Characteristics of Circulating CD4+ T Cell Subsets in Patients with Mycobacterium avium Complex Pulmonary Disease.

Although prevalence of Mycobacterium avium complex pulmonary disease (MAC-PD) is increasing, limited data are available regarding vulnerability to Mycobacterium avium complex (MAC) infections. To understand the pathobiology of interaction between MAC and host-immunity, it is important to understand the characteristics for circulating T cells in terms of the immunological phenotype and functional correlates in MAC-PD. We aimed to characterize immunophenotype, cytokine profile, and immune inhibitory receptors of circulating CD4+ T cells in MAC-PD patients. We enrolled 71 MAC-PD and 20 control individuals. Flow cytometric analysis was performed to determine T cell subsets and immune checkpoint markers. Ex vivo cytokine productions in response to MAC were determined using enzyme-linked immunosorbent assay. The frequencies of CD4+ T cells and CD4+IL-17+ T cells decreased, while CD4+IL-4+ T cells and CD4+CD25+Foxp3+ T cells increased in peripheral blood mononuclear cells (PBMCs) of MAC-PD individuals upon MAC stimulation compared with those cells in healthy donor-PBMCs. Additionally, we found increased PD-1, CTLA-4, and TIM-3-expressing T cells in MAC- PD individuals in response to MAC-stimulation, indicating that suppressed T cell-mediated response is associated with the susceptibility to MAC infection. These results may help to explain impaired T cell-mediated responses and pave the way for better strategies to achieve protective immunity against MAC infection.

miRNA Expression Profiles and Potential as Biomarkers in Nontuberculous Mycobacterial Pulmonary Disease.

Pulmonary disease (PD) due to nontuberculous mycobacteria (NTM) is increasing globally, but specific biomarkers for NTM-PD have not been established. As circulating miRNAs are promising biomarkers for various diseases, we investigated whether miRNAs have potential as NTM-PD biomarkers. Sera from 12 NTM-PD patients due to Mycobacterium avium, M. intracellulare, M. abscessus, or M. massiliense and three healthy controls were initially evaluated via small RNA sequencing. Multiple miRNAs showed significant differences in expression in patients compared to in healthy controls, with some expression differences unique to PD caused by a specific mycobacterial species. Notably, 14 miRNAs exhibited significant expression differences in PD associated with all four mycobacteria. Validation by quantitative reverse-transcription-PCR in an additional 40 patients with NTM-PD and 40 healthy controls confirmed that four differentially expressed miRNAs (hsa-miR-484, hsa-miR-584-5p, hsa-miR-625-3p, and hsa-miR-4732-5p) showed significantly higher serum expressions in NTM-PD patients than in controls. Receiver operating characteristic curve analysis of these four miRNAs supported the discriminative potential for NTM-PD and their combination provided an improved diagnostic value for NTM-PD. Furthermore, bioinformatics analysis revealed their 125 target genes, which were mostly associated with immune responses. Collectively, this study identified four miRNAs as potential biomarkers for NTM-PD and provided insight into NTM-PD pathophysiology.

The effects of myokines on osteoclasts and osteoblasts.

Recently, muscle has received much attention as an endocrine organ regulating other biological targets, including the pancreas, liver, and adipose tissue. Although there is a possibility that muscle-secreting factors biochemically affect bone metabolism in a paracrine manner, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. Therefore, we performed in vitro as well as animal experiments using conditioned media (CM) collected from C2C12 myoblast and myotube cultures to better understand the interactions between muscle and bone. Compared with non-CM (i.e., control) and myoblast CM, myotube CM markedly inhibited in vitro bone resorption through the suppression of osteoclast differentiation and resorptive activity of individual osteoclasts. Consistently, the expressions of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), decreased with myotube CM. Myotube CM significantly stimulated preosteoblast viability and migration and reduced apoptosis, thereby resulting in an increase in calvaria bone formation. Importantly, systemic treatment with myotube CM for 4 weeks increased bone per tissue volume by 30.7% and 19.6% compared with control and myoblast CM, respectively. These results support the hypothesis that muscle plays beneficial roles in bone health via secretion of anabolic factors, in addition to mechanical stimuli, and importantly indicate that muscle-derived factors can be potential therapeutic targets against metabolic bone diseases.

