α-Glucosidase inhibitor miglitol attenuates glucose fluctuation, heart rate variability and sympathetic activity in patients with type 2 diabetes and acute coronary syndrome: a multicenter randomized controlled (MACS) study.

BACKGROUND: Little is known about clinical associations between glucose fluctuations including hypoglycemia, heart rate variability (HRV), and the activity of the sympathetic nervous system (SNS) in patients with acute phase of acute coronary syndrome (ACS). This pilot study aimed to evaluate the short-term effects of glucose fluctuations on HRV and SNS activity in type 2 diabetes mellitus (T2DM) patients with recent ACS. We also examined the effect of suppressing glucose fluctuations with miglitol on these variables. METHODS: This prospective, randomized, open-label, blinded-endpoint, multicenter, parallel-group comparative study included 39 T2DM patients with recent ACS, who were randomly assigned to either a miglitol group (n = 19) or a control group (n = 20). After initial 24-h Holter electrocardiogram (ECG) (Day 1), miglitol was commenced and another 24-h Holter ECG (Day 2) was recorded. In addition, continuous glucose monitoring (CGM) was performed throughout the Holter ECG. RESULTS: Although frequent episodes of subclinical hypoglycemia (≤4.44 mmo/L) during CGM were observed on Day 1 in the both groups (35% of patients in the control group and 31% in the miglitol group), glucose fluctuations were decreased and the minimum glucose level was increased with substantial reduction in the episodes of subclinical hypoglycemia to 7.7% in the miglitol group on Day 2. Holter ECG showed that the mean and maximum heart rate and mean LF/HF were increased on Day 2 in the control group, and these increases were attenuated by miglitol. When divided 24-h time periods into day-time (0700-1800 h), night-time (1800-0000 h), and bed-time (0000-0700 h), we found increased SNS activity during day-time, increased maximum heart rate during night-time, and glucose fluctuations during bed-time, which were attenuated by miglitol treatment. CONCLUSIONS: In T2DM patients with recent ACS, glucose fluctuations with subclinical hypoglycemia were associated with alterations of HRV and SNS activity, which were mitigated by miglitol, suggesting that these pathological relationships may be a residual therapeutic target in such patients. Trial registration Unique Trial Number, UMIN000005874 ( https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000006929 ).

Neutrophil priming occurs in a sequential manner and can be visualized in living animals by monitoring IL-1ß promoter activation.

Rapid enhancement of phagocyte functionality is a hallmark of neutrophil priming. GeneChip analyses unveiled elevated CD54, dectin-2, and IL-1ß mRNA expression by neutrophils isolated from inflammatory sites. In fact, CD54 and dectin-2 protein expression was detected on neutrophils recovered from skin, peritoneal, and lung inflammation lesions but not on those in bone marrow or peripheral blood. Neutrophils increased CD54 and dectin-2 mRNA during migration in Boyden chambers and acquired CD54 and dectin-2 surface expression after subsequent exposure to GM-CSF. Neutrophils purified from IL-1ß promoter-driven DsRed-transgenic mice acquired DsRed signals during cell migration or exposure to GM-CSF. CD54 and dectin-2 were expressed by DsRed(+) (but not DsRed(-)) neutrophils in GM-CSF-supplemented cultures, and neutrophils recovered from inflammatory sites exhibited strong DsRed signals. The dynamic process of neutrophil priming was studied in chemically induced inflammatory skin lesions by monitoring DsRed expression using confocal microscopy. A majority (>80%) of Ly6G(+) neutrophils expressed DsRed, and those DsRed(+)/Ly6G(+) cells exhibited crawling motion with a higher velocity compared with their DsRed(-)/Ly6G(+) counterparts. This report unveils motile behaviors of primed neutrophils in living animals. We propose that neutrophil priming occurs in a sequential manner with rapid enhancement of phagocyte functionality, followed by CD54 and dectin-2 mRNA and protein expression, IL-1ß promoter activation, and accelerated motility. Not only do these findings provide a new conceptual framework for our understanding of the process of neutrophil priming, they also unveil new insights into the pathophysiology of many inflammatory disorders that are characterized by neutrophil infiltration.

IL-1α Gene Deletion Protects Oligodendrocytes after Spinal Cord Injury through Upregulation of the Survival Factor Tox3.

Spinal cord injury (SCI) causes the release of danger signals by stressed and dying cells, a process that leads to neuroinflammation. Evidence suggests that inflammation plays a role in both the damage and repair of injured neural tissue. We show that microglia at sites of SCI rapidly express the alarmin interleukin (IL)-1α, and that infiltrating neutrophils and macrophages subsequently produce IL-1ß. Infiltration of these cells is dramatically reduced in both IL-1α(-/-) and IL-1ß(-/-) mice, but only IL-1α(-/-) mice showed rapid (at day 1) and persistent improvements in locomotion associated with reduced lesion volume. Similarly, intrathecal administration of the IL-1 receptor antagonist anakinra restored locomotor function post-SCI. Transcriptome analysis of SCI tissue at day 1 identified the survival factor Tox3 as being differentially regulated exclusively in IL-1α(-/-) mice compared with IL-1ß(-/-) and wild-type mice. Accordingly, IL-1α(-/-) mice have markedly increased Tox3 levels in their oligodendrocytes, beginning at postnatal day 10 (P10) and persisting through adulthood. At P10, the spinal cord of IL-1α(-/-) mice showed a transient increase in mature oligodendrocyte numbers, coinciding with increased IL-1α expression in wild-type animals. In adult mice, IL-1α deletion is accompanied by increased oligodendrocyte survival after SCI. TOX3 overexpression in human oligodendrocytes reduced cellular death under conditions mimicking SCI. These results suggest that IL-1α-mediated Tox3 suppression during the early phase of CNS insult plays a crucial role in secondary degeneration. SIGNIFICANCE STATEMENT: The mechanisms underlying bystander degeneration of neurons and oligodendrocytes after CNS injury are ill defined. We show that microglia at sites of spinal cord injury (SCI) rapidly produce the danger signal interleukin (IL)-1α, which triggers neuroinflammation and locomotor defects. We uncovered that IL-1α(-/-) mice have markedly increased levels of the survival factor Tox3 in their oligodendrocytes, which correlates with the protection of this cell population, and reduced lesion volume, resulting in unprecedented speed, level, and persistence of functional recovery after SCI. Our data suggest that central inhibition of IL-1α or Tox3 overexpression during the acute phase of a CNS insult may be an effective means for preventing the loss of neurological function in SCI, or other acute injuries such as ischemia and traumatic brain injuries.

Harnessing DCs by substance P.

In this issue of Blood, Janelsins et al report that substance P (SP)­treated dendritic cells (DCs) efficiently home to lymph nodes, where they induce inflammatory DCs to produce interleukin-12 (IL-12), thereby promoting type 1 polarized immunity.

Neutrophil differentiation into a unique hybrid population exhibiting dual phenotype and functionality of neutrophils and dendritic cells.

Neutrophils have been reported to acquire surface expression of MHC class II and co-stimulatory molecules as well as T-cell stimulatory activities when cultured with selected cytokines. However, cellular identity of those unusual neutrophils showing antigen presenting cell (APC)-like features still remains elusive. Here we show that both immature and mature neutrophils purified from mouse bone marrow differentiate into a previously unrecognized "hybrid" population showing dual properties of both neutrophils and dendritic cells (DCs) when cultured with granulocyte macrophage-colony-stimulating factor but not with other tested growth factors. The resulting hybrid cells express markers of both neutrophils (Ly6G, CXCR2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86). They also exhibit several properties typically reserved for DCs, including dendritic morphology, probing motion, podosome formation, production of interleukin-12 and other cytokines, and presentation of various forms of foreign protein antigens to naïve CD4 T cells. Importantly, they retain intrinsic abilities of neutrophils to capture exogenous material, extrude neutrophil extracellular traps, and kill bacteria via cathelicidin production. Not only do our results reinforce the notion that neutrophils can acquire APC-like properties, they also unveil a unique differentiation pathway of neutrophils into neutrophil-DC hybrids that can participate in both innate and adaptive immune responses.

Emergence, origin, and function of neutrophil-dendritic cell hybrids in experimentally induced inflammatory lesions in mice.

Although unusual neutrophils expressing major histocompatibility complex class II (MHC II) and costimulatory molecules have been detected at inflammatory sites in mice and humans, their identity, origin, and function remain unclear. We have demonstrated that, when cultured with granulocyte macrophage-colony-stimulating factor, neutrophils can give rise to a unique hybrid population exhibiting dual phenotypic and functionality of neutrophils and dendritic cells (DCs). Here we report that hybrid cells expressing surface markers of neutrophils (Ly6G, L-selectin, CXC chemokines receptor 2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86) become detectable in the peritoneal cavity, skin, lung, and lymph nodes under inflammatory conditions. Importantly, 20% to 30% of the adoptively transferred neutrophils acquired CD11c and MHC II expression when recovered from inflammatory lesions, demonstrating neutrophil → hybrid conversion in living animals. Using Escherichia coli strains expressing green fluorescent protein and ovalbumin, we further show hybrids play dual protective roles by rapidly clearing bacteria and presenting bacterial antigens to CD4 T cells. These results indicate that some of the neutrophils recruited to inflammatory lesions can differentiate into neutrophil-DC hybrids, thus challenging the classic view of neutrophils as terminally differentiated leukocytes destined to die or to participate primarily in host innate immunity.

Reduced ratio of eicosapentaenoic acid and docosahexaenoic acid to arachidonic acid is associated with early onset of acute coronary syndrome.

BACKGROUND: The hospitalization rate for acute coronary syndrome (ACS) for people aged ≤50 has remained stable over the past decade. Increased serum levels of n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are associated with a decreased incidence of cardiovascular events and mortality in older patients; however, it is currently unknown whether reduced serum levels of n-3 PUFAs is also a risk factor for ACS in patients aged ≤50 years. METHODS AND RESULTS: We retrospectively reviewed 102 (male/ female 73/29) Japanese ACS patients whose serum levels of EPA/arachidonic acid (AA) and DHA/AA were evaluated on admission. The EPA/AA ratio was the lowest in patients aged ≤50 compared to patients aged 51-74 and ≥75. Pearson correlation analysis showed that early ACS onset was associated with low EPA/AA and DHA/AA ratios, and multiple regression analysis determined that decreased ratios of EPA/AA and DHA/AA, and male sex, current smoker status, increased body mass index and triglyceride levels, independently correlated with early ACS onset. Conversely, low-density and high-density lipoproteins, glycated hemoglobin, and hypertension did not correlate with early ACS onset. Subgroup analyses of male patients revealed that decreased ratios of EPA/AA and DHA/AA independently correlated with early ACS onset. CONCLUSION: Decreased EPA/AA and DHA/AA ratios may be risk factors for early onset of ACS, suggesting that reduced EPA/AA and DHA/AA may represent targets for preventing ACS in Japanese young people.

Neutrophil-derived IL-1ß is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1ß/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1ß at the site of infection. Furthermore, neutrophil-derived IL-1ß was essential for host defense since adoptive transfer of IL-1ß-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1ß-deficient mice. S. aureus-induced IL-1ß production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1ß and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1ß production by neutrophils.

Cardiac rehabilitation reduces serum levels of oxidized low-density lipoprotein.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) levels have been found to play an important role in the progression of atherosclerosis. However, methods for effectively reducing oxLDL levels have not been established. Comprehensive cardiac rehabilitation (CR) with exercise training prevents the progression of atherosclerosis, and might reduce oxLDL levels. METHODS AND RESULTS: We measured the serum levels of malondialdehyde-modified LDL (MDA-LDL), a marker of oxLDL, in 136 patients who were enrolled in a 6-month CR program. Peak oxygen consumption (V̇O2) and MDA-LDL levels were analyzed, before and 6 months after enrolment. In total, 67 patients completed the CR program (CR group) and 69 patients failed to complete the program (non-CR group). Peak V̇O2increased significantly in the CR group (P<0.01). The levels of MDA-LDL decreased significantly in the CR group (P<0.01) but not in the non-CR group. ∆V̇O2(peak V̇O2after CR-peak V̇O2before CR) was negatively associated with ∆MDA-LDL (MDA-LDL after CR-MDA-LDL before CR) (R(2)=0.11, P=0.01). Multiple regression analysis showed that continuing CR was an independent determining factor for lowering MDA-LDL levels. CONCLUSIONS: CR decreases oxLDL levels in patients with cardiovascular diseases. Moreover, CR may prevent cardiovascular events through an antioxidative effect.

Serum concentration of eicosapentaenoic acid is associated with cognitive function in patients with coronary artery disease.

BACKGROUND: Recent studies have shown that intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with reduced risk of cognitive impairment and coronary artery disease (CAD); however, it is currently unknown whether reduced serum n-3 PUFA is associated with cognitive impairment in patients with CAD. METHODS: We retrospectively evaluated cognitive function with the mini-mental state examination (MMSE), serum levels of PUFAs (including eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA], dihomogammalinolenic acid [DGLA], and arachidonic acid [AA]), cardiovascular risk factors (hypertension, dyslipidemia, diabetes mellitus, cerebrovascular disease, and history of current/previous smoking), and parameters of cardiac function (left ventricular ejection fraction and brain natriuretic peptide levels) in 146 Japanese CAD patients. The associations between the MMSE scores and the other parameters were evaluated. RESULTS: Pearson correlation analysis showed that EPA (R = 0.25, P <0.01), EPA/AA ratio (R = 0.22, P = 0.01), and left ventricular ejection fraction (R = 0.15, P = 0.04) were positively associated with MMSE score, and that age (R = -0.20, P <0.01) and brain natriuretic peptide levels (R = -0.28, P <0.01) were inversely associated with MMSE score. Multiple regression analysis showed that age (P <0.05) was negatively associated with MMSE score, while EPA (P <0.01) and EPA/AA ratio (P <0.05) were positively associated with MMSE score; however, sex; body mass index; left ventricular ejection fraction; levels of DHA, AA, and DGLA; DHA/AA ratio; brain natriuretic peptide; and presence of hypertension, dyslipidemia, diabetes mellitus, cerebrovascular disease, and history of current/previous smoking were statistically excluded. CONCLUSIONS: Serum EPA concentration is associated with cognitive function in patients with CAD, suggesting that a low serum EPA level is a risk factor for cognitive impairment independent of cardiac function, including left ventricular ejection fraction. This correlation potentially lends further support to a role of dietary n-3 PUFAs in preventing the cognitive decline in CAD patients.

Low MICA Gene Expression Confers an Increased Risk of Graves' Disease: A Mendelian Randomization Study.

Background: Expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) plays a major role as a "danger signal" on stressed cells to promote removal of the latter by NKG2D-expressing cytotoxic lymphocytes. NKG2DL expression has been found in peripheral immune cells as well, such as in macrophages; however, the effect of this expression is yet to be determined. Methods: We determined instrumental variables (IVs; R2 <0.01 in linkage disequilibrium), explaining the major variance in major histocompatibility complex class I chain-related protein A (MICA) and B (MICB) gene expression levels from the expression-quantitative trait locus (eQTL) of NKG2DLs based on the RNA-seq analysis of peripheral blood mononuclear cells (PBMCs) from 381 Japanese. Simultaneously, the target outcomes were filtered by PheWAS from 58 health risks, using a community-based cohort study composed of 44,739 Japanese residents. Finally, we estimated the causal effect of gene expression levels on the outcomes using the Mendelian randomization approach. Results: We determined nine and four IVs, explaining 87.6% and 33.0% of MICA and MICB gene expression levels, respectively. In the association test, we identified 10 or 13 significant outcomes associated with the MICA or MICB eQTLs, respectively, as well as the causal effect of MICA expression on Graves' disease (GD) (p = 4.2 × 10-3; odds ratio per 1 S.D. difference in the expression: 0.983 [confidence interval: 0.971-0.995]), using the weighted median estimator, without significant pleiotropy (p > 0.05), and the results were consistent across the sensitivity analyses. Conclusions: Our study provide novel evidence associating NKG2DL expression with GD, an autoimmune thyroiditis; direction of the effect indicated the immunoregulatory role of MICA expression in PBMCs, suggesting the importance of further functional assays in inflammatory diseases.

Evaluation of short-term epigenetic age fluctuation.

Considerable effort has been spent on lowering and maintaining the epigenetic age. However, the extent to which epigenetic age fluctuates under normal conditions is poorly understood. Therefore, we analyzed methylation data from monocytes and peripheral blood mononuclear cells collected from two Japanese men. The ranges of the Pan-tissue, Skin and blood, and DNAm PhenoAge epigenetic age during 3 months were ≥ 5.62, ≥ 3.04, and ≥ 8.23 years, and the maximum daily changes were 5.21, 3.20, and 6.53 years, respectively. These fluctuations were not suppressed by correcting for cell-type composition. Although the underlying biological mechanism remains unclear, there was a nonnegligible degree of age fluctuation which should inform personalized clinical applications.

Identification of crassin acetate as a new immunosuppressant triggering heme oxygenase-1 expression in dendritic cells.

By screening 720 natural compounds in a standard 2-way allogeneic mixed leukocyte reaction assay, we identified a potent immunosuppressive capacity of crassin acetate (CRA), a coral-derived cembrane diterpenoid. CRA efficiently inhibited allogeneic mixed leukocyte reaction as well as antigen-specific activation of CD4 T cells by bone marrow-derived dendritic cells (DCs). With regard to cellular targets, CRA suppressed not only mitogen-triggered T-cell activation, but also lipopolysaccharide-induced DC maturation, indicating dual functionality. Treatment with CRA at nontoxic doses induced heme oxygenase-1 (HO-1) mRNA/protein expression and HO-1 enzymatic activity in DCs, suggesting a unique mechanism of action. In fact, lipopolysaccharide-induced DC maturation was also inhibited by structurally unrelated compounds known to induce HO-1 expression or carbon monoxide (CO) release. Allergic contact hypersensitivity response to oxazolone and oxazolone-induced Langerhans cell migration from epidermis were both prevented almost completely by systemic administration of CRA. Not only do our results support the recent concept that HO-1/CO system negatively regulates immune responses, they also form both conceptual and technical frameworks for a more systematic, large-scale drug discovery effort to identify HO-1/CO-targeted immunosuppressants with dual target specificity.

CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness.

CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39-/- mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39-/- mice. Paradoxically, T cell-mediated allergic contact hypersensitivity was severely attenuated in CD39-/- mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39-/- DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC-T cell communication in antigen presentation.

Congenital Hypogonadotropic Hypogonadism with Early-Onset Coronary Artery Disease.

The patient with congenital hypogonadotropic hypogonadism (HH) shows low serum levels of androgen, which is a group of sex hormones including testosterone, caused by the decreased gonadotropin release in the hypothalamus. Recent reports showed androgens exert protective effects against insulin resistance or atherosclerotic diseases, such as diabetes mellitus or coronary artery disease. However, whether the juvenile hypogonadism affects the diabetes or cardiovascular disease is unclear. We report a case of a middle-aged man with congenital HH who had severe coronary artery disease complicated with metabolic disorders. J. Med. Invest. 68 : 189-191, February, 2021.

Stimulus-dependent deacylation of bacterial lipopolysaccharide by dendritic cells.

We describe here a previously unrecognized property of dendritic cells (DCs), the ability to deacylate the lipid A moiety of gram-negative bacterial LPSs. Both immature DCs of the XS52 cell line and bone marrow-derived DCs produce acyloxyacyl hydrolase, an enzyme that detoxifies LPS by selectively removing the secondary acyl chains from lipid A. Acyloxyacyl hydrolase expression decreased when DCs were incubated with IL-4, IL-1 beta, TNF alpha, and an agonistic CD40 antibody (maturation cocktail), and increased after treatment with LPS, CpG oligodeoxynucleotides, or a gram-positive bacterium (Micococcus luteus). Maturation cocktail treatment also diminished, whereas LPS treatment enhanced or maintained the cells' ability to kill Escherichia coli, deacylate LPS, and degrade bacterial protein. Enzymatic deacylation of LPS is an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist.