Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders

Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy body (DLB). We have employed a multiplex quantitative proteomics method, iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), in conjunction with multidimensional chromatography, followed by tandem mass spectrometry (MS/MS), to simultaneously measure relative changes in the proteome of cerebrospinal fluid (CSF) obtained from patients with AD, PD, and DLB compared to healthy controls. The diagnosis of AD and DLB was confirmed by autopsy, whereas the diagnosis of PD was based on clinical criteria. The proteomic findings showed quantitative changes in AD, PD, and DLB as compared to controls; among more than 1,500 identified CSF proteins, 136, 72, and 101 of the proteins displayed quantitative changes unique to AD, PD, and DLB, respectively. Eight unique proteins were confirmed by Western blot analysis, and the sensitivity at 95% specificity was calculated for each marker alone and in combination. Several panels of unique makers were capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity. Although these preliminary findings must be validated in a larger and different population of patients, they suggest that a roster of proteins may be generated and developed into specific biomarkers that could eventually assist in clinical diagnosis and monitoring disease progression of AD, PD and DLB.     

Interleukin-10 and interferon-gamma gene polymorphisms in patients with nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a multifactorial disease. Cytokines driving the immune response seem to be disturbed in NPC patients. Since interleukin-10 (IL-10) is known to reduce the production of interferon-gamma (IFN-gamma), we supposed that genetic differences in IL-10 and IFN-gamma expression could be a mechanism by which NPC cells escape antitumour immune response. As the production of each cytokine is affected by the genetic background, we investigated the possible association between single nucleotide polymorphisms in genes of IL-10 and IFN-gamma with NPC. Different IL-10 -1082 G/A and IFN-gamma+874 Tau/Alpha genotypes were determined in 160 patients with nasopharyngeal carcinoma and 197 healthy controls. No association was found either for each SNP studied alone or for the combined analysis for both IL-10 and IFN-gamma polymorphisms among NPC patients in comparison with controls. Compared with individuals from high incidence countries, we noted huge significant differences in genotype distribution between individuals from low and intermediate NPC incidence countries. Polymorphisms of the IL-10 and IFN-gamma do not appear to be associated with NPC risk in the Tunisian population. Nevertheless, we strongly believe that the relationship between cytokines polymorphisms and NPC susceptibility deeply depends on the ethnicity.     

Regional variability, processing methods, and biophysical properties of human fascia lata extracellular matrix

This study aims to assess the regional variability, processing methods, mechanical, biochemical, and cellular properties of human fascia lata as a scaffold for soft tissue repair and tissue engineering applications. Ten pairs of fascia lata (donor age 18-55) were used. One fascia patch from each pair was used to assess the geometric and biomechanical variability of fresh fascia. The other from each pair was subjected to 1 of 2 allograft processing methods: antibiotic soak alone or acellularization plus antibiotic soak. Stiffness, modulus, hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan (CSDS GAG), and DNA content were quantified in fascia from fresh and treated groups. The effect of location was not significant for thickness or stiffness within a 6 x 12 cm2 region of the iliotibial tract of fresh human fascia lata. Processing did not significantly change the stiffness, modulus, or CSDS GAG content of fascia ECM. However, hydroxyproline (collagen) content is significantly reduced in acellularized fascia, probably reflecting a removal of soluble collagen during the treatment (p < 0.02). Processing reduced the DNA content of fresh fascia approximately 10-fold (p < 0.001). The mechanical, chemical and ultrastructural similarities between fascia lata and tendon may make fresh or processed fascia an attractive ECM scaffold for soft tissue, particularly tendon, repair.     

cis-3, 4′, 5-Trimethoxy-3′-aminostilbene disrupts tumor vascular perfusion without damaging normal organ perfusion

Purpose  Targeting tumor vasculature by colchicine site microtubule inhibitors is a new approach in cancer therapy. Here we investigate cis-3, 4′, 5-trimethoxy-3′-aminostilbene (stilbene 5c) in its effect on tumor vascular perfusion, pharmacokinetics, toxicity and therapeutic efficacy in a mouse xenograft model. Methods  Tumor xenograft model was established with subcutaneous injection of UCI-101 ovarian cancer cells into nude mice. Tumor blood perfusion was investigated by dynamic contrast-enhanced (DCE) MRI studies. Pharmacokinetic studies were performed by LC/MS/MS to quantify the concentrations of stilbene 5c in plasma. Tumor size was measured by the long and short axes of tumor to calculate tumor volume. Mouse cardiac function study was determined by Doppler echocardiography using the Vevo770TM imaging system. Microvascular density was determined by CD34 staining of tissue sections. Results  Stilbene 5c selectively suppresses tumor perfusion without damaging normal organ perfusion in DCE-MRI studies. Histological sections of normal organs treated with stilbene 5c do not reveal any major toxicity in H&E staining. Microvascular density determined by CD34 staining is unchanged in normal organs, but significantly decreased in tumor after stilbene 5c treatment. Biodistribution study shows that stilbene 5c is not detectable in heart and lung, rapidly decreased in brain, liver, and kidney, but remains high in tumor for more than 3 h after IV injection of stilbene 5c, suggesting preferential accumulation in tumor. Mice treated with 5 days of stilbene 5c had negligible cardiac toxicity based on their normal left ventricular ejection fraction. In vivo efficacy study of stilbene 5c showed that it only suppresses tumor growth by 40% if used alone, but combination with bevacizumab is significantly better. Conclusion  Stilbene 5c is a useful vascular disrupting agent and combination with bevacizumab could be a promising therapy for cancer.     

