Review of the Clinical Evidences of Modulated Electro-Hyperthermia (mEHT) Method: An Update for the Practicing Oncologist.

Background: Modulated electro-hyperthermia (mEHT) is a variation of the conventional hyperthermia which selectively targets the malignant cell membranes in order to heat the malignant tissue and sensitize the tissue to oncology treatments. Although widely applied, the formulation of guidelines for the use thereof is still in progress for many tumors. Aim: In this paper we review the literature on the effects of mEHT in cancer patients on local disease control and survival. Methodology: Our review on data presents the collected experience with capacitive hyperthermia treatments with the EHY-2000+ device (OncoTherm Ltd., Germany). A literature search was conducted in Pubmed and articles were grouped and discussed according to: trial type, animal studies, in vitro studies, and reviews. Search results from Conference Abstracts; Trial Registries; Thesis and Dissertations and the Oncothermia Journal were included in the discussions. Results: Modulated electro-hyperthermia is a safe form of hyperthermia which has shown to effectively sensitizes deep tumors, regardless of the thickness of the adipose layers. The technology has demonstrated equal benefits compared to other forms of hyperthermia for a variety of tumors. Given the effective heating ability to moderate temperatures, the improved tumor perfusion, and ability to increase drug absorption, mEHT is a safe and effective heating technology which can be easily applied to sensitize tumors which have demonstrated benefits with the addition of hyperthermia. Modulated electro-hyperthermia also appears to improve local control and survival rates and appears to induce an abscopal (systemic) response to ionizing radiation. Conclusion: Based on clinical studies, the method mEHT is a feasible hyperthermia technology for oncological applications. Concomitant utilization of mEHT is supported by the preclinical and clinical data.

Nicotine interferes with purinergic signaling in smooth muscle cells isolated from urinary bladders of patients with lower urinary tract symptoms.

In patients with outlet obstruction, the contraction of the base is reduced compared to that of healthy individuals, while the contraction of the dome is not affected. Here, we investigated the cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the urinary bladder. Smooth muscle cells either from the base or from the dome of human urinary bladders were cultured to determine the contribution of cholinergic and purinergic mechanisms to their Ca2+ homeostasis. While ATP evoked Ca2+ transients in all the cells, nicotine and carbachol induced Ca2+ transients only in 56% and 44% of the cells, respectively. When ATP was administered together with nicotine or carbachol, the amplitudes of the Ca2+ transients recorded from cells prepared from the base of bladders were significantly smaller (42 ± 6% with nicotine and 56 ± 9% with carbachol) than those evoked by ATP alone. This inhibition was much less apparent in the dome of bladders. The inhibition between the cholinergic and purinergic signaling pathways reported in this work may decrease the strength of the contraction of the base of the urinary bladder in patients with outlet obstruction during voiding.

Hyperthermia versus Oncothermia: Cellular Effects in Complementary Cancer Therapy.

Hyperthermia means overheating of the living object completely or partly. Hyperthermia, the procedure of raising the temperature of a part of or the whole body above normal for a defined period of time, is applied alone or as an adjunctive with various established cancer treatment modalities such as radiotherapy and chemotherapy. However, hyperthermia is not generally accepted as conventional therapy. The problem is its controversial performance. The controversy is originated from the complications of the deep heating and the focusing of the heat effect. The idea of oncothermia solves the selective deep action on nearly cellular resolution. We would like to demonstrate the force and perspectives of oncothermia, as a highly specialized hyperthermia in clinical oncology. Our aim is to prove the ability of oncothermia to be a candidate to become a widely accepted modality of the standard cancer care. We would like to show the proofs and the challenges of the hyperthermia and oncothermia applications to provide the presently available data and summarize the knowledge in the topic. Like many early stage therapies, oncothermia lacks adequate treatment experience and long-range, comprehensive statistics that can help us optimize its use for all indications.

Rehabilitation of stroke patients using Yamamoto New Scalp Acupuncture: a pilot study.

OBJECTIVES: The aim of this study was to investigate whether acupuncture, especially Yamamoto's New Scalp Acupuncture (YNSA), is of value in additional to standard poststroke motor rehabilitation. DESIGN: A prospective, assessor-blinded randomized control trial was carried out in an inpatient stroke rehabilitation unit with day hospital service. After inclusion, patients were stratified into control group and acupuncture group, randomly. OUTCOME MEASURES: The Barthel Index, the Rivermead Scale Index, and the Visual Analogue Scale were used to follow the efficacy of treatment. RESULTS: In the acupuncture group, all the sensory, motor, and functional scores improved significantly during the examination period until 2 years after injury. The Barthel Index is increased from 4±2 to 95±4 in the acupuncture group. This index also increased in the control group (from 4±2 to 75±4), but the changes were significantly less than in the acupuncture group. A significant spontaneous recovery during the 2-year follow-up was found, but the YNSA treatment facilitated the functional recovery. Improved moving function and more flexible joints and ligaments were observed in comparison to the patients' condition prior to treatment. CONCLUSIONS: The data suggest that the YNSA is a useful method to treat stroke patients and enhance their quality of life.

A possible role of the cholinergic and purinergic receptor interaction in the regulation of the rat urinary bladder function.

The contractile activation of the upper (dome) and lower (base) parts of the urinary bladder show some differences. Cellular mechanisms that might be responsible for cholinergic effects blocking non-adrenergic non-cholinergic contractions in the base of the rat urinary bladder were investigated. Smooth muscle cells were thus freshly isolated or cultured both from the dome and the base of the rat urinary bladder and the contribution from cholinergic and purinergic pathways to their Ca(2+) homeostasis was examined. The expression of nicotinic acetylcholine (nAChR) and P2X2 purinergic receptors on the cultured cells and on tissue sections was investigated. The ATP-evoked Ca(2+) transients in rat smooth muscle cells did not show any desensitization. However, when ATP was administered together with carbamylcholine (CCh), the latter essentially prevented ATP from evoking Ca(2+) transients in smooth muscle cells from the base (suppression to 12 ± 2.5% of control, n = 57; p < 0.01), but not from the dome (99 ± 5% of control, n = 52; p > 0.05) of the rat urinary bladder. While atropine was unable to modify (6 ± 3% of control, n = 14; p < 0.05), α-bungarotoxin (118 ± 12% of control, n = 20; p > 0.05) blocked the inhibitory effects of CCh. Additionally, α7 subunits of nAChR and P2X2 purinergic receptors were identified using immunocytochemistry, immunohistochemistry, and Western blot in cultured urinary bladder smooth muscle cells, in urinary bladder sections, and in urinary bladder muscle strips, respectively, suggesting that the activation of nAChR modifies the action of ATP.

Altered expression of triadin 95 causes parallel changes in localized Ca2+ release events and global Ca2+ signals in skeletal muscle cells in culture.

The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome- or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization-evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin-specific immunoreactivity. Functional experiments on these cells revealed that while caffeine-evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.

Activation of cholinergic receptors blocks non-adrenergic non-cholinergic contractions in the rat urinary bladder.

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 microM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to alpha,beta-methylene ATP (alpha,beta-mATP) (10 microM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 microM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100 microM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with alpha,beta-mATP) decreased the NANC contractions and the direct contractile response to alpha,beta-mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.

Hypotonic stress influence the membrane potential and alter the proliferation of keratinocytes in vitro.

