A multilectin affinity approach for comparative glycoprotein profiling of rheumatoid arthritis and spondyloarthropathy

Background

Arthritis refers to inflammation of joints and includes common disorders such as rheumatoid arthritis (RA) and spondyloarthropathies (SpAs). These diseases differ mainly in terms of their clinical manifestations and the underlying pathogenesis. Glycoproteins in synovial fluid might reflect the disease activity status in the joints affected by arthritis; yet they have not been systematically studied previously. Although markers have been described for assisting in the diagnosis of RA, there are currently no known biomarkers for SpA.

Materials and methods

We sought to determine the relative abundance of glycoproteins in RA and SpA by lectin affinity chromatography coupled to iTRAQ labeling and LC-MS/MS analysis. We also used ELISA to validate the overexpression of VCAM-1, one of the candidate proteins identified in this study, in synovial fluid from RA patients.

Results and discussion

We identified proteins that were previously reported to be overexpressed in RA including metalloproteinase inhibitor 1 (TIMP1), myeloperoxidase (MPO) and several S100 proteins. In addition, we discovered several novel candidates that were overexpressed in SpA including Apolipoproteins C-II and C-III and the SUN domain-containing protein 3 (SUN3). Novel molecules found overexpressed in RA included extracellular matrix protein 1 (ECM1) and lumican (LUM). We validated one of the candidate biomarkers, vascular cell adhesion molecule 1 (VCAM1), in 20 RA and SpA samples using ELISA and confirmed its overexpression in RA (p-value <0.01). Our quantitative glycoproteomic approach to study arthritic disorders should open up new avenues for additional proteomics-based discovery studies in rheumatological disorders.     

Deaths from Symptomatically Identifiable Furious Rabies in India: A Nationally Representative Mortality Survey

Background

It is estimated that India has more deaths from rabies than any other country. However, existing estimates are indirect and rely on non-representative studies.

Methods and Principal Findings

We examined rabies deaths in the ongoing Million Death Study (MDS), a representative survey of over 122,000 deaths in India that uses enhanced types of verbal autopsy. We estimated the age-specific mortality rates of symptomatically identifiable furious rabies and its geographic and demographic distributions. A total of 140 deaths in our sample were caused by rabies, suggesting that in 2005 there were 12,700 (99% CI 10,000 to 15,500) symptomatically identifiable furious rabies deaths in India. Most rabies deaths were in males (62%), in rural areas (91%), and in children below the age of 15 years (50%). The overall rabies mortality rate was 1.1 deaths per 100,000 population (99%CI 0.9 to 1.4). One third of the national rabies deaths were found in Uttar Pradesh (4,300) and nearly three quarters (8,900) were in 7 central and south-eastern states: Chhattisgarh, Uttar Pradesh, Odisha, Andhra Pradesh, Bihar, Assam, and Madhya Pradesh.

Conclusions and Significance

Rabies remains an avoidable cause of death in India. As verbal autopsy is not likely to identify atypical or paralytic forms of rabies, our figure of 12,700 deaths due to classic and clinically identifiable furious rabies underestimates the total number of deaths due to this virus. The concentrated geographic distribution of rabies in India suggests that a significant reduction in the number of deaths or potentially even elimination of rabies deaths is possible.     

Quantitative Proteomic Analysis of Serum from Pregnant Women Carrying a Fetus with Conotruncal Heart Defect Using Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Labeling

Objective

To identify differentially expressed proteins from serum of pregnant women carrying a conotruncal heart defects (CTD) fetus, using proteomic analysis.

Methods

The study was conducted using a nested case-control design. The 5473 maternal serum samples were collected at 14–18 weeks of gestation. The serum from 9 pregnant women carrying a CTD fetus, 10 with another CHD (ACHD) fetus, and 11 with a normal fetus were selected from the above samples, and analyzed by using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS). The differentially expressed proteins identified by iTRAQ were further validated with Western blot.

Results

A total of 105 unique proteins present in the three groups were identified, and relative expression data were obtained for 92 of them with high confidence by employing the iTRAQ-based experiments. The downregulation of gelsolin in maternal serum of fetus with CTD was further verified by Western blot.

Conclusions

The identification of differentially expressed protein gelsolin in the serum of the pregnant women carrying a CTD fetus by using proteomic technology may be able to serve as a foundation to further explore the biomarker for detection of CTD fetus from the maternal serum.     

