Unbalanced repeated-measures models with structured covariance matrices

The question of how to analyze unbalanced or incomplete repeated-measures data is a common problem facing analysts. We address this problem through maximum likelihood analysis using a general linear model for expected responses and arbitrary structural models for the within-subject covariances. Models that can be fit include standard univariate and multivariate models with incomplete data, random-effects models, and models with time-series and factor-analytic error structures. We describe Newton-Raphson and Fisher scoring algorithms for computing maximum likelihood estimates, and generalized EM algorithms for computing restricted and unrestricted maximum likelihood estimates. An example fitting several models to a set of growth data is included.     

Characteristics of xanthan gum-based biodegradable superporous hydrogel

A novel biopolymer-based superporous hydrogel (SPH) was synthesized through chemical crosslinking by graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) on to xanthan gum (XG) via redox initiator system of ammonium persulfate (APS) and N, N, N′, N′-tetramethylethylenediamine (TMED), in the presence of N, N′-methylenebisacrylamide (MBA) crosslinking agent, sodium bicarbonate foaming agent, a triblock copolymer of polyoxyethylene/polyoxypropylene/polyoxyethylene as a foam stabilizer. Characterization of SPH was done by FT-IR, TGA, SEM, HPC and GCMS. The effects of pH and salinity on the swelling aptitude of the SPH were investigated along with its degradability in Streptococcus bovis medium.     

Biocompatible and biodegradable dual-drug release system based on silk hydrogel containing silk nanoparticles

We developed a facile and quick ethanol-based method for preparing silk nanoparticles and then fabricated a biodegradable and biocompatible dual-drug release system based on silk nanoparticles and the molecular networks of silk hydrogels. Model drugs incorporated in the silk nanoparticles and silk hydrogels showed fast and constant release, respectively, indicating successful dual-drug release from silk hydrogel containing silk nanoparticles. The release behaviors achieved by this dual-drug release system suggest to be regulated by physical properties (e.g., β-sheet contents and size of the silk nanoparticles and network size of the silk hydrogels), which is an important advantage for biomedical applications. The present silk-based system for dual-drug release also demonstrated no significant cytotoxicity against human mesenchymal stem cells (hMSCs), and thus, this silk-based dual-drug release system has potential as a versatile and useful new platform of polymeric materials for various types of dual delivery of bioactive molecules.     

Of blood cells and the nervous system

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Extended release of bevacizumab by thermosensitive biodegradable and biocompatible hydrogel

The antibody bevacizumab (Avastin) has been used clinically to treat intraocular neovascular diseases based on its antivascular endothelial growth factor (VEGF) character. The anti-VEGF strategy for retinal neovascular diseases is limited by the short half-life of bevacizumab and thus requires frequent injections. This Article reports the sustained release of bevacizumab from a biocompatible material that is composed of a triblock copolymer of poly(2-ethyl-2-oxazoline)-b-poly(ε-caprolactone)-b-poly(2-ethyl-2-oxazoline) (PEOz-PCL-PEOz). The amphiphilic PEOz-PCL-PEOz triblock copolymer was synthesized in three steps. First, the PEOz was polymerized by methyl p-toluenesulfonate and 2-ethyl-2-oxazoline (EOz), and the living end was terminated by potassium hydroxide methanolic solution. Subsequently, the hydroxyl-PEOz was used as a macroinitiator for the ring-opening polymerization of ε-caprolactone using a Tin(II) octoate catalyst to synthesize the telechelic hydroxylated PEOz-PCL. Finally, the PEOz-PCL-PEOz triblock copolymer was obtained using the 1,6-hexamethylene diisocyanateas a coupling reagent. The PEOz-PCL-PEOz was chemically and molecularly characterized by GPC, (1)H NMR, and FTIR, and its aqueous solution (ECE hydrogel) showed a reversible sol (room temperature)-gel (physiological temperature) phase transition, which serves as an easy antibody-packing system with extended release. The biodegradability of ECE hydrogel was assessed by the porosity formation at different periods by scanning electron microscopy. The ECE hydrogel had no in vitro cytotoxicity on the human retinal pigment epithelial cell line by flow cytometry. The histomorphology and electrophysiology of the rabbit neuroretina were preserved after 2 months of intravitreal injection. In conclusion, the ECE hydrogel has a temperature-sensitive sol-gel phase transition and is effective in vitro. Its intraocular biocompatibility demonstrated its great potential to be widely used in biomedical applications for extended drug release.     