Assessment of 7 trace elements in serum of patients with nontuberculous mycobacterial lung disease.

Nontuberculous mycobacterial (NTM) lung diseases are an emerging cause of pulmonary infection, becoming more common in the clinical setting as incidence of NTM lung diseases steadily increases worldwide. Trace elements are essential micronutrients and are known to play many important roles in infectious diseases. We investigated the concentrations of trace elements in patients with NTM lung disease and compared these values to patients with pulmonary tuberculosis and healthy controls. A case-control study was conducted to evaluate the serum trace element concentrations in 95 patients with NTM lung disease, 97 patients with pulmonary tuberculosis, and 99 healthy control subjects. The serum concentrations of 7 trace elements (cobalt, copper, chromium, manganese, molybdenum, selenium, and zinc) were measured using inductively coupled plasma-mass spectrometry. We also analyzed demographic data, clinical outcomes, and other biochemical parameters. The median serum concentrations of copper and molybdenum were higher in patients with NTM lung disease (109 vs. 91 µg/dL, p < 0.001 and 1.70 vs. 0.96 µg/L, p < 0.001). In contrast, the median serum concentrations of selenium and zinc were significantly lower in patients with NTM lung disease than in healthy controls (105 vs. 115 µg/L, p < 0.001 and 94 vs. 102 µg/dL, p < 0.001). Compared to patients with pulmonary tuberculosis, the serum concentrations of molybdenum and zinc were higher in patients with NTM lung disease, while cobalt and copper concentrations were lower (p < 0.001). Correlations among trace element concentrations were observed (copper and zinc, r = -0.367; cobalt and molybdenum, r = -0.360; selenium and zinc, r = 0.335; and manganese and zinc, r = 0.327, respectively). None of the 7 trace elements were associated with treatment outcomes. Patients with NTM lung disease showed different serum trace element concentrations. Our study indicates that altered trace element status is associated with mycobacterial disease. Further study investigating the clinical significance of individual trace elements and their association with nutritional status in patients with NTM lung disease would be required.

Severe Hypophosphatemia-Induced Acute Toxic-Metabolic Encephalopathy in Continuous Renal Replacement Therapy.

Acute toxic-metabolic encephalopathy (TME) is an acute condition of global cerebral dysfunction in the absence of primary structural brain disease. Severe hypophosphatemia leads to muscle weakness and involves the diaphragm but hypophosphatemia-induced TME is very rare. Herein, we report the case of a 43-year-old woman with encephalopathy with severe hypophosphatemia during continuous renal replacement therapy. She presented with features of oliguric acute kidney injury on diabetic kidney disease due to volume depletion. At admission, her mental status was alert but gradually changed to stupor mentation during continuous renal replacement therapy. Her phosphate level was less than 0.41 mEq/L and Glasgow coma scale decreased from 15 to 5. After phosphate intravenous replacement and administration of phosphate-containing replacement solution, the phosphate level increased to 2.97 mEq/L and mental state returned to alert state. This case demonstrates that the level of phosphorus should be observed during continuous renal replacement therapy.

The effect of platelet-rich plasma on the differentiation of synovium-derived mesenchymal stem cells.

Platelet-rich plasma (PRP), the plasma portion of blood with a high platelet concentration, has been reported to be helpful in stem cell chondrogenesis due to large amount of growth factors it contains. Here, we examined the influence of PRP on the differentiation of synovium-derived stem cells (SDSCs) and also evaluated if PRP alone was sufficient to induce SDSCs differentiation. First, the cell proliferation in various differentiation media was analyzed using the MTT assay and it was significantly higher in groups cultured with media that contained PRP. Then, We performed Safranin-O staining and type I, II, and X collagen immunohistochemistry (chondrogenesis), von Kossa staining (osteogenesis), and Oil Red O staining (adipogenesis). The staining was most prominent in groups cultured with optimized differentiation media without PRP in all three lineages of differentiation. The mRNA expression levels of typical differentiation markers were also analyzed using reverse transcription quantitative polymerase chain reaction. Although, culture in optimized differentiation media increased the mRNA expression of the typical differentiation marker genes, they were significantly reduced when cultured in the media supplemented with PRP. PRP has negative effects on SDSC differentiation in all three differentiation lineages and PRP alone does not induce SDSC differentiation.

Effects of CD14 macrophages and proinflammatory cytokines on chondrogenesis in osteoarthritic synovium-derived stem cells.

We investigated the effects of CD14 macrophages and proinflammatory cytokines on chondrogenic differentiation of osteoarthritic synovium-derived stem cells (SDSCs). Osteoarthritic synovial fluid was analyzed for interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and IL-6. Levels of stem cell surface markers in osteoarthritic SDSCs were evaluated using flow cytometry. CD14-negative cells were obtained using magnetically activated cell sorting. We compared chondrogenic potentials between whole cells and CD14-negative cells in CD14(low) cells and CD14(high) cells, respectively. To assess whether nuclear factor-κB (NF-κB) and CCAAT/enhancer-binding protein ß (C/EBPß) modulate IL-1ß-induced alterations in chondrogenic potential, we performed small interfering RNA transfection. We observed a significant correlation between the CD14 ratio in osteoarthritic SDSCs and IL-1ß and TNF-α in osteoarthritic synovial fluid. Phenotypic characterization of whole cells and CD14-negative cells showed no significant differences in levels of stem cell markers. mRNA expression of type II collagen was higher in CD14-negative cell pellets than in whole cell pellets. Immunohistochemical staining indicated higher levels of type II collagen in the CD14-negative cell pellets of CD14(high) cells than in whole cell pellets of CD14(high) cells. As expected, IL-1ß and TNF-α significantly inhibited the expression of chondrogenic-related genes in SDSCs, an effect which was antagonized by knockdown of NF-κB and C/EBPß. Our results suggest that depletion of CD14(+) synovial macrophages leads to improved chondrogenic potential in CD14(high) cell populations in osteoarthritic SDSCs, and that NF-κB (RelA) and C/EBPß are critical factors mediating IL-1ß-induced suppression of the chondrogenic potential of human SDSCs.

Synovium-derived mesenchymal stem cells encapsulated in a novel injectable gel can repair osteochondral defects in a rabbit model.

We developed a novel injectable type I collagen/hyaluronic acid/fibrinogen (COL/HA/FG) composite gel that encapsulated synovium-derived mesenchymal stem cells (SDSCs) for the repair of damaged articular cartilage. We first analyzed the suitability of the composite gel as a three-dimensional injectable cell carrier in vitro. In an in vivo rabbit model, the COL/HA/FG composite gel displayed the potential to regenerate and repair osteochondral defects in the knee. Culture of the SDSCs encapsulated COL/HA/FG composite gel in a chondrogenic medium resulted in high viability of the SDSCs and high expressions of type II collagen, aggrecan, and sox 9 mRNA. Moreover, glycosaminoglycans and type II collagen were accumulated within the extracellular matrix. In the animal model, the SDSCs encapsulated COL/HA/FG composite gel produced a hyaline-like cartilage construct. Twenty-four weeks after transplantation, the defects had been repaired with hyaline cartilage-like tissue that was densely stained by safranin-O and immunostained by a type II collagen antibody. This data suggest that the SDSC-encapsulated COL/HA/FG composite gel can be a good therapeutic candidate/strategy for repairing of damaged articular cartilage.