Judit Boczán

Vanilloid receptor-1 (TRPV1) expression and function in the vasculature of the rat.

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.

Miller Fisher syndrome--a presenting clinical manifestation of lung cancer in a previously apparently healthy individual.

Sirs: A 54 year-old white male complained of clumsiness of the right upper extremity, diplopia and unsteadiness of gait starting about 6 days before admission. The history was negative except for smoking an average of 2 packs of cigarettes per day in the preceding 40 years. On admission he had right sided complete oculomotor nerve lesion, severe bilateral lower motoneuron facial palsy (Fig. 1A and 1B), absent Achilles reflexes, diminished other deep tendon reflexes and severe gait ataxia. The patient did not have objective sensory loss, major paresis or signs of upper motor neuron lesion. Except for an elevated erythrocyte sedimentation rate (46 mm/hour) and a borderline serum glucose level the routine blood tests were normal. The cerebrospinal fluid (CSF) had an extremely elevated protein content (3.115 g/L) associated with some pleocytosis (256 cells per microliters). The CSF cells were lymphocytes (20 %), macrophages (48 %, a few of them signet-ring cells), monocytes (12 %), and 20 % of the cells were intensely stained, atypical giant cells with large round marginal nuclei. These cells were PAS positive and were also cytokeratin positive with CK7 immunocytochemistry (Fig. 2A). Nerve conduction studies revealed mild axonal sensorimotor neuropathy. On computed tomography contrast enhancement was seen in the right Sylvian fissure (Fig. 2B). MRI detected several small (< 10 mm) contrast enhancing lesions mostly in the cerebral cortex (Fig. 2C, arrows), and in some other CSF-adjacent regions (cerebellar surface, basal ganglia adjacent to the lateral ventricle, periaqueductal gray matter) as well. Chest CT identified a small tumor in the right lung (Fig. 2D). The tumor was removed and appeared to be a carcinoma with adenomatous structure staining with PAS. With immunohistochemical examination the tumor cells were CK7 positive, CK20 negative, exhibited nuclear positivity with TTF-1, and about 5 % of the cells had nuclear positivity with Mib-1. A histological diagnosis of grade 3 bronchial adenocarcinoma was established. Miller Fisher described a syndrome of ophthalmoplegia, ataxia and areflexia as a variant of the Guillain-Barré syndrome [2]. In a series of 50 cases [4] facial palsy was present in about one third of the cases. Although a cerebrospinal fluid (CSF) cell count over 50/μl is rare in Guillain-Barré syndrome, it has been reported in several cases [6]. Axonal, predominantly, sensory neuropathy was found in 5 of 6 patients with Miller Fisher syndrome (MFS) [9]. Therefore, from the clinical signs the diagnosis of MFS could have been considered for our case. In leptomeningeal carcinomatosis the incidence of clinical signs at presentation were 11 % for oculomotor lesion, 11 % for facial nerve involvement and 15–15 % for cerebellar signs and polyradiculopathy [1]. Therefore the probability of the coincidence of the individual signs of MFS is low. There are only 4 published cases where the syndrome was associated with malignant diseases. Leptomeningeal infiltration was described by Guarino et al. [3] in 2 cases. One of them developed the syndrome 6 months after gastrectomy for cancer and the other patient 4 years after thyroidectomy for cancer and 2 years after the diagnosis of acute LETTER TO THE EDITORS

New perspectives in the renin-angiotensin-aldosterone system (RAAS) I: endogenous angiotensin converting enzyme (ACE) inhibition.

