Enzyme-like Immunosorbent Assay Research Articles

New preclinical biomarkers for prion diseases in the cerebrospinal fluid proteome revealed by mass spectrometry

Current diagnostic methods for prion diseases only work in late stages of the disease when neurodegeneration is irreversible. Therefore, biomarkers that can detect the disease before the onset of clinical symptoms are necessary. High-throughput discovery proteomics is of great interest in the search for such molecules. Here we used mass spectrometry to analyse the cerebrospinal fluid proteome in an animal prion disease: preclinical and clinical sheep affected with natural scrapie, and healthy sheep. Interestingly, we found 46 proteins in the preclinical stage that were significantly altered (p < 0.01) compared to healthy sheep, mainly associated with biological processes such as stress and inflammatory responses. Five of them were selected for validation by enzyme-like immunosorbent assay: synaptotagmin binding, cytoplasmic RNA interacting protein (SYNCRIP), involved in nucleic acid metabolism; phospholipase D3 (PLD3) and cathepsin D (CTSD), both related to lysosomal apoptosis; complement component 4 (C4), an element of the classical immune response; and osteopontin (SPP1), a proinflammatory cytokine. These proteins significantly increased in the preclinical stage and maintained their levels in the clinical phase, except for CTSD, whose concentration returned to basal levels in the clinical group. Further research is ongoing to explore their potential as preclinical biomarkers of prion diseases.

Are Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 Useful as Markers in Diagnostic Management of Children with Newly Diagnosed Ulcerative Colitis?

Matrix Metaloproteinase-9 (MMP-9) and Tissue Inhibitor of Metaloproteinase-1 (TIMP-1), enzymes involved in tissue remodelling, have been previously reported to be overexpressed in the colonic mucosa of patients with Ulcerative colitis (UC). The aim of this study was to determine the relation of MMP-9 and TIMP-1 with UC phenotypes, the disease activity index and routinely tested inflammatory markers in newly diagnosed paediatric patients. The study group comprised 35 children diagnosed with UC and 20 control groups. Serum and faecal concentrations of MMP-9 and TIMP-1 were estimated using enzyme-like immunosorbent assay kits and correlated to the disease activity index (Paediatric Ulcerative Colitis Activity Index, PUCAI), UC phenotype (Paris Classification), inflammatory markers and endoscopic score (Mayo score). Children with UC presented with significantly higher serum and faecal concentrations of studied markers compared to the control group. Both serums, MMP-9 and TIMP-1, were higher in children with more extended and severe lesions in the colon. Furthermore, serum MMP-9 correlated with the Mayo score, Paris classification and C-reactive protein (CRP) levels. Serum TIMP-1 showed correlation with PUCAI, Paris Classification, CRP levels and the erythrocyte sedimentation rate. Serum and faecal levels of MMP-9 and TIMP-1 are useful in discriminating UC patients and non-invasive assessments of disease phenotypes. It seemed that simultaneous measurement of these proteins in combination with other common markers of inflammation could be applied in clinical practice.

Accuracy of Serum Golgi Protein 73 and Alpha Fetoprotein (AFP) to Diagnose Hepatocellular Carcinoma

Background: Hepatocellular Carcinoma (HCC) is one of the most common malignancy in the liver. Modalities of diagnostic are often an obstacle in HCC surveillance. Alpha fetoprotein (AFP) is one of protein that often used in the diagnostic of HCC in chronic liver disease. Golgi protein 73 (GP73), is one of the candidate biomarkers in early diagnostic of HCC and found in biliary epithelial cells but rarely expressed by normal hepatocytes. Expression of GP73 was reported to be increased in a large number of malignancies. Aims of this study to evaluate differences in Golgi protein 73 serum (sGP73) and AFP in diagnosing hepatocellular carcinoma in patients with liver cirrhosis. Materials and Methods: This cross-sectional study was conducted at Haji Adam Malik Hospital in 2020. Serum level of GP73 and others biomarker was detected using enzyme-like immunosorbent assay. Results: From 90 subjects, Liver cirrhosis and HCC group had significantly higher AFP than the control group. AFP was superior in determining HCC to GP73. At a cut off value of &gt; 394.5.00 ng/mL, AFP yielded a sensitivity of 83.3% and specificity of 67%, for discriminating liver cirrhosis and HCC (AUC 0.84), while GP73 with cut off value of &gt; 82.5 ng/mL, sensitivity of 70% and specificity of 57% (AUC 0.74). Conclusion:GP73 was significantly higher in HCC patients in comparison to non-HCC patients and healthy population. Compared with alpha fetoprotein, GP73 was superior in discriminating HCC in healthy population but inferior in group of liver cirrhosis. Keywords: Golgi Protein 73, Alpha Fetoprotein, Hepatocellular carcinoma.

The cytokine alterations/abnormalities and oxidative damage in the pancreas during hypertension development

The aim of the present study was to compare the content of cytokines, chemokines, and oxidative stress markers in the pancreas of spontaneously hypertensive rats (SHRs) and Wistar Kyoto Rats (WKYs) serving as controls. Enzyme-like immunosorbent assay (ELISA) and biochemical methods were used to measure pancreatic levels of interleukin-1ß, interleukin-6, tumor necrosis factor α, transforming growth factor β, RANES, monocyte chemoattractant protein 1, interferon gamma-induced protein 10, malondialdehyde, and sulfhydryl groups. The results showed that the pancreatic concentrations of all studied cytokines and chemokines did not differ between 5-week-old SHRs and WKYs, except RANTES which was significantly reduced in juvenile SHRs. In 10-week-old animals, except interleukin-1ß, the levels of all these proteins were significantly reduced in SHRs. The pancreatic levels of malondialdehyde were significantly reduced in 5-week-old SHRs and significantly elevated in 10-week-old SHRs while the contents of sulfhydryl groups were similar in both rat strains at any age studied. In conclusion, these data provide evidence that in maturating SHRs, the pancreatic levels of cytokines and chemokines are significantly reduced, while malondialdehyde significantly elevated. This suggests that in the pancreas of mature SHRs, the inflammation process is suppressed but there is ongoing oxidative damage.

Ginkgetin exerts anti-inflammatory effects on cerebral ischemia/reperfusion-induced injury in a rat model via the TLR4/NF-κB signaling pathway.

Ginkgo biloba, a natural biflavonoid isolated from Ginkgo biloba leaves, is reported to have strong anti-inflammatory and immunosuppressive properties. The aim of this study is to investigate the potential anti-inflammatory mechanisms of ginkgo flavonoids on cerebral ischemia/reperfusion (I/R) injury. Inflammatory-associated cytokines in cerebral ischemic hemispheres were determined by immunohistochemical staining, Western blot and enzyme-like immunosorbent assay (ELISA). Our results indicated that treatment with Ginkgetin significantly restored rat brain I/R-induced neurological deficit scores. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in Ginkgetin treatment group (100 mg/kg) also significantly reduced. The expression inflammation-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8) was also decreased in Ginkgetin treatment group. However, the expression of interleukin-10 (IL-10) was remarkably increased. Thus, this study demonstrates that Ginkgetin protects neurons from I/R-induced rat injury by down-regulating pro-inflammatory cytokines and blocking the TLR4/NF-κB pathway.

Lycopene improves the efficiency of anti-PD-1 therapy via activating IFN signaling of lung cancer cells

BackgroundMonoclonal antibodies targeting programmed death-1 receptor (PD-1) and its ligand (PD-L1) have been developed to treat cancers including lung cancer. In this study, we aimed to investigate whether lycopene could promote the effect of anti-PD-1 treatment on lung cancer.MethodsTumor formation assay was conducted. Immune reactions were assessed by detecting several cytokine levels using enzyme-like immunosorbent assay. T cell activity was analyzed using cytometry. The mechanism of lycopene action was investigated using Western blot, quantitative real-time polymerase chain reaction and bisulfite sequencing analysis.ResultsAfter the mice injected with Lewis lung carcinoma (LLC) cells were sacrificed, we found that combined lycopene and anti-PD-1 reduced the tumor volume and weight compared to control treatment. Cell apoptosis in the tumor tissues was significantly enhanced in mice with combined lycopene and anti-PD-1 treatment in comparison with those of either lycopene or anti-PD-1 alone. Furthermore, lycopene could assist anti-PD-1 to elevate the levels of interleukin (IL)-1 and interferon (IFN) γ while reduce the levels of IL-4 and IL-10 in the spleen of mice injected with LLC cells. Lycopene treatment increased the CD4+/CD8+ ratio in the spleen and promoted IFNγ-expressing CD8+ T cells in tumor tissues. Upon IFNγ stimulation, lycopene diminished PD-L1 expression via activating JAK and repressing phosphorylation of AKT.ConclusionOur results have demonstrated that lycopene could be used as a potential adjuvant drug to synergistically improve the efficiency of anti-PD-1 therapy.

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Immunological disturbances have been implicated in the pathogenesis of some neurodegenerative disorders like Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Adhesion molecules are markers of activated endothelial cells upregulated by action of cytokinesTo investigate the activation or not of the vascular cells in AD and ALS, serum soluble intercellular adhesion molecule-1 (ICAM-1) and soluble E-selectin were evaluated (enzyme-like immunosorbent assay, ELISA) in 22 patients with Alzheimer's disease (AD), 20 patients with amyotrophic lateral sclerosis (ALS), 34 patients with non-inflammatory neurological diseases (NIND) and 15 control subjects.Patients with AD had higher s-ICAM-1 levels compared to NIND patients and control subjects (p<0.0027 and p<0.04, respectively). Patients with ALS had not higher s-ICAM-1 levels compared to NIND patients and control subjects (p<0.21 and p<0.31, respectively). Soluble-E-selectin levels in AD and ALS patients were not statistically different compared to NIND patients and controls (p<0.4, p<0.9 and p<0.3, p<0.19, respectively).The presence of high s-ICAM values may be related to immunological processes involved in pathogenetic mechanisms of AD. The not statistically significant values of s-E selectin, a glycoprotein considered an exclusive marker of endothelial activation, seem to suggest the neural rather than the endothelial s-ICAM origin in patients with AD. The low values of s-ICAM-1 and sE-selectin in the serum of ALS patients do not exclude the presence of an unconventional immunological abnormality in this disorder.

Anti-inflammation Effects of Oxysophoridine on Cerebral Ischemia-Reperfusion Injury in Mice.

Oxysophoridine (OSR) is a bioactive alkaloid extracted from the Sophora alopecuroides Linn. Our aim is to explore the potential anti-inflammation mechanism of OSR in cerebral ischemic injury. Mice were intraperitoneally pretreated with OSR (62.5, 125, and 250mg/kg) or nimodipine (Nim) (6mg/kg) for 7days followed by cerebral ischemia. The inflammatory-related cytokines in cerebral ischemic hemisphere tissue were determined by immunohistochemistry staining, Western blot and enzyme-like immunosorbent assay (ELISA). OSR-treated groups observably suppressed the nuclear factor kappa B (NF-κB), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). OSR-treated group (250mg/kg) markedly reduced the inflammatory-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8). Meanwhile, it dramatically increased the interleukin-10 (IL-10). Our study revealed that OSR protected neurons from ischemia-induced injury in mice by downregulating the proinflammatory cytokines and blocking the NF-κB pathway.

Maternal serum concentrations of angiogenic and antiangiogenic factors in threatened miscarriage at 7–12weeks’ gestation and the risk of adverse pregnancy outcomes

Abstract Objective To assess the ability of first trimester maternal serum (FTMS) soluble Fms-like tyrosine kinase (sFlt-1)/placental growth factor (PlGF) ratio in the first trimester threatened miscarriage (FTTM) to predict fetal loss in early pregnancy and adverse pregnancy outcomes when pregnancy continued to ⩾30 weeks to start suitable antenatal care as early as possible. Design A case control prospective cross-sectional study. Material and methods 320 cases of TM, 7–12 weeks’ gestation and 320 normal controls 7–12 weeks’ gestation without vaginal bleeding and other manifestations of FTTM and delivered at ⩾30 weeks’ gestation were enrolled in the study. All were primigravidae. Estimation of FTMS sFlt-1 and PlGF in the first trimester of TM and controls by enzyme-like immunosorbent assay (ELISA) and follow up of them till delivery were done. Adverse pregnancy outcomes encountered were gestational hypertension (GH), preeclampsia (PE), intrauterine growth restriction (IUGR), abruptio placentae, spontaneous preterm labor (PTL), placenta previa, preterm premature rupture of membranes (PPROM) and retained placenta. Results All adverse pregnancy outcomes were significantly ( P 74.6 (cut-off point) predicted these adverse pregnancy outcomes with sensitivity 88.70%, specificity 95.40, positive predictive value 90.16% and negative predictive value 94.68% – adverse pregnancy outcomes not related to placental impairment as PLT >34 weeks’ gestation, PPROM, placenta previa and retained placenta were not predicted by sFlt-1/PlGF ratio because in these conditions FTMS concentration of these factors were normal. Conclusions 1-Adverse outcomes late in pregnancy (⩾30 weeks’ gestation) were more common in FTTM. 2-Diminished FTMS concentration of sFlt-1 and PlGF as well as sFlt-1/PlGF ratio 74.6 may predict development of adverse outcomes characterized by impaired placentation. 4-Adverse pregnancy outcomes not related to impaired placentation were associated with normal FTMS concentration of sFlt-1 and PlGF and were not predicted by sFlt-1/PlGF ratio.

Protein oxidation in a group of subjects with metabolic syndrome

AimsTo examine the protein oxidation, marker of the oxidative stress, in metabolic syndrome (MS). MethodsWe enrolled 106 subjects (45 women and 61 men) with MS of which 43 (14 women and 27 men) were with diabetes mellitus and 63 (31 women and 32 men) were without diabetes mellitus, and 54 subjects (19 women and 35 men) as control group. The protein oxidation, expressed as carbonyl groups, was measured by an enzyme-like immunosorbent assay (ELISA) kit (BioCell PC test kit, Enzo Life Sciences AG, Switzerland). ResultsIn the whole group of MS subjects, in comparison with control group, a significant increase in carbonyl groups was present. The same datum was also evident between control group and diabetic subjects with MS and between control group and nondiabetic subjects with MS. No difference was observed between the two subgroups (diabetic and nondiabetic subjects with MS) about NOx. Few information were obtained examining the linear regression among carbonyl groups, age, BMI, waist circumference, blood pressure values and metabolic pattern of MS subjects. ConclusionsIn MS subject we observed an increase of protein oxidation not influenced by diabetes mellitus. Several strategies may be employed to reduce this parameter.

Proinflammatory cytokines in serum and cerebrospinal fluid of CIDP patients

Introduction: Little is known about the role of cytokines in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Interleukin (IL)-12 and IL-15 are the major growth and differentiation factors for Th-1 cells and IL-17 is a marker of Th-17 cell expansion and activation, a high proinflammatory new subset of T cells that induce severe autoimmunity.Patients and methods: We measured by enzyme-like immunosorbent assay serum and cerebrospinal fluid (CSF) levels of IL-15, IL-12, and IL-17 in 24 patients with CIDP and 12 patients with other non-inflammatory neurological disorders and serum levels in 16 healthy subjects.Results: We found a positive association of CSF IL-12 (P = 0·012) with CIDP presence (P<0·001).Conclusions: Our findings suggest that IL-12 may be involved as potential marker of immune activation in CIDP. The increase in its levels in CSF may be a marker of initiation of Th-1 cell-mediated immunity.

Anti-inflammatory and antinociceptive activities of the ethanolic extract of Bougainvillea xbuttiana

Ethnopharmacological relevanceBougainvillea xbuttiana is widely distributed in Mexico and it is used as an analgesic in folk medicine. Aim of the studyIn the present study the in vivo antinociceptive and anti-inflammatory effects of the Bougainvillea xbuttiana ethanolic extract have been studied in mice. Materials and methodsThe phytochemical analysis was performed. Antinociceptive activity was evaluated through writhing and formalin test in mice. The anti-inflammatory activity was determined with the carrageenan-induced mice paw oedema model. IL-6, IL-10 and IFN-γ levels were determined by enzyme-like immunosorbent assay, whereas TNF and nitrite levels were detected by standard assay with L929 cells and colorimetric Griess reactive, respectively. ResultsThe results showed that the ethanolic extract of the Bougainvillea xbuttiana has significant anti-inflammatory and antinociceptive activities, by inhibition of nociception induced by acetic acid and paw oedema. This extract also induced a decrease in TNF levels and an increase of IL-6, IFN-γ and NO levels that we observed up to 2h. The highest levels of IL-10 were observed up to 4h. The ratios of pro-/anti-inflammatory cytokines in sera from mice injected with the ethanolic extract, may be manifesting an anti-inflammatory status. ConclusionsThe present study provides convincing evidences that Bougainvillea xbuttiana extract possesses significant anti-nociceptive and anti-inflammatory effects.

Cyprinus carpio Decoction Improves Nutrition and Immunity and Reduces Proteinuria through Nephrin and CD2AP Expressions in Rats with Adriamycin-Induced Nephropathy.

Cyprinus carpio decoction (CCD) is a well-known Chinese food medicine formula, accepted widely as a useful therapy in preventing edema and proteinuria caused by renal disease. However, the mechanism underlying this effect remains unclear. The current study investigated the potential mechanism of CCD in alleviating nephropathy induced by adriamycin (ADR) in rats. 70 eight-week-old Wistar rats were randomly divided into normal, model, fosinopril, YD, YG groups. All rats except for the normal group received 6.5 mg/kg·bw of ADR injection into the vena caudalis once. Different doses of CCD (11.3 and 22.5 g kg−1) were lavaged to rats in YD and YG groups, respectively. Then the serum biochemical values of the total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (Cr), electrolyte levels, and the urinary protein (UP) content in 12 hr urine were measured. Interleukin-4 (IL-4) and interferon (INF-γ) were measured by enzyme-like immunosorbent assay (ELISA). The pathomorphological analysis was observed using light and electron microscopy, and the expressions of nephrin and CD2-associated protein (CD2AP) in renal tissues were determined by immunohistochemical assay. The results indicated that CCD can relieve ADR-induced nephropathy (ADN) by improving the nutrition status, regulating the immunity, and inhibiting proteinuria by increasing nephrin and CD2AP expressions.

Pattern recognition receptors involved in the inflammatory attenuating effects of soybean isoflavone in β-amyloid peptides 1-42 treated rats

Pattern recognition receptors (PRRs) play important roles in the inflammatory responses to Alzheimer's disease (AD). Our previous study indicated that soybean isoflavone (SIF) exhibited anti-inflammatory effect in rats treated by β-amyloid peptides1-42 (Aβ1-42). In present study, we further detected the effects of SIF against inflammation caused by Aβ1-42 treatment in rats. Serum inflammatory mediators and neurotrophic factors including transforming growth factor-β (TGF-β), inducible nitric oxide synthase (iNOS), brain-derived neurotrophic factor (BDNF) and S100β were detected by enzyme-like immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR) and western blot methods were applied for detecting mRNA and protein expression of interleukin-1β (IL-1β), iNOS, tumor necrosis factor-α (TNF-α), TGF-β, BDNF, S100β, myeloid differentiation factor88 (Myd88), Toll-like receptor2 (TLR2), formyl peptide receptors (FPRs), inhibitor κB kinase (IKK) and inhibitor κB-α (IκB-α) in rat's brain tissue. Our results indicated that SIF could reduce the production of IL-1β, TNF-α and iNOS induced by Aβ1-42 in serum and brain of rats. SIF also significantly reversed Aβ1-42-induced up-regulation of TLR2, FPR, Myd88, IKK and decreased IκB-α mRNA and protein expressions in rats. These results suggested that TLR2 and FPR might involve in the inflammatory process induced by Aβ1-42 treatment, and SIF was an efficiency compound in reversing the inflammation caused by Aβ1-42 treatment.