Glycolytic metabolism in cultured cells of the nervous system

Pyruvate and lactate efflux from C-6 glioma cells has been found to be regulated by both the medium glucose concentration and the medium concentration of the two acids. Each moves down a concentration gradient until the extracellular level is in equilibrium with the intracellular. Long-term growth studies demonstrated that the cells preferentially utilize glucose but that once it is depleted, they will take up first pyruvate, followed by lactate, for further metabolism. Changes in the intracellular levels of the two metabolites correspond to those seen in the medium. The rate of glycogen breakdown parallels that of medium glucose ultilization. Preliminary results with the C-1300 neuroblastoma cells showed pyruvate and lactate efflux rates comparable to those of the glioma cells.     

Glycolytic metabolism in cultured cells of the nervous system

The transport and metabolism of glucose was examined in monolayers of C-6 glioma cells. 1) Glucose transport appeared to have both a low (Km = 7.74 mM) and a high (Km = 1.16 mM) affinity site in C-6cells; whereas 2-deoxyglucose had only one (Km = 3.7 mM). 2) A large portion of the accumulated glucose was rapidly metabolized to the two glycolytic end products, lactate and pyruvate, and then extruded into the medium. The temperature-dependent efflux of lactate and pyruvate was linear up to 2 hrs with 6 to 10 times more lactate being extruded into the medium than pyruvate. 3) The efflux of lactate and pyruvate increased with increasing extracellular (medium) pH. The presence of 5 percent CO2 not only inhibited the acid efflux but also inhibited the short-term uptake of glucose. The CO2 effect was attributed to a lowering of the medium pH since bicarbonate alone either increased or did not inhibit efflux. 4) Valinomycin increased the levels of cellular lactate but not those of pyruvate by almost three-fold. Lactate efflux was stimulated while that of pyruvate was inhibited. The addition of 5 percent CO2 increased the cellular levels of both lactate and pyruvate, but unlike valinomycin decreased the acid efflux. Idoacetate inhibited the acid efflux by 50 percent suggesting that glycolysis is necessary for efflux.     

Glycolytic metabolism in cultured cells of the nervous system

The effects of thiamine deficiency and of the antithiamine drug pyrithiamine on the C-6 glioma and the C-1300 neuroblastoma cell lines have been studied. Thiamine deficiency increased the doubling time of the neuroblastoma cells without affecting that of the glioma cells. Pyrithiamine prevented both cell lines from doubling even once. (hiamine deficiency had only slight effects on intracellular pyruvate and lactate levels or on efflux rates for the acids, but pyrithiamine treatment resulted in large increases in both the intracellular levels and the efflux in both cell lines. For comparison, the pyruvate and lactate levels in mouse brain were measured. The levels from thiamine-deficient mouse brain were essentially unchanged from controls while pyrithiamine treatment caused a significant elevation only of the pyruvate concentration.     

The mast cells of the mammalian central nervous system

Summary The uptake and turnover of the precursors of heparin and heparan sulphate (³⁵S), and of serotonin (³H-5-hydroxytryptophan; ³H-5-HTP) by mast cells (MCs) and neurolipomastocytoid cells (NLMs) of the mammalian CNS were studied. Rats of varying age from 1 day to early adulthood were injected with ³⁵S (as a solution of sodium sulphate) and ³H-5-HTP, and allowed to survive for different periods. Several fixatives, as well as lengths of exposure to photographic emulsion, were tested. The monoamine oxidase inhibitor, nialamide, needed to be given before uptake of ³H-5-HTP could be adequately demonstrated especially in the CNS. ³⁵S was taken up by structures known to contain a great deal of sulphate, viz., cartilage and goblet cells, as well as by MCs of adult liver and thymus, but not by MCs of adult CNS. All of these structures, including the MCs of CNS, took it up much more avidly in babies than in adults. ³H-5-HTP had a similar effect in that the MCs of younger animals took it up more strongly than did those of adults. In the MCs of the CNS uptake seemed to increase up to 15 days of age but then to decrease as maturity was reached. The MCs are located in the leptomeninges of the cerebral hemispheres as well as the choroid fissures and dorsal thalamus. The NLMs, ubiquitously distributed in the leptomeninges as well as perivascularly, showed less radioactivity with both markers in fewer cells and only in babies. The possible significance of these results is discussed. It is concluded that MCs, and to a lesser extent NLMs, of the CNS do permit entry of these markers, and that the more immature the cells, the heavier the load that enters. Adult cells do not seem to take up precursor suggesting little or no turnover.Supported in part by a grant from the Incentive Plan of the Medical School, American University of Beirut, and by Research Support Grant MA-004 from the College of Graduate Studies, University of Kuwait     

Mechanism of stem cells in the central nervous system

Injury to the central nervous system (CNS) can result in severe functional impairment. The brain and spinal cord, which constitute the CNS, have been viewed for decades as having a very limited capacity for regeneration. However, over the last several years, the body of evidence supporting the concept of regeneration and continuous renewal of neurons in specific regions of the CNS has increased. This evidence has significantly altered our perception of the CNS and has offered new hope for possible cell therapy strategies to repair lost function. Transplantation of stem cells or the recruitment of endogenous stem cells to repair specific regions of the brain or spinal cord is the next exciting research challenge. However, our understanding of the existing stem cell pool in the adult CNS remains limited. This review will discuss the identification and characterization of CNS stem cells in the adult brain and spinal cord.     