Angiotensin-converting enzyme (ACE) inhibitors represent the fifth most often prescribed drugs. ACE inhibitors decrease 5-year mortality by approximately one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors, which endogenous inhibitory effects are much less characterized than that for the clinically administered ACE inhibitors. Here we aimed to investigate this endogenous ACE inhibition in human sera. It was hypothesized that ACE activity is masked by an endogenous inhibitor, which dissociates from the ACE when its concentration decreases upon dilution. ACE activity was measured by FAPGG hydrolysis first. The specific (dilution corrected) enzyme activities significantly increased by dilution of human serum samples (23.2±0.7 U/L at 4-fold dilution, 51.4±0.3 U/L at 32-fold dilution, n = 3, p = 0.001), suggesting the presence of an endogenous inhibitor. In accordance, specific enzyme activities did not changed by dilution when purified renal ACE was used, where no endogenous inhibitor was present (655±145 U/L, 605±42 U/L, n = 3, p = 0.715, respectively). FAPGG conversion strongly correlated with angiotensin I conversion suggesting that this feature is not related to the artificial substrate. Serum samples were ultra-filtered to separate ACE (MW: 180 kDa) and the hypothesized inhibitor. Filtering through 50 kDa filters was without effect, while filtering through 100 kDa filters eliminated the inhibiting factor (ACE activity after <100 kDa filtering: 56.4±2.4 U/L, n = 4, control: 26.4±0.7 U/L, n = 4, p<0.001). Lineweaver-Burk plot indicated non-competitive inhibition of ACE by this endogenous factor. The endogenous inhibitor had higher potency on the C-terminal active site than N-terminal active site of ACE. Finally, this endogenous ACE inhibition was also present in mouse, donkey, goat, bovine sera besides men (increasing of specific ACE activity from 4-fold to 32-fold dilution: 2.8-fold, 1.7-fold, 1.5-fold, 1.8-fold, 2.6-fold, respectively). We report here the existence of an evolutionary conserved mechanism suppressing circulating ACE activity, in vivo, similarly to ACE inhibitory drugs.

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) II: Albumin Suppresses Angiotensin Converting Enzyme (ACE) Activity in Human

About 8% of the adult population is taking angiotensin-converting enzyme (ACE) inhibitors to treat cardiovascular disease including hypertension, myocardial infarction and heart failure. These drugs decrease mortality by up to one-fifth in these patients. We and others have reported previously that endogenous inhibitory substances suppress serum ACE activity, in vivo, similarly to the ACE inhibitor drugs. Here we have made an effort to identify this endogenous ACE inhibitor substance. ACE was crosslinked with interacting proteins in human sera. The crosslinked products were immunoprecipitated and subjected to Western blot. One of the crosslinked products was recognized by both anti-ACE and anti-HSA (human serum albumin) antibodies. Direct ACE-HSA interaction was confirmed by binding assays using purified ACE and HSA. HSA inhibited human purified (circulating) and human recombinant ACE with potencies (IC50) of 5.7±0.7 and 9.5±1.1 mg/mL, respectively. Effects of HSA on the tissue bound native ACE were tested on human saphenous vein samples. Angiotensin I evoked vasoconstriction was inhibited by HSA in this vascular tissue (maximal force with HSA: 6.14±1.34 mN, without HSA: 13.54±2.63 mN), while HSA was without effects on angiotensin II mediated constrictions (maximal force with HSA: 18.73±2.17 mN, without HSA: 19.22±3.50 mN). The main finding of this study is that HSA was identified as a potent physiological inhibitor of the ACE. The enzymatic activity of ACE appears to be almost completely suppressed by HSA when it is present in its physiological concentration. These data suggest that angiotensin I conversion is limited by low physiological ACE activities, in vivo.

Vascular metabolism of anandamide to arachidonic acid affects myogenic constriction in response to intraluminal pressure elevation

AIMS We hypothesized that arachidonic acid produced by anandamide breakdown contributes to the vascular effects of anandamide. MAIN METHODS Isolated, pressurized rat skeletal muscle arteries, which possess spontaneous myogenic tone, were treated with anandamide, arachidonic acid, capsaicin (vanilloid receptor agonist), WIN 55-212-2 (cannabinoid receptor agonist), URB-597 (FAAH inhibitor), baicalein (lipoxygenase inhibitor), PPOH (cytochrome P450 inhibitor), and indomethacin (cyclooxygenase inhibitor). Changes in the arteriolar diameter in response to the various treatments were measured. To assess the effect of anandamide metabolism, anandamide was applied for 20 min followed by washout for 40 min. This protocol was used to eliminate other, more direct effects of anandamide in order to reveal how anandamide metabolism may influence vasodilation. KEY FINDINGS Anandamide at a low dose (1μM) evoked a loss of myogenic tone, while a high dose (30 μM) not only attenuated the myogenic response but also evoked acute dilation. Both of these effects were inhibited by the FAAH inhibitor URB-597 and were mimicked by arachidonic acid. The CB1 and CB2 agonist R-WIN 55-212-2 and the vanilloid receptor agonist capsaicin were without effect on the myogenic response. The inhibition of the myogenic response by anandamide was blocked by indomethacin and PPOH, but not by baicalein or removal of the endothelium. FAAH expression in the smooth muscle cells of the blood vessels was confirmed by immunohistochemistry. SIGNIFICANCE Anandamide activates the arachidonic acid pathway in the microvasculature, affecting vascular autoregulation (myogenic response) and local perfusion.

