Intrathecal kappa free light chains as markers for multiple sclerosis.

Cerebrospinal fluid (CSF) kappa free light chain (KFLC) index has been described as a reliable marker of intrathecal IgG synthesis to diagnose multiple sclerosis (MS). Our aims were: (1) to compare the efficiency of KFLC through different interpretation approaches in diagnosing MS. (2) to evaluate the prognostic value of KFLC in radiologically and clinically isolated syndromes (RIS-CIS). We enrolled 133 MS patients and 240 with other neurological diseases (93 inflammatory including 18 RIS-CIS, 147 non-inflammatory). Albumin, lambda free light chain (LFLC) and KFLC were measured in the CSF and serum by nephelometry. We included two groups of markers: (a) corrected for blood-CSF barrier permeability: immunoglobulin G (IgG), KFLC and LFLC indexes. (b) CSF ratios (not including albumin and serum-correction): CSF KFLC/LFLC, CSF KFLC/IgG, CSF LFLC/IgG. KFLC were significantly higher in MS patients compared to those with other diseases (both inflammatory or not). KFLC index and CSF KFLC/IgG ratio showed high sensitivity (93% and 86.5%) and moderate specificity (85% and 88%) in diagnosing MS. RIS-CIS patients who converted to MS showed greater KFLC index and CSF KFLC/IgG. Despite OB are confirmed to be the gold-standard to detect intrathecal IgG synthesis, the KFLC confirmed their accuracy in MS diagnosis. A "kappa-oriented" response characterizes MS and has a prognostic impact in the RIS-CIS population.

Kappa free light chains could predict early disease course in multiple sclerosis.

BACKGROUND: Cerebrospinal fluid (CSF) kappa free light chains (KFLC) have been suggested as quantitative alternative to oligoclonal bands (OB) in multiple sclerosis (MS) diagnosis. Despite OB have been associated to poor disease prognosis, little is known on KFLC in predicting MS early progression. Our aim is to evaluate the prognostic value of KFLC in a cohort of Italian MS patients. METHODS: 100 patients (64 females) underwent CSF analysis during their diagnostic MS work-up. We collected clinical/paraclinical features (gender, age at onset, clinical course, early MS treatments (within 1 year), gadolinium-enhancing (Gd+) lesions), calculated K index (ratio CSF-serum KFLC and albumin), and MS severity score (MSSS) at last follow up (minimum 1 year). Statistical analysis included Mann-Whitney descriptive analysis, Spearman correlation for independent samples, and linear regression for significant predictors. RESULTS: K index resulted a significant predictor for disability over time being higher in patients who developed greater MSSS. Accordingly, K index was also significantly increased in patients undergoing early versus delayed treatment (N = 50/100, p = 0.046). A similar role in predicting MS disability was confirmed for age at onset. No other factors were retained in our regression model. Of note, K index was not associated to known MS prognostic markers such as gender, age at onset, and Gd+ lesions (N = 31/96). CONCLUSION: Our study suggests KFLC as a CSF quantitative marker to predict early disability in MS (despite not being a substitute for OB).

Role of the laboratory in monitoring patients receiving dual antiplatelet therapy.

INTRODUCTION: The increasing demand for therapeutic monitoring in patients receiving antiplatelet therapy has been paralleled by the development of instruments and tests whose clinical usefulness is still under debate. We devised a laboratory approach to detect patients with antiplatelet resistance at risk to develop thrombotic events. METHODS: One hundred and eighty patients, under aspirin and clopidogrel after angioplasty and stent implantation, were studied by PFA100(®) with collagen/epinephrine (CoEPI, cutoff 165s) cartridge and by Multiplate(®) using arachidonic acid (ASPItest, pos < 862AUC), ADP (ADPtest, pos < 417AUC), and collagen (COLtest, pos < 607AUC). RESULTS: Only 67 of 173 patients with ASPI < 862 displayed a prolonged CoEPI and up to 65 patients had normal CoEPI despite ASPI < 300. Patients with ASPI < 300 had significantly lower COL than patients with ASPI > 300. One hundred and thirty-eight patients displaying ADP < 417 had significantly lower COL than those with ADP > 417. Association between COL and ADP remained after ASPI stratification: in patients with suboptimal (ASPI 300-892) or maximal (ASPI < 300) response to aspirin, having ADP < 417 (clopidogrel responsive) increased COL positivity, respectively, from 9.5 to 58.8% and from 47.6 to 82.7%. CONCLUSION: A combination of specific tests may be useful in identifying higher-risk patients with poor compliance or drug resistance who potentially may benefit from therapy change.

Renoprotective effects of telmisartan in patients with advanced chronic kidney disease.

BACKGROUND: Angiotensin II receptor blockers (ARBs) provide renoprotective effects in patients with mild-to-moderate chronic kidney disease (CKD). However, there have been few reports regarding whether ARBs show clinical efficacy and safety in patients with advanced CKD. METHODS: Seventy-two hypertensive patients with Stages 3 - 4 CKD receiving no ARBs were enrolled in this study and observed up to 48 months. Telmisartan was added to conventional antihypertensive agents (n = 36, mean estimated glomerular filtration ratio [eGFR] 19.7 ml/min/1.73 m2) whilst the remaining control patients were not treated with ARBs (n = 36, mean eGFR 19.2 ml/min/1.73 m2). Urinary protein excretion, kidney function, and the occurrence of end-stage renal disease requiring renal replacement therapy, hyperkalemia, and death were analyzed. RESULTS: Baseline characteristics of each group were similar. During the observation period, the blood pressures of each group decreased at similar rates. In the telmisartan group, 17 patients (47.2%) were introduced to renal replacement therapy, as compared with 31 patients (86.1%) in the control group (relative risk 0.55, 95% confidence interval 0.19 - 0.92, p < 0.05). Telmisartan significantly reduced proteinuria levels (from 3.47 +/- 3.00 to 2.41 +/- 2.46 g/g . creatinine, p < 0.05) and was associated with a reduction of 49.6% in the decline rate of eGFR. The incidence of major adverse events in both groups was similar. CONCLUSIONS: The addition of telmisartan to conventional antihypertensive therapy is associated with significant improvement in kidney outcome without increased incidence of adverse effects, even in patients with advanced CKD.

Antibodies against oxidized phospholipids in laboratory tests exploring lupus anti-coagulant activity.

Lupus anti-coagulants (LA) are a variety of anti-phospholipid antibodies characterized by their capacity to interfere with phospholipid-dependent coagulation assays. LA are increasingly recognized as important predictors of thrombosis. However, the antigen specificity of LA is still poorly characterized. Growing evidence indicates that oxidized phospholipids are among the targets of anti-phospholipid antibodies. This prompted us to investigate the role of IgG directed against different oxidized phospholipids in 164 subjects without clotting factor defects that were tested for the presence of LA using a LA-sensitive activate partial thromboplastin time (aPTT-FSL) and a screening/confirmation assay based on diluted Russell's viper venom test (dRVVT-PL). The response to aPTT-FSL was significantly (P < 0.0005) associated with high titres of IgG against oxidized phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol, whereas positivity to dRVVT-PL was associated with the elevation of IgG against oxidized phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine (P < 0.0005) and phosphatidylinositol (P < 0.01). No difference in reactivity against oxidized cardiolipin was evident between the different groups. Positivity to the dRVVT-PL test was also associated significantly (P < 0.005) with the elevation of anti-cardiolipin and anti-beta(2)-glycoprotein-1 IgG. However, stepwise logistic regression demonstrated that IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylcholine were the only independent predictors of the response to dRVVT-PL assay, while IgG recognizing oxidized phosphatidylethanolamine and oxidized phosphatidylinositol were independent predictors of the response to aPTT-FSL test. In conclusion, autoantibodies against defined oxidized phospholipids are independent predictors of LA detection by aPTT-FSL or dRVVT-PL assays and might contribute to the variability often observed in the responses to the functional tests detecting LA.

Prednisolone inhibits hyperosmolarity-induced expression of MCP-1 via NF-kappaB in peritoneal mesothelial cells.

The mechanism of peritoneal fibrosis in patients on continuous ambulatory peritoneal dialysis (CAPD) is poorly elucidated. We investigated the cellular mechanism of high-glucose-induced expression of monocyte chemoattractant protein-1 (MCP-1), which is important in recruiting monocytes into the peritoneum and progression of peritoneal fibrosis, and examined the inhibitory mechanism of glucocorticoids. Rat peritoneal mesothelial cells were cultured in high-glucose-containing medium and then analyzed for phosphorylation levels of p42/44 and p38 mitogen-activated protein (MAP) kinases (MAPK), MAPK or extracellular signal-regulated kinase kinase (MEK)1/2, c-Jun N-terminal kinase (JNK)1/2, and protein kinase C (PKC) by Western blotting. Expression of MCP-1 was examined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. DNA-binding activity of nuclear factor (NF)-kappaB was measured by electrophoretic mobility shift assay. High glucose increased MCP-1 mRNA and MCP-1 protein expression. Although glucose increased phosphorylation of MEK1/2, p42/44 MAPK, p38 MAPK, JNK1/2, and PKC, and DNA-binding activity of NF-kappaB, its effect on MCP-1 expression was suppressed only by PKC and NF-kappaB inhibitors. Mannitol caused a similar increase in PKC and NF-kappaB activation and MCP-1 synthesis. Prednisolone increased I-kappaB-alpha expression and inhibited glucose/mannitol-induced NF-kappaB DNA binding and MCP-1 expression without affecting PKC phosphorylation. The inhibitory effects of prednisolone on MCP-1 expression were reversed by mifepristone, a glucocorticoid receptor antagonist. Our results indicate that glucose induces MCP-1 mainly through hyperosmolarity by activating PKC and its downstream NF-kappaB, and that such effect was inhibited by prednisolone, suggesting the efficacy of prednisolone in preventing peritoneal fibrosis in patients on CAPD.

Cannabinoids modulate synaptic activity in the rat supraoptic nucleus.

In the present study, we investigated the effects of the cannabinoid receptor agonist CP55,940 on excitatory and inhibitory synaptic transmission in the rat supraoptic nucleus. Whole-cell patch clamp recordings were performed on supraoptic neurones in in vitro brain slice preparations. CP55,940 significantly reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents in a concentration-dependent manner. These changes were potently reversed by the CB1 receptor antagonist AM251. The results indicate that cannabinoids modulate the activity of magnocellular neurosecretory neurones by presynaptic inhibition of both excitatory and inhibitory synaptic transmission.

Intermittent footshock facilitates dendritic vasopressin release but suppresses vasopressin synthesis within the rat supraoptic nucleus.

Emotional stress inhibits vasopressin release from the pituitary but may facilitate its release from the dendrites in the hypothalamus. We examined effects of intermittently applied footshock upon the amount of vasopressin heteronuclear RNA in the hypothalamus. The footshock decreased plasma vasopressin concentration but increased its extracellular concentration within the supraoptic nucleus. The contents of the vasopressin heteronuclear RNA in the supraoptic nucleus were significantly decreased after the shock. These data suggest that intermittent footshock decreases not only vasopressin release from the axon terminals in the pituitary, but also vasopressin synthesis in the cell bodies in the hypothalamus while the stimulus facilitates vasopressin release from the dendrites in the hypothalamus. The data also suggest differential control of dendritic vasopressin release and synthesis in the hypothalamus.

Increased levels of hypothalamic neuronal nitric oxide synthase and vasopressin in salt-loaded Dahl rat.

The plasma concentration of arginine vasopression (AVP) and the expression level of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular nucleus (PVN) and the Supraoptic nucleus (SON) of Sprague-Dawley (SD). Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats on a high salt diet were examined by radioimmunoassay for AVP and in situ hybridization histochemistry for nNOS. The high salt diet containing 8.0% NaCl was given for 4 weeks. The concentrations of AVP in hypertensive Dahl S rats were significantly increased in comparison with those in SD rats and Dahl R rats on a high salt diet. The levels of nNOS mRNA and NADPH-diaphorase activity in the PVN and SON of hypertensive Dahl S rats were greater than those in Dahl R rats on a high salt diet. The antihypertensive drugs, either nicardipine or captopril were administered to the Dahl S rats for 2 weeks beginning 2 weeks after the start of the high salt diet The nNOS mRNA in the PVN and SON of Dahl S rats given a high salt diet was not upregulated by treatment with nicardipine, while the nNOS mRNA in salt loaded Dahl S rats was greater upregulated by treatment with captopril to that greater than without the antihypertensive drug. Our results suggest that the increased NO production in the PVN and SON of hypertensive Dahl S rats may be ineffective in decreasing blood pressure or inhibiting AVP secretion.

Activation of hypothalamic neuronal nitric oxide synthase in lithium-induced diabetes insipidus rats.

The expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) in rats with lithium (Li)-induced polyuria was examined by using in situ hybridization histochemistry. The state of the thyroid axis in these rats was also examined by in situ hybridization histochemistry for thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) mRNAs and radioimmunoassay for circulating thyroid hormones. Adult male Wistar rats consuming a diet that contained LiCl (60 mmol/kg) for 4 weeks developed remarkable polyuria. The urine in the Li-treated rats was hypotonic and had a large volume and low ionic concentration. The nNOS mRNA in the PVN and SON was significantly increased in the Li-treated rats in comparison with that in control. The increased levels of the nNOS mRNA in the PVN and SON were confirmed by NADPH-diaphorase histochemical staining. There were no differences of TRH mRNA in the PVN, TSH mRNA in the anterior pituitary and plasma concentrations of free T3 and free T4 between Li-treated rats and control rats. These results suggest that Li-induced diabetes insipidus may activate nNOS in the PVN and SON without change of the thyroid axis.

