Uncovering the potential of CD44v/SYNE1/miR34a axis in salivary fluids of oral cancer patients.

OBJECTIVES: Late-stage diagnosis is one of the major confounders for poor prognosis of patients with oral cancer owing to lack of a biomarker to diagnose this disease at an early stage. Moreover, till date, invasive biopsies are the only option to assess disease occurrence and progression in this malignancy. Thus, this study aims to identify and assess potential salivary markers in OSCC patients in order to open newer avenues in the field of non-invasive biopsies. METHODOLOGY: Bioinformatic-based analysis was performed to identify potential biomarkers that could be assessed in OSCC patients. The expression patterns of CD44v and its genetic and epigenetic modulators were assessed in saliva of OSCC patients, leukoplakia, and controls using real-time and methylation-specific PCR. Statistical analysis was conducted to understand the significance of these markers in terms of their clinical relevance. RESULTS: CD44v/SYNE1/miR34a axis was identified using bioinformatic analysis, and the expression profile of these markers was assessed in saliva of OSCC patients. CD44v6 and CD44v10 demonstrated a significantly increased expression, whereas SYNE1 and miR34a depicted a significantly decreased expression in OSCC patients. Statistical analysis suggested a probable role of CD44v6, SYNE1, and miR34a in early stages of the malignancy, whereas a strong association was observed between CD44v6, CD44v10, and miR34a expression with locoregional aggressiveness and histopathological conditions. CONCLUSION: Collectively, these findings suggested a plausible role of CD44v/SYNE1/miR34a axis as non-invasive salivary biomarkers to diagnose this disease at an early stage and predict the early onset of metastasis.

Diagnostic accuracy of BP180 NC16a and BP230-C3 ELISA in serum and saliva of patients with bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is a subepidermal blistering disease, characterized by autoantibodies directed against BP180 and BP230. Collecting saliva is an easy and painless way of obtaining biological samples, and can be used for diagnosis of autoimmune diseases. AIM: To compare the diagnostic accuracy of serum and salivary BP180-NC16a and BP230-C3 in the initial diagnosis of BP. METHODS: We assessed 50 patients newly diagnosed with BP and 50 healthy controls. The diagnosis of BP was confirmed based on clinical, histopathological and immunofluorescence findings. Serum and saliva samples were collected from both groups, and BP180 and BP230 titres were assessed using commercially available ELISA kits. RESULTS: Using serum, the sensitivity of the serum BP180 and BP230 ELISA assays was 88% and 48%, respectively, and the specificity of both was 96%. Using saliva with the cutoff value proposed by the manufacturer, sensitivity was 56.2% and 14.6%, and specificity was 98% and 100%, respectively. Using the best calculated cutoff for saliva, sensitivity increased to 87.5% and 77.1%, and specificity to 96% and 62%, respectively. There was a significant correlation between serum and saliva BP180 levels and the severity of skin disease. Both serum and saliva BP230 levels were significantly higher in patients with mucosal involvement. CONCLUSION: Serum BP180 NC16a ELISA is a sensitive and specific test for the initial diagnosis of BP, whereas serum BP230-C3 ELISA is highly specific, but less sensitive. Saliva may be a noninvasive and convenient alternative for use in the BP180 NC16a ELISA to diagnose BP.

Involuntary movement in stiff-person syndrome with amphiphysin antibodies: A case report.

RATIONALE: Stiff-person syndrome (SPS) is a rare neurological immune disorder characterized by progressive axial and proximal limb muscle rigidity, stiffness, and painful muscle spasms. Amphiphysin antibodies are positive in approximately 5% of SPS patients. To date, there have been no relevant reports on involuntary movement in cases of SPS with amphiphysin antibodies. PATIENT CONCERNS: We describe the case of a 69-year-old man with a 2-year history of progressive stiffness in the neck, bilateral shoulders, and chest muscles, and a more-than-a-year history of dyspnea accompanied by mandibular involuntary movement. The patient was a vegetarian and had good health in the past. The family's medical history was unremarkable. DIAGNOSES: He was diagnosed with SPS based on the progressive muscle stiffness, the amphiphysin antibody seropositivity, the continuous motor activity on electromyography, and the effective treatment with benzodiazepines. INTERVENTIONS: The patient was orally administered clonazepam and baclofen, and corticosteroid IV followed by prednisone orally. OUTCOMES: In the hospital, after treatment with methylprednisolone, clonazepam, and baclofen, the patient's rigidity, stiffness, and dyspnea significantly improved. The involuntary movement of the mandible persisted throughout the treatment process. Currently, under oral treatment with baclofen and clonazepam, the patient's symptoms of muscle stiffness and dyspnea exist, and follow-up is continued. LESSONS: We report a rare and novel case of involuntary movement in SPS with amphiphysin antibodies. The present report explores the relationship between SPS and involuntary movement and expands the spectrum of clinical manifestations of SPS.

Nesfatin-1 and ghrelin levels in serum and saliva of epileptic patients: hormonal changes can have a major effect on seizure disorders.

Nesfatin-1 and ghrelin are the two recently discovered peptide hormones involved in the control of appetite. Besides its main appetite-control function, ghrelin also has anticonvulsant effects, while nesfatin-1 causes depolarization in the paraventricular nucleus (PVN). The aims of this study, therefore, were to investigate: (i) whether there are differences in the concentrations of nesfatin-1 and ghrelin in saliva and serum samples between eplilepsy patients and normal controls and (ii) whether salivary glands produce nesfatin-1. The study included a total of 73 subjects: 8 patients who were newly diagnosed with primary generalized seizures and had recently started antiepileptic drug therapy; 21 who had primary generalized seizures and were continuing with established antiepileptic drug therapy; 24 who had partial seizures (simple: n = 12 or complex: n = 12) and were continuing with established antiepileptic drug therapy; and 20 controls. Salivary gland tissue samples were analyzed for nesfatin-1 expression by immunochemistry and ELISA. Saliva and serum ghrelin levels were measured by ELISA and RIA, and nesfatin-1 levels by ELISA. Nesfatin-1 immunoreactivity was detected in the striated and interlobular parts of the salivary glands and the ducts. The nesfatin-1 level in the brain was around 12 times higher than in the salivary gland. Before antiepileptic treatment, both saliva and serum nesfatin-1 levels were around 160-fold higher in patients who are newly diagnosed with primary generalized epilepsy (PGE) than in controls; these levels decreased with treatment but remained about 10 times higher than the control values. Saliva and serum nesfatin-1 levels from patients with PGE and partial epilepsies who were continuing antiepileptic drugs were also 10-fold higher than control values. Serum and saliva ghrelin levels were significantly (twofold) lower in epileptic patients before treatment than in controls; they recovered somewhat with treatment but remained below the control values. These results suggest that the low ghrelin and especially the dramatically elevated nesfatin-1 levels might contribute to the pathophyisology of epilepsy. Therefore, serum and saliva ghrelin and especially the remarkably increased nesfatin-1 might be candidate biomarkers for the diagnosis of epilepsy and for monitoring the response to anti-epileptic treatment.

Establishment of an enzyme immunoassay system for gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF).

A two-site enzyme immunoassay for gliostatin (GLS)/platelet-derived endothelial cell growth factor (PD-ECGF) has been developed. The detection limit of gliostatin/PD-ECGF was 30 pg/well, and the optimal assay range was 0.1 to ng/well. This assay system enabled us to confirm the immunochemical identity of both factors and to detect immunoreactive gliostatin/PD-ECGF (IR-GLS/PD-ECGF) in human biological body fluids. The age-related analysis from newborn to 69 years revealed that the serum IR-GLS/PD-ECGF level was high in infants younger than 1 year old (1.8 ng/ml) and in the 20-year-old age group (1.8 ng/ml), and highest in the umbilical cord blood (2.1 ng/ml). Curiously high concentrations were detected in saliva with a significant sex difference (11.3 ng/ml for males and 48.7 ng/ml for females), and in synovial fluids (3.7 ng/ml). A number of human tumor cells, gastric cancer cells, MKN-74, neuroblastoma cells, GOTO, as well as epidermoid carcinoma cells, A431, were found to produce a significant amount of IR-GLS/PD-ECGF (0.2 to 21.8 ng/mg protein), and some of them secreted the IR-GLS/PD-ECGF in the conditioned medium (approximately 0.5 ng/ml). The enzyme immunoassay system is sufficiently sensitive for the basic and clinical study of gliostatin/PD-ECGF in human body fluids, tissues and organs.

Impaired thrombin generation in Reelin-deficient mice: a potential role of plasma Reelin in hemostasis.