Different desensitization patterns for sensory and vascular TRPV1 populations in the rat: Expression, localization and functional consequences

Background and purpose TRPV1 is expressed in sensory neurons and vascular smooth muscle cells, contributing to both pain perception and tissue blood distribution. Local desensitization of TRPV1 in sensory neurons by prolonged, high dose stimulation is re-engaged in clinical practice to achieve analgesia, but the effects of such treatments on the vascular TRPV1 are not known. Experimental approach Newborn rats were injected with capsaicin for five days. Sensory activation was measured by eye wiping tests and plasma extravasation. Isolated, pressurized skeletal muscle arterioles were used to characterize TRPV1 mediated vascular responses, while expression of TRPV1 was detected by immunohistochemistry. Key results Capsaicin evoked sensory responses, such as eye wiping (3.6±2.5 versus 15.5±1.4 wipes, p<0.01) or plasma extravasation (evans blue accumulation 10±3 versus 33±7 µg/g, p<0.05) were reduced in desensitized rats. In accordance, the number of TRPV1 positive sensory neurons in the dorsal root ganglia was also decreased. However, TRPV1 expression in smooth muscle cells was not affected by the treatment. There were no differences in the diameter (192±27 versus 194±8 µm), endothelium mediated dilations (evoked by acetylcholine), norepinephrine mediated constrictions, myogenic response and in the capsaicin evoked constrictions of arterioles isolated from skeletal muscle. Conclusion and implications Systemic capsaicin treatment of juvenile rats evokes anatomical and functional disappearance of the TRPV1-expressing neuronal cells but does not affect the TRPV1-expressing cells of the arterioles, implicating different effects of TRPV1 stimulation on the viability of these cell types.

New perspectives in the renin-angiotensin-aldosterone system (RAAS) III: endogenous inhibition of angiotensin converting enzyme (ACE) provides protection against cardiovascular diseases.

ACE inhibitor drugs decrease mortality by up to one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors. Here we investigated the clinical significance of this potential endogenous ACE inhibition. ACE concentration and activity was measured in patients serum samples (n = 151). ACE concentration was found to be in a wide range (47–288 ng/mL). ACE activity decreased with the increasing concentration of the serum albumin (HSA): ACE activity was 56±1 U/L in the presence of 2.4±0.3 mg/mL HSA, compared to 39±1 U/L in the presence of 12±1 mg/mL HSA (values are mean±SEM). Effects of the differences in ACE concentration were suppressed in human sera: patients with ACE DD genotype exhibited a 64% higher serum ACE concentration (range, 74–288 ng/mL, median, 155.2 ng/mL, n = 52) compared to patients with II genotype (range, 47–194 ng/mL, median, 94.5 ng/mL, n = 28) while the difference in ACE activities was only 32% (range, 27.3–59.8 U/L, median, 43.11 U/L, and range 15.6–55.4 U/L, median, 32.74 U/L, respectively) in the presence of 12±1 mg/mL HSA. No correlations were found between serum ACE concentration (or genotype) and cardiovascular diseases, in accordance with the proposed suppressed physiological ACE activities by HSA (concentration in the sera of these patients: 48.5±0.5 mg/mL) or other endogenous inhibitors. Main implications are that (1) physiological ACE activity can be stabilized at a low level by endogenous ACE inhibitors, such as HSA; (2) angiotensin II elimination may have a significant role in angiotensin II related pathologies.

Three novel mutations and genetic epidemiology analysis of the Gap Junction Beta 1 (GJB1) gene among Hungarian Charcot-Marie-Tooth disease patients.

Pathogenic variants of the gap junction beta 1 (GJB1) gene are responsible for the Charcot-Marie-Tooth neuropathy X type 1 (CMTX1). In this study, we report the mutation frequency of GJB1 in 210 Hungarian CMT patients and the phenotype comparison between male and female CMTX1 patients. Altogether, 13 missense substitutions were found in the GJB1 gene. Among them, 10 have been previously described as pathogenic variants (p.Arg15Trp, p.Val63Ile, p.Leu89Val, p.Ala96Gly, p.Arg107Trp, p.Arg142Gln, p.Arg164Trp, p.Arg164Gln, p.Pro172Ala and p.Asn205Ser), while 3 were novel, likely pathogenic alterations (p.Val13Glu, p.Glu186Gly, p.Met194Ile). These variants were not present in controls and were predicted as disease causing by in silico analysis. The frequency of the variants was 6.7% in our cohort which refers to a common cause of hereditary neuropathy among Hungarian patients. In addition to the classical phenotype, CNS involvement was proved in 26.1% of the CMTX1 patients. GJB1 pathogenic alterations were found mainly in males but we also detected them in female probands. The statistical analysis of CMTX1 patients revealed a significant difference between the two genders regarding the age of onset, Charcot-Marie-Tooth neuropathy and examination scores.

[Therapy for anti-MuSK antibody positive myasthenia gravis].

The authors report the case of a 27-year-old woman with muscle-specific receptor tyrosine kinase antibody positive myasthenia with predominantly ocular and bulbar symptoms. Both edrophonium and low dose (4×30 mg/day) pyridostigmin resulted in cholinergic side effects including fasciculation mainly in the facial and neck muscles, and excessive salivation. The patient responded well to a relatively high dose of chronic corticosteroid treatment (methyprednisolone 64mg/day), but the decrease of the corticosteroid dose below 16 mg/day induced exacerbation of the clinical symptoms. Immunosuppression with azathioprine and methotrexate failed to maintain the clinical improvement. However, plasma exchange was always very effective, and all clinical symptoms improved significantly. The authors conclude that patients with muscle-specific receptor tyrosine kinase antibody positive myasthenia gravis should have an individual treatment protocol differing from those used in patients who do not have this antibody but are positive for acetylcholine-receptor antibody. Identification of the pathogenic antibody in the early stage of myasthenia gravis may help to develop the optimal, individualized treatment strategy, to avoid severe side effects, and to achieve fast improvement.A szerzők egy 27 eves fiatal nő esetet ismertetik, aki dontően ocularis-bulbaris tuneteket okozo izomspecifikus kinazellenes antitest pozitiv myasthenia gravisban szenvedett. Mind az intravenas edrophonium, mind a szajon at alkalmazott kis dozisu (4×30 mg/nap) pyridostigmin kolinerg mellekhatasokat, a mimikai es nyakizmok kellemetlen fasciculatiojat, hypersalivatiot okozott. A beteg tartos, viszonylag nagy dozisu kortikoszteroidkezelesre (64 mg/nap metilprednisolon) jol reagalt, azonban a napi adag 16 mg ala csokkentesekor a klinikai tunetek gyorsan kiujultak. Az azathioprinnel es methotrexattal tortenő immunszuppresszio hatasa nem volt latvanyos. A plazmaferezis azonban minden alkalommal gyors javulast, csaknem teljes tunetmentesseget valtott ki. A szerzők kozlemenyukkel arra kivanjak felhivni a figyelmet, hogy az izomspecifikus kinazellenes antitest pozitiv myasthenias betegek kezelesi protokollja elter az izomspecifikus kinazellenes antitest negativ es acetilkolin receptor ellenes antitest pozitiv betegek kezelesetől. A korkep etiologiajaban szerepet jatszo antitest azonositasa mar a betegseg korai stadiumaban lehetőve teszi a myasthenias betegek individualis optimalis kezelesi strategiajanak felallitasat, a sulyos mellekhatasok, mint peldaul a kolinergias krizisek megelőzeset es a tunetek gyors javulasat. Orv. Hetil., 2011, 152, 1586–1589. | The authors report the case of a 27-year-old woman with muscle-specific receptor tyrosine kinase antibody positive myasthenia with predominantly ocular and bulbar symptoms. Both edrophonium and low dose (4×30 mg/day) pyridostigmin resulted in cholinergic side effects including fasciculation mainly in the facial and neck muscles, and excessive salivation. The patient responded well to a relatively high dose of chronic corticosteroid treatment (methyprednisolone 64mg/day), but the decrease of the corticosteroid dose below 16 mg/day induced exacerbation of the clinical symptoms. Immunosuppression with azathioprine and methotrexate failed to maintain the clinical improvement. However, plasma exchange was always very effective, and all clinical symptoms improved significantly. The authors conclude that patients with muscle-specific receptor tyrosine kinase antibody positive myasthenia gravis should have an individual treatment protocol differing from those used in patients who do not have this antibody but are positive for acetylcholine-receptor antibody. Identification of the pathogenic antibody in the early stage of myasthenia gravis may help to develop the optimal, individualized treatment strategy, to avoid severe side effects, and to achieve fast improvement. Orv. Hetil., 2011, 152, 1586–1589.