Proteomic Analysis of the Human Skin Proteome after In Vivo Treatment with Sodium Dodecyl Sulphate

Background

Skin has a variety of functions that are incompletely understood at the molecular level. As the most accessible tissue in the body it often reveals the first signs of inflammation or infection and also represents a potentially valuable source of biomarkers for several diseases. In this study we surveyed the skin proteome qualitatively using gel electrophoresis, liquid chromatography tandem mass spectrometry (GeLC-MS/MS) and quantitatively using an isobaric tagging strategy (iTRAQ) to characterise the response of human skin following exposure to sodium dodecyl sulphate (SDS).

Results

A total of 653 skin proteins were assigned, 159 of which were identified using GeLC-MS/MS and 616 using iTRAQ, representing the most comprehensive proteomic study in human skin tissue. Statistical analysis of the available iTRAQ data did not reveal any significant differences in the measured skin proteome after 4 hours exposure to the model irritant SDS.

Conclusions

This study represents the first step in defining the critical response to an irritant at the level of the proteome and provides a valuable resource for further studies at the later stages of irritant exposure.     

Comparative Proteomics Analysis of Placenta from Pregnant Women with Intrahepatic Cholestasis of Pregnancy

Introduction

Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach.

Methods

The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to separate differentially expressed placental proteins from 4 pregnant women with ICP and 4 healthy pregnant women. Bioinformatics analysis was used to find the relative processes that these differentially expressed proteins were involved in. Three apoptosis related proteins ERp29, PRDX6 and MPO that resulted from iTRAQ-based proteomics were further verified in placenta by Western blotting and immunohistochemistry. Placental apoptosis was also detected by TUNEL assay.

Results

Proteomics results showed there were 38 differentially expressed proteins from pregnant women with ICP and healthy pregnant women, 29 were upregulated and 9 were downregulated in placenta from pregnant women with ICP. Bioinformatics analysis showed most of the identified proteins was functionally related to specific cell processes, including apoptosis, oxidative stress, lipid metabolism. The expression levels of ERp29, PRDX6 and MPO were consistent with the proteomics data. The apoptosis index in placenta from ICP patients was significantly increased.

Conclusion

This preliminary work provides a better understanding of the proteomic alterations of placenta from pregnant women with ICP and may provide us some new insights into the pathophysiology and potential novel treatment targets for ICP.     

Evidence That SOX2 Overexpression Is Oncogenic in the Lung

Background

SOX2 (Sry-box 2) is required to maintain a variety of stem cells, is overexpressed in some solid tumors, and is expressed in epithelial cells of the lung.

Methodology/Principal Findings

We show that SOX2 is overexpressed in human squamous cell lung tumors and some adenocarcinomas. We have generated mouse models in which Sox2 is upregulated in epithelial cells of the lung during development and in the adult. In both cases, overexpression leads to extensive hyperplasia. In the terminal bronchioles, a trachea-like pseudostratified epithelium develops with p63-positive cells underlying columnar cells. Over 12–34 weeks, about half of the mice expressing the highest levels of Sox2 develop carcinoma. These tumors resemble adenocarcinoma but express the squamous marker, Trp63 (p63).

Conclusions

These findings demonstrate that Sox2 overexpression both induces a proximal phenotype in the distal airways/alveoli and leads to cancer.     

Lower Fetuin-A,Retinol Binding Protein 4 and Several Metabolites after Gastric Bypass Compared to Sleeve Gastrectomy in Patients with Type 2 Diabetes

Background

Bypass of foregut secreted factors promoting insulin resistance is hypothesized to be one of the mechanisms by which resolution of type 2 diabetes (T2D) follows roux-en-y gastric bypass (GBP) surgery.

Aim

To identify insulin resistance-associated proteins and metabolites which decrease more after GBP than after sleeve gastrectomy (SG) prior to diabetes remission.

Methods

Fasting plasma from 15 subjects with T2D undergoing GBP or SG was analyzed by proteomic and metabolomic methods 3 days before and 3 days after surgery. Subjects were matched for age, BMI, metformin therapy and glycemic control. Insulin resistance was calculated using homeostasis model assessment (HOMA-IR). For proteomics, samples were depleted of abundant plasma proteins, digested with trypsin and labeled with iTRAQ isobaric tags prior to liquid chromatography-tandem mass spectrometry analysis. Metabolomic analysis was performed using gas chromatography-mass spectrometry. The effect of the respective bariatric surgery on identified proteins and metabolites was evaluated using two-way analysis of variance and appropriate post-hoc tests.

