How I do it: optic canal unroofing in surgery for tuberculum sellae meningiomas with compression of the optic nerve.

BACKGROUND: Tuberculum sellae meningiomas are challenging to treat when accompanied with altered vision due to compression of the optic nerve. These tumors mostly refer to be benign; therefore, gross total removal and excellent functional recovery are desired. METHOD: We describe the microsurgical treatment of tuberculum sellae meningioma with altered vision function on the left eye. Intradural unroofing of the optic canal with gross total resection of the tumor led to immediate excellent recovery. Intraoperative video highlights key steps of our surgical approach. CONCLUSION: Optic canal unroofing is in our opinion safe and mandatory when treating tuberculum sellae meningiomas with compression of optic nerve.

Low serum vitamin D levels are associated with a low percentage of TREM-2+ monocytes in low-grade gliomas and poorer overall survival in patients with high-grade gliomas.

INTRODUCTION: Anti-inflammatory effect of vitamin D (VD) could be beneficial in improving the survival of glioma patients. The aim of our study was to analyse the serum levels of vitamin D in glioma patients and to find an association with the prognosis of glioma patients and other investigated parameters. MATERIAL AND METHODS: The study included 63 patients with gliomas. Percentage of CD14+ monocytes, TREM-1+ and TREM-2+ monocytes were determined by flow cytometry, serum levels of 25(OH)D were evaluated by electrochemiluminescent binding test. RESULTS: Six patients out of 63 had normal levels of VD. A significant difference in the overall survival (OS) in the patients with severe VD deficiency, VD deficiency and insufficiency in grade IV was found. In grade II and III, the levels of vitamin D positively correlated with the percentage of TREM-2+ monocytes, and in grade II also a negative correlation of VD with TREM-1/TREM-2 ratio was observed. CONCLUSION: Levels of VD could influence the prognosis of patients with high-grade gliomas. Serum level of 25(OH)D in low-grade gliomas positively correlated with the percentage of anti-inflammatory acting TREM-2+ monocytes and negatively with TREM-1/TREM-2 ratio. This could be protective against the progression to high-grade glioma, because TREM-2 is associated with protective functions such as: tissue repair, control of local inflammation, or phagocytosis (Tab. 4, Fig. 4, Ref. 79).

TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg2+ and divalent metal cations such as Ca2+, Zn2+, Cd2+, Mn2+, and Ni2+. TRPM7 channels are inhibited by cytosolic Mg2+, rendering them largely inactive in intact cells. The dependence of channel activity on extracellular Mg2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg2+ concentrations varying between 400 nM and 1.4 mM for 1-3 days, obtaining an IC50 value of 54 µM. Maintaining the cells in 400 nM or 8 µM [Mg2+]o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg2+ depletion. A total of 1.4 mM [Mg2+]o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg2+ depletion but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni2+, Zn2+, Cd2+, Co2+, Mn2+, Sr2+, and Ba2+ requires TRPM7 channel activity. Mg2+ loading modestly reduced cytotoxicity of Zn2+, Co2+, Ni2+, and Mn2+ but not of Cd2+. Channel blocker NS8593 reduced Co2+ and Mn2+ but not Cd2+ or Zn2+ cytotoxicity and interfered with Mg2+ loading as evaluated by TRPM7 channel basal activity. Ba2+ and Sr2+ were neither detectably toxic nor permeant through the plasma membrane. These results indicate that in Jurkat T cells, entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg2+ entry requires TRPM7 channels.

Correction to: TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

The original article was published with an error. In Figure 9b there are 3 typographical errors: instead of the Greek mu letter it shows the unconverted data.

TREM-1 and TREM-2 Expression on Blood Monocytes Could Help Predict Survival in High-Grade Glioma Patients.