Value and limitations of the duke criteria for the diagnosis of infective endocarditis

OBJECTIVES

The purpose of this study was to assess the value and limitations of Duke criteria for the diagnosis of infective endocarditis (IE).

BACKGROUND

Duke criteria have been shown to be more sensitive in diagnosing IE than the von Reyn criteria, but the diagnosis of IE remains difficult in some patients.

METHODS

Both classifications were applied in 93 consecutive patients with pathologically proven IE. Blood cultures, and transthoracic and transesophageal echocardiography were performed in all patients.

RESULTS

Sensitivities for the diagnosis of IE were 56% and 76% for von Reyn and Duke criteria, respectively. Fifty-two patients were correctly classified as “probable IE” by von Reyn and “definite IE” by Duke criteria (group 1). However, discrepancies were observed in 41 patients. Eleven patients (group 2) were misclassified as “rejected” by von Reyn, but were “definite IE” by Duke criteria; this difference could be explained by negative blood cultures and positive echocardiogram in all patients. In eight patients (group 3), the diagnosis of IE was “possible” by von Reyn but “definite” by Duke criteria. This difference was essentially explained by the failure of the von Reyn classification to consider echocardiographic abnormalities as major criteria. Twenty-two patients (group 4) were misclassified as possible IE using Duke criteria, being false negative of this classification. Echocardiographic major criteria were present in 19 patients, but blood cultures were negative in 21 patients. The cause of negative blood cultures was prior antibiotic therapy in 11 patients and Q-fever endocarditis diagnosed by positive serology in three cases.

CONCLUSIONS

Twenty-four percent of patients with proved IE remain misclassified as “possible IE” despite the use of Duke criteria, especially in cases of culture-negative and Q-fever IE. Increasing the diagnostic value of echographic criteria in patients with prior antibiotic therapy and typical echocardiographic findings and considering the serologic diagnosis of Q fever as a major criterion would further improve the clinical diagnosis of IE.     

Evaluation of serum levels of IgG subclasses and anti-ribosyl-ribitolphosphate IgG and IgG2 in children with Haemophilus influenzae b meningitis.

In 40 children with Haemophilus influenzae b (Hib) meningitis, we determined serum levels (mg/dl) of IgG subclasses using the radial immunodiffusion method; 67.8 per cent of these children were less than 24 months old. In 14 children of the sample we measured serum IgG and IgG2 anti-ribosyl-ribitolphosphate (anti-PRP) (by enzyme-linked immunosorbent assay, ELISA) in the acute and convalescent phases of the disease. Lower IgG2 levels than those of the control group were obtained in all age ranges: 3-12 months, 1-2 years (p < 0.01), and 2-5 years (p < 0.001). IgG4 was also present in lower levels in patients of all age ranges (p < 0.05, p < 0.001, and p < 0.01 respectively). Serum levels of IgG anti-PRP and IgG2 anti-PRP measured were very low in the acute phase of the disease in all age ranges and there was no notable increase in levels during the convalescent phase of the disease. This result indicates that children less than 24 months old do not produce sufficient levels of IgG and IgG2 anti-PRP even after Hib meningitis.     

Recurrent miscarriages as an indication for percutaneous tricuspid valvuloplasty during pregnancy

Isolated balloon tricuspid valvuloplasty was successfully performed in a pregnant woman with a history of recurrent miscarriages. The course of pregnancy was uneventful, and a healthy baby was delivered. This report highlights the unusual indication for the procedure, which was recurrent miscarriage, and outlines some technical aspects of tricuspid valvuloplasty. Cathet. Cardiovasc. Diagn. 40:283–286, 1997. © 1997 Wiley-Liss, Inc.     

Detection and genotyping of the hepatitis C RNA in tear fluid from patients with chronic hepatitis C

Tear fluid from 51 patients with chronic hepatitis C virus (HCV) infection was analyzed for the presence of the hepatitis C RNA to assess the potential role of this fluid in virus transmission. HCV sequences were amplified from sera and tear fluids by nested polymerase chain reaction using primers from the 5′ non coding region of the virus genome. Positive samples were genotyped by the LiPA^TM procedure. HCV RNA was detected in 76.5% (39/51) of the sera and in 9.8% (5/51) of the tear fluid samples. The presence of the RNA in the tear fluid was independent of the severity of the hepatitis and of the viral load as measured by the branched DNA^TM assay. The genotypes of the tears and serum isolates were different for two patients. For another patient, the HCV RNA was positive in the tear sample but negative in the serum sample. These findings suggest that tear fluid may transmit HCV but the source of HCV RNA in this fluid needs to be better understood. J. Med. Virol. 51:231–233, 1997. © Wiley-Liss, Inc.