicted, animals in the positive contrast condition were hyperphagic compared to no contrast animals. Animals in the negative contrast (high fat to chow) condition were hypophagic compared to no contrast animals. A dopamine agonist specific to the dopamine three receptor, ((+/-)-7-Hydroxy-dipropylaminotetralin HBr) and the dopamine-2 receptor antagonist raclopride were administered in equimolar doses peripherally to assess the involvement of the dopamine receptor subtypes in the violation of expectancy food intake effects. 7-Hydroxy-dipropylaminotetralin HBr blocked the hyperphagia associated with positive contrast and did not disrupt intake in the negative contrast or no contrast paradigm. Raclopride was ineffective at disrupting food intake. These results support the hypothesis that the dopamine-3 receptor is involved in the hyperphagia of an unexpected high fat meal.

A role for shoot protein in shoot-root dry matter allocation in higher plants.

BACKGROUND AND AIMS: It is stated in many recent publications that nitrate (NO3-) acts as a signal to regulate dry matter partitioning between the shoot and root of higher plants. Here we challenge this hypothesis and present evidence for the viewpoint that NO3- and other environmental effects on the shoot:root dry weight ratio (S:R) of higher plants are often related mechanistically to changes in shoot protein concentration. METHODS: The literature on environmental effects on S:R is reviewed, focusing on relationships between S:R, growth and leaf NO3- and protein concentrations. A series of experiments carried out to test the proposal that S:R is dependent on shoot protein concentration is highlighted and new data are presented for tobacco (Nicotiana tabacum). KEY RESULTS/EVIDENCE: Results from the literature and new data for tobacco show that S:R and leaf NO3- concentration are not significantly correlated over a range of environmental conditions. A mechanism involving the relative availability of C and N substrates for growth in shoots can explain how shoot protein concentration can influence shoot growth and hence root growth and S:R. Generally, results in the literature are compatible with the hypothesis that macronutrients, water, irradiance and CO2 affect S:R through changes in shoot protein concentration. In detailed studies on several species, including tobacco, a linear regression model incorporating leaf soluble protein concentration and plant dry weight could explain the greater proportion of the variation in S:R within and between treatments over a wide range of conditions. CONCLUSIONS: It is concluded that if NO3- can influence the S:R of higher plants, it does so only over a narrow range of conditions. Evidence is strong that environmental effects on S:R are often related mechanistically to their effects on shoot protein concentration.