Upregulation of synapsin IIa and IIb mRNAs in the paraventricular and supraoptic nuclei in chronic salt loaded and lactating rats.

The effects of chronic salt loading (2% saline to drink for 5 and 10 days), gestation, lactation and adrenalectomy on the expression of synapsin IIa and IIb genes were examined in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. In each control, synapsin IIa and IIb genes were moderately expressed in the magnocellular division of the PVN and SON, while few transcripts of synapsin IIa and IIb were observed in the parvocellular division of the PVN. Chronic salt loading, gestation on day 21 and lactation on day 10 caused significant increases in synapsin IIa and IIb transcripts in the magnocellular division of the PVN and SON, compared to each control. Although corticotropin-releasing hormone transcripts in the parvocellular division of the PVN were significantly increased in the adrenalectomized rats, no changes in the transcripts of synapsin IIa and IIb were observed throughout the PVN. These results suggest that physiological stimuli such as osmotic challenge and lactation potently increase synapsin IIa and IIb mRNAs in the magnocellular neurons of the PVN and SON.

Centrally administered orexin/hypocretin activates HPA axis in rats.

The effects of i.c.v. administration of orexin/hypocretin on plasma ACTH, corticosterone and c-fos mRNA in the paraventricular nucleus (PVN) of the rat were examined. Plasma ACTH levels were markedly increased at 30 min after i.c.v. administration of orexin-A. Plasma corticosterone levels were significantly increased in a dose-related manner 30 min after i.c.v. administration of orexin-A and orexin-B. In situ hybridization histochemistry revealed that the induction of the c-fos mRNA in the parvocellular division of the PVN was increased in a dose-related manner 30 min after i.c.v. administration of orexin-A and orexin-B. These results suggest that central orexin/hypocretin activates hypothalamo-pituitary-adrenal (HPA) axis and may be involved in stress-induced activation of the HPA axis.

Postnatal development of orexin/hypocretin in rats.

We examined developmental changes of orexins/hypocretins and their receptors (OX1R and OX2R) in the rat hypothalamus from postnatal day 0 to 10 weeks, using in situ hybridization histochemistry for the prepro-orexin, OX1R and OX2R mRNAs and immunohistochemistry for orexin-A and orexin-B. The prepro-orexin mRNA was weakly detected in the lateral hypothalamic area (LHA) from days 0 to 15. Orexin-A- and -B-like immunopositive cells and fibers were not detected from days 0 to 10, but they were observed after day 15. The prepro-orexin mRNA in the LHA markedly increased between days 15 and 20. The OX1R mRNA was detected in the ventromedial hypothalamic area (VMH) at day 0. The OX2R mRNA was not detected in the paraventricular nucleus (PVN) at days 0 and 1, but weakly observed on day 5. The OX1R mRNA in the VMH and OX2R mRNA in the PVN gradually increased throughout the postnatal period. Next, we examined the effects of milk deprivation and intraperitoneal (i.p.) administration of leptin on the hypothalamic prepro-orexin mRNA in pups. Although 24-h milk deprivation did not affect the level of the prepro-orexin mRNA at days 5 and 10, i.p. administration of leptin from days 0 to 3 caused a significant increase in the prepro-orexin mRNA on days 5 and 10. These results suggest that the development of orexins may be associated with developmental changes such as increase of leptin, weaning, feeding and sleep/wakefulness states.

Induction of pituitary adenylate cyclase-activating polypeptide mRNA in the medial parvocellular part of the paraventricular nucleus of rats following kainic-acid-induced seizure.

We examined the effects of kainic acid (KA)-induced seizure on the expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) gene in the paraventricular nucleus (PVN) of rats using in situ hybridization histochemistry. Subcutaneous administration of KA (12 mg/kg) in adult male Sprague-Dawley rats caused a progressive development of seizure behavior. An induction of the PACAP gene expression in the medial parvocellular part of the PVN (mpPVN) was observed 3, 6, 12, 24 and 48 h after subcutaneous administration of KA. From a nearly undetectable level, PACAP gene expression increased in the mpPVN and reached maximum 12 h after subcutaneous administration of KA. PACAP gene expression returned to near basal level 48 h after stimulation with KA. Using a specific monoclonal PACAP antibody, PACAP immunoreactivity (-IR) gradually increased during the following 24 h after KA administration. In controls, PACAP-IR was located exclusively in nerve fibers of the mpPVN, whereas KA administration induced PACAP-IR in cell bodies of the mpPVN, and a dense accumulation of PACAP-IR nerve fibers in the external zone of the median eminence was observed. Induction of the PACAP gene expression following KA-induced seizure was significantly reduced by pretreatment with diazepam or MK-801 (nonselective N-methly-D-aspartate receptor antagonist). These results suggest that PACAP in the hypothalamo-adenohypophysial system may have a hypophysiotropic role during KA-induced seizure.