Keratinocyte proliferation and differentiation is strongly influenced by mechanical forces. We investigated the effect of osmotic changes in the development of HaCaT cells in culture using intracellular calcium measurements, electrophysiological recordings and molecular biology techniques. The application of hypotonic stress (174 mOsmol/l) caused a sustained hyperpolarization of HaCaT cells from a resting potential of -27 +/- 4 to -51 +/- 9 mV. This change was partially reversible. The surface membrane channels involved in the hyperpolarization were identified as chloride channels due to the lack of response in the absence of the anion. Cells responded with an elevation of intracellular calcium concentration to hypotonic stress, which critically depended on external calcium. The presence of phorbol-12-myristate-13-acetate in the culture medium for 12 h augmented the subsequent response to hypotonic stress. A sudden switch from iso- to hypotonic solution increased cell proliferation and suppressed the production of involucrin, filaggrin and transglutaminase, markers of keratinocyte differentiation. It is concluded that sudden mechanical forces increase the proliferation of keratinocytes through alterations in their membrane potential and intracellular calcium concentration. These changes together with additional modifications in channel expression and intracellular signalling mechanisms could underlie the increased proliferation of keratinocytes in hyperproliferative skin diseases.

Functional genomics of calcium channels in human melanoma cells.

Ca(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca(2+) channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X(7) channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X(7), RyR2 does not function as a calcium channel. On the other hand, the P2X(7) receptor has an antiapoptotic function in melanoma cells, since ATP-activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.

Differential expression of potassium currents in Deiters cells of the guinea pig cochlea.

Among the supporting cells, Deiters cells are in intimate contact with outer hair cells (OHCs) in the inner ear. The aim of this study was to characterize the outward rectifying K+ current of Deiters cells in conjunction with cellular morphological characteristics. In the majority of cells, the K+ current had a biphasic inactivation kinetics (tau1 and tau2 were 2,735+/-90 (n=77) and 160+/-14 ms (n=72), respectively). The rapidly inactivating current component was more sensitive to Charybdotoxin (ChTx, 10 nM) block whereas the slowly inactivating current could be blocked more efficiently by tetraethylammonium (1 mM). All these point toward the existence of two distinct potassium channel types in these cells. Deiters cells attached to shorter OHCs had more voluminous, whereas those attached to longer OHCs had lanky cell bodies. The inactivation kinetics was slower in cells having corpulent cell bodies due to the increased proportion of the slowly inactivating current component (0.736+/-0.033, n=27) as compared to the one determined for lanky cells (0.522+/-0.023, n=36). The average peak K+ current was higher in Deiters cells connected to OHCs (5,417+/-541 pA, n=40) than in isolated ones (3,527+/-410, n=37). Deiters cells having different cell shapes and showing different K+ channel expression may contribute to the active mechanism of the cochlea to various degrees.

Changes in purinoceptor distribution and intracellular calcium levels following noise exposure in the outer hair cells of the guinea pig.

Among the cells of the inner ear, the outer hair cells (OHCs) are the most important targets of noise-induced effects, being the most sensitive cell types. The aim of this study was to examine the effects of noise (50 Hz-20 kHz, 80 dB sound pressure level, 14 days) on intracellular calcium levels and on the expression pattern of purinoceptors in the membrane of the OHCs of the guinea pig and to measure the stiffness changes of the lateral membrane of these cells. In noise-exposed animals, the resting intracellular calcium concentration increased compared to nontreated animals and was slightly higher in the cells of the basal (219 +/- 29 nM: ) than in the apical (181 +/- 24 nM: ) turns of the cochlea. After application of 180 muM: adenosine triphosphate, the intracellular calcium level rose by 60 +/- 22 nM: in cells from the apical and by 44 +/- 10 nM: in cells from the basal turns, significantly less than in nontreated animals. Expression of the P(2X1), P(2X2), P(2X4), P(2X7), P(2Y1) and P(2Y4) receptor subtypes was suppressed, while expression of the P(2Y2) subtype did not decrease in either of the two preparations. In parallel with the increase in intracellular calcium concentration, the stiffness of the lateral wall of the OHCs was increased. Noise-induced changes in intracellular calcium homeostasis and subsequently in the calcium-dependent regulatory mechanisms may modify OHC lateral wall stiffness and may lead to reduction of the efficacy of the cochlear amplifier.