Results

HOMA-IR improved, albeit not significantly, in both groups after surgery. Proteomic analysis yielded seven proteins which decreased significantly after GBP only, including Fetuin-A and Retinol binding protein 4, both previously linked to insulin resistance. Significant decrease in Fetuin-A and Retinol binding protein 4 after GBP was confirmed using ELISA and immunoassay. Metabolomic analysis identified significant decrease of citrate, proline, histidine and decanoic acid specifically after GBP.

Conclusion

Greater early decrease was seen for Fetuin-A, Retinol binding protein 4, and several metabolites after GBP compared to SG, preceding significant weight loss. This may contribute to enhanced T2D remission observed following foregut bypass procedures.     

Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background

The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues.

Methods

We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay.

Results

The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR.

Conclusions

RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.     

Dog bites in humans and estimating human rabies mortality in rabies endemic areas of Bhutan

Background

Dog bites in humans are a public health problem worldwide. The issues of increasing stray dog populations, rabies outbreaks, and the risk of dogs biting humans have been frequently reported by the media in Bhutan. This study aimed to estimate the bite incidence and identify the risk factors for dog bites in humans, and to estimate human deaths from rabies in rabies endemic south Bhutan.

Methods

A hospital-based questionnaire survey was conducted during 2009–2010 among dog bites victims who visited three hospitals in Bhutan for anti-rabies vaccine injection. Decision tree modeling was used to estimate human deaths from rabies following dog bite injuries in two rabies endemic areas of south Bhutan.

Results

Three hundred and twenty four dog bite victims were interviewed. The annual incidence of dog bites differed between the hospital catchment areas: 869.8 (95% CI: 722.8–1022.5), 293.8 (240–358.2) and 284.8 (251.2–323) per 100,000 people in Gelephu, Phuentsholing and Thimphu, respectively. Males (62%) were more at risk than females (P<0.001). Children aged 5–9 years were bitten more than other age groups. The majority of victims (71%) were bitten by stray dogs. No direct fatal injury was reported. In two hospital areas (Gelephu and Phuentsholing) in south Bhutan the annual incidence of death from rabies was 3.14 (95% CI: 1.57–6.29) per 100,000 population. The decision tree model predicted an equivalent annual incidence of 4.67 (95% CI: 2.53–7.53) deaths/100,000 population at risk. In the absence of post exposure prophylaxis, the model predicted 19.24 (95% CI: 13.69–25.14) deaths/year in these two areas.

Conclusions

Increased educational awareness of people about the risk of dog bites and rabies is necessary, particularly for children in rabies endemic areas of Bhutan.     

Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli

Background

Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties.

Methods and Findings

Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors.

Conclusions

Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.     

Refolding process of cysteine-rich proteins:Chitinase as a model

Background:

Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.

Methods:

Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.

Results:

After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.

Conclusions:

By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.Key Words: Chitinase, Cysteine-rich proteins, Protein refolding, Protein solubilization     

Aberrant Mucin5B expression in lung adenocarcinomas detected by iTRAQ labeling quantitative proteomics and immunohistochemistry

Background

Lung cancer is the number one cause of cancer-related deaths in the United States and worldwide. The complex protein changes and/or signature of protein expression in lung cancer, particularly in non-small cell lung cancer (NSCLC) has not been well defined. Although several studies have investigated the protein profile in lung cancers, the knowledge is far from complete. Among early studies, mucin5B (MUC5B) has been suggested to play an important role in the tumor progression. MUC5B is the major gel-forming mucin in the airway. In this study, we investigated the overall protein profile and MUC5B expression in lung adenocarcinomas, the most common type of NSCLCs.

Methods

Lung adenocarcinoma tissue in formalin-fixed paraffin-embedded (FFPE) blocks was collected and microdissected. Peptides from 8 tumors and 8 tumor-matched normal lung tissue were extracted and labeled with 8-channel iTRAQ reagents. The labeled peptides were identified and quantified by LC-MS/MS using an LTQ Orbitrap Velos mass spectrometer. MUC5B expression identified by iTRAQ labeling was further validated using immunohistochemistry (IHC) on tumor tissue microarray (TMA).