A physiological role for adrenomedullin in rats; a potent hypotensive peptide in the hypothalamo-neurohypophysial system.

Adrenomedullin, a potent hypotensive peptide, was originally isolated from human phaeochromocytoma. Adrenomedullin immunoreactivity and gene expression are found not only in peripheral organs but also in the central nervous system. Adrenomedullin labelled cells were localised in the hypothalamus, including in the paraventricular and supraoptic nuclei, in rats. Abundant adrenomedullin-immunoreactive fibres and varicosities were found in the hypothalamo-neurohypophysial tract and the internal zone of the median eminence in colchicine-treated and hypophysectomized rats, whereas in control rats few adrenomedullin-labelled fibres were observed. We examined the effects of intracerebroventricular administration of adrenomedullin on neurosecretory cells in the paraventricular and supraoptic nuclei of rats, using immunohistochemistry for Fos protein and in situ hybridisation histochemistry for c-fos mRNA. Intracerebroventricular administration of adrenomedullin caused a marked induction of Fos-like immunoreactivity in the paraventricular nucleus and the dorsal part of the supraoptic nucleus. In the paraventricular and supraoptic nuclei, nuclear Fos-like immunoreactivity was predominantly in oxytocin-immunoreactive cells rather than vasopressin-immunoreactive cells. The induction of c-fos mRNA in the paraventricular and supraoptic nuclei was increased in a dose-related manner 30 min after intracerebroventricular administration of adrenomedullin. This induction was reduced by pre-treatment with the adrenomedullin receptor antagonist, human adrenomedullin-(22-52)-NH2. Intracerebroventricular administration of adrenomedullin also caused a marked increase in the plasma concentration of oxytocin. Extracellular recordings from magnocellular neurosecretory cells in the paraventricular nucleus revealed that putative oxytocin-secreting cells were activated by intracerebroventricular administration of adrenomedullin. These results suggest that central adrenomedullin preferentially stimulates the secretion of oxytocin by activating hypothalamic oxytocin-secreting cells and may have an important role in salt appetite and body fluid homeostasis in rats.

Effects of food restriction on the hypothalamic prepro-orexin gene expression in genetically obese mice.

Orexins, which are identical to hypocretins, are novel hypothalamic orexigenic peptides. We examined the effects of food restriction on the expression of the prepro-orexin gene in control (C57Bl/6J) and genetically obese mice (ob/ob and db/db), using in situ hybridization histochemistry. Dry food was given 3 g/day to each obese mouse for 2 weeks. Food restriction caused a significant increase of the prepro-orexin gene expression in obese mice in comparison with ad libitum fed animals. Although the levels of the expression of the prepro-orexin gene in obese mice were significantly lower than those in C57Bl/6J mice during feeding ad libitum, food restriction caused an increase in the expression of the prepro-orexin gene in the hypothalamus of obese mice. The expression of the neuropeptide Y (NPY) gene was increased significantly in the arcuate nucleus of obese mice compared to that of control mice during feeding ad libitum. Food restriction for 2 weeks also caused a significant increase of the expression in the NPY gene in all groups. These results indicate that the hypothalamic prepro-orexin gene could be upregulated by food restriction without leptin signal in genetically obese mice.

Increase of urocortin-like immunoreactivity in the supraoptic nucleus of Dahl rats given a high salt diet.

Urocortin-like immunoreactivity (Ucn-LI) in the supraoptic nucleus (SON) of Dahl rats was examined. Dahl salt-sensitive (S) rats fed with a high salt diet developed hypertension. Numbers of Ucn-LI neurons in the SON in Dahl S on a high salt diet were markedly increased, compared with those in Dahl salt-resistant (R) rats on the same. Sporadic Ucn-LI neurons were found in the SON of both Dahl S and R on a normal diet. Numbers of Ucn-LI neurons in the SON of spontaneously hypertensive rat (SHR) and stroke-prone SHR, genetic models of hypertension, and control rats (Sprague-Dawley and Wistar-Kyoto) were similar. These results suggest that Ucn in the SON is associated with salt loading-induced hypertension rather than spontaneous hypertension.