Citrullinated human and murine MOG35-55 display distinct biophysical and biochemical behavior.

The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied extensively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullination of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential pathogenic mechanism underlying this disease. The immunodominant region of MOG is highly conserved between species, with the only difference between the murine and human protein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrullinated murine MOG35-55 is fundamentally different for human and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immunized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this unexpected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoimmune encephalomyelitis is necessary.

Rapidly progressive amyotrophic lateral sclerosis is associated with microglial reactivity and small heat shock protein expression in reactive astrocytes.

AIMS: Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive loss of motor neurons, muscle weakness, spasticity, paralysis and death usually within 2-5 years of onset. Neuroinflammation is a hallmark of ALS pathology characterized by activation of glial cells, which respond by upregulating small heat shock proteins (HSPBs), but the exact underlying pathological mechanisms are still largely unknown. Here, we investigated the association between ALS disease duration, lower motor neuron loss, TARDNA-binding protein 43 (TDP-43) pathology, neuroinflammation and HSPB expression. METHODS: With immunohistochemistry, we examined HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 expression in cervical, thoracic and sacral spinal cord regions in 12 ALS cases, seven with short disease duration (SDD), five with moderate disease duration (MDD), and ten age-matched controls. Expression was quantified using ImageJ to examine HSP expression, motor neuron numbers, microglial and astrocyte density and phosphorylated TDP-43 (pTDP-43+) inclusions. RESULTS: SDD was associated with elevated HSPB5 and 8 expression in lateral tract astrocytes, while HSP16.2 expression was increased in astrocytes in MDD cases. SDD cases had higher numbers of motor neurons and microglial activation than MDD cases, but similar levels of motor neurons with pTDP-43+ inclusions. CONCLUSIONS: Increased expression of several HSPBs in lateral column astrocytes suggests that astrocytes play a role in the pathogenesis of ALS. SDD is associated with increased microgliosis, HSPB5 and 8 expression in astrocytes, and only minor changes in motor neuron loss. This suggests that the interaction between motor neurons, microglia and astrocytes determines neuronal fate and functional decline in ALS.

Heat shock proteins are differentially expressed in brain and spinal cord: implications for multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. In this study we have compared brain and spinal cord white (WM) and grey (GM) matter from MS and control tissues, focusing on small heat shock proteins (HSPB) and HSP16.2. Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age-matched non-neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ. Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed, although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal-appearing GM and type 1 GM lesions. The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents.

Increased expression of distinct galectins in multiple sclerosis lesions.

AIMS: Multiple sclerosis (MS) is a chronic progressive degenerative disorder of the central nervous system, characterized by inflammation, demyelination, ultimate failure of remyelination and axonal loss. Current research identifies galectins, adhesion/growth-regulatory effectors binding ß-galactosides, peptide motifs and lipids, as important immunomodulators in diverse inflammatory diseases. However, little is known about their expression, cellular localization and role in human central nervous system tissue. To identify a potential role of galectins in MS, their expression and localization in control white matter (CWM) and demyelinated MS lesions were examined. METHODS: qPCR, Western blot and immunohistochemical analyses were performed on human post mortem CWM and MS lesions at different stages. Cultured astrocytes, derived from healthy subjects and MS patients, were analysed similarly. RESULTS: Among 11 different galectins tested, galectins-1, -3, -8 and -9 were present at detectable levels in CWM, and, interestingly, significantly enhanced in active MS lesions. On the cellular level, galectins localized to microglia/macrophages, astrocytes and endothelial cells. Intriguingly, galectin-9 displayed a distinctly different intracellular localization in microglia/macrophages when comparing active and inactive MS lesions, being restricted to the nuclei in active lesions, and primarily localizing in the cytoplasm in inactive lesions. Furthermore, enhanced levels of galectin-1, detected as dimers in Western blot analysis, were released by cultured astrocytes from MS patients. CONCLUSIONS: This study provides a detailed analysis of galectins in MS lesions and assigns distinct galectins to different aspects of the disease. Thus, besides being known as modulators of inflammatory processes, our findings suggest additional association of distinct galectins with MS pathology.

Primate flicker sensitivity: psychophysics and electrophysiology.

A quantitative comparison is made between the psychophysical flicker response of man and similar data obtained electrophysiologically from the cones of macaque monkeys. When the psychophysical data are obtained from an eye that is strongly light-adapted, there is excellent agreement between the two sets of data at high frequencies. Under this condition, both kinds of data fit a distributed-parameter model, whose time constant also agrees with that derived from studies of the phosphenes elicited by electrical stimulation of the human eye. On the other hand, psychophysical data obtained with fully modulated stimuli (which minimally adapt the eye) yield a longer time constant for the same model. These results imply that the psychophysical flicker thresholds are normally controlled by a distributed filtering process that is proximal to the receptor stage. This slower, psychophysical process is evidently desensitized by intense adapting lights, so that the faster one that governs the electrophysiological responses can be detected.

Expression of dominant-negative and chimeric subunits reveals an essential role for beta1 integrin during myelination.

Myelination represents a remarkable example of cell specialization and cell-cell interaction in development. During this process, axons are wrapped by concentric layers of cell membrane derived either from central nervous system (CNS) oligodendrocytes or peripheral nervous system Schwann cells. In the CNS, oligodendrocytes elaborate a membranous extension with an area of more than 1000 times that of the cell body. The mechanisms regulating this change in cell shape remain poorly understood. Signaling mechanisms regulated by cell surface adhesion receptors of the integrin family represent likely candidates. Integrins link the extracellular environment of the cell with both intracellular signaling molecules and the cytoskeleton and have been shown to regulate the activity of GTPases implicated in the control of cell shape. Our previous work has established that oligodendrocytes and their precursors express a limited repertoire of integrins. One of these, the alpha6beta1 laminin receptor, can interact with laminin-2 substrates to enhance oligodendrocyte myelin membrane formation in cell culture. However, these experiments do not address the important question of integrin function during myelination in vivo, nor do they define the respective roles of the alpha and beta subunits in the signaling pathways involved. Here, we use a dominant-negative approach to provide, for the first time, evidence that beta1 integrin function is required for myelination in vivo and use chimeric integrins to dissect apart the roles of the extracellular and cytoplasmic domains of the alpha6 subunit in the signaling pathways of myelination.

Molecular cloning and characterization of human and murine DNase II.

We have cloned and sequenced novel cDNAs that encode human and murine DNase II, the acidic deoxyribonuclease. Sequence analysis predicts that huDNase II contains an N-terminal signal sequence and that mature DNase II has 344 residues with a calculated molecular mass of 38 032 Da. DNase II is a novel enzyme with no homologies to proteins of known function. Surprisingly, C. elegans appears to possess a family of DNase II homologs. Unlike DNase I-like enzymes that have tissue-specific expression patterns, huDNase II is ubiquituously expressed at low levels. When huDNase II is expressed in human 293 cells, we observe secretion of a novel 42-44 kDa glycoprotein; approximately 20-30% of recombinant human DNase II activity is secreted in this system. The secreted enzyme possesses DNA hydrolytic activity and shares biochemical properties with purified DNase II obtained from other species. We also show that the mechanism by which DNase II cuts DNA is similar to DNase I in that the enzyme produces nicks rather than double-strand cuts.

Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages.

We have cloned human and murine DNase I-like cDNAs, termed LS-DNase, which are expressed at high levels in liver and spleen tissues. LS-DNase expression is highly specific to macrophage populations within these and other tissues. Mature LS-DNase from both species is a secreted, non-glycosylated protein containing 285 residues, with a calculated molecular mass of 33 kDa and a basic isoelectric point. Human and murine LS-DNase are highly conserved and share 83% identity. Sequence analysis reveals that LS-DNase shares 46% amino acid sequence identity with DNase I. However, several residues identified as important for interaction of human DNase I with actin are not conserved in both human and murine LS-DNase. Consistent with this observation, recombinant human LS-DNase possesses a DNA hydrolytic activity which, unlike DNase I, is not inhibited by G-actin. The existence of a family of DNase I-like molecules that have tissue-specific expression patterns and the possible role of a macrophage specific DNase are discussed.

Cone difference signal in foveal local electroretinogram of primate.

Psychophysical and electrophysiological studies have shown that the perception of color is in part dependent upon an opponent signal between the long (R) and middle (G) wavelength--sensitive cone systems. Models of human color vision hypothesize that this signal is derived from the difference between sensitivities of the R and G classes of cones. We report here a slow potential in the foveal local electroretinogram (LERG) of primate that correlates well with the absolute logarithmic difference between psychophysically deduced R and G primaries. The foveal LERG is recorded from cynomolgus macaque monkeys with the use of low-frequency sinusoidally flickering stimuli. Responses obtained at the neural wavelength, typically in the region between 540 and 570 nm, or less like log-saturated sinusoids, whereas responses obtained to other wavelength stimuli have a negative component. The amplitude of the negative-going component is deduced by fitting waveforms obtained at the neutral wavelength to responses obtained to the other wavelengths. The validity of this nonlinear analysis is supported by fitting the deduced hyperpolarizing response vs. intensity (RvI) functions with the relationship, V/Vmax = I/(I r sigma), as previously found for single retinal units. The negative component RvI function does not follow this relationship--as expected for an R-G difference signal; a decrease in amplitude at high illuminances could account for perceptual luminance dependent hue shifts.

Field sensitivity of the "red" mechanism derived from primate local electroretinogram.

Using the local ERG in response to a long-wavelength stimulus as an indicator, field sensitivity functions have been obtained from cynomolgus macaque monkeys (Macaca fascicularis) with flashed and sinusoidally flickering test stimuli. These functions show the reciprocal of the relative radiance, for various adapting wavelengths, required to reduce a 667-nm test response to a criterion level. The resulting functions resemble both Stiles's pi 5 and the SR function of Smith and Pokorny, provided that pi 5 and SR are displaced about 7 nm toward longer wavelengths, in agreement with microspectrophotometric evidence. When field sensitivity functions are obtained with a 20 Hz sinusoidal test stimulus, using a continuous change of field wavelength, the direction of a slow spectral traverse has a large effect upon the shape of the sensitivity and phase functions--a hysteresis effect These effects do not occur at 5 Hz. The test light is proven to be ineffective upon G cones, and it appears unlikely that measurably significant signals are significantly induced into G cones as an indirect result of the modulation of R cones. Therefore, the steady background light absorbed in the G cones seems to be influencing the response of the R cones.

Sex hormone binding globulin deficiency due to a homozygous missense mutation.

CONTEXT: SHBG is known as the major sex steroid binding protein in plasma, and it regulates the bioavailability of both T and estradiol levels required for effects on target tissues. We identified a man with an undetectable SHBG concentration in combination with low total T. He presented with a 7-year history of muscle weakness, fatigue, and a low libido. OBJECTIVES: To determine the cause of the SHBG deficiency, we employed both genetic analysis of the SHBG gene and transgene SHBG expression. RESULTS: Genetic analysis identified a novel homozygous missense mutation that was predicted to be deleterious for protein function. Transgene expression showed that the mutation resulted in a block in SHBG secretion accompanied by increased expression of the endoplasmic reticulum molecular chaperone HSPA5. The mutation results in accumulation of the mutant SHBG within the cell and failure to secrete the mutant protein. Screening of family members identified one sister who was also deficient for SHBG. CONCLUSIONS: We have identified a family with a missense mutation within the SHBG gene, which results in a complete deficiency of plasma SHBG in the homozygous state. Although total T level was low in the male patient, it did not interfere with normal gonadal development and spermatogenesis, suggesting a limited role of SHBG in sexual maturation and male physiology.

Polyunsaturated fatty acid supplementation stimulates differentiation of oligodendroglia cells.

Dietary polyunsaturated fatty acids (PUFAs) have been postulated as alternative supportive treatment for multiple sclerosis, since they may promote myelin repair. We set out to study the effect of supplementation with n-3 and n-6 PUFAs on OLN-93 oligodendroglia and rat primary oligodendrocyte differentiation in vitro. It appeared that OLN-93 cells actively incorporate and metabolise the supplemented PUFAs in their cell membrane. The effect of PUFAs on OLN-93 differentiation was further assessed by morphological and Western blot evaluation of markers of oligodendroglia differentiation: 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), zonula occludens-1 (ZO-1) and myelin-associated glycoprotein (MAG). Supplementation of the OLN-93 cells with n-3 and n-6 PUFAs increased the degree of differentiation determined by morphological analysis. Moreover, CNP protein expression was significantly increased by gamma-linolenic acid (GLA, 18:3n-6) supplementation. In accordance with the OLN-93 results, studies with rat primary oligodendrocytes, a more advanced model of cell differentiation, showed GLA supplementation to promote oligodendrocyte differentiation. Following GLA supplementation, increased numbers of proteolipid protein (PLP)-positive oligodendrocytes and increased myelin sheet formation was observed during differentiation of primary oligodendrocytes. Moreover, increased CNP, and enhanced PLP and myelin basic protein expression were found after GLA administration. These studies provide support for the dietary supplementation of specific PUFAs to support oligodendrocyte differentiation and function.

Cyclorotation impacts on toric contact lens fitting and performance.

The paper provides: (1) a review of the ocular cyclorotation literature; (2) an overview of the environmental factors such as posture and viewing conditions, including biocularity and distance, that can produce ocular cyclorotation; and (3) a discussion of how ocular cyclorotation can affect toric contact lens fitting and performance.

Predicting visual performance following excimer photorefractive keratectomy.

BACKGROUND: A duplex optical image is created when the ablation zone formed by excimer photorefractive keratectomy is smaller than the entrance pupil. Visual performance and secondary effects are analyzed using a theoretical model of the optical image. METHODS: A point-spread function having a centered in-focus component surrounded by an annular out-of-focus component is calculated from pupil size, ablation size, refractive error, and photoreceptor directional sensitivity. The line-spread, edge-spread, and optical transfer functions are derived. RESULTS: In the line- and edge-spread functions, secondary maxima and curvilinear ramps are most evident with low refractive errors. The half-height widths of the point- and line-spread functions change little. The optical transfer function is reduced in proportion to the distribution of light between the image components. CONCLUSIONS: Stable point and line half-height widths explain why Snellen visual acuity is insensitive to annular blur. Contrast sensitivity correlates with symptoms of haze and fog. Halos and ghost images are associated with secondary optical maxima and curvilinear ramps. Neither visual acuity nor contrast sensitivity can predict halos or ghost images. Halos and ghost images will be most prevalent in low illumination and for low refractive corrections. High refractive errors will produce fewer visual side effects than low refractive errors.

Sinusoidal flicker characteristcis of primate cones in response to heterochromatic stimuli.

Electrophysiological recordings of primate photorecptors have been obtained and frequency response characteristics of the red, green and blue cones have been determined and compared to previous psychophysical findings. Using cynomolgus monkeys, we recorded the foveal local electroretinogram, which is dominated by the late receptor potential, and obtained criterion-response threshold data for sinusoidally flickered test stimuli complementary chromatic adapting backgrounds. Our results support the hypotheses that (a) the shapes of the MTFs of the red and green cone systems are identical and are determined solely by the photoreceptors at high frequencies, and (b) the blue cones have an MTF with a lower corner frequency than the red- and green-cone systems.

Response of primate cones to sinusoidally flickering homochromatic stimuli.

1. The response of the primate cone photoreceptors to sinusoidally flickering stimuli has been obtained by monitoring the late receptor potential (LRP). 2. By comparing the response characteristics of the foveal local electroretinogram (LERG) before and after the intraocular infusion of sodium aspartate, it was found that the b-wave in the foveal LERG does not affect the monitoring of the LRP to steady-state flicker. 3. Functions describing the supra-threshold frequency response characteristics of the photoreceptors were obtained. 4. Linearity was found to hold for low amplitude responses, and temporal modulation transfer functions (MTFs) were obtained for the photoreceptors at various adaptation levels. 5. The cone photoreceptors were found to act approximately as passive low pass filters compounded with some low frequency attenuation. 6. The high frequency response of the photoreceptors at various adaptation levels tends toward a common high frequency asymptote, much like human psychophysical findings, and can be described by a diffusion model. 7. Non-linearities (convexity-upwards) suggest modest positive feedback at the level of the photoreceptors. 8. Mechanisms limiting the magnitude of the receptor response at low frequencies have little effect on the phase lag of the response.