Results

A total of 1288 peptides from 210 proteins were identified and quantified in tumor tissues. Twenty-two proteins showed a greater than 1.5-fold differences between tumor and tumor-matched normal lung tissues. Fifteen proteins, including MUC5B, showed significant changes in tumor tissues. The aberrant expression of MUC5B was further identified in 71.1% of lung adenocarcinomas in the TMA.

Discussions

A subset of tumor-associated proteins was differentially expressed in lung adenocarcinomas. The differential expression of MUC5B in lung adenocarcinomas suggests its role as a potential biomarker in the detection of adenocarcinomas.     

Rabies post-exposure prophylaxis in the Philippines: health status of patients having received purified equine F(ab')(2) fragment rabies immunoglobulin (Favirab)

Background

Recommended treatment for severe rabies exposure in unvaccinated individuals includes wound cleaning, administration of rabies immunoglobulins (RIG), and rabies vaccination. We conducted a survey of rabies treatment outcomes in the Philippines.

Methods

This was a case series involving 7,660 patients (4 months to 98 years of age) given purified equine RIG (pERIG) at the Research Institute for Tropical Medicine (Muntinlupa, Philippines) from July 2003 to August 2004 following Category II or III exposures. Data on local and systemic adverse reactions (AR) within 28 days and biting animal status were recorded; outcome data were obtained by telephone or home visit 6–29 months post-exposure.

Results

Follow-up data were collected for 6,464 patients. Of 151 patients with laboratory-confirmed rabies exposure, 143 were in good health 6–48 months later, seven could not be contacted, and one 4-year-old girl died. Of 16 deaths in total, 14 were unrelated to rabies exposure or treatment. Two deaths were considered PEP failures: the 4-year old girl, who had multiple deep lacerated wounds from a rabid dog of the nape, neck, and shoulders requiring suturing on the day of exposure, and an 8-year-old boy who only received rabies PEP on the day of exposure.

Conclusions

This extensive review of outcomes in persons with Category III exposure shows the recommended treatment schedule at RITM using pERIG is well tolerated, while survival of 143 laboratory-confirmed rabies exposures confirms the intervention efficacy. Two PEP intervention failures demonstrate that sustained education and training is essential in rabies management.     

Geographical Analysis of the Distribution and Spread of Human Rabies in China from 2005 to 2011

Background

Rabies is a significant public health problem in China in that it records the second highest case incidence globally. Surveillance data on canine rabies in China is lacking and human rabies notifications can be a useful indicator of areas where animal and human rabies control could be integrated. Previous spatial epidemiological studies lacked adequate spatial resolution to inform targeted rabies control decisions. We aimed to describe the spatiotemporal distribution of human rabies and model its geographical spread to provide an evidence base to inform future integrated rabies control strategies in China.

Methods

We geo-referenced a total of 17,760 human rabies cases of China from 2005 to 2011. In our spatial analyses we used Gaussian kernel density analysis, average nearest neighbor distance, Spatial Temporal Density-Based Spatial Clustering of Applications with Noise and developed a model of rabies spatiotemporal spread.

Findings

Human rabies cases increased from 2005 to 2007 and decreased during 2008 to 2011 companying change of the spatial distribution. The ANN distance among human rabies cases increased between 2005 and 2011, and the degree of clustering of human rabies cases decreased during that period. A total 480 clusters were detected by ST-DBSCAN, 89.4% clusters initiated before 2007. Most of clusters were mainly found in South of China. The number and duration of cluster decreased significantly after 2008. Areas with the highest density of human rabies cases varied spatially each year and in some areas remained with high outbreak density for several years. Though few places have recovered from human rabies, most of affected places are still suffering from the disease.

Conclusion

Human rabies in mainland China is geographically clustered and its spatial extent changed during 2005 to 2011. The results provide a scientific basis for public health authorities in China to improve human rabies control and prevention program.     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Presence of Virus Neutralizing Antibodies in Cerebral Spinal Fluid Correlates with Non-Lethal Rabies in Dogs

Background

Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans.

Methodology/Principal Findings

Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS.

Conclusions/Significance

Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.