Age-dependence of the spontaneous activity of the rat urinary bladder.

Abnormal mechanical function of the bladder is manifested in a number of ways including higher frequency of involuntary detrusor contractions associated with reduced compliance of the bladder that is responsible for an increase in intraluminal pressure during filling. There are basically two ways to approach experimentally these problems: (1) by studying the neural control of the lower urinary tract function, and (2) by measuring the properties of smooth muscle cells in the bladder wall. Studies on smooth muscle function often do not take the origin of smooth muscle cells into account i.e., whether they were harvested from normal or overactive bladders. Although, this simplistic view may be beneficial to understanding the generation of the spontaneous activity of the bladder, however, it does not sufficiently explain the cell-to-cell propagation of the spontaneous smooth muscle activity. The spontaneous activity of smooth muscle is an important factor that works against the bladder compliance in the filling phase, and may inversely affect the neurally evoked response during micturition. The intensity of spontaneous activity is the age-dependent; it is high in neonatal bladders it is small or almost non-existent in adults and reemerges in older bladders. This review focuses on these age-dependent alterations of spontaneous bladder contractions and describes the possible mechanisms which may have important role in regulating the spontaneous contractions using the rat as an animal model.

Computer model of action potential of mouse ventricular myocytes.

We have developed a mathematical model of the mouse ventricular myocyte action potential (AP) from voltage-clamp data of the underlying currents and Ca2+ transients. Wherever possible, we used Markov models to represent the molecular structure and function of ion channels. The model includes detailed intracellular Ca2+ dynamics, with simulations of localized events such as sarcoplasmic Ca2+ release into a small intracellular volume bounded by the sarcolemma and sarcoplasmic reticulum. Transporter-mediated Ca2+ fluxes from the bulk cytosol are closely matched to the experimentally reported values and predict stimulation rate-dependent changes in Ca2+ transients. Our model reproduces the properties of cardiac myocytes from two different regions of the heart: the apex and the septum. The septum has a relatively prolonged AP, which reflects a relatively small contribution from the rapid transient outward K+ current in the septum. The attribution of putative molecular bases for several of the component currents enables our mouse model to be used to simulate the behavior of genetically modified transgenic mice.

Developmental changes in spontaneous smooth muscle activity in the neonatal rat urinary bladder.

Changes in spontaneous activity of the urinary bladder during postnatal development were examined in muscle strips from the base and dome of bladders from 1- to 5-wk-old rats. Activity was analyzed using fast Fourier transformation (FFT), nonlinear cross prediction, and the Shannon entropy test. Spontaneous activity was not detected in strips from 1- to 5-day-old rats but was observed in 50% of strips from 6- to 7-day-old rats and was prominent in strips from 2-wk-old animals. FFT analysis revealed one peak in activity, which was significantly faster in the bladder base (0.21 +/- 0.03 Hz) than in the dome (0.08 +/- 0.01 Hz). A second peak at approximately 0.5 Hz was detected at 3-5 wk of age. Atropine but not tetrodotoxin decreased the amplitude of spontaneous contractions, whereas carbachol, a muscarinic agonist, unmasked or stimulated spontaneous activity. These data suggest that slow rhythmic activity observed previously in neonatal whole bladders is generated by pacemaker cells in the bladder base or dome. The emergence of faster activity in bladders from older animals may reflect the development of multiple pacemaker sites, which would reduce coordination within the bladder wall and improve storage function in the mature bladder.

The role of region IVS5 of the human cardiac calcium channel in establishing inactivated channel conformation: use-dependent block by benzothiazepines.

