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Optogenetics gives us unprecedented power to investigate brain connectivity. The ability to activate neural circuits with single cell resolution and its ease of application has provided a wealth of knowledge in brain function. More recently, optogenetics has shown tremendous utility in prosthetics applications, including vision restoration for patients with retinitis pigmentosa. One of the disadvantages of optogenetics, however, is its poor temporal bandwidth, i.e. the cell's inability to fire at a rate that matches the optical stimulation rate at high frequencies (>30 Hz). This research proposes a new strategy to overcome the temporal limits of optogenetic stimulation. Using whole-cell current clamp recordings in mouse retinal ganglion cells expressing channelrhodopsin-2 (H134R variant), we observed that randomizing inter-pulse intervals can significantly increase a retinal ganglion cell's temporal response to high frequency stimulation.Clinical Relevance- A significant disadvantage of optogenetic stimulation is its poor temporal dynamics which prohibit its widespread use in retinal prosthetics. We have shown that randomizing the interval between stimulation pulses reduces adaptation in retinal ganglion cells. This stimulation strategy may contribute to new levels of functional restoration in therapeutics which incorporate optogenetics.

We determined the retinal ganglion cell types projecting to the medial subdivision of inferior pulvinar (PIm) and the superior colliculus (SC) in the common marmoset monkey, Callithrix jacchus. Adult marmosets received a bidirectional tracer cocktail into the PIm (conjugated to Alexa fluor 488), and the SC (conjugated to Alexa fluor 594) using an MRI-guided approach. One SC injection included the pretectum. The large majority of retrogradely labelled cells were obtained from SC injections, with only a small proportion obtained after PIm injections. Retrogradely labelled cells were injected intracellularly in vitro using lipophilic dyes (DiI, DiO). The SC and PIm both received input from a variety of ganglion cell types. Input to the PIm was dominated by broad thorny (41%), narrow thorny (24%) and large bistratified (25%) ganglion cells. Input to the SC was dominated by parasol (37%), broad thorny (24%) and narrow thorny (17%) cells. Midget ganglion cells (which make up the large majority of primate retinal ganglion cells) and small bistratified (blue-ON/yellow OFF) cells were never observed to project to SC or PIm. Small numbers of other wide-field ganglion cell types were also encountered. Giant sparse (presumed melanopsin-expressing) cells were only seen following the tracer injection which included the pretectum. We note that despite the location of pulvinar complex in dorsal thalamus, and its increased size and functional importance in primate evolution, the retinal projections to pulvinar have more in common with SC projections than they do with projections to the dorsal lateral geniculate nucleus., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma., (© 2021. The Author(s).)

The middle temporal (MT) area of the extrastriate visual cortex has long been studied in adulthood for its distinctive physiological properties and function as a part of the dorsal stream, yet interestingly it possesses a similar maturation profile as the primary visual cortex (V1). Here, we examined whether an early-life lesion in MT of marmoset monkeys (six female, two male) altered the dorsal stream development and the behavioral precision of reaching-to-grasp sequences. We observed permanent changes in the anatomy of cortices associated with both reaching (parietal and medial intraparietal areas) and grasping (anterior intraparietal area), as well as in reaching-and-grasping behaviors. In addition, we observed a significant impact on the anatomy of V1 and the direction sensitivity of V1 neurons in the lesion projection zone. These findings indicate that area MT is a crucial node in the development of primate vision, affecting both V1 and areas in the dorsal visual pathway known to mediate visually guided manual behaviors. SIGNIFICANCE STATEMENT Previous studies have identified a role for the MT area of the visual cortex in perceiving motion, yet none have examined its central role in the development of the visual cortex and in the establishment of visuomotor behaviors. To address this, we used a unilateral MT lesion model in neonatal marmosets before examining the anatomic, physiological, and behavioral consequences. In adulthood, we observed perturbations in goal-orientated reach-and-grasp behavior, altered direction selectivity of V1 neurons, and changes in the cytoarchitecture throughout dorsal stream areas. This study highlights the importance of MT as a central node in visual system development and consequential visuomotor activity., (Copyright © 2021 the authors.)

Infants and adults respond differently to brain injuries. Specifically, improved neuronal sparing along with reduced astrogliosis and glial scarring often observed earlier in life, likely contributes to improved long-term outcomes. Understanding the underlying mechanisms could enable the recapitulation of neuroprotective effects, observed in infants, to benefit adults after brain injuries. We reveal that in primates, Eph/ ephrin signaling contributes to age-dependent reactive astrocyte behavior. Ephrin-A5 expression on astrocytes was more protracted in adults, whereas ephrin-A1 was only expressed on infant astrocytes. Furthermore, ephrin-A5 exacerbated major hallmarks of astrocyte reactivity via EphA2 and EphA4 receptors, which was subsequently alleviated by ephrin-A1. Rather than suppressing reactivity, ephrin-A1 signaling shifted astrocytes towards GAP43+ neuroprotection, accounting for improved neuronal sparing in infants. Reintroducing ephrin-A1 after middle-aged focal ischemic injury significantly attenuated glial scarring, improved neuronal sparing and preserved circuitry. Therefore, beneficial infant mechanisms can be recapitulated in adults to improve outcomes after CNS injuries., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In rodents, innate and learned fear of predators depends on the medial hypothalamic defensive system, a conserved brain network that lies downstream of the amygdala and promotes avoidance via projections to the periaqueductal gray. Whether this network is involved in primate fear remains unknown. To address this, we provoked flight responses to a predator (moving snake) in the marmoset monkey under laboratory conditions. We combined c-Fos immunolabeling and anterograde/retrograde tracing to map the functional connectivity of the ventromedial hypothalamus, a core node in the medial hypothalamic defensive system. Our findings demonstrate that the ventromedial hypothalamus is recruited by predator exposure in primates and that anatomical connectivity of the rodent and primate medial hypothalamic defensive system are highly conserved.

The medial pulvinar (PM) is a multimodal associative thalamic nucleus, recently evolved in primates. PM participates in integrative and modulatory functions, including directed attention, and consistently exhibits alterations in disorders such as schizophrenia and autism. Despite essential cognitive functions, the cortical inputs to the PM have not been systematically investigated. To date, less than 20 cortices have been demonstrated to project to PM. The goal of this study was to establish a comprehensive map of the cortical afferents to PM in the marmoset monkey. Using a magnetic resonance imaging-guided injection approach, we reveal 62 discrete cortices projecting to the adult marmoset PM. We confirmed previously reported connections and identified further projections from discrete cortices across the temporal, parietal, retrosplenial-cingulate, prefrontal, and orbital lobes. These regions encompass areas recipient of PM efferents, demonstrating the reciprocity of the PM-cortical connectivity. Moreover, our results indicate that PM neurones projecting to distinct cortices are intermingled and form multimodal cell clusters. This microunit organization, believed to facilitate cross-modal integration, contrasts with the large functional subdivisions usually observed in thalamic nuclei. Altogether, we provide the first comprehensive map of PM cortical afferents, an essential stepping stone in expanding our knowledge of PM and its function., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Retinotopic specializations in the ventral visual stream, especially foveal adaptations, provide primates with high-acuity vision in the central visual field. However, visual field specializations have not been studied in the dorsal visual stream, dedicated to processing visual motion and visually guided behaviors. To investigate this, we injected retrograde neuronal tracers occupying the whole visuotopic representation of the middle temporal (MT) visual area in marmoset monkeys and studied the distribution and morphology of the afferent primary visual cortex (V1) projections. Contrary to previous reports, we found a heterogeneous population of V1-MT projecting neurons distributed in layers 3C and 6. In layer 3C, spiny stellate neurons were distributed mainly in foveal representations, while pyramidal morphologies were characteristic of peripheral eccentricities. This primate adaptation of the V1 to MT pathway is arranged in a way that we had not previously understood, with abundant stellate projection neurons in the high-resolution foveal portions, suggesting rapid relay of motion information to visual area MT. We also describe that the medial portion of the inferior pulvinar (PIm), which is the main thalamic input to area MT, shows a retinotopic organization, likely reflecting the importance of this pathway during development and the establishment of area MT topography., Competing Interests: The authors declare no competing interest.

The primate visual brain possesses a myriad of pathways, whereby visual information originating at the retina is transmitted to multiple subcortical areas in parallel, before being relayed onto the visual cortex. The dominant retinogeniculostriate pathway has been an area of extensive study, and Vivien Casagrande's work in examining the once overlooked koniocellular pathway of the lateral geniculate nucleus has generated interest in how alternate subcortical pathways can contribute to visual perception. Another subcortical visual relay center is the inferior pulvinar (PI), which has four subdivisions and numerous connections with other subcortical and cortical areas and is directly recipient of retinal afferents. The complexity of subcortical connections associated with the PI subdivisions has led to differing results from various groups. A particular challenge in determining the exact connectivity pattern has been in accurately targeting the subdivisions of the PI with neural tracers. Therefore, in the present study, we used a magnetic resonance imaging (MRI)-guided stereotaxic injection system to inject bidirectional tracers in the separate subdivisions of the PI, the superior layers of the superior colliculus, the retina, and the lateral geniculate nucleus. Our results have determined for the first time that the medial inferior pulvinar (PIm) is innervated by widefield retinal ganglion cells (RGCs), and this pathway is not a collateral branch of the geniculate and collicular projecting RGCs. Furthermore, our tracing data shows no evidence of collicular terminations in the PIm, which are confined to the centromedial and posterior PI., (© 2018 Wiley Periodicals, Inc.)

