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Abstract Background Cytokines secreted by immune cells and activated glia play central roles in both the pathogenesis of and protection from damage to the central nervous system (CNS) in multiple sclerosis (MS). Methods We have used gene array analysis to identify the initial direct effects of cytokines on CNS glia by comparing changes in early gene expression in CNS glial cultures treated for 6 hours with cytokines typical of those secreted by Th1 and Th2 lymphocytes and monocyte/macrophages (M/M). Results In two previous papers, we summarized effects of these cytokines on immune-related molecules, and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. In this paper, we present the effects of the cytokines on molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. Many of the changes in gene expression were similar to those seen in ischemic preconditioning and in early inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), related to ion homeostasis, mitochondrial function, neurotransmission, vitamin D metabolism and a variety of transcription factors and signaling pathways. Among the most prominent changