CNS Penetration of Intrathecal-Lumbar Idursulfase in the Monkey, Dog and Mouse: Implications for Neurological Outcomes of Lysosomal Storage Disorder

A major challenge for the treatment of many central nervous system (CNS) disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome) is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S). I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a) warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b) may have broader implications for CNS treatment with biopharmaceuticals

Expression of GLUT1 and GLUT3 Glucose Transporters in Endometrial and Breast Cancers

Cancer cells have accelerated metabolism and high glucose requirements. The up-regulation of specific glucose transporters may represent a key mechanism by which malignant cells may achieve increased glucose uptake to support the high rate of glycolysis. In present study we analyzed the mRNA and protein expression of GLUT1 and GLUT3 glucose transporters by quantitative real-time polymerase chain reaction (Q-PCR) and Western blotting technique in 76 cases of endometrial carcinoma and 70 cases of breast carcinoma. SLC2A1 and SLCA2A3 mRNAs expression was found, respectively in 100% and 97.4% samples of endometrial cancers and only in 50% and 40% samples of breast cancers. In endometrial cancers GLUT1 and GLUT3 protein expression was identified in 67.1% and 30.3% of cases. Analogously, in breast cancers in 48.7% and 21% of samples, respectively. The results showed that both endometrial and breast poorly differentiated tumors (grade 2 and 3) had significantly higher GLUT1 and GLUT3 expression than well-differentiated tumors (grade 1). Statistically significant association was found between SLCA2A3 mRNA expression and estrogen and progesterone receptors status in breast cancers. GLUT1 has been reported to be involved in the uptake of glucose by endometrial and breast carcinoma cells earlier and the present study determined that GLUT3 expression is also involved. GLUT1 and GLUT3 seem to be important markers in endometrial and breast tumors differentiation

Post-Transcriptional Regulation of 5-Lipoxygenase mRNA Expression via Alternative Splicing and Nonsense-Mediated mRNA Decay

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression

In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

<p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.</p> <p>Methods</p> <p>Mice carrying human (FaDu_dd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.</p> <p>Results</p> <p>In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu_dd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.</p> <p>Conclusions</p> <p>The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.</p

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent

Complement in the pathogenesis of Alzheimer's disease

The emergence of complement as an important player in normal brain development and pathological remodelling has come as a major surprise to most scientists working in neuroscience and almost all those working in complement. That a system, evolved to protect the host against infection, should have these unanticipated roles has forced a rethink about what complement might be doing in the brain in health and disease, where it is coming from, and whether we can, or indeed should, manipulate complement in the brain to improve function or restore homeostasis. Complement has been implicated in diverse neurological and neuropsychiatric diseases well reviewed elsewhere, from depression through epilepsy to demyelination and dementia, in most complement drives inflammation to exacerbate the disease. Here, I will focus on just one disease, the most common cause of dementia, Alzheimer’s disease. I will briefly review the current understanding of what complement does in the normal brain, noting, in particular, the many gaps in understanding, then describe how complement may influence the genesis and progression of pathology in Alzheimer’s disease. Finally, I will discuss the problems and pitfalls of therapeutic inhibition of complement in the Alzheimer brain

Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences

Efficient processing of information by the central nervous system (CNS) represents an important evolutionary advantage. Thus, homeostatic mechanisms have developed that provide appropriate circumstances for neuronal signaling, including a highly controlled and stable microenvironment. To provide such a milieu for neurons, extracellular fluids of the CNS are separated from the changeable environment of blood at three major interfaces: at the brain capillaries by the blood-brain barrier (BBB), which is localized at the level of the endothelial cells and separates brain interstitial fluid (ISF) from blood; at the epithelial layer of four choroid plexuses, the blood-cerebrospinal fluid (CSF) barrier (BCSFB), which separates CSF from the CP ISF, and at the arachnoid barrier. The two barriers that represent the largest interface between blood and brain extracellular fluids, the BBB and the BCSFB, prevent the free paracellular diffusion of polar molecules by complex morphological features, including tight junctions (TJs) that interconnect the endothelial and epithelial cells, respectively. The first part of this review focuses on the molecular biology of TJs and adherens junctions in the brain capillary endothelial cells and in the CP epithelial cells. However, normal function of the CNS depends on a constant supply of essential molecules, like glucose and amino acids from the blood, exchange of electrolytes between brain extracellular fluids and blood, as well as on efficient removal of metabolic waste products and excess neurotransmitters from the brain ISF. Therefore, a number of specific transport proteins are expressed in brain capillary endothelial cells and CP epithelial cells that provide transport of nutrients and ions into the CNS and removal of waste products and ions from the CSF. The second part of this review concentrates on the molecular biology of various solute carrier (SLC) transport proteins at those two barriers and underlines differences in their expression between the two barriers. Also, many blood-borne molecules and xenobiotics can diffuse into brain ISF and then into neuronal membranes due to their physicochemical properties. Entry of these compounds could be detrimental for neural transmission and signalling. Thus, BBB and BCSFB express transport proteins that actively restrict entry of lipophilic and amphipathic substances from blood and/or remove those molecules from the brain extracellular fluids. The third part of this review concentrates on the molecular biology of ATP-binding cassette (ABC)-transporters and those SLC transporters that are involved in efflux transport of xenobiotics, their expression at the BBB and BCSFB and differences in expression in the two major blood-brain interfaces. In addition, transport and diffusion of ions by the BBB and CP epithelium are involved in the formation of fluid, the ISF and CSF, respectively, so the last part of this review discusses molecular biology of ion transporters/exchangers and ion channels in the brain endothelial and CP epithelial cells