BACKGROUND: Reelin is a large extracellular glycoprotein that is present in the peripheral blood. That Reelin interacts with the coagulation components and elicits a functional role in hemostasis has not yet been elucidated. OBJECTIVES: The hemostatic activity of Reelin is investigated and defined in this study. METHODS: The interplay of Reelin with coagulation components was elucidated by far-Western and liposome/platelet binding assays. In vivo and ex vivo hemostasis-related analyses of Reelin-deficient mice and plasma were also performed. RESULTS: Reelin interacted with the liposomes containing phosphatidylserine (PS) or phosphatidylcholine. Instead of interacting with known Reelin receptors (ApoE receptor 2, very low density lipoprotein receptor and integrin ß1), Reelin interacted with PS of the activated platelets. The interaction between Reelin and the coagulation factors of thrombin and FXa was also demonstrated with the Kd of 11.7 and 21.2 nm, respectively. Reelin-deficient mice displayed a prolonged bleeding time and an increase in rebleeding rate. Despite the fact that Reelin deficiency had no significant effect on the clotting time of prothrombin and activated partial thromboplastin time, the fibrin clot formation was abnormal and the fibrin clot structure was relatively loosened with reduced clot strength. Abnormal fibrinogen expression did not account for the hemostatic defects associated with Reelin deficiency. Instead, thrombin generation was impaired concomitant with an altered prothrombin cleavage pattern. CONCLUSIONS: By interacting with platelet phospholipids and the coagulation factors, thrombin and FXa, Reelin plays a selective role in coagulation activation, leading to thrombin generation and formation of a normal fibrin clot.

Purification and identification of brain-derived neurotrophic factor from human serum.

Brain-derived neurotrophic factor (BDNF), a 27-kDa noncovalently linked homodimer with subunits of approximately 13.5 kDa as viewed by SDS-PAGE, is thought to be primarily produced in the central nervous system. We report here the isolation of BDNF from pooled normal human sera, using a two-step purification process followed by SDS-PAGE, transfer to a polyvinylidene difluoride membrane, and subsequent identification of the protein by sequence analysis of the appropriate band(s) from the membrane. The level of BDNF in pooled human sera was estimated to be approximately 15 ng/ml as determined by an enzyme-linked immunosorbant assay. The average for six individuals was 18.9 +/- 5.7 ng/ml. There is an approximately 200-fold increase in the levels of BDNF in serum relative to plasma. Results from experiments using differential centrifugation suggest that the source of this increase is due to release from platelets. The presence of high levels of BDNF in serum suggests a role for this neurotrophin either in nerve repair at sites of injured tissue or in nonneuronal functions.

Profile of autoantibody to basement membrane zone proteins in patients with mucous membrane pemphigoid: long-term follow up and influence of therapy.

Mucous membrane pemphigoid (MMP) is an autoimmune mucocutaneous blistering disease characterized by autoantibodies to components within the basement membrane zone. In this study, we report the titers of autoantibodies to antigens in the BMZ, in the sera of 13 patients, treated with intravenous immunoglobulin as monotherapy over a consecutive 18-month period. Using bovine gingiva lysate as substrate in an immunoblot assay, autoantibodies to human bullous pemphigoid antigens (BPAg1 and BPAg2), human beta4 integrin, and laminin 5 were measured. A statistically significant (P < 0.05) decline in the autoantibody titers to beta4-integrin was observed after 3.42 months of initiating the IVIg therapy. These titers were undetectable after 13 months of therapy. The titers of antibodies to BPAg1 and BPAg2 did not correlate with disease activity or response to therapy. Antibodies to laminins were not detected. In patients with MMP, autoantibody titers to beta4-integrin correlate with disease activity and response to therapy.

Responses of plasma concentrations of A type natriuretic peptide and B type natriuretic peptide to alacepril, an angiotensin-converting enzyme inhibitor, in patients with congestive heart failure.

BACKGROUND: Plasma concentrations of A type or atrial natriuretic peptide (ANP) and B type or brain natriuretic peptide (BNP) are increased in patients with congestive heart failure (CHF). OBJECTIVE: To examine the haemodynamic and hormonal responses, especially of ANP and BNP, to oral administration of an angiotensin-converting enzyme (ACE) inhibitor in patients with CHF and in controls. PATIENTS: 12 patients with CHF and 11 controls. METHODS: Haemodynamic variables and plasma concentrations of ANP, BNP, and other hormones were serially measured for 24 hours after alacepril (37.5 mg) was given by mouth. RESULTS: Pulmonary capillary wedge pressure and systemic vascular resistance decreased significantly in both groups. The cardiac index increased only in the CHF group. In patients with CHF pulmonary capillary wedge pressure, systemic vascular resistance, and cardiac index were significantly changed from 1 to 12 hours after alacepril administration. Plasma ANP and BNP decreased significantly after alacepril was given to the CHF group: neither concentration changed in the control group. In the CHF group plasma ANP was significantly lower between 1 and 6 hours and was highly significantly correlated with pulmonary capillary wedge pressure. Plasma BNP, however, was significantly lower between 6 and 24 hours after alacepril and was not correlated with pulmonary capillary wedge pressure. CONCLUSIONS: The response of plasma BNP after alacepril administration occurred later and lasted longer than the plasma ANP response. This may indicate that the mechanisms of synthesis, secretion, or degradation of the two peptides are different.