M9.3 Association of maternal, paternal and fetal single nucleotide polymorphisms in vascular endothelial growth factor family genes with preeclampsia

To assess the ability of first trimester maternal serum (FTMS) soluble Fms-like tyrosine kinase (sFlt-1)/placental growth factor (PlGF) ratio in the first trimester threatened miscarriage (FTTM) to predict fetal loss in early pregnancy and adverse pregnancy outcomes when pregnancy continued to ⩾30 weeks to start suitable antenatal care as early as possible.A case control prospective cross-sectional study.320 cases of TM, 7–12 weeks’ gestation and 320 normal controls 7–12 weeks’ gestation without vaginal bleeding and other manifestations of FTTM and delivered at ⩾30 weeks’ gestation were enrolled in the study. All were primigravidae. Estimation of FTMS sFlt-1 and PlGF in the first trimester of TM and controls by enzyme-like immunosorbent assay (ELISA) and follow up of them till delivery were done. Adverse pregnancy outcomes encountered were gestational hypertension (GH), preeclampsia (PE), intrauterine growth restriction (IUGR), abruptio placentae, spontaneous preterm labor (PTL), placenta previa, preterm premature rupture of membranes (PPROM) and retained placenta.All adverse pregnancy outcomes were significantly (P < 0.05) higher in FTTM than in controls (44.52% vs. 17.5%). In FTTM MS concentrations of sFlt-1 and PlGF and sFlt-1/PlGF ratio were markedly reduced if fetal loss was encountered. sFlt-1/PlGF ratio <51.71 (cut-off point) predicted fetal loss with sensitivity 90.32%, specificity 98.47%, positive predicted value 93.33% and negative predictive value 97.60%. Patients that developed adverse pregnancy outcomes related to impaired placentation as GH, PE, IUGR, PTL <34 weeks’ gestation and abruptio placentae had reduced FTMS PlGF and increased sFlt-1/PlGF ratio, in both FTTM and controls. Soluble Flt-1/PlGF ratio >74.6 (cut-off point) predicted these adverse pregnancy outcomes with sensitivity 88.70%, specificity 95.40, positive predictive value 90.16% and negative predictive value 94.68% – adverse pregnancy outcomes not related to placental impairment as PLT >34 weeks’ gestation, PPROM, placenta previa and retained placenta were not predicted by sFlt-1/PlGF ratio because in these conditions FTMS concentration of these factors were normal.1-Adverse outcomes late in pregnancy (⩾30 weeks’ gestation) were more common in FTTM. 2-Diminished FTMS concentration of sFlt-1 and PlGF as well as sFlt-1/PlGF ratio <51.71 may predict fetal loss in FTTM. 3-In FTTM and normal controls FTMS PlGF was reduced and sFlt-1/PlGF ratio >74.6 may predict development of adverse outcomes characterized by impaired placentation. 4-Adverse pregnancy outcomes not related to impaired placentation were associated with normal FTMS concentration of sFlt-1 and PlGF and were not predicted by sFlt-1/PlGF ratio.

Circulating interleukin-15 and RANTES chemokine in MS patients: effect of treatment with methylprednisolone in patients with relapse

Introduction: Interleukin-15 (IL-15) is a proinflammatory cytokine. RANTES is a member of the beta chemokines subfamily with strong chemoattractant activity for T lymphocytes and monocytes.Materials and methods: We measured by enzyme-like immunosorbent assay (ELISA) serum levels of IL-15 and RANTES in 24 patients with MS in relapse, 27 patients with stable MS and 21 healthy subjects. Serum levels of IL-15 and RANTES were also measured before, 5 days and 1 month after onset of treatment with methylprednisolone i.v.Results: IL-15 serum levels were higher in patients with relapse compared with patients in stable stage of the disease and healthy subjects (p=0·001 and p=0·008 respectively). RANTES serum levels were increased in patients with relapse and stable disease as compared to healthy subjects (p=0·01). IL-15 and RANTES levels were not decreased after treatment with corticosteroids.Conclusions: Our findings suggest a possible role of IL-15 and RANTES in MS. Treatment with methylprednisolone in relapse had no effect on serum IL-15 and RANTES levels.

Interleukin-17 and interleukin-23 are elevated in serum and cerebrospinal fluid of patients with ALS: a reflection of Th17 cells activation?

There is evidence that immunological factors may involved in pathogenetic mechanisms of amyotrophic lateral sclerosis (ALS). Th17 cells are characterized by predominant production of IL-17 and are suggested to be crucial in destructive autoimmunity. Interleukin-23 (IL-23) appears to play a supporting role in the continued stimulation and survival of Th17. We measured by enzyme-like immunosorbent assay (ELISA) serum and cerebrospinal fluid (CSF) levels of IL-17 and IL-23 in 22 patients with ALS and 19 patients with other non-inflammatory neurological disorders (NIND) studied as a control group. IL-17 and IL-23 serum and CSF levels were also correlated with duration of the disease, the disability level and the clinical subtype of the disease onset in patients with ALS. IL-17 and IL-23 serum levels were higher in patients with ALS as compared with patients with NIND (P = 0.015 and P = 0.002 respectively). IL-17 and IL-23 CSF levels were also increased in patients with ALS (P = 0.0006 and P = 0.000001 respectively). IL-17 and IL-23 levels were not correlated with disease duration, disability scale or clinical subtype of the disease onset in ALS patients. Our findings suggest that these molecules may be involved in the pathogenetic mechanisms acting as potential markers of Th17 cells activation in ALS.

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva

Background It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. Methods In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). Results AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. General significance AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M 3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M 3 mAChR agonist's action on human salivary glands.

Effect of treatment with methylprednisolone on the serum levels of IL-12, IL-10 and CCL2 chemokine in patients with multiple sclerosis in relapse

Objectives Interleukin-12 (IL-12), a proinflammatory cytokine produced by Th1 cells, and interleukin-10 (IL-10), a product of Th2 cells, are involved in the pathogenetic mechanisms of multiple sclerosis (MS). CCL2 chemokine expression is induced by Th2 cytokines and is decreased in MS relapse. The mechanisms responsible for the beneficial effects of IVmethylprednisolone in attacks are not clearly established and the duration of the effect of this treatment remains controversial. Patients and methods We measured by enzyme-like immunosorbent assay (ELISA) serum levels of IL-12, IL-10 and CCL2 before, 5 days and 1 month after the initiation of treatment with IVMP in 20 patients with MS in relapse. Results A significant increase of IL-10 and decrease of CCL2 serum levels was observed ( p = 0.0028 and 0.045 respectively) five days after the onset of steroid treatment but not after one month. Steroid treatment had no influence in serum levels of IL-12. Conclusions The clinical improvement of our MS patients with relapse following the treatment with methylprednisolone may be associated with an immediate but not a long-term modification of serum levels of IL-10 and CCL2. IL-12 may not be influenced by steroid treatment.

Changes in maternal plasma macrophage-colony stimulating factor levels during normal pregnancy.

An enzyme-like immunosorbent assay and a blood autoanalyser were used to determine macrophage-colony stimulating factor (M-CSF) levels and the absolute number and percentage of circulating monocytes in 80 normal women with singleton pregnancies at 12-40 weeks' gestation, and ten healthy non-pregnant volunteers. The mean values of M-CSF and absolute number and percentage of circulating monocytes of the control group were 367 U/ml (SD 43) and 389 x 10(6)/l (SD 180) and 5.3% (SD 1.7) respectively. In pregnancy, M-CSF was significantly higher than non-pregnant controls only after 28 weeks' gestation. The absolute number and the percentage of circulating monocytes increased significantly with gestation after 16 weeks. There was no significant association between the concentration of M-CSF and the number or percentage of circulating monocytes. These data suggest that during pregnancy there is an up-regulation of M-CSF and monocytes.