Serum Hsp70 Antigen: Early Diagnosis Marker in Perinatal Asphyxia

Background: Perinatal asphyxia is an important cause of mortality and permanent neurological and developmental deficit. Early and accurate diagnosis would help to establish the likely prognosis and may also help in determining the most appropriate treatment. Studies in experimental animal models suggest that a protein called Hsp70 may be a good and potentially useful marker of cellular stress that may be clinically useful in determining the presence of neonatal asphyxia. Objectives: Regarding the importance of early and accurate diagnosis of asphyxia, we conducted this study, which is the first investigation of the comparison of the serum Hsp70 antigen level between asphyxiated and healthy infants. Patients and Methods: In this observational study, the serum concentrations of Hsp70 antigen were compared between neonates suffering from perinatal asphyxia (n = 50) and normal neonates (n = 51). The inclusion criteria for the cases were neonates who had reached term and had at least two clinical criteria of asphyxia. Exclusion criteria were babies with gestational age < 37 weeks, infants with congenital abnormalities or positive blood culture. Exclusion criteria in this group were the requirement to hospital stay during first week of the life or babies whose mothers had difficulties during pregnancy or delivery. Term neonates without major anomalies who had asphyxia during delivery were enrolled in the first six hours after delivery, and control group consisted of healthy term neonates without problems and normal delivery process in the first week of life. The cord blood was taken during labor to measure Hsp70 antigen level by using an in-house ELISA (The enzyme-linked immunosorbent assay). Results: The median values of serum anti Hsp70 titers were significantly higher in asphyxiated neonates compared with non-asphyxiated neonates (0.36 [0.04 - 1.14] vs 0.24 [0.01 - 0.63]). At cutoff point = 0.3125 ng/mL, sensitivity was 58% and specificity 76% based on ROC curve. Conclusions: A significant difference between the serum concentrations of Hsp70 of the control and patient group was observed in this study. It is inferred serum concentrations of Hsp70 antigen may be a useful marker for the early diagnosis of that prenatal hypoxia.

Reduced platelet amyloid precursor protein ratio (APP ratio) predicts conversion from mild cognitive impairment to Alzheimer's disease

Studies have shown that platelet APP ratio (representing the percentage of 120-130 kDa to 110 kDa isoforms of the amyloid precursor protein) is reduced in patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). In the present study, we sought to determine if baseline APP ratio predicts the conversion from MCI to AD dementia after 4 years of longitudinal assessment. Fifty-five older adults with varying degrees of cognitive impairment (34 with MCI and 21 with AD) were assessed at baseline and after 4 years. MCI patients were re-classified according to the conversion status upon follow-up: 25 individuals retained the diagnostic status of MCI and were considered as stable cases (MCI-MCI); conversely, in nine cases the diagnosis of dementia due to AD was ascertained. The APP ratio (APPr) was determined by the Western blot method in samples of platelets collected at baseline. We found a significant reduction of APPr in MCI patients who converted to dementia upon follow-up. These individuals had baseline APPr values similar to those of demented AD patients. The overall accuracy of APPr to identify subjects with MCI who will progress to AD was 0.74 +/- A 0.10, p = 0.05. The cut-off of 1.12 yielded a sensitivity of 75 % and a specificity of 75 %. Platelet APPr may be a surrogate marker of the disease process in AD, with potential implications for the assessment of abnormalities in the APP metabolism in patients with and at risk for dementia. However, diagnostic accuracy was relatively low. Therefore, studies in larger samples are needed to determine whether APPr may warrant its use as a biomarker to support the early diagnosis of AD.

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Evaluation of Calretinin as a New Marker in the Diagnosis of Hirschsprung Disease

Background: Hirschsprung’s disease (HD) is a congenital intestinal motility disorder with absence of ganglion cells in the colonic wall. Diagnosis of the disease is mainly based on the identification of the lack of ganglion cells in the pathology sections of the colon which is very difficult and time consuming and also needs several serial cut sections. There are many proposed markers in this field in the literature but none of them has been satisfactory. Calretinin immunohistochemistry (IHC) has been introduced as a new diagnostic marker to overcome the problems in diagnosis of this disease about 5 years ago. However there are few studies regarding the benefits and pitfalls of this marker. Objectives: The aim of this study is to determine the diagnostic value of calretinin IHC in detecting aganglionosis (HD). Patients and Methods: 27 HD patients and 28 non-Hirschsprung’s disease (NHD) patients were collected in a prospective study and calretinin IHC was performed on 31 aganglionic and 51 normoganglionic full wall thickness sections of colectomies (some of the cases had more than 1 section). The IHC slides were evaluated by two pathologists and the diagnostic value was calculated in comparison with gold standard which is the presence or absence of ganglion cells in serial Hematoxylin and Eosin (HE) stained sections of the colectomies. Results: There was great concordance between the final diagnosis of both pathologists and gold standard (k > 0.9). Calretinin immunostaining showed 100% specificity and positive predictive value and more than 90% sensitivity and negative predictive value. High agreement was present between the two pathologists (k > 0.9). Conclusions: Calretinin IHC is a very convenient, useful and valuable method to demonstrate aganglionosis in HD patients. Loss of calretinin immunostaining in lamina propria and submucosa is characteristic of HD.