Treatment of anti-MuSK antibody positive myasthenia gravis

The authors report the case of a 27-year-old woman with muscle-specific receptor tyrosine kinase antibody positive myasthenia with predominantly ocular and bulbar symptoms. Both edrophonium and low dose (4×30 mg/day) pyridostigmin resulted in cholinergic side effects including fasciculation mainly in the facial and neck muscles, and excessive salivation. The patient responded well to a relatively high dose of chronic corticosteroid treatment (methyprednisolone 64mg/day), but the decrease of the corticosteroid dose below 16 mg/day induced exacerbation of the clinical symptoms. Immunosuppression with azathioprine and methotrexate failed to maintain the clinical improvement. However, plasma exchange was always very effective, and all clinical symptoms improved significantly. The authors conclude that patients with muscle-specific receptor tyrosine kinase antibody positive myasthenia gravis should have an individual treatment protocol differing from those used in patients who do not have this antibody but are positive for acetylcholine-receptor antibody. Identification of the pathogenic antibody in the early stage of myasthenia gravis may help to develop the optimal, individualized treatment strategy, to avoid severe side effects, and to achieve fast improvement.A szerzők egy 27 eves fiatal nő esetet ismertetik, aki dontően ocularis-bulbaris tuneteket okozo izomspecifikus kinazellenes antitest pozitiv myasthenia gravisban szenvedett. Mind az intravenas edrophonium, mind a szajon at alkalmazott kis dozisu (4×30 mg/nap) pyridostigmin kolinerg mellekhatasokat, a mimikai es nyakizmok kellemetlen fasciculatiojat, hypersalivatiot okozott. A beteg tartos, viszonylag nagy dozisu kortikoszteroidkezelesre (64 mg/nap metilprednisolon) jol reagalt, azonban a napi adag 16 mg ala csokkentesekor a klinikai tunetek gyorsan kiujultak. Az azathioprinnel es methotrexattal tortenő immunszuppresszio hatasa nem volt latvanyos. A plazmaferezis azonban minden alkalommal gyors javulast, csaknem teljes tunetmentesseget valtott ki. A szerzők kozlemenyukkel arra kivanjak felhivni a figyelmet, hogy az izomspecifikus kinazellenes antitest pozitiv myasthenias betegek kezelesi protokollja elter az izomspecifikus kinazellenes antitest negativ es acetilkolin receptor ellenes antitest pozitiv betegek kezelesetől. A korkep etiologiajaban szerepet jatszo antitest azonositasa mar a betegseg korai stadiumaban lehetőve teszi a myasthenias betegek individualis optimalis kezelesi strategiajanak felallitasat, a sulyos mellekhatasok, mint peldaul a kolinergias krizisek megelőzeset es a tunetek gyors javulasat. Orv. Hetil., 2011, 152, 1586–1589. | The authors report the case of a 27-year-old woman with muscle-specific receptor tyrosine kinase antibody positive myasthenia with predominantly ocular and bulbar symptoms. Both edrophonium and low dose (4×30 mg/day) pyridostigmin resulted in cholinergic side effects including fasciculation mainly in the facial and neck muscles, and excessive salivation. The patient responded well to a relatively high dose of chronic corticosteroid treatment (methyprednisolone 64mg/day), but the decrease of the corticosteroid dose below 16 mg/day induced exacerbation of the clinical symptoms. Immunosuppression with azathioprine and methotrexate failed to maintain the clinical improvement. However, plasma exchange was always very effective, and all clinical symptoms improved significantly. The authors conclude that patients with muscle-specific receptor tyrosine kinase antibody positive myasthenia gravis should have an individual treatment protocol differing from those used in patients who do not have this antibody but are positive for acetylcholine-receptor antibody. Identification of the pathogenic antibody in the early stage of myasthenia gravis may help to develop the optimal, individualized treatment strategy, to avoid severe side effects, and to achieve fast improvement. Orv. Hetil., 2011, 152, 1586–1589.