OBJECTIVE: In recent years, the role of the modern inflammatory markers TREM-1 (triggering receptors expressed on myeloid cells) and HMGB1 (high mobility group box 1 protein) in tumorigenesis has begun to be studied. Their role in gliomas is not clear. The aim of our study was to find the role of inflammation in gliomas. Patients and Methods. In 63 adult patients with gliomas and 31 healthy controls, the expressions of TREM-1 and TREM-2 on CD14+ blood cells (method: flow cytometry) and the levels of soluble sTREM-1, HMGB1, IL-6, and IL-10 (Elisa tests) were analyzed. RESULTS: Cox proportional hazard analysis showed that a TREM-1/TREM-2 ratio was associated with reduced overall survival (HR = 1.001, P = 0.023). Patients with a TREM-1/TREM-2 ratio above 125 survived significantly shorter than patients with a TREM-1/TREM-2 ratio below 125. The percentage of CD14+ TREM-1+ cells was strongly associated with a plasma IL-6/IL-10 ratio (positively) and with IL-10 (negatively). Conversely, we found a higher percentage of CD14+ TREM-2+ monocytes in better surviving patients; these cells could downregulate the exaggerated inflammation and potentiate the phagocytosis in the tumor. The serum levels of HMGB1 negatively correlated with the percentage of CD14+ TREM-1+ cells and with the TREM-1/TREM-2 ratio. The positive correlation between the serum levels of a late proinflammatory cytokine HMGB1 with the percentage of TREM2+ CD14+ monocytes can be explained as an effort for suppression of systemic inflammation by anti-inflammatory acting CD14+ TREM-2+ cells. CONCLUSION: We showed that the TREM-1/TREM-2 ratio (expression on the surface of blood monocytes) could help predict prognosis in patients with gliomas, especially in high-grade gliomas, and that systemic inflammation has an impact on the patient's overall survival. This is the first study that showed that TREM expression on monocytes in peripheral blood could help predict prognosis in patients with gliomas.

Depletion of plasma membrane-associated phosphoinositides mimics inhibition of TRPM7 channels by cytosolic Mg2+, spermine, and pH.

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg2+, protons, and polyamines. Currents through these channels (ITRPM7) are robustly potentiated when the cell interior is exchanged with low Mg2+-containing buffers. ITRPM7 is also potentiated by phosphatidyl inositol bisphosphate (PI(4,5)P2) and suppressed by its hydrolysis. Here we characterized internal Mg2+- and pH-mediated inhibition of TRPM7 channels in HEK293 cells overexpressing WT voltage-sensing phospholipid phosphatase (VSP) or its catalytically inactive variant VSP-C363S. VSP-mediated depletion of membrane phosphoinositides significantly increased channel sensitivity to Mg2+ and pH. Proton concentrations that were too low to inhibit ITRPM7 when the VSP-C363S variant was expressed (pH 8.2) became inhibitory in WT VSP-expressing cells. At pH 6.5, protons inhibited ITRPM7 both in WT and VSP C363S-expressing cells but with a faster time course in the WT VSP-expressing cells. Inhibition by 150 µm Mg2+ was also significantly faster in the WT VSP-expressing cells. Cellular PI(4,5)P2 depletion increased the sensitivity of TRPM7 channels to the inhibitor 2-aminoethyl diphenyl borinate, which acidifies the cytosol. Single substitutions at Ser-1107 of TRPM7, reducing its sensitivity to Mg2+, also decreased its inhibition by spermine and acidic pH. Furthermore, these channel variants were markedly less sensitive to VSP-mediated PI(4,5)P2 depletion than the WT. We conclude that the internal Mg2+-, polyamine-, and pH-mediated inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and resultant disruption of PI(4,5)P2-channel interactions.

Neurenteric cysts, incidence and surgical treatment.

OBJECTIVES: Neurenteric cysts (NCs) of the central nervous system (CNS) are cystic congenital lesions that may occur anywhere along the neural tube. They are most common in the spinal region, in the lower part of the cervical and upper thoracic spine. Intracranial NCs occur rarely and there are only small series of patients published in literature worldwide. Microsurgical resection is the treatment of choice. We present our experience in treatment of NCs and review of literature. METHODS: Seven patients with NC of CNS who were operated at the Department of Neurosurgery of Comenius University at University Hospital Bratislava within nine years (2010-2018) were included in the study. The series was retrospectively evaluated with an emphasis on symptomatology, surgery and postoperative course. RESULTS: In three of the seven patients, NC was localized intracranially, in the other four, NC was in the spinal canal. In three patients, a complete removal of NC was achieved (2 intracranial NC, 1 spinal NC). In other patients, a portion of the cyst wall was left to prevent the development of a postoperative neurological deficit. After surgery, the neurological symptoms were completely resolved in six patients, while in one patient, they were alleviated. In one patient, a complication occurred during the postoperatove course. There was no recurrence during the follow-up (3-111 months, mean duration 39 months). CONCLUSION: In our series of patients with intracranial and intraspinal tumors, the incidence of NC was higher than presented in the published data. Our own surgical experience has shown that complete tumor resection is not always possible for intimate adherence to the surrounding structures. Leaving a tiny portion of the cyst wall allowed us to achieve good clinical results with no recurrence. Long-term follow-up of patients is required due to the risk of recurrence. However, it can already be stated that an adequate extent of resection leads to good clinical results (Tab. 1, Fig. 4, Ref. 33).