Corneal sensitivity as an ophthalmic marker of diabetic neuropathy

Purpose. The objective of this study was to explore the discriminative capacity of non-contact corneal esthesiometry (NCCE) when compared with the neuropathy disability score (NDS) score—a validated, standard method of diagnosing clinically significant diabetic neuropathy. Methods. Eighty-one participants with type 2 diabetes, no history of ocular disease, trauma, or surgery and no history of systemic disease that may affect the cornea were enrolled. Participants were ineligible if there was history of neuropathy due to non-diabetic cause or current diabetic foot ulcer or infection. Corneal sensitivity threshold was measured on the eye of dominant hand side at a distance of 10 mm from the center of the cornea using a stimulus duration of 0.9 s. The NDS was measured producing a score ranging from 0 to 10. To determine the optimal cutoff point of corneal sensitivity that identified the presence of neuropathy (diagnosed by NDS), the Youden index and “closest-to-(0,1)” criteria were used. Results. The receiver-operator characteristic curve for NCCE for the presence of neuropathy (NDS ≥3) had an area under the curve of 0.73 (p = 0.001) and, for the presence of moderate neuropathy (NDS ≥6), area of 0.71 (p = 0.003). By using the Youden index, for an NDS ≥3, the sensitivity of NCCE was 70% and specificity was 75%, and a corneal sensitivity threshold of 0.66 mbar or higher indicated the presence of neuropathy. When NDS ≥6 (indicating risk of foot ulceration) was applied, the sensitivity was 52% with a specificity of 85%. Conclusions. NCCE is a sensitive test for the diagnosis of minimal and more advanced diabetic neuropathy and may serve as a useful surrogate marker for diabetic and perhaps other neuropathies.

Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR

Introduction The ability to screen blood of early stage operable breast cancer patients for circulating tumour cells is of potential importance for identifying patients at risk of developing distant relapse. We present the results of a study of the efficacy of the immunobead RT-PCR method in identifying patients with circulating tumour cells. Results Immunomagnetic enrichment of circulating tumour cells followed by RT-PCR (immunobead RT-PCR) with a panel of five epithelial specific markers (ELF3, EPHB4, EGFR, MGB1 and TACSTD1) was used to screen for circulating tumour cells in the peripheral blood of 56 breast cancer patients. Twenty patients were positive for two or more RT-PCR markers, including seven patients who were node negative by conventional techniques. Significant increases in the frequency of marker positivity was seen in lymph node positive patients, in patients with high grade tumours and in patients with lymphovascular invasion. A strong trend towards improved disease free survival was seen for marker negative patients although it did not reach significance (p = 0.08). Conclusion Multi-marker immunobead RT-PCR analysis of peripheral blood is a robust assay that is capable of detecting circulating tumour cells in early stage breast cancer patients.

The effect of the ecstasy 'come-down' on the diagnosis of ecstasy dependence

Background: The existence of an ecstasy dependence syndrome is controversial. We examined whether the acute after-effects of ecstasy use (i.e., the “come-down”) falsely lead to the identification of ecstasy withdrawal and the subsequent diagnosis of ecstasy dependence. Methods: The Structured Clinical Interview for DSM-IV-TR Disorders: Research Version (SCID-RV) was administered to 214 Australian ecstasy users. Ecstasy withdrawal was operationalized in three contrasting ways: (i) as per DSM-IV criteria; (ii) as the expected after effects of ecstasy (a regular come-down); or (iii) as a substantially greater or longer come-down than on first use (intense come-down). These definitions were validated against frequency of ecstasy use, readiness to change and ability to resist the urge to use ecstasy. Confirmatory factor analyses were used to see how they aligned with the overall dependence syndrome. Results: Come-down symptoms increased the prevalence of withdrawal from 1% (DSM-IV criterion) to 11% (intense come-downs) and 75% (regular come-downs). Past year ecstasy dependence remained at 31% when including the DSM-IV withdrawal criteria and was 32% with intense come-downs, but increased to 45% with regular come-downs. Intense come-downs were associated with lower ability to resist ecstasy use and loaded positively on the dependence syndrome. Regular come-downs did not load positively on the ecstasy dependence syndrome and were not related to other indices of dependence. Conclusion: The acute after-effects of ecstasy should be excluded when assessing ecstasy withdrawal as they can lead to a false diagnosis of ecstasy dependence. Worsening of the ecstasy come-down may be a marker for dependence.

Current trends in the etiology and diagnosis of HPV-related head and neck cancers

Human papilloma virus (HPV) infection is a major risk factor for a distinct subset of head and neck squamous cell carcinoma (HNSCC). The current review summarizes the epidemiology of HNSCC and the disease burden, the infectious cycle of HPV, the roles of viral oncoproteins, E6 and E7, and the downstream cellular events that lead to malignant transformation. Current techniques for the clinical diagnosis of HPV-associated HNSCC will also be discussed, that is, the detection of HPV DNA, RNA, and the HPV surrogate marker, p16 in tumor tissues, as well as HPV-specific antibodies in serum. Such methods do not allow for the early detection of HPV-associated HNSCC and most cases are at an advanced stage upon diagnosis. Novel noninvasive approaches using oral fluid, a clinically relevant biological fluid, allow for the detection of HPV and cellular alterations in infected cells, which may aid in the early detection and HPV-typing of HNSCC tumors. Noninvasive diagnostic methods will enable early detection and intervention, leading to a significant reduction in mortality and morbidity associated with HNSCC.

Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

Progression of diffuse gliomas - from the first diagnosis to recurrence

Gliomas are the most frequent primary brain tumours. The cardinal features of gliomas are infiltrative growth pattern and progression from low-grade tumours to a more malignant phenotype. These features of gliomas generally prevent their complete surgical excision and cause their inherent tendency to recur after initial treatment and lead to poor long-term prognosis. Increasing knowledge about the molecular biology of gliomas has produced new markers that supplement histopathological diagnostics. Molecular markers are also used to evaluate the prognosis and predict therapeutic response. The purpose of this thesis is to study molecular events involved in the malignant progression of gliomas. Gliomas are highly vascularised tumours. Contrast enhancement in magnetic resonance imaging (MRI) reflects a disrupted blood-brain barrier and is often seen in malignant gliomas. In this thesis, 62 astrocytomas, oligodendrogliomas and oligoastrocytomas were studied by MRI and immunohistochemistry. Contrast enhancement in preoperative MRI was associated with angiogenesis, tumour cell proliferation and histological grade of gliomas. Activation of oncogenes by gene amplification is a common genetic aberration in gliomas. EGFR amplification on chromosome 7p12 occurs in 30-40% of glioblastomas. PDGFRA, KIT and VEGFR2 are receptor tyrosine kinase genes located on chromosome 4q12. Amplification of these genes was studied using in situ hybridisation in the primary and recurrent astrocytomas, oligodendrogliomas and oligoastrocytomas of 87 patients. PDGFRA, KIT or VEGFR2 amplification was found in 22% of primary tumours and 36% of recurrent tumours including low-grade and malignant gliomas. The most frequent aberration was KIT amplification, which occurred in 10% of primary tumours and in 27% of recurrent tumours. The expression of ezrin, cyclooxygenase 2 (COX-2) and HuR was studied immunohistochemically in a series of primary and recurrent gliomas of 113 patients. Ezrin is a cell membrane-cytoskeleton linking-protein involved in the migration of glioma cells. The COX-2 enzyme is implicated in the carcinogenesis of epithelial neoplasms and is overexpressed in gliomas. HuR is an RNA-stabilising protein, which regulates the expression of several proteins including COX-2. Ezrin, COX-2 and HuR were associated with histological grade and the overall survival of glioma patients. However, in multivariate analysis they were not independent prognostic factors. In conclusion, these results suggest that contrast enhancement in MRI can be used as a surrogate marker for the proliferative and angiogenic potential of gliomas. Aberrations of PDGFRA, KIT and VEGFR2 genes, as well as the dysregulated expression of ezrin, COX-2 and HuR proteins, are linked to the progression of gliomas.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

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Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data. (C) 2002 Elsevier Science B.V. All rights reserved.