The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims

The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.

Methods

Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.

Results

Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, respectively, but not affected by Cav channels antagonists.

Conclusions

Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.     

Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6

Background

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.

Methods

We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 and the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.

Results

OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, {"type":"entrez-nucleotide","attrs":{"text":"R59949","term_id":"830644","term_text":"R59949"}}R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, a blocker of DAG synthesis, inhibited acute HPV whereas {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125"}}U73343, the inactive form of {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, had no effect on HPV.

Conclusion

These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.     

Profilin-1 is expressed in human atherosclerotic plaques and induces atherogenic effects on vascular smooth muscle cells

Background

Profilin-1 is an ubiquitous actin binding protein. Under pathological conditions such as diabetes, profilin-1 levels are increased in the vascular endothelium. We recently demonstrated that profilin-1 overexpression triggers indicators of endothelial dysfunction downstream of LDL signaling, and that attenuated expression of profilin-1 confers protection from atherosclerosis in vivo.

Methodology

Here we monitored profilin-1 expression in human atherosclerotic plaques by immunofluorescent staining. The effects of recombinant profilin-1 on atherogenic signaling pathways and cellular responses such as DNA synthesis (BrdU-incorporation) and chemotaxis (modified Boyden-chamber) were evaluated in cultured rat aortic and human coronary vascular smooth muscle cells (VSMCs). Furthermore, the correlation between profilin-1 serum levels and the degree of atherosclerosis was assessed in humans.

Principal Findings

In coronary arteries from patients with coronary heart disease, we found markedly enhanced profilin expression in atherosclerotic plaques compared to the normal vessel wall. Stimulation of rat aortic and human coronary VSMCs with recombinant profilin-1 (10⁻⁶ M) in vitro led to activation of intracellular signaling cascades such as phosphorylation of Erk1/2, p70^S6 kinase and PI3K/Akt within 10 minutes. Furthermore, profilin-1 concentration-dependently induced DNA-synthesis and migration of both rat and human VSMCs, respectively. Inhibition of PI3K (Wortmannin, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or Src-family kinases (SU6656, PP2), but not PLCγ ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122), completely abolished profilin-induced cell cycle progression, whereas PI3K inhibition partially reduced the chemotactic response. Finally, we found that profilin-1 serum levels were significantly elevated in patients with severe atherosclerosis in humans (p<0.001 vs. no atherosclerosis or control group).

Conclusions

Profilin-1 expression is significantly enhanced in human atherosclerotic plaques compared to the normal vessel wall, and the serum levels of profilin-1 correlate with the degree of atherosclerosis in humans. The atherogenic effects exerted by profilin-1 on VSMCs suggest an auto-/paracrine role within the plaque. These data indicate that profilin-1 might critically contribute to atherogenesis and may represent a novel therapeutic target.     

Intracerebroventricular injection of leukotriene B4 attenuates antigen-induced asthmatic response via BLT1 receptor stimulating HPA-axis in sensitized rats

Background

Basic and clinical studies suggest that hypothalamic-pituitary-adrenal (HPA) axis is the neuroendocrine-immnue pathway that functionally regulates the chronic inflammatory disease including asthma. Our previous studies showed corresponding changes of cytokines and leukotriene B₄ (LTB₄) between brain and lung tissues in antigen-challenged asthmatic rats. Here, we investigated how the increased LTB₄ level in brain interacts with HPA axis in regulating antigen-induced asthmatic response in sensitized rats.

Methods

Ovalbumin-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB₄ or {"type":"entrez-nucleotide","attrs":{"text":"U75302","term_id":"1857248","term_text":"U75302"}}U75302 (a selective LTB₄ BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v) 30 min before challenge. Lung resistance (R_L) and dynamic lung compliance (C_dyn) were measured before and after antigen challenge. Inflammatory response in lung tissue was assessed 24 h after challenge. Expression of CRH mRNA and protein in hypothalamus were evaluated by RT-PCR and Western Blot, and plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using the ELISA kits.

Results

Antigen challenge decreased pulmonary function and induced airway inflammation, evoked HPA axis response in sensitized rats. Administration of LTB₄ via i.c.v markedly attenuated airway contraction and inflammation. Meanwhile, LTB₄ via i.c.v markedly increased CORT and ACTH level in plasma before antigen challenge, and followed by further increases in CORT and ACTH levels in plasma after antigen challenge in sensitized rats. Expression of CRH mRNA and protein in hypothalamus were also significantly increased by LTB₄ via i.c.v in sensitized rats after antigen challenge. These effect were completely blocked by pre-treatment with BLT1 receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"U75302","term_id":"1857248","term_text":"U75302"}}U75302 (10 ng), but not by BLT2 antagonist {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172","term_text":"LY255283"}}LY255283.

Conclusions

LTB₄ administered via i.c.v down-regulates the airway contraction response and inflammation through activation of the HPA axis via its BLT1 receptor. This study expands our concept of the regulatory role of intracranial inflammatory mediators in inflammatory diseases including asthma. The favourable effects of LTB₄ on the HPA axis may help to explain the phenomenon of self-relief after an asthmatic attack.     

Nitrogen-containing bisphosphonates inhibit RANKL- and M-CSF-induced osteoclast formation through the inhibition of ERK1/2 and Akt activation

Background

Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7.

Results

It was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation.

Conclusions

This indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.     

Safety and T cell modulating effects of high dose vitamin D3 supplementation in multiple sclerosis

Background

A poor vitamin D status has been associated with a high disease activity of multiple sclerosis (MS). Recently, we described associations between vitamin D status and peripheral T cell characteristics in relapsing remitting MS (RRMS) patients. In the present study, we studied the effects of high dose vitamin D₃ supplementation on safety and T cell related outcome measures.

Methodology/Principal Findings

Fifteen RRMS patients were supplemented with 20 000 IU/d vitamin D₃ for 12 weeks. Vitamin D and calcium metabolism were carefully monitored, and T cell characteristics were studied by flowcytometry. All patients finished the protocol without side-effects, hypercalcaemia, or hypercalciuria. The median vitamin D status increased from 50 nmol/L (31–175) at week 0 to 380 nmol/L (151–535) at week 12 (P<0.001). During the study, 1 patient experienced an exacerbation of MS and was censored from the T cell analysis. The proportions of (naïve and memory) CD4⁺ Tregs remained unaffected. Although Treg suppressive function improved in several subjects, this effect was not significant in the total cohort (P = 0.143). An increased proportion of IL-10⁺ CD4⁺ T cells was found after supplementation (P = 0.021). Additionally, a decrease of the ratio between IFN-γ⁺ and IL-4⁺ CD4⁺ T cells was observed (P = 0.035).

Conclusion/Significance

Twelve week supplementation of high dose vitamin D₃ in RRMS patients was well tolerated and did not induce decompensation of calcium metabolism. The skewing towards an anti-inflammatory cytokine profile supports the evidence on vitamin D as an immune-modulator, and may be used as outcome measure for upcoming randomized placebo-controlled trials.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00940719","term_id":"NCT00940719"}}NCT00940719     

Calcium Supplementation Increases Blood Creatinine Concentration in a Randomized Controlled Trial

Background

Calcium supplements are widely used among older adults for osteoporosis prevention and treatment. However, their effect on creatinine levels and kidney function has not been well studied.

Methods

We investigated the effect of calcium supplementation on blood creatinine concentration in a randomized controlled trial of colorectal adenoma chemoprevention conducted between 2004–2013 at 11 clinical centers in the United States. Healthy participants (N = 1,675) aged 45–75 with a history of colorectal adenoma were assigned to daily supplementation with calcium (1200 mg, as carbonate), vitamin D₃ (1000 IU), both, or placebo for three or five years. Changes in blood creatinine and total calcium concentration were measured after one year of treatment and multiple linear regression was used to estimate effects on creatinine concentrations.

Results

After one year of treatment, blood creatinine was 0.013±0.006 mg/dL higher on average among participants randomized to calcium compared to placebo after adjustment for other determinants of creatinine (P = 0.03). However, the effect of calcium treatment appeared to be larger among participants who consumed the most alcohol (2–6 drinks/day) or whose estimated glomerular filtration rate (eGFR) was less than 60 ml/min/1.73 m² at baseline. The effect of calcium treatment on creatinine was only partially mediated by a concomitant increase in blood total calcium concentration and was independent of randomized vitamin D treatment. There did not appear to be further increases in creatinine after the first year of calcium treatment.

Conclusions

Among healthy adults participating in a randomized clinical trial, daily supplementation with 1200 mg of elemental calcium caused a small increase in blood creatinine. If confirmed, this finding may have implications for clinical and public health recommendations for calcium supplementation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00153816","term_id":"NCT00153816"}}NCT00153816     

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes

Background

The ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca²⁺-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/Principal Findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca²⁺-handling proteins. Detailed laser confocal Ca²⁺ imaging demonstrated spontaneous whole-cell [Ca²⁺]_i transients. These transients required Ca²⁺ influx via L-type Ca²⁺ channels, as demonstrated by their elimination in the absence of extracellular Ca²⁺ or by administration of the L-type Ca²⁺ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca²⁺ store, contributing to [Ca²⁺]_i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca²⁺) and ryanodine (decreasing [Ca²⁺]_i). Similarly, the importance of Ca²⁺ reuptake into the SR via the SR Ca²⁺ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca²⁺]_i transients elimination. Finally, the presence of an IP3-releasable Ca²⁺ pool in hiPSC-CMs and its contribution to whole-cell [Ca²⁺]_i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122.

Conclusions/Significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca²⁺ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca²⁺]_i transients in hiPSC-CMs on both sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and intracellular store Ca²⁺ release.     

Complex dynamics of osteoclast formation and death in long-term cultures

Background

Osteoclasts, cells responsible for bone resorption, contribute to the development of degenerative, metabolic and neoplastic bone diseases, which are often characterized by persistent changes in bone microenvironment. We aimed to investigate the dynamics of osteoclast formation and death in cultures that considerably exceeded the length of standard protocol and to design a mathematical model describing osteoclastogenesis.

Methodology/Principal Findings

RAW 264.7 monocytic cells fuse to form multinucleated osteoclasts upon treatment with pro-resorptive cytokine RANKL. We have found that in long-term experiments (15–26 days), the dynamics of changes in osteoclast numbers was remarkably complex and qualitatively variable in different experiments. Whereas 19 of 46 experiments exhibited single peak of osteoclast formation, in 27 experiments we observed development of successive waves of osteoclast formation and death. Periodic changes in osteoclast numbers were confirmed in long-term cultures of mouse bone marrow cells treated with M-CSF and RANKL. Because the dynamics of changes in osteoclast numbers was found to be largely independent of monocytes, a two-species model of ordinary differential equations describing the changes in osteoclasts and monocytes was ineffective in recapitulating the oscillations in osteoclast numbers. Following experimental observation that medium collected from mature osteoclasts inhibited osteoclastogenesis in fresh cultures, we introduced a third variable, factor f, to describe osteoclast-derived inhibitor. This model allowed us to simulate the oscillatory changes in osteoclasts, which were coupled to oscillatory changes in the factor f, whereas monocytes changed exponentially. Importantly, to achieve the experimentally observed oscillations with increasing amplitude, we also had to assume that osteoclast presence stimulates osteoclast formation.

Conclusions/Significance

This study identifies the critical role for osteoclast autocrine regulation in controlling long-term dynamic of osteoclast formation and death and describes the complementary roles for negative and positive feedback mediators in determining the sharp dynamics of activation and inactivation of osteoclasts.     

Effects of Intermittent IL-2 Alone or with Peri-Cycle Antiretroviral Therapy in Early HIV Infection: The STALWART Study

Background

The Study of Aldesleukin with and without antiretroviral therapy (STALWART) evaluated whether intermittent interleukin-2 (IL-2) alone or with antiretroviral therapy (ART) around IL-2 cycles increased CD4⁺ counts compared to no therapy.

Methodology

Participants not on continuous ART with ≥300 CD4⁺ cells/mm³ were randomized to: no treatment; IL-2 for 5 consecutive days every 8 weeks for 3 cycles; or the same IL-2 regimen with 10 days of ART administered around each IL-2 cycle. CD4⁺ counts, HIV RNA, and HIV progression events were collected monthly.

Principal Findings

A total of 267 participants were randomized. At week 32, the mean CD4⁺ count was 134 cells greater in the IL-2 alone group (p<0.001), and 133 cells greater in the IL-2 plus ART group (p<0.001) compared to the no therapy group. Twelve participants in the IL-2 groups compared to 1 participant in the group assigned to no therapy experienced an opportunistic event or died (HR 5.84, CI: 0.59 to 43.57; p = 0.009).

Conclusions

IL-2 alone or with peri-cycle HAART increases CD4⁺ counts but was associated with a greater number of opportunistic events or deaths compared to no therapy. These results call into question the immunoprotective significance of IL-2-induced CD4⁺ cells.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00110812","term_id":"NCT00110812"}}NCT00110812     

IL-32 and IL-17 interact and have the potential to aggravate osteoclastogenesis in rheumatoid arthritis

Introduction

Interleukin (IL)-32 and IL-17 play critical roles in pro-inflammatory responses and are highly expressed in the synovium of patients with rheumatoid arthritis (RA). We investigated the relations between these two cytokines (IL-17 and IL-32) for their ability to induce each other and to stimulate osteoclasts in RA fibroblast-like synoviocytes (FLSs) and T cells.

Methods

FLSs were isolated through surgical synovectomy obtained from patients with RA or osteoarthritis (OA). Real-time PCR were performed to evaluate the expression of IL-32, IL-17 and osteoclast-related genes. Immunohistochemical staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to determine the distribution of inflammatory cytokines and the presence of osteoclastogenesis.

Results

IL-17 induced the expression of IL-32 in the FLSs from RA patients, as assessed by microarray. IL-32 production was increased by IL-17. IL-32 in the FLSs from RA patients induced the production of IL-17 in CD4⁺ T cells. IL-32 and IL-17 were colocalized near TRAP-positive areas in joint specimens. IL-17 and IL-32 synergistically induced the differentiation of osteoclasts, as demonstrated by the expression of osteoclast-related genes. IL-32 and IL-17 also could induce resorption by osteoclasts in a RANKL-dependent manner.

Conclusions

IL-17 affected the expression of IL-32 in FLSs of RA patients and IL-32 induced the production of IL-17 in CD4+ T cells. Both IL-17 and IL-32 cytokines can reciprocally influence each other''s production and amplify the function of osteoclastogenesis in the in RA synovium. Separately, IL-17 and IL-32 each stimulated osteoclastogenesis without RANKL. Together, the two cytokines synergistically amplified the differentiation of osteoclasts, independent of RANKL stimulation.     

Vitamin E isoforms differentially regulate intercellular adhesion molecule-1 activation of PKCα in human microvascular endothelial cells

Aims

ICAM-1-dependent leukocyte recruitment in vivo is inhibited by the vitamin E isoform d-α-tocopherol and elevated by d-γ-tocopherol. ICAM-1 is reported to activate endothelial cell signals including protein kinase C (PKC), but the PKC isoform and the mechanism for ICAM-1 activation of PKC are not known. It is also not known whether ICAM-1 signaling in endothelial cells is regulated by tocopherol isoforms. We hypothesized that d-α-tocopherol and d-γ-tocopherol differentially regulate ICAM-1 activation of endothelial cell PKC.

Results

ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ in TNFα-pretreated human microvascular endothelial cells. ICAM-1 activation of PKCα was blocked by the PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, ERK1/2 inhibitor PD98059, and xanthine oxidase inhibitor allopurinol. ERK1/2 activation was blocked by inhibition of XO and PLC but not by inhibition of PKCα, indicating that ERK1/2 is downstream of XO and upstream of PKCα during ICAM-1 signaling. During ICAM-1 activation of PKCα, the XO-generated ROS did not oxidize PKCα. Interestingly, d-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol.

Conclusions

Crosslinking ICAM-1 stimulated XO/ROS which activated ERK1/2 that then activated PKCα. ICAM-1 activation of PKCα was inhibited by d-α-tocopherol and this inhibition was ablated by the addition of d-γ-tocopherol. These tocopherols regulated ICAM-1 activation of PKCα without altering the upstream signal ERK1/2. Thus, we identified a mechanism for ICAM-1 activation of PKC and determined that d-α-tocopherol and d-γ-tocopherol have opposing regulatory functions for ICAM-1-activated PKCα in endothelial cells.     

miR-31 controls osteoclast formation and bone resorption by targeting RhoA

Introduction

Increased activity of osteoclasts is responsible for bone loss and joint destruction in rheumatoid arthritis. For osteoclast development and bone resorption activity, cytoskeletal organization must be properly regulated. MicroRNAs (miRNAs) are endogenous small noncoding RNAs that suppress expression of their target genes. This study was conducted to identify crucial miRNAs to control osteoclasts.

Methods

miRNA expression in the bone marrow-derived macrophages (BMM) with or without receptor activator of nuclear factor κB ligand (RANKL) stimulation was analyzed by miRNA array. To examine the role of specific miRNAs in osteoclast formation, bone resorption activity and actin ring formation, the BMM were retrovirally transduced with miRNA antagomirs. To confirm whether the suppressive effects on osteoclastogenesis by miR-31 inhibition were mediated by targeting RhoA, osteoclast formation was analyzed in the presence of the RhoA inhibitor, exoenzyme C3.

Results

miR-31 was identified as one of the highly upregulated miRNAs during osteoclast development under RANKL stimulation. Inhibition of miR-31 by specific antagomirs suppressed the RANKL-induced formation of osteoclasts and bone resorption. Phalloidin staining of osteoclasts revealed that actin ring formation at the cell periphery was severely impaired by miR-31 inhibition, and clusters of small ringed podosomes were observed instead. In these osteoclasts, expression of RhoA, one of the miR-31 target genes, was upregulated by miR-31 inhibition in spite of the impaired osteoclastogenesis. Treatment with the RhoA inhibitor, exoenzyme C3, rescued the osteoclastogenesis impaired by miR-31 inhibition.

Conclusions

miR-31 controls cytoskeleton organization in osteoclasts for optimal bone resorption activity by regulating the expression of RhoA.     

Effects of High Doses of Cholecalciferol in Normal Subjects: A Randomized Double-Blinded,Placebo-Controlled Trial

Background

Vitamin D repletion with high doses of vitamin D is often recommended to patients and healthy subjects. The safety, especially concerning changes in urinary calcium excretion is of great importance.

Methods

In a double-blinded, placebo-controlled study in 40 healthy volunteers, we examined the changes in mineral metabolism during supplementation with 3000 IU of oral cholecalciferol daily during 4 months.

Results

Both 25(OH)vitamin D and 1,25(OH)₂vitamin D increased significantly in the active treated group as compared to the placebo group (186% versus 14% (P<0.001) and 28% versus – 8% (P<0.001)). No change was observed in urinary calcium excretion in the active group compared to the placebo group (P = 0.891). Fibroblast growth factor 23 increased significantly by 10% (P<0.018) in the active group. However, there was no difference in changes in FGF23 between treatment groups (P = 0.457).

Conclusion

High dose cholecalciferol significantly increases 25(OH)vitamin D and 1,25(OH)₂vitamin D levels compared to placebo. No changes in urinary calcium excretion or other measured components of the mineral metabolism were found between groups.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00952562","term_id":"NCT00952562"}}NCT00952562.     

Enhanced interleukin-1 activity contributes to exercise intolerance in patients with systolic heart failure

Background

Heart failure (HF) is a complex clinical syndrome characterized by impaired cardiac function and poor exercise tolerance. Enhanced inflammation is associated with worsening outcomes in HF patients and may play a direct role in disease progression. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that becomes chronically elevated in HF and exerts putative negative inotropic effects.

Methods and Results

We developed a model of IL-1β-induced left ventricular (LV) dysfunction in healthy mice that exhibited a 32% reduction in LV fractional shortening (P<0.001) and a 76% reduction in isoproterenol response (P<0.01) at 4 hours following a single dose of IL-1β 3 mcg/kg. This phenotype was reproducible in mice injected with plasma from HF patients and fully preventable by pretreatment with IL-1 receptor antagonist (anakinra). This led to the design and conduct of a pilot clinical to test the effect of anakinra on cardiopulmonary exercise performance in patients with HF and evidence of elevated inflammatory signaling (n = 7). The median peak oxygen consumption (VO₂) improved from 12.3 [10.0, 15.2] to 15.1 [13.7, 19.3] mL·kg^–1·min^–1 (P = 0.016 vs. baseline) and median ventilator efficiency (V_E/VCO₂ slope) improved from 28.1 [22.8, 31.7] to 24.9 [22.9, 28.3] (P = 0.031 vs. baseline).

Conclusions

These findings suggest that IL-1β activity contributes to poor exercise tolerance in patients with systolic HF and identifies IL-1β blockade as a novel strategy for pharmacologic intervention.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01300650","term_id":"NCT01300650"}}NCT01300650     

Umbilical Cord Blood IL-6 as Predictor of Early-Onset Neonatal Sepsis in Women with Preterm Prelabour Rupture of Membranes

Objective

To evaluate umbilical cord interleukin (IL)-6 and funisitis as independent predictors of early-onset neonatal sepsis (EONS) in preterm prelabor rupture of membranes (PPROM).

Design

Prospective cohort study.

Setting

Evaluation of umbilical cord IL-6 and funisitis as predictors of early-onset neonatal sepsis in PPROM.

Population

176 women with PPROM between 23+0−36+6 weeks of gestation.

Methods

Umbilical cord IL-6 was assayed by ELISA. Funisitis was defined according to the Salafia classification. Data was adjusted by gestational age at delivery and prenatal administration of corticosteroids and antibiotics.

Main Outcome Measures

Binary logistic regression was performed to assess the independence of umbilical cord IL-6 and funisitis to predict EONS in women complicated with PPROM.

Results

The rate of EONS was 7%. Funisitis was present in 18% of women. Umbilical cord IL-6 was significantly higher in women complicated with EONS than without [median (range) 389.5 pg/mL (13.9–734.8) vs 5.2 (0.1–801–4), p<0.001]. Umbilical cord IL-6 was the only independent predictor of early-onset neonatal sepsis (odds ratio 13.6, p = 0.004).

Conclusion

Umbilical cord IL-6 was the only predictor of early-onset neonatal sepsis in PPROM. Contrary to what is reported, funisitis was not.     

IL-10 Dependent Suppression of Type 1, Type 2 and Type 17 Cytokines in Active Pulmonary Tuberculosis

Background

Although Type 1 cytokine responses are considered protective in pulmonary tuberculosis (PTB), their role as well as those of Type 2, 17 and immunoregulatory cytokines in tuberculous lymphadenitis (TBL) and latent tuberculosis (LTB) have not been well studied.

Aim and Methods

To identify cytokine responses associated with pulmonary tuberculosis (TB), TB lymphadenitits and latent TB, we examined mycobacterial antigen-specific immune responses of PTB, TBL and LTB individuals. More specifically, we examined ESAT-6 and CFP-10 induced Type 1, Type 2 and Type 17 cytokine production and their regulation using multiplex ELISA.

Results

PTB individuals exhibited a significantly lower baseline as well as antigen-specific production of Type 1 (IFNγ, TNFα and IL-2); Type 2 (IL-4) and Type 17 (IL-17A and IL-17F) cytokines in comparison to both TBL and LTB individuals. TBL individuals exhibited significantly lower antigen-specific IFNγ responses alone in comparison to LTB individuals. Although, IL-10 levels were not significantly higher, neutralization of IL-10 during antigen stimulation resulted in significantly enhanced production of IFNγ, IL-4 and IL-17A in PTB individuals, indicating that IL-10 mediates (at least partially) the suppression of cytokine responses in PTB.

Conclusion

Pulmonary TB is characterized by an IL-10 dependent antigen-specific suppression of Type 1, Type 2 and Type 17 cytokines, reflecting an important association of these cytokines in the pathogenesis of active TB.     

Decreased Frequencies of Circulating CD4+ T Follicular Helper Cells Associated with Diminished Plasma IL-21 in Active Pulmonary Tuberculosis

Background

Circulating T follicular helper (Tfh) cells represent a distinct subset of CD4⁺ T cells and are important in immunity to infections. Although they have been shown to play a role in experimental models of tuberculosis infection, their role in human tuberculosis remains unexplored.

Aims/Methodology

To determine the distribution of circulating Tfh cells in human TB, we measured the frequencies of Tfh cells ex vivo and following TB - antigen or polyclonal stimulation in pulmonary TB (PTB; n = 30) and latent TB (LTB; n = 20) individuals, using the markers CXCR5, PD-1 and ICOS.

Results

We found that both ex vivo and TB - antigen induced frequencies of Tfh cell subsets was significantly lower in PTB compared to LTB individuals. Similarly, antigen induced frequencies of Tfh cells expressing IL-21 was also significantly lower in PTB individuals and this was reflected in diminished circulating levels of IL-21 and IFNγ. This was not accompanied by diminished frequencies of activated or memory B cell subsets. Finally, the diminution in frequency of Tfh cells in PTB individuals was dependent on IL-10, CTLA-4 and PD-L1 in vitro.

Conclusions

Thus, PTB is characterized by adiminution in the frequency of Tfh cell subsets.