Isolation of mononuclear cells from the central nervous system of rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.     

Plasticity and stem cells in the vertebrate nervous system

How and when do vertebrate neural precursor cells choose their fates? While some studies suggest a series of commitments on the road to fate choice, many recent experiments indicate that precursor fate choices can often be changed. Additionally, the identification of common gene control mechanisms in precursors suggest that these cells share fundamental properties throughout development.     

Glia as antigen-presenting cells in the central nervous system

The contribution of the cells within the central nervous system (CNS) toward adaptive immune responses is emerging and incompletely understood. Recent findings indicate important functional interactions between T-cells and glial cells within the CNS that may contribute to disease and neuropathology through antigen presentation. Although glia are not classically considered antigen-presenting cell (APC) types, there is growing evidence indicating that glial antigen presentation plays an important role in several neurological diseases. This review discusses these findings which incriminate microglia, astrocytes, and oligodendrocyte lineage cells as CNS-resident APC types with implications for understanding disease.     

Cancer stem cells in the mammalian central nervous system

Malignant tumours intrinsic to the central nervous system (CNS) are among the most difficult of neoplasms to treat effectively. The major biological features of these tumours that preclude successful therapy include their cellular heterogeneity, which renders them highly resistant to both chemotherapy and radiotherapy, and the propensity of the component tumour cells to invade, diffusely, the contiguous nervous tissues. The tumours are classified according to perceived cell of origin, gliomas being the most common generic group. In the 1970s transplacental administration of the potent neurocarcinogen, N-ethyl-N-nitrosourea (ENU), enabled investigation of the sequential development of brain and spinal neoplasms by electron microscopy and immunohistochemistry. The significance of the primitive cells of the subependymal plate in cellular origin and evolution of a variety of glial tumours was thereby established. Since then, the development of new cell culture methods, including the in vitro growth of neurospheres and multicellular tumour spheroids, and new antigenic markers of stem cells and glial/neuronal cell precursor cells, including nestin, Mushashi-1 and CD133, have led to a reappraisal of the histological classification and origins of CNS tumours. Moreover, neural stem cells may also provide new vectors in exciting novel therapeutic strategies for these tumours. In addition to the gliomas, stem cells may have been identified in paediatric tumours including cerebellar medulloblastoma, thought to be of external granule cell neuronal derivation. Interestingly, while the stem cell marker CD133 is expressed in these primitive neuroectodermal tumours (PNETs), the chondroitin sulphate proteoglycan neuronal/glial 2 (NG2), which appears to denote increased proliferative, but reduced migratory activity in adult gliomas, is rarely expressed. This is in contrast to the situation in the histologically similar supratentorial PNETs. A possible functional 'switch' between proliferation and migration in developing neural tumour cells may exist between NG2 and ganglioside GD3. The divergent pathways of differentiation of CNS tumours and the possibility of stem cell origin, for some, if not all, such neoplasms remain a matter for debate and continued research, but the presence of self-renewing neural stem cells in the CNS of both children and adults strongly suggests a role for these cells in tumour initiation and resistance to current therapeutic strategies.     

Stem cells in the adult mammalian central nervous system

Over the past year, evidence has accrued that adult CNS stem cells are a widespread progenitor cell type. These cells may normally replace neurons and/or glia in the adult brain and spinal cord. Advances have been made in understanding the signals that regulate stem cell proliferation and differentiation. A deeper understanding of the structure of germinal zones has helped us move towards identifying stem cells in vivo. Recent studies suggest that the fate of stem cell progeny in vivo may be linked to the complexity of the animal's environment.     

Circadian rhythms and glial cells of the central nervous system

Glial cells are the most abundant cells in the central nervous system and play crucial roles in neural development, homeostasis, immunity, and conductivity. Over the past few decades, glial cell activity in mammals has been linked to circadian rhythms, the 24-h chronobiological clocks that regulate many physiological processes. Indeed, glial cells rhythmically express clock genes that cell-autonomously regulate glial function. In addition, recent findings in rodents have revealed that disruption of the glial molecular clock could impact the entire organism. In this review, we discuss the impact of circadian rhythms on the function of the three major glial cell types – astrocytes, microglia, and oligodendrocytes – across different locations within the central nervous system. We also review recent evidence uncovering the impact of glial cells on the body's circadian rhythm. Together, this sheds new light on the involvement of glial clock machinery in various diseases.