Raman spectroscopic analysis of high molecular weight proteins in solution - considerations for sample analysis and data pre-processing.

This study explores the potential of Raman spectroscopy, coupled with multivariate regression techniques and a protein separation technique (ion exchange chromatography), to quantitatively monitor diagnostically relevant changes in high molecular weight proteins in liquid plasma. Measurement protocols to detect the imbalances in plasma proteins as an indicator of various diseases using Raman spectroscopy are optimised, such that strategic clinical applications for early stage disease diagnostics can be evaluated. In a simulated plasma protein mixture, concentrations of two proteins of identified diagnostic potential (albumin and fibrinogen) were systematically varied within physiologically relevant ranges. Scattering from the poorly soluble fibrinogen fraction is identified as a significant impediment to the accuracy of measurement of mixed proteins in solution, although careful consideration of pre-processing methods allows construction of an accurate multivariate regression prediction model for detecting subtle changes in the protein concentration. Furthermore, ion exchange chromatography is utilised to separate fibrinogen from the rest of the proteins and mild sonication is used to improve the dispersion and therefore quality of the prediction. The proposed approach can be expeditiously employed for early detection of pathological disorders associated with high or low plasma/serum proteins.

The role of claudin-1 and claudin-7 in cervical tumorigenesis.

BACKGROUND/AIM: The claudin family of proteins are key constituents of tight junctions and the aberrant expression of these proteins can contribute to de-stabilisation of tight junctions and thus to loss of cell polarity and cohesion. Increased expression of claudin-1 and claudin-7 has been observed in pre-invasive cervical lesions and cervical carcinomas. The present study attempted to assess the effect of claudin-1 and claudin-7 overexpression on the HeLa cervical carcinoma cell line, in terms of cell proliferation/viability, permeability, invasion and migration. MATERIALS AND METHODS: HeLa cells were stably transfected with expression vectors containing the claudin-1 and claudin-7 genes to produce two separate stable cell lines expressing claudin-1 and claudin-7, respectively. The stable cell lines were examined with regard to their invasion and migration abilities, cell permeability and cell proliferation/viability and compared to non-claudin-1 or -7 transfected HeLa. RESULTS: The present study found that claudin-1 and claudin-7 affected the migratory ability of HeLa cells, reducing their ability to migrate in a gap closure assay compared to non-claudin-transfected HeLa cells. Monolayers of claudin-1 and claudin-7 transfected cells also displayed an increased transepithelial electrical resistance indicating decreased permeability compared to non-claudin-transfected HeLa. The study found that claudin-1 or claudin-7 expression had no effect on the proliferation or viability of HeLa cells. Claudin-1 or -7 expression also did not affect the invasive ability of HeLa cells with both stable cells lines and non-claudin-transfected HeLa cells all showing low invasive ability. CONCLUSION: The results of the present study indicate that claudin-1 and claudin-7 overexpression alone does not contribute to increased tumorigenesis in cervical carcinoma, instead claudin-1 and - 7 expression in HeLa cells contribute to reducing the migratory ability of cells and decrease their permeability.

Wild-type measles virus infection upregulates poliovirus receptor-related 4 and causes apoptosis in brain endothelial cells by induction of tumor necrosis factor-related apoptosis-inducing ligand.

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

RNAi-mediated reversible opening of the blood-brain barrier.

BACKGROUND: The blood-brain barrier (BBB) contains tight junctions (TJs) which reduce the space between adjacent endothelial cells lining the fine capillaries of the microvasculature of the brain to form a selective and regulatable barrier. METHODS: Using a hydrodynamic approach, we delivered siRNA targeting the TJ protein claudin-5 to the endothelial cells of the BBB in mice. RESULTS: We have shown a significant decrease in claudin-5 mRNA levels 24 and 48 hours post-delivery of siRNA, with levels of protein expression decreasing up to 48 hours post-injection compared to uninjected, phosphate-buffered saline (PBS)-injected and non-targeting siRNA-injected mice. We observed increased permeability at the BBB to molecules up to 742 Da, but not 4400 Da, using tracer molecule perfusion and MRI analysis. To illustrate the functional efficacy of size-selective and transient barrier opening, we have shown that enhanced delivery of the small neuropeptide thyrotropin-releasing hormone (TRH) (MW 360 Da) to the brains of mice 48 hours post-injection of siRNA targeting claudin-5 significantly modifies behavioural output. CONCLUSIONS: These data demonstrate that it is now possible to transiently and size-selectively open the BBB in mice, allowing in principle the delivery of a wide range of agents for the establishment and treatment of experimental mouse models of neurodegenerative, neuropsychiatric and malignant diseases.

Feline immunodeficiency virus infection: a valuable model to study HIV-1 associated encephalitis.

Feline immunodeficiency virus (FIV), like human immunodeficiency virus (HIV)-1, is a neurotropic lentivirus and is associated with neuropathology in natural and experimental infections. FIV enters the brain early following experimental infection, and virus has been proposed to enter the brain via the blood-brain barrier and blood-CSF barrier, within infected lymphocytes and monocytes/macrophages. However the entry of cell-free virus or the direct infection of brain endothelial cells and astrocytes of the blood-brain barrier may also contribute to CNS infection. This review explores the role played by the FIV model in the elucidation of mechanism of lentiviral entry to the brain and viral interactions with the CNS, particularly in relation to lymphotropic lentiviruses.

Altered expression and interaction of adherens junction proteins in the developing OLM of the Rho(-/-) mouse.

Retinitis Pigmentosa (RP) represents a major cause of progressive retinal disease worldwide and comprises a heterogeneous group of inherited diseases that are characterised by primary degeneration of rod photoreceptors and secondary degeneration of cone photoreceptors in the retina. The outer limiting membrane (OLM) which allows for the interaction of photoreceptors with surrounding photoreceptors and Müller cells is compromised in many degenerative retinal diseases. Using indirect immunostaining of retinal cryosections from C-129 Wild Type (WT) and C-129 Rho(-/-) mice, we have determined levels of expression of the adherens junction associated proteins ZO-1, beta-catenin and p120-catenin at the OLM from newborn and 1, 2, 3, 4 and 5-week old animals. We have also used immunoprecipitation analysis to determine changes in the association of E-cadherin with ZO-1, beta-catenin and p120-catenin and the association of alpha-catenin with ZO-1 and beta-catenin at these time points in WT and Rho(-/-) mice. We have found that ZO-1 expression at the OLM is present in WT and Rho(-/-) mice after 2weeks, but that levels of expression at the OLM decrease after this time point in the Rho(-/-) mice. beta-catenin expression in the Rho(-/-) mice became compromised at the OLM after 3 weeks, showing a distinct change in staining pattern after 4 weeks and no staining at the OLM after 5 weeks. Moreover, we have shown that p120-catenin expression is not evident at the OLM of the Rho(-/-) mice at the 4 or 5 week time point. To complement this data, we have performed immunoprecipitation analysis on neural retinal lysates from WT and Rho(-/-) mice and herein report fluctuations in the association of E-cadherin with ZO-1, and beta-catenin, while showing that the interaction of E-cadherin with p120-catenin is not established in the retina of C-129 WT and Rho(-/-) mice until 4 weeks after birth and remains un-changed up to and including 5 weeks after birth. Meanwhile, we report that the association of alpha-catenin with ZO-1 is decreased in retinas of the Rho(-/-) from newborn animals up to and including 5 weeks after birth. We have also shown that the association of alpha-catenin and beta-catenin is not well established in WT and Rho(-/-) mice until at least 5 weeks after birth. We hypothesize that these retinal changes at the OLM may contribute significantly to the pathogenesis of retinal degenerations and may represent a unique therapeutic target for intervention in conditions involving rapid photoreceptor cell death.

Differences in expression of junctional adhesion molecule-A and beta-catenin in multiple sclerosis brain tissue: increasing evidence for the role of tight junction pathology.

Previously we have employed antibodies to the tight junction (TJ)-associated proteins ZO-1 and occludin to describe endothelial tight junction abnormalities, in lesional and normal appearing white matter, in primary and secondary progressive multiple sclerosis (MS). This work is extended here by use of antibodies to the independent TJ-specific proteins and junctional adhesion molecule A & B (JAM-A, JAM-B). We have also assessed the expression in MS of beta-catenin, a protein specific to the TJ-associated adherens junction. Immunocytochemistry and semiquantitative confocal microscopy for JAM-A and beta-catenin was performed on snap-frozen sections from MS cases (n=11) and controls (n=6). Data on 1,443 blood vessels was acquired from active lesions (n=13), inactive lesions (n=13), NAWM (n=20) and control white matter (n=13). In MS abnormal JAM-A expression was found in active (46%) and inactive lesions (21%), comparable to previous data using ZO-1. However, a lower level of TJ abnormality was found in MS NAWM using JAM-A (3%) compared to ZO-1 (13%). JAM-B was strongly expressed on a small number of large blood vessels in control and MS tissues but at too low a level for quantitative analysis. By comparison with the high levels of abnormality observed with the TJ proteins, the adherens junction protein beta-catenin was normally expressed in all MS and control tissue categories. These results confirm, by use of the independent marker JAM-A, that TJ abnormalities are most frequent in active white matter lesions. Altered expression of JAM-A, in addition to affecting junctional tightness may also both reflect and affect leukocyte trafficking, with implications for immune status within the diseased CNS. Conversely, the adherens junction component of the TJ, as indicated by beta-catenin expression is normally expressed in all MS and control tissue categories.

Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function.

PURPOSE: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF165-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. METHODS: C57/Bl6 mice were injected with VEGF165 and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF165 and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. RESULTS: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF165. It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF165-mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF165-mediated permeability compared with control (untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF165. CONCLUSIONS: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF165-induced paracellular permeability.

Growth and characterisation of a cell culture model of the feline blood-brain barrier.

An in vitro model of the feline blood-brain barrier was developed using primary cultures of brain capillary endothelial cells derived from adult cats. They were grown in the presence of astrocytes obtained from newborn kittens. Feline endothelial cell cultures were characterised by uptake of DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) and expression of von Willebrand factor. Astrocytes were characterised based on their expression of glial fibrillary acidic protein (GFAP). Electron microscopy revealed junctional specialisation between endothelial cells. Occludin and ZO-1 expression by the endothelial cell cultures was detected by Western blot analysis. Barrier function of co-cultured endothelial cells and astrocytes was confirmed by a transendothelial electrical resistance (TEER) value of 30-35 Omegacm2 and apparent permeability coefficients (Papp) for FD-40 (FITC-dextran, 40 kDa) of 4x10(-6) cm/s and for FD-4 (4kDa) of 1.92x10(-5) cm/s. In endothelial cell monolayers grown with astrocyte-conditioned medium, the TEER value was lower (20-25 Omegacm2), and Papp of FD-40 and FD-4 was higher at 6.27x10(-6) and 3.96x10(-5) cm/s, respectively. This model should have useful applications in the examination of events occurring at the BBB early in FIV infection, and may provide knowledge applicable to HIV infection.

Neuropathology associated with feline immunodeficiency virus infection highlights prominent lymphocyte trafficking through both the blood-brain and blood-choroid plexus barriers.

Feline immunodeficiency virus (FIV) infection in the cat is a well-evaluated model of human immunodeficiency virus (HIV)-1 infection in man with both viruses associated with significant neuropathology. Although studies in both HIV and FIV infections have shown that virus enters the brain in the acute stages of disease, little is known of the mechanisms of viral entry. The dissection of this stage is fundamental to the development of therapies that may prevent or modulate central nervous system (CNS) infection. The present study was designed to characterize the early sequential neuropathological changes following infection with FIV(GL8), a strain known to enter the CNS in acute infection. Cats were infected either by the intraperitoneal (n = 13) or intravenous (n = 12) route with 2000 cat infectious units of virus. Histopathological assessments following intraperitoneal infections were at 4 (n = 2), 5 (n = 1), 8 (n = 3), 10 (n = 1), 16 (n = 1), 32 (n = 2), 52 (n = 2), and 104 (n = 1) weeks post infection whereas animals infected intravenously were examined (n = 3) at 1, 4, 10, and 23 weeks post infection. The most significant lesions following both routes of infection were lymphocyte-rich perivascular infiltrates within cerebral and cerebellar meninges, in choroid plexus and spinal cord dura mater and within epineurium of the sciatic nerve. In addition, following intravenous infection perivascular infiltrations were noted in parenchymal blood vessels primarily of cerebral white matter. Infiltrates were composed of CD79+ B cells and CD3+ T cells. The latter population contained a mixture of CD4+ and CD8+ cells. The severity of lesions increased in intensity in the 8-to 16-week period following infection and then began to wane. The evaluation of this large group of cats at multiple time points revealed pathology comparable with that of early stage HIV-1-associated encephalitis. Moreover, in contrast to previous FIV neuropathology studies, transient meningeal, choroid plexus, and parenchymal vascular pathology were consistent significant findings suggesting that, as in HIV-1 infection, blood-brain barrier and choroid plexus brain barrier integrity are both compromised in early infection.