Recent evidence demonstrates that the pulvinar nuclei play a critical role in shaping the connectivity and function of the multiple cortical areas they connect. Surprisingly, however, little is known about the development of this area, the largest corpus of the thalamic nuclei, which go on to occupy 40% of the adult thalamus in the human. It was proposed that the nonhuman primate and the human pulvinar develop according to very different processes, with a greatly reduced neurogenic period in nonhuman primate compared to human and divergent origins. In the marmoset monkey, we demonstrate that neurons populating the pulvinar are generated throughout gestation, suggesting that this aspect of development is more similar to the human than first predicted. While we were able to confirm the diencephalic source of pulvinar neurons, we provide new evidence contesting the presence of an additional niche in the telencephalon. Finally, our study defines new molecular markers that will simplify future investigations in the development and evolution of the pulvinar., (© 2018 Wiley Periodicals, Inc.)

An evolutionary hallmark of anthropoid primates, including humans, is the use of vision to guide precise manual movements. These behaviors are reliant on a specialized visual input to the posterior parietal cortex. Here, we show that normal primate reaching-and-grasping behavior depends critically on a visual pathway through the thalamic pulvinar, which is thought to relay information to the middle temporal (MT) area during early life and then swiftly withdraws. Small MRI-guided lesions to a subdivision of the inferior pulvinar subnucleus (PIm) in the infant marmoset monkey led to permanent deficits in reaching-and-grasping behavior in the adult. This functional loss coincided with the abnormal anatomical development of multiple cortical areas responsible for the guidance of actions. Our study reveals that the transient retino-pulvinar-MT pathway underpins the development of visually guided manual behaviors in primates that are crucial for interacting with complex features in the environment., Competing Interests: The authors declare no conflict of interest.

The development of the neocortex requires co-ordination between proliferation and differentiation, as well as the precise orchestration of neuronal migration. Eph/ephrin signaling is crucial in guiding neurons and their projections during embryonic development. In adult ephrin-A2 knockout mice we consistently observed focal patches of disorganized neocortical laminar architecture, ranging in severity from reduced neuronal density to a complete lack of neurons. Loss of ephrin-A2 in the pre-optic area of the diencephalon reduced the migration of neocortex-bound interneurons from this region. Furthermore, ephrin-A2 participates in the creation of excitatory neurons by inhibiting apical progenitor proliferation in the ventricular zone, with the disruption of ephrin-A2 signaling in these cells recapitulating the abnormal neocortex observed in the knockout. The disturbance to the architecture of the neocortex observed following deletion of ephrin-A2 signaling shares many similarities with defects found in the neocortex of children diagnosed with autism spectrum disorder.

The New World marmoset monkey (Callithrix jacchus) has a relatively short gestational period compared with other primates but possesses a retina at a similar stage of maturation by birth. Previous studies have highlighted that the complex fovea of the marmoset undergoes a more rapid postnatal development in comparison with the Macaca monkey, reaching a mature stage earlier than these species. In this current study, we examined the prenatal proliferation profile of cells in the entire retina employing the thymidine analogs and also determined their phenotype by double-label immunocytochemistry using type-specific markers. Akin to other primate species, we demonstrate a centroperipheral gradient in the emergence of both neurons and Müller glia with cones, ganglion cells, and horizontal cells generated first in the fovea at fetal day (Fd)70-74 and with the last generated at the retinal edge at Fd115. Rods, bipolar cells, amacrine cells, displaced amacrine cells, and Müller glia were generated between Fd76 and Fd135 along the same gradient. Similar to foveal development, marmoset neuronal generation was rapid, only taking 51% of gestation whereas in Macaca this takes 81%., (© 2015 Wiley Periodicals, Inc.)

Traditional "textbook" theory suggests that the development and maturation of visual cortical areas occur as a wave from V1. However, more recent evidence would suggest that this is not the case, and the emergence of extrastriate areas occurs in a non-hierarchical fashion. This proposition comes from both physiological and anatomical studies but the actual developmental sequence of extrastriate areas remains unknown. In the current study, we examined the development and maturation of the visual cortex of the marmoset monkey, a New World simian, from embryonic day 130 (15 days prior to birth) through to adulthood. Utilizing the well-described expression characteristics of the calcium-binding proteins calbindin and parvalbumin, and nonphosphorylated neurofilament for the pyramidal neurons, we were able to accurately map the sequence of development and maturation of the visual cortex. To this end, we demonstrated that both V1 and middle temporal area (MT) emerge first and that MT likely supports dorsal stream development while V1 supports ventral stream development. Furthermore, the emergence of the dorsal stream-associated areas was significantly earlier than ventral stream areas. The difference in the temporal development of the visual streams is likely driven by a teleological requirement for specific visual behavior in early life.

Background: Conscious vision is believed to depend upon an intact primary visual cortex (V1), although injury in early life is often accompanied by the preservation of visual capacity, unlike in adulthood. The middle temporal area (MT) receives input from the retinorecipient koniocellular layers of the lateral geniculate nucleus (LGN) and the more recently described medial subdivision of the inferior pulvinar (PIm) of the thalamus, pathways that potentially contribute to preservation of vision after early damage to V1., Results: We examined the potential of these pathways to the long-term preservation of vision after permanent lesions of primate V1 in early and adult life by using a combination of neural tracing and diffusion MRI. We show that early-life V1 lesions lead to less pruning of the retina-pulvinar-MT pathway than is observed in control or adult lesion animals., Conclusions: These findings suggest that sustained visual input through the pulvinar to MT following a lesion of V1 in early life has the capacity to afford improved visual outcomes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Retrograde transneuronal degeneration (RTD) of retinal ganglion cells and dorsal lateral geniculate (LGN) neurons are well described following a lesion of the primary visual cortex (V1) in both Old World monkeys and humans. Based on previous studies of New World monkeys and prosimians, it was suggested that these species displayed no RTD following a lesion of V1. In this study of the New World marmoset monkey, 1 year after a unilateral V1 lesion either in adults or at 14 days after birth, we observed ~20 % ganglion cell (GC) loss in adult but ~70 % in infants. This finding is similar to the RTD previously described for Old World Macaca monkeys. Furthermore, in infants we find a similar amount of RTD at 3 weeks and 1 year following lesion, demonstrating that RTD is very rapid in neonates. This highlights the importance of trying to prevent the rapid onset of RTD following a lesion of V1 in early life as a strategy for improved functional recovery. Despite differences in GC loss, there was little difference between LGN degeneration in infant versus adult lesions. A wedge on the horizontal meridian corresponding to the LGN foveal representation revealed extensive neuronal loss. Retinal afferent input was labeled by cholera toxin B subunit. Input to the degenerated parvocellular layers was difficult to detect, while input to magnocellular and koniocellular layers was reduced but still apparent. Our demonstration that the New World marmoset monkey shares many of the features of neuroplasticity with Old World Macaca monkeys and humans emphasizes the opportunity and benefit of marmosets as models of visual cortical injury.

The hierarchical development of the primate visual cortex and associated streams remains somewhat of a mystery. While anatomical, physiological, and psychological studies have demonstrated the early maturation of the dorsal "where"/"how" or motion cortical stream, little is known about the circuitry responsible. The influence of the retinogeniculostriate pathway has been investigated, but little attention has been paid to the role of two more recently described disynaptic retinothalamic projections to the middle temporal (MT) area, an early maturing dorsal stream cortical field, and which bypass the primary visual cortex (V1). These pathways are via the koniocellular layers of the lateral geniculate nucleus (LGN) and the medial portion of the inferior pulvinar (PIm). Both have been demonstrated in the adult nonhuman primate, but their influence during the maturation of the visual cortex is unknown. We used a combination of neural tracing and immunohistochemistry to follow the development of LGN and PIm inputs to area MT in the marmoset monkey. Our results revealed that the early maturation of area MT is likely due to the disynaptic retinopulvinar input and not the retinogeniculate input or the direct projection from V1. Furthermore, from soon after birth to adulthood, there was a dynamic shift in the ratio of input from these three structures to area MT, with an increasing dominance of the direct V1 afference.

The versatility of perfluorophenyl azide (PFPA) derivatives makes them useful for attaching a wide variety of biomolecules and polymers to surfaces. Herein, a single molecule force spectroscopy (SMFS) study of the concanavalin A/mannose interaction was carried out using PFPA immobilization chemistry. SMFS of the concanavalin A/mannose interaction yielded an average unbinding force of 70-80 pN for loading rates between 8000 and 40,000 pN/s for mannose surfaces on aminated glass, and an unbinding force of 57 ± 20 pN at 6960 pN/s for mannose surfaces on gold-coated glass. Dynamic force spectroscopy was used to determine the dissociation rate constant, k(off), for this interaction to be 0.16 s(-1).

In recent years, the temporal clustering analysis (TCA) method has been introduced to analyze functional MRI (fMRI) data without prior information about the activation patterns or experimental paradigms. It has been successfully applied to situations under which the timing of events of interest is not known. However, useful information regarding the spatial correlation of activation pixels with their neighbors is not taken into account in the original TCA (OTCA) method. In this study, we propose a new method called 'STCA' (spatial-TCA) which incorporates spatial information with the TCA method to improve the sensitivity in detecting the time window. The spatial information is defined as the correlation coefficient of the time activity curve between each pixel and its neighbors. The inclusion of spatial information can effectively reduce the contribution from noisy pixels and enhance the sensitivity. Both simulated data and in vivo fMRI experiments are employed to verify the method. Preliminary results show that the proposed method has increased the sensitivity significantly for in vivo fMRI data in detecting the activation response time as compared to both OTCA and modified TCA (MTCA). The OTCA/MTCA was applied to spatially smoothed data for various contrast-noise ratios and compared to STCA. The SNR improvements of both OCTA/MTCA are obvious but blurring effects are also visible. The STCA does not have this artifact.

Background: The relationship between monochromatic aberrations of the human eye and myopia has been investigated extensively over the past decade or so but the results are inconclusive. Wide age range, narrow refractive error range and large individual variations are common confounding factors in previous studies. To address these shortcomings, we conducted a two-part study. The first part set out to determine whether the monochromatic aberrations in myopic eyes are different from those in the non-myopic eyes in adults. Subjects were drawn from a narrow age band to minimise the confounding effect of age. The second part of this study compared the monochromatic aberrations of the more myopic and less myopic eyes of anisometropes., Methods: Monochromatic aberrations were measured for 5 mm pupils using the Complete Ophthalmic Analysis System (COAS) Wavefront Analyzer in 116 subjects with refractive spherical equivalent between -10.38 D and +1.38 D and in 26 anisometropes. Measurements were done in a dark room with natural pupils. The refractive errors, corneal curvatures and axial lengths of the eyes were measured under natural accommodation., Results: Highly myopic eyes had significantly smaller root mean square (RMS) values of fourth order (p = 0.015) and spherical aberrations (p = 0.009) than non-myopic eyes. The correlation coefficient was 0.2354 (p = 0.011) between fourth-order aberrations and refractive spherical equivalents and 0.2817 (p = 0.002) between spherical aberration and refractive spherical equivalents. Less myopic eyes of the anisometropes showed significantly larger total higher-order, third-order and spherical aberrations than the more myopic eyes (p < 0.05, p < 0.05 and p < 0.01, respectively)., Conclusions: Our study shows that spherical aberration is associated with refractive error. More myopic eyes tended to have smaller amounts of spherical aberration, however, the 'cause or effect' question remains. Longitudinal studies are needed to further investigate the relationship between monochromatic aberrations and refractive development.

Perfluorinated surfactants have emerged as priority environmental contaminants due to recent reports of their detection in environmental and biological matrices as well as concerns regarding their persistence and toxicity. In June 2000, 22000 L of fire retardant foam containing perfluorinated surfactants was accidentally released at L. B. Pearson International Airport, Toronto, ON, and subsequently entered into Etobicoke Creek, a tributary to Lake Ontario. A suite of analytical tools that include liquid chromatography/tandem mass spectrometry (LC/MS/MS) and 19F NMR were employed to characterize fish (common shiner, Notropus cornutus) and surface water samples collected following the discharge of the perfluorinated material. Total perfluoroalkanesulfonate (4, 6, and 8 carbons) concentrations in fish liver samples ranged from 2.00 to 72.9 microg/g, and total perfluorocarboxylate (5-14 carbons) concentrations ranged from 0.07 to 1.02 microg/g. In addition to fish samples, total perfluoroalkanesulfonate (6 and 8 carbons) concentrations were detected in creek water samples by LC/MS/MS over a 153 day sampling period with concentrations ranging from <0.017 to 2260 microg/L; perfluorooctanoate concentrations (<0.009-11.3 microg/L) were lower than those observed for the perfluoroalkane-sulfonates. By 19F NMR, the total perfluorinated surfactant concentrations in surface water samples ranged from < 10 to 17000 microg/L. A bioaccumulation factor range of 6300-125000 was calculated for perfluorooctanesulfonate, based on concentrations in fish liver and surface water. The residence time of perfluorooctanesulfonate in Etobicoke Creek as well as the high bioaccumulation in fish liver suggests that perfluorinated surfactants will persist and bioaccumulate following release into the aquatic environment.

The ubiquitous detection of perfluorooctane sulfonate (PFOS) in humans and animals has produced a need for sensitive and compound-specific analytical methods to determine the environmental distribution of fluorinated organic contaminants. A suite of potential PFOS precursors (sulfonamides) and fluorotelomer alcohols (FTOHs) were separated by gas chromatography and detected by chemical ionization mass spectrometry (GC/CI-MS). Full-scan spectra were collected in both positive and negative chemical ionization (PCI and NCI, respectively) mode to determine retention time windows and fragmentation patterns. In selected ion monitoring (SIM) mode, instrumental detection limits ranged from 0.2 to 20 pg for individual analytes, depending on ionization mode. PCI mode was preferred for routine analysis because of the simple mass spectra produced, typified by the presence of a major molecular ion [M + H]+. High-volume air samplers collected gaseous and particle-bound fluoroorganics on composite media consisting of XAD-2, polyurethane foam (PUF), and quartz-fiber filters. The combined collection efficiency for individual analytes was 87 to 136% in breakthrough experiments. Application of the method to the analysis of ambient air from urban and rural sites confirmed the presence of six novel fluorinated atmospheric contaminants at picogram per meter3 concentrations. Low concentrations of fluoroorganics were consistently detected in blanks (<4 pg m(-3)); however, this did not prevent confirmation or quantification of environmental concentrations.

Perfluorinated surfactants are an important class of specialty chemicals that have received recent attention as a result of their persistence in the environment. Two analytical methods for the determination of perfluorinated surfactants in aqueous samples were developed in order to investigate a spill of 22000 L of fire retardant foam containing perfluorinated surfactants into Etobicoke Creek (Toronto, Ontario). With the first method, aliquots of surface water (0.2-200 mL) were preconcentrated using solid-phase extraction. Liquid chromatography/tandem mass spectrometry was employed for identification and quantification of each perfluorinated surfactant. Total perfluorinated surfactant concentrations in surface water samples ranged from 0.011 to 2270 microg/L, and perfluorooctanesulfonate was the predominant surfactant observed. Interestingly, perfluorooctanoate was detected in surface water sampled upstream of the spill. A second method employing 19F NMR was developed for the determination of total perfluorinated surfactant concentrations in aqueous samples (2-100 mL). By 19F NMR, the surface water concentrations ranged from nondetect (method detection limit, 10 microg/L for a 100-mL sample) to 17000 microg/L. These methods permit comprehensive evaluation of aqueous samples for the presence of perfluorinated surfactants and have applicability to other sample matrixes.

The aim of this study was to determine how oxidized LDL affects endothelin secretion by endothelial cells, monocytes and macrophages. It was found that different degrees of oxidation of LDL had different effects on endothelin production. Extensively oxidized LDL inhibited endothelin secretion from cultured endothelial cells. It also attenuated endothelin secretion from phorbol ester-activated macrophages. The inhibitory effect on endothelin secretion required a substantial degree of LDL oxidation as reflected by an increase in absorbance at 234 nm (conjugated diene) of 0.7 AU with 125 nM LDL and a two- to three-fold increase in migration distance on electrophoresis. Oxidized LDL inhibited thymidine incorporation in porcine aortic endothelial cells, hence in these cells cytotoxicity may account for at least part of the inhibition of endothelin secretion. Acetyl LDL slightly increased basal endothelin release by endothelial cells, but native LDL or mildly oxidized LDL had no significant effect. Overall, the present findings argue against a stimulatory effect of oxidized LDL on endothelin release as contributing to increased vasoreactivity in atherosclerosis. In fact, the apparent inhibition of endothelin release by extensively oxidized LDL might tend to attenuate vasoreactivity near atherosclerotic lesions.

Celiac disease (CD) occurs as a result of an abnormal immune response, within the mucosa of the small bowel, to dietary gliadin peptides. To further characterize the intramucosal lymphocytes in patients with untreated CD, we compared T cell receptor (TCR) variable region gene expression in small bowel biopsies from patients with CD to that of normal small bowel. We also assessed TCR genotypes, using restriction fragment length polymorphisms (RFLPs) spanning the V beta gene locus, comparing 59 CD patients to 64 normals. The abnormal immune response in CD is polyclonal, without evidence of restriction or significantly increased expression of any TCR variable region gene families, compared to normal small bowel. No significant association was found between TCR genotypes, as defined by TCR V beta RFLPs, and CD.

Among the important pleiotropic responses to gamma interferon (IFN-gamma) during the activation of macrophages (M phi) is the increased expression of major histocompatibility complex class II genes. In the present study, infection with Leishmania donovani was shown to inhibit in parallel the induction by IFN-gamma of H-2 A beta gene transcription, class II mRNA accumulation, and H-2 Ad protein expression in cells of the murine macrophage cell line P388D1. Treatment of P388D1 cells with either the adenylate cyclase activator cholera toxin or the protein kinase A activator N6-2'-O-dibutyryl cyclic AMP (dibutyryl cAMP) similarly inhibited the induction by IFN-gamma of class II protein expression, and in parallel with Leishmania infection, cholera toxin inhibited the induction of mRNA for the H-2 A alpha and H-2 A beta proteins. Concentrations of intracellular cAMP were significantly increased in cholera toxin-treated cells but not in leishmania-infected cells. These findings indicate that at least one mechanism by which Leishmania infection attenuates the activation of M phi by IFN-gamma involves selective, transcriptional inhibition of major histocompatibility complex class II genes via a cAMP-independent mechanism.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.

To define further the basis of T cell function in the inductive and effector limbs of the normal intestinal immune system, the capacity of mucosal lymphocytes to produce and use lymphokines and their effects on regulation of Ig production were determined in normal nonhuman primates. Northern blots of RNA from mitogen-activated lamina propria T cells contained more mRNA for IL-2 and IFN-gamma than did mesenteric lymph node T cells. In comparison with lymphocytes from peripheral sites, there was high expression of IL-4 and IL-5 mRNA in both mesenteric lymph node and lamina propria T cells. In studies of lymphokine utilization, T cells from lamina propria had high IL-2-induced but no IL-4-induced proliferative responses. In contrast, mesenteric lymph node T cells had high IL-4-induced and lower IL-2-induced proliferative responses compared with lamina propria T cells. Lamina propria T cells had higher helper activity in PWM-stimulated cultures and exhibited less inhibition by IL-4 than did mesenteric lymph node T cells. These data and previous studies suggest that T cells in an inductive site such as the mesenteric lymph node are a mixed population containing both "naive" cells with low potential for IFN-gamma and IL-2 production and differentiated cells with high potential for IL-4 and IL-5 production. In contrast, the data suggest that T cells in the effector compartment of the lamina propria are comprised primarily of differentiated "memory" cells that produce high levels of IL-2, IFN-gamma, IL-4, and IL-5, have high helper activity, and have a more limited ability to proliferate in response to lymphokines such as IL-4.

A case of kappa light-chain myeloma with pleural involvement is reported. The diagnosis was made by pleural biopsy, pleural fluid cytology, and urine immunoelectrophoresis. A review of the literature on pleural involvement in multiple myeloma revealed that this is the first reported case in patients with light-chain myeloma.

Uptake of cholesterol-containing lipoproteins by macrophages in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. There are a number of possible mechanisms by which macrophages might accumulate cholesterol, and one that has attracted much interest recently involves the uptake of oxidatively modified low density lipoprotein (LDL) via a specific cell surface receptor, termed the scavenger or acetyl-LDL receptor. Previous studies have shown that chemical derivatization of LDL with reagents that result in neutralization of the charge of lysine amino groups also allows recognition by this receptor. As well, it has been shown that oxidation of LDL is accompanied by a decrease in free lysine groups and binding of lipid products to apolipoprotein B. The present studies were done to further characterize the receptor-binding domain on oxidized LDL. It was found that LDL could be modified by incubation with water-soluble products derived from autoxidized unsaturated fatty acids under conditions that inhibited oxidation of the LDL itself. The LDL modified in this way had increased electrophoretic mobility but showed no evidence of the oxidative damage that typifies LDL oxidized by exposure to metal ions. Furthermore, the oxidation product-modified LDL was rapidly degraded by cultured macrophages through the scavenger receptor pathway. Bovine albumin modified by oxidation products also showed greatly accelerated degradation by macrophages. When analyzed by reverse-phase high pressure liquid chromatography, the reactive oxidation products appeared less polar than fatty acids or simple medium-chain aldehydes. When treated with the carbonyl reagent 2,4-dinitrophenylhydrazine, the reactive fractions yielded derivatives, some of which were identified by mass spectrometry as hydrazones of nonenal, heptenal, pentenal, and crotonaldehyde. A series of 2-unsaturated aldehydes (acrolein to 2-nonenal) were all found to modify LDL, but none of these aldehyde-modified LDLs were recognized by the scavenger receptor of macrophages and all were degraded much more slowly by these cells than LDL modified with oxidation products. Furthermore, copper-oxidized LDL had only very slight immunoreactivity toward a panel of antibodies specific for adducts of simple 2-unsaturated aldehydes. Analysis of underivatized autoxidized fatty acids by coupled liquid chromatography/thermospray mass spectrometry revealed compounds with m/z corresponding to M+17, M+31, and 2M+31 in fractions that were capable of modifying LDL. The unoxidized fatty acids showed a dominant peak at M-1. These results indicate that the scavenger receptor of macrophages can recogn

Massive small intestinal resection in the rat results in both structural and functional changes in the residual small bowel. Sodium-dependent D-glucose transport was examined in brush-border membrane vesicles derived from the proximal small bowel mucosa of male Sprague-Dawley rats 2 and 6 wk after a 66% distal jejunoileal resection or jejunoileal transection. Kinetic characteristics for the low-affinity, high-capacity system and high-affinity, low-capacity system were defined with rapid filtration under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Mucosal weight, protein, and deoxyribonucleic acid content were increased in the residual intestinal segment compared to transected controls and morphometric studies revealed increased villus and crypt heights as well as an increased mitotic index. Postresection mean kinetic parameters for D-glucose transport at 2 wk (low-affinity system: Km, 177.5 +/- 45.1 microM; Vmax, 3.73 +/- 0.99 nmol X mg protein-1 X min-1; and high-affinity system: Km, 6.2 +/- 1.9 microM; Vmax, 0.12 +/- 0.06 nmol X mg protein-1 X min-1) and 6 wk (low-affinity system: Km, 267.8 +/- 83.1 microM; Vmax, 0.06 +/- 0.01 nmol X mg protein-1 X min-1; and high-affinity system: Km, 6.5 +/- 1.1 microM; Vmax, 0.06 +/- 0.01 nmol X mg protein-1 X min-1) were similar to values post-transection at 2 wk (low-affinity system: Km, 280.4 +/- 53.7 microM; Vmax, 3.05 +/- 0.32 nmol X mg protein-1 X min-1; and high-affinity system: Km, 9.1 +/- 1.3 microM; Vmax, 0.17 +/- 0.01 nmol X mg protein-1 X min-1) and 6 wk (low-affinity system: Km, 271.7 +/- 17.5 microM; Vmax, 4.69 +/- 0.23 nmol X protein-1 X min-1; and high-affinity system: Km, 10.6 +/- 4.2 microM; Vmax, 0.16 +/- 0.09 nmol X mg protein-1 X min-1). These kinetic data suggest that the hyperplastic response in adapting proximal small bowel after distal resection is accompanied by a persistence of the membrane functional characteristics for both sodium-dependent D-glucose transport systems despite an altered pattern of enterocyte proliferation and differentiation.

Massive small intestinal resection results in both structural and functional changes in the residual small bowel. Sodium-dependent D-glucose transport was examined in brush-border membrane vesicles derived from the terminal 20-30 cm of ileal mucosa of male Sprague-Dawley rats, 2 and 6 wk after 66% proximal jejunoileal resection or jejunoileal transection. Kinetic characteristics for sodium-dependent D-glucose transport were investigated with rapid filtration under conditions of a zero-trans, 100 mM cis-NaSCN gradient. Mucosal weight, protein, and DNA content were increased in the residual terminal intestinal segment compared with transected controls, whereas morphometric studies revealed increased villus and crypt heights as well as an increased mitotic index. Mean kinetic transport parameters at 6 wk after proximal small bowel resection revealed two saturable systems in the distal residual ileum: first, a low-affinity, high-capacity system with a Km of 0.19 +/- 0.03 mM and a Vmax of 0.48 +/- 0.04 nmol.mg protein-1.min-1; and second, a high-affinity, low-capacity system with a Km of 0.009 +/- 0.001 mM and a Vmax of 0.105 +/- 0.016 nmol.mg protein-1.min-1. In contrast, negligible sodium-dependent D-glucose transport was detected in the most distal ileum in control animals or animals 2 wk after resection or 2 and 6 wk after transection. Thus adaptational changes including mucosal hyperplasia and the appearance of two sodium-dependent D-glucose brush-border membrane vesicle transport systems occur in the residual distal intestine after massive proximal small bowel resection.

Trauma, chest surgery or other invasive procedures and underlying lung disease are often found to precede clostridial empyema. A case is described in which empyema caused by Clostridium perfringens was not associated with any of these factors.