The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. Single channel properties of mutants that lost use dependence toward diltiazem were characterized by drastically elongated mean open times and distinctly slower time constants of open time distribution. Mutation of individual residues of the IVMLF segment in IVS5 did not mimic the complete loss of use dependence as observed for the replacement of the whole stretch. These results establish evidence that amino acids that govern inactivation and the drug-binding site and other amino acids that are located distal from the putative drug-binding site contribute significantly to the function of the benzothiazepine receptor region. The data are consistent with a complex "pocket" conformation that is responsive to a specific class of L-type calcium channel inhibitors. The data allow for a concept that multiple sites within regions of the alpha(1) subunit contribute to auto-regulation of the L-type Ca(2+) channel.

Altered excitation-contraction coupling in myocytes from remodeled myocardium after chronic myocardial infarction.

Following myocardial infarction (MI), the left ventricle undergoes progressive dilatation and eccentric hypertrophy, i.e., remodeling, which is greater in the adjacent than the remote region. The cellular mechanisms underlying these regional differences were studied. One (n=5) and 8 weeks (n=8) after anteroapical MI in sheep, cardiac myocytes were isolated from the adjacent and remote regions. At 8 weeks after MI, myocyte function in the remote region was not different from values either in sham controls (n=3) or animals 1 week after MI. At 8 weeks after MI, myocyte contractile function (% contraction) was decreased, P<0.01, in the adjacent region (6.4+/-0.4%), as compared with the remote region (8.8+/-0.5%) and was associated with decreased amplitude of Ca(2+)transients (adjacent, 0.69+/-0.09 v remote, 1.08+/-0.20, P<0.05) and L-type Ca(2+)current density (adjacent, 3.6+/-0.2 v remote, 4.8+/-0.2 pA/pF, P<0.05). Relaxation was also impaired significantly in myocytes from the adjacent region, associated with decreased protein levels of SERCA2a. The myocytes were hypertrophied more in the adjacent region than the remote region. Furthermore, focal areas of central myofibrillar lysis and increased glycogen deposition were observed in the adjacent region. These results indicate that impaired excitation-contraction coupling underlies dysfunction of myocytes from the adjacent non-infarcted myocardium after chronic MI, even in the absence of heart failure. Hypertrophy is implicated as the mechanism, since these changes were noted at 8 weeks, but not at 1 week after MI.

Use of antisense oligonucleotides to verify the role of the alpha(1A)-adrenergic receptor in the contractility of the rat uterus post partum.

The adrenergic system plays a major role in the regulation of the contractility of the uterus during pregnancy. This study investigated the role of the alpha(1A)-adrenergic receptor (AR) in this regulation. The use of partial phosphorothioate antisense oligodeoxynucleotides (AONs) permitted the sequence-selective inhibition of AR gene expression. AONs were injected together with a cationic liposomal carrier agent into the post partum rat uterus. Incubation for 12 or 24 h with the most effective AON (480-AON) caused a 58.7 or 53.0% inhibition, respectively, of the expression of the alpha(1A)-AR density, whereas incubation for 36 or 48 h resulted in only a 38.8 or 26.7% inhibition, respectively. The decrease of the alpha(1A)-AR density by 480-AON was demonstrated by Western blot analysis and a radioreceptor binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus after AON treatment were measured on isolated rat uterine tissue by electric field stimulation. The significant decrease in the ability of the uterus to contract was indicated by the area under the curve method. The electric field studies revealed that the specific alpha(1A)-blockers 5-methylurapidil and WB 4101 inhibited the rhythmic contraction by about 74 and 70% in the control uteri and by 25 and 20% in 480-AON-treated uteri, respectively. The curves for the beta-mimetic (terbutaline) and alpha(1D)-antagonist (BMY7370) inhibitors were unchanged after 480-AON treatment of the uteri. These results suggest the importance of the alpha(1A)-AR in the tocolytic effect exerted by the alpha(1)-antagonist, although high concentrations of antagonists can not exclude the role of alpha(1D)-ARs, too. Additionally, these prove that the knockdown transformation by AONs offers a useful animal model for the investigation of receptors controlling the function of uterine tissue.