99mTc-antitumor necrosis factor-alpha scintigraphy for the detection of inflammatory activity in rheumatoid arthritis.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-α) is an important inflammatory cytokine. 99mTc-anti-TNF-α antibody scintigraphy has proven to be a viable alternative to MRI in specific cases. The objective of this study was to evaluate the performance of scintigraphy with 99mTc-anti-TNF-α in the identification of inflammatory foci in individuals diagnosed with rheumatoid arthritis using MRI as the gold standard. METHODS: This cross-sectional, descriptive and analytical-qualitative clinical study compared the performance of 99mTc-anti-TNF-α scintigraphy with that of MRI with intravenous administration of gadolinium (used as the gold standard) and a clinical examination (Disease Activity Score 28) in 220 joints of 20 patients with a diagnosis of rheumatoid arthritis and one healthy control. RESULTS: The concordance of scintigraphy with MRI in individuals with a diagnosis of rheumatoid arthritis was 79%. The accuracy, sensitivity and specificity of scintigraphy for distinguishing between inflammatory and noninflammatory sites were 92, 89, and 93%, respectively. No adverse reactions to the examinations were reported. CONCLUSIONS: Scintigraphy with 99mTc-anti-TNF-α was well-tolerated and had a good ability to distinguish between inflammatory and noninflammatory lesions in patients with rheumatoid arthritis.

The Manufacture of GMP-Grade Bone Marrow Stromal Cells with Validated In Vivo Bone-Forming Potential in an Orthopedic Clinical Center in Brazil.

In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of 21.93 ± 12.81 × 106 cells, while at passage 2, it was of 83.05 ± 114.72 × 106 cells. All final cellular products were free from contamination with aerobic/anaerobic pathogens, mycoplasma, and bacterial endotoxins. The expanded BMSCs expressed CD73, CD90, CD105, and CD146 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Most importantly, nine out of 10 of the cell products formed bone when transplanted in vivo. These validated procedures will serve as the basis for in-house BMSC manufacturing for use in clinical applications in our center.

Eicosapentaenoic acid potentiates the therapeutic effects of adipose tissue-derived mesenchymal stromal cells on lung and distal organ injury in experimental sepsis.

BACKGROUND: Even though mesenchymal stromal cells (MSCs) mitigate lung and distal organ damage in experimental polymicrobial sepsis, mortality remains high. We investigated whether preconditioning with eicosapentaenoic acid (EPA) would potentiate MSC actions in experimental sepsis by further decreasing lung and distal organ injury, thereby improving survival. METHODS: In C57BL/6 mice, sepsis was induced by cecal hligation and puncture (CLP); sham-operated animals were used as control. Twenty-four hours after surgery, CLP mice were further randomized to receive saline, adipose tissue-derived (AD)-MSCs (105, nonpreconditioned), or AD-MSCs preconditioned with EPA for 6 h (105, EPA-preconditioned MSCs) intravenously. After 24 h, survival rate, sepsis severity score, lung mechanics and histology, protein level of selected biomarkers in lung tissue, cellularity in blood, distal organ damage, and MSC distribution (by technetium-99m tagging) were analyzed. Additionally, the effects of EPA on the secretion of resolvin-D1 (RvD1), prostaglandin E2 (PGE2), interleukin (IL)-10, and transforming growth factor (TGF)-ß1 by MSCs were evaluated in vitro. RESULTS: Nonpreconditioned and EPA-preconditioned AD-MSCs exhibited similar viability and differentiation capacity, accumulated mainly in the lungs and kidneys following systemic administration. Compared to nonpreconditioned AD-MSCs, EPA-preconditioned AD-MSCs further reduced static lung elastance, alveolar collapse, interstitial edema, alveolar septal inflammation, collagen fiber content, neutrophil cell count as well as protein levels of interleukin-1ß and keratinocyte chemoattractant in lung tissue, and morphological abnormalities in the heart (cardiac myocyte architecture), liver (hepatocyte disarrangement and Kupffer cell hyperplasia), kidney (acute tubular necrosis), spleen (increased number of megakaryocytes and lymphocytes), and small bowel (villi architecture disorganization). EPA preconditioning of MSCs resulted in increased secretion of pro-resolution and anti-inflammatory mediators (RvD1, PGE2, IL-10, and TGF-ß). CONCLUSIONS: Compared to nonpreconditioned cells, EPA-preconditioned AD-MSCs yielded further reductions in the lung and distal organ injury, resulting in greater improvement in sepsis severity score and higher survival rate in CLP-induced experimental sepsis. This may be a promising therapeutic approach to improve outcome in septic patients.

Olive Oil, Palm Oil, and Hybrid Palm Oil Distinctly Modulate Liver Transcriptome and Induce NAFLD in Mice Fed a High-Fat Diet.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent worldwide. The most severe form is nonalcoholic steatohepatitis (NASH). Among risk factors for the development of NAFLD is excessive lipid intake. Since palm (P) oil is the most consumed oil in the world, we aimed to investigate the effects of high-fat diets made with P oil, hybrid palm (HP) oil, or olive (O) oil in liver. Twenty-four male mice (C57Bl/6J) were fed a high-fat diet (41% fat) containing P, HP, or O oils for 8 weeks and compared to a control (C) group fed a chow diet. Adiposity was measured with computed tomography. Body, adipose tissue, and liver weights, as well as liver fat (Bligh⁻Dyer), blood lipid profile, glucose, and liver enzymes were measured. Liver histology (hematoxylin⁻eosin) and transcriptome (microarray-based) were performed. ANOVA tests with Newman⁻Keuls were used. Body weight was increased in the P group (p < 0.001) and body fat in the O group (C vs. O p ≤ 0.01, P vs. O p ≤ 0.05, HP vs. O p ≤ 0.05). All high-fat diets disturbed the blood lipid profile and glucose, with marked effects of HP on very low-density lipoprotein cholesterol (VLDL), triglycerides, and alkaline phosphatase (p ≤ 0.001). HP had the highest liver fat (42.76 ± 1.58), followed by P (33.94 ± 1.13). O had a fat amount comparable to C (16.46 ± 0.34, 14.71 ± 0.70, respectively). P and HP oils induced hepatocyte ballooning. Transcriptome alterations of the O group were related to amino acid metabolism and fatty acid (FA) metabolism, the P group to calcium ion homeostasis, and HP oil to protein localization. Both P and HP oils induced NASH in mice via disturbed hepatocyte transcription. This raises concerns about the content of these oils in several industrialized foods.

Bone Marrow-Derived Mononuclear Cell Therapy Accelerates Renal Ischemia-Reperfusion Injury Recovery by Modulating Inflammatory, Antioxidant and Apoptotic Related Molecules.

BACKGROUND/AIMS: We investigated the regenerative capacity of intravenous administration of bone marrow-derived mononuclear cells (BMMCs) in a rat model of bilateral renal ischemia/reperfusion (IR) injury and the involvement of inflammatory anti-inflammatory and other biological markers in this process. METHODS: Rats were subjected to 1h bilateral renal pedicle clamping. BMMCs were injected i.v 1h after reperfusion and tracked by 99mTc and GFP+ BMMCs. Twenty-four hours after reperfusion, renal function and histological changes were evaluated. The mRNA (real time PCR) and protein (ELISA and immuno-staining) expression of biological markers were analyzed. RESULTS: Renal function and structure improved after infusion of BMMCs in the IR group (IR-C). Labeled BMMCs were found in the kidneys after therapy. The expression of inflammatory and biological markers (TLR-2, TRL-4, RAGE, IL-17, HMGB-1, KIM-1) were reduced and the expression of anti-inflammatory and antioxidant markers (IL-10, Nrf2, and HO-1) were increased in IR-C animals compared with IR untreated animals (IR-S). The apoptotic index diminished and the proliferation index increased in IR-C compared with IR-S. CONCLUSION: The results contribute to our understanding of the role of different biological players in morphofunctional renal improvement and cytoprotection in a post-ischemic reperfusion kidney injury model subjected to cellular therapy.

Infusion of bone marrow mononuclear cells reduces lung fibrosis but not inflammation in the late stages of murine silicosis.

We hypothesized that infusion of bone marrow mononuclear cells (BMMCs) in the late stages of silica-induced damage would reduce the remodelling process in a murine model of silicosis. C57BL/6 mice were assigned to 2 groups. In the SIL group, mice were instilled with a silica particle suspension intratracheally. Control (C) mice received saline under the same protocol. On the 40th day, some of the animals from both groups were killed. The others were treated with either saline or BMMCs (1×10(6) cells) intravenously (C+BMMC and SIL+BMMC), and the mice were killed 70 days after the start of the protocol. In the mice in the SIL+BMMC group, collagen deposition, the presence of silica particles inside nodules, the presence of macrophages and cells reactive for inducible nitric oxide synthase were reduced. Lung parameters also improved. Beyond that, the total and differential cellularity of bronchoalveolar lavage fluid, immunoexpression of transforming growth factor-ß, the number of T regulatory cells and apoptosis were increased. However, the presence of male donor cells in lung tissue was not observed using GFP+ cells (40d) or Y chromosome DNA (70d). Therefore, BMMC therapy in the late stages of experimental silicosis improved lung function by diminishing fibrosis but inflammatory cells persisted, which could be related to expansion of T regulatory cells, responsible for the beneficial effects of cell therapy.

Heavy metal tolerance (Cr, Ag AND Hg) in bacteria isolated from sewage.

Samples of sewage from a university hospital and a chemistry technical school were analysed for the percentage of bacterial tolerance to chromium (Cr), silver (Ag) and mercury (Hg). Additionally, we investigated the effect of these metals on pigmentation and on some enzymatic activities of the metal tolerant strains isolated, as well as antimicrobial resistance in some metal tolerant Enterobacteriaceae strains. Tolerance to Cr was observed mainly in Gram positive bacteria while in the case of Ag and Hg the tolerant bacteria were predominately Gram negative. Hg was the metal for which the percentage of tolerance was significantly higher, especially in samples from the hospital sewage (4.1%). Mercury also had the most discernible effect on color of the colonies. Considering the effect of metals on the respiratory enzymes, one strain of Ag-tolerant Bacillus sp. and one of Hg-tolerant P. aeruginosa were unable to produce oxidase in the presence of Ag and Hg, respectively, while the expression of gelatinase was largely inhibited in various Gram negative strains (66% by Cr). Drug resistance in Hg-tolerant Enterobacteriaceae strains isolated from the university hospital sewage was greater than 80%, with prevalence of multiple resistance, while the Ag-tolerant strains from the same source showed about 34% of resistance, with the predominance of mono-resistance. Our results showed that, despite the ability of metal tolerant strains to survive and grow in the presence of these elements, the interactions with these metals may result in metabolic or phisiological changes in this group of bacteria.

(99m)Tc-anti-TNF-α scintigraphy in RA: a comparison pilot study with MRI and clinical examination.

OBJECTIVE: To compare the use of radiolabelled human monoclonal anti-TNF-α scintigraphy with clinical examination and MRI of hands and wrists joints in patients with active RA. METHODS: Eight patients with active RA, 28-joint DAS (DAS-28) ≥ 3.2 and a healthy volunteer underwent whole body and hand/wrist scintigraphy after the administration of anti-human TNF-α labelled with technetium-99m ((99m)Tc). One hundred and ninety-eight joints were examined. Patients were also given clinical examinations in addition to MRI of the hands and wrists. RESULTS: Of the 198 joints examined, signs of inflammation were detected by MRI in 49 (24.7%) and by scintigraphy in 48 (24.2%) joints, with agreement between the two methods in 44 joints. In five joints, MRI was positive and scintigraphy negative. In another four joints, scintigraphy was positive and MRI negative for signs of inflammation. MRI and scintigraphy were in agreement for negative results for 145 joints. The sensitivity and specificity of scintigraphy was 89.8 and 97.3%, respectively. When clinical parameters (presence of swelling and tenderness of joints) were compared with the MRI findings, lower correlation coefficients were observed (sensitivity of 59.2% and 65.3%, respectively). CONCLUSIONS: Scintigraphy using (99m)Tc-anti-TNF-α showed high correlation with the presence of inflammatory signs detected by MRI in the hands and wrists of patients with active RA, and demonstrated a greater sensitivity than clinical examination. These results can assist in better understanding of anti-cytokine therapy and support the achievement of evidence-based biologic therapy.