[Lumbar sympathectomy literature review over the past 15 years]. / Lumbálna sympatektómia prehlad svetovej literatúry za posledných 15 rokov.

INTRODUCTION: Lumbar sympathectomy (LS) irreversibly damages a part of the sympathetic trunk and adjacent ganglia between L1 and L5, typically between L2 and L4. The first LS was performed in 1923. Initially, it used to be performed very often; however, with the progress of vascular and endovascular surgery its importance gradually continues to decline. The aim of the paper is to present literature review focusing on LS over the past 15 years. METHOD: Literature review of 113 academic articles found in academic journal databases. PATHOPHYSIOLOGY: Irreversible interruption of the efferent innervation leads to relative vasodilation of small vessels in lower extremities (α1-receptors blockade), and it reduces the volume of sweat due to inactivation of eccrine glands and nociception from lower limbs. INDICATION: Raynaud´s phenomenon, thromboangitis obliterans, non-revascularizable peripheral arterial disease (PAD) (Fontain grade III-IV), hyperhidrosis, persistent pain in lower extremities, chronic pain of amputation stump, frostbites, chilblains.Effect: The three largest studies showed a positive effect in 63.6-93.4% cases of PAD and in 97%100% cases of hyperhidrosis. The positive effect was defined as warmer lower extremities, increased blood flow, acceleration of chronic defects healing, sweating disappearance and pain reduction. CONCLUSION: Lumbar sympathectomy still remains a useful method in the treatment of above mentioned diseases if properly indicated. KEY WORDS: lumbar sympathectomy - Raynaud´s phenomenon - thromboangitis obliterans -peripheral arterial disease - hyperhidrosis.

Reconstruction of eyelids with Washio flap in anophthalmia.

The authors present a case report of a patient with anophthalmia in whom retroauriculo-temporal flap (Washio flap) was used for reconstruction of eyelids. This flap, which is mostly used for reconstructions of nasal defects, was not used in this way according to available literature.

Use of tetraethylammonium (TEA) and Tris loading for blocking TRPM7 channels in intact cells.

Tetraethylammonium (TEA), a quaternary ammonium compound, is a well-known blocker of potassium channels belonging to various subfamilies, such as KV1-3, KCa1, 2 and prokaryotic KcsA. In many cases, TEA acts from the extracellular side by open pore blockade. TEA can also block transient receptor potential (TRP) cation channels, such as TRPM7, in a voltage-dependent manner. In human T lymphocytes, intracellular (cytosolic) TEA and its analog TMA (tetramethylammonium) inhibit TRPM7 channel currents in the outward but not inward direction. By contrast, intracellular Mg2+, protons and polyamines inhibit both outward and inward current components equally. Likewise, the majority of available pharmacological tools inhibit TRPM7 channels in a voltage-independent manner. Since TRPM7 is a steeply outwardly rectifying conductance, voltage-dependent blockers can be useful for studying the cellular functions of this channel. TRPM7 protein is endogenously expressed in diverse cell lines, including HEK, HeLa, CHO, RBL and Jurkat. Using patch-clamp electrophysiology, we found that incubating HEK293 and Jurkat T cells overnight in the presence of 20 mM TEA-Cl, resulted in the nearly complete blockade of whole-cell TRPM7 outward current, measured at break-in. By contrast, the inward current was unchanged in TEA-loaded cells. The blockade was fully reversible after washout of intracellular solution in whole-cell but not in perforated-patch recording configurations. Overnight incubation with 20 mM TMA-Cl resulted in a more modest blockade of the outward TRPM7 current. Internal 129 mM TMA and TEA eliminated most of the outward current. TEA uptake in transfected HEK293 cells led to blockade of recombinant murine TRPM7 and the Mg2+ and pH insensitive Ser1107Arg variant. Unexpectedly, Tris-HCl, a widely used pH buffer, could similarly be loaded into Jurkat and HEK cells, and preferentially blocked outward TRPM7 currents. 20 mM and 129 mM Tris in the internal solution blocked TRPM7 current in outward but not inward direction. Voltage-dependent channel blockade by TEA, TMA and Tris loading will be useful for studying the properties and functions of TRPM7-mediated ion transport in intact cells.

Discovery of selective Orai channel blockers bearing an indazole or a pyrazole scaffold.

The calcium release activated calcium (CRAC) channel is highly expressed in T lymphocytes and plays a critical role in regulating T cell proliferation and functions including activation of the transcription factor nuclear factor of activated T cells (NFAT), cytokine production and cytotoxicity. The CRAC channel consists of the Orai pore subunit and STIM (stromal interacting molecule) endoplasmic reticulum calcium sensor. Loss of CRAC channel mediated calcium signaling has been identified as an underlying cause of severe combined immunodeficiency (SCID), leading to drastically weakened immunity against infections. Gain-of-function mutations in Orai and STIM have been associated with tubular aggregated myopathy (TAM), a skeletal muscle disease. While a number of small molecules have shown activity in inhibiting the CRAC signaling pathway, the usefulness of those tool compounds is limited by their off-target activity against TRPM4 and TRPM7 ion channels, high lipophilicity, and a lack of understanding of their mechanism of action. We report structure-activity relationship (SAR) studies that resulted in the characterization of compound 4k [1-(cyclopropylmethyl)-N-(3-fluoropyridin-4-yl)-1H-indazole-3-carboxamie] as a fast onset, reversible, and selective CRAC channel blocker. 4k fully blocked the CRAC current (IC50: 4.9 µM) and the nuclear translocation of NFAT at 30 and 10 µM, respectively, without affecting the electrophysiological function of TRPM4 and TRPM7 channels. Computational modeling appears to support its direction binding to Orai proteins that form the transmembrane CRACchannel.

Sensitivity of TRPM7 channels to Mg2+ characterized in cell-free patches of Jurkat T lymphocytes.

Transient receptor potential melastatin 7 (TRPM7) channels were originally identified electrophysiologically when depletion of cytosolic Mg(2+) resulted in the gradual development of an outwardly rectifying cation current. Conversely, inclusion of millimolar Mg(2+) in internal solutions prevented activation of these channels in whole cell patch clamp. We recently demonstrated that the Jurkat T-cell whole cell TRPM7 channels are inhibited by internal Mg(2+) in a biphasic manner, displaying high [IC(50(1)) ≈ 10 µM] and low [IC(50(2)) ≈ 165 µM] affinity inhibitor sites. In that study, we had characterized the dependence of the maximum cell current density on intracellular Mg(2+) concentration. To characterize Mg(2+) inhibition in Jurkat T cells in more detail and compare it to whole cell results, we recorded single TRPM7 channels in cell-free membrane patches and investigated the dependence of their activity on Mg(2+) added on the cytoplasmic side. We systematically varied free Mg(2+) from 265 nM to 407 µM and evaluated the extent of channel inhibition in inside-out patch for 58 patches. We found that the TRPM7 channel shows two conductance levels of 39.0 pS (γ(1)) and 18.6 pS (γ(2)) and that both are reversibly inhibited by internal Mg(2+). The 39.0-pS conductance is the dominant state of the channel, observed most frequently in this recording configuration. The dose-response relation in inside-out patches shows a steeper Mg(2+) dependence than in whole cell, yielding IC(50(1)) of 25.1 µM and IC(50(2)) of 91.2 µM.. Single-channel analysis shows that the primary effect of Mg(2+) in multichannel patches is a reversible reduction of the number of conducting channels (N(o)). Additionally, at high Mg(2+) concentrations, we observed a saturating 20% reduction in unitary conductance (γ(1)). Thus Mg(2+) inhibition in whole cell can be explained by a drop in individual participating channels and a modest reduction in conductance. We also found that TRPM7 channels in some patches were not sensitive to this ion at submaximal Mg(2+) concentrations. Interestingly, Mg(2+) inhibition showed the property of use dependence: with repeated applications, Mg(2+) effect became gradually more potent, which suggests that Mg(2+) sensitivity of the channel is a dynamic characteristic that depends on other membrane factors.

Detailed examination of Mg2+ and pH sensitivity of human TRPM7 channels.

TRPM7 channel kinase is a protein highly expressed in cells of hematopoietic lineage, such as lymphocytes. Studies performed in native and heterologous expression systems have shown that TRPM7 forms nonselective cation channels functional in the plasma membrane and activated on depletion of cellular Mg(2+). In addition to internal Mg(2+), cytosolic pH and the phospholipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] are potent physiological regulators of this channel: protons inhibit, while PI(4,5)P(2) is required for TRPM7 channel activity. These channels are also inhibited from inside by other metal cations and polyamines. While the regulation of TRPM7 channels by internal metal ions, acidic pH, and PI(4,5)P(2) is voltage independent, extracellular metal cations and polyamines block voltage dependently at micromolar concentrations and appear to occupy a distinct blocking site. In the present study we investigated intracellular Mg(2+) and pH dependence of native TRPM7 currents using whole cell patch-clamp electrophysiology in human Jurkat T lymphocytes and HEK293 cells. Our main findings are 1) Mg(2+) inhibition involves not one but two separate sites of high (∼10 µM) and low (∼165 µM) affinity; and 2) while sharing certain characteristics with Mg(2+) inhibition, protons most likely inhibit through one inhibitory site, corresponding to the low-affinity Mg(2+) site, with an estimated IC(50) of pH 6.3. Additionally, we present data on amplitude distribution of preactivated TRPM7 currents in Jurkat T lymphocytes in the absence of prior Mg(2+) or proton depletion.

STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane.

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

NSAIDs Naproxen, Ibuprofen, Salicylate, and Aspirin Inhibit TRPM7 Channels by Cytosolic Acidification.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used for relieving pain and inflammation accompanying numerous disease states. The primary therapeutic mechanism of these widely used drugs is the inhibition of cyclooxygenase 1 and 2 (COX1, 2) enzymes that catalyze the conversion of arachidonic acid into prostaglandins. At higher doses, NSAIDs are used for prevention of certain types of cancer and as experimental treatments for Alzheimer's disease. In the immune system, various NSAIDs have been reported to influence neutrophil function and lymphocyte proliferation, and affect ion channels and cellular calcium homeostasis. Transient receptor potential melastatin 7 (TRPM7) cation channels are highly expressed in T lymphocytes and are inhibited by Mg2+, acidic pH, and polyamines. Here, we report a novel effect of naproxen, ibuprofen, salicylate, and acetylsalicylate on TRPM7. At concentrations of 3-30mM, they reversibly inhibited TRPM7 channel currents. By measuring intracellular pH with the ratiometric indicator BCECF, we found that at 300µM to 30mM, these NSAIDs reversibly acidified the cytoplasm in a concentration-dependent manner, and propose that TRPM7 channel inhibition is a consequence of cytosolic acidification, rather than direct. NSAID inhibition of TRPM7 channels was slow, voltage-independent, and displayed use-dependence, increasing in potency upon repeated drug applications. The extent of channel inhibition by salicylate strongly depended on cellular PI(4,5)P2 levels, as revealed when this phospholipid was depleted with voltage-sensitive lipid phosphatase (VSP). Salicylate inhibited heterologously expressed wildtype TRPM7 channels but not the S1107R variant, which is insensitive to cytosolic pH, Mg2+, and PI(4,5)P2 depletion. NSAID-induced acidification was also observed in Schneider 2 cells from Drosophila, an organism that lacks orthologous COX genes, suggesting that this effect is unrelated to COX enzyme activity. A 24-h exposure to 300µM-10mM naproxen resulted in a concentration-dependent reduction in cell viability. In addition to TRPM7, the described NSAID effect would be expected to apply to other ion channels and transporters sensitive to intracellular pH.

Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.

STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.

[Navigation-assisted sonography for soft tissues in the head and neck region]. / Navigationsunterstützte Weichteilsonographie für den Kopf-Hals-Bereich.

BACKGROUND: In soft tissue surgery of the head and neck region tissue shifts limit the usefulness of conventional CT/MRI-based navigation procedures. Furthermore, changes caused by invasive measures cannot be visualized. METHODS: A novel navigation device for sonography of soft tissues was developed. This consists of a navigated ultrasound scanner, a navigated surgical instrument, and a personal computer with custom-made software. Its use makes an additional visualization by means of CT or MRI dispensable. RESULTS: The system deviation (three-dimensional error) of this newly developed prototype was less than 1 mm. The practical application in a model setup showed good handling properties of the system. Orientation and approach of the surgical instrument to the sonographically visualized target structure were rapid and accurate. CONCLUSION: This new navigation system does not require additional CT or MRI images. The navigated ultrasound probe shows tissue changes in real time. This navigation system is especially suitable for invasive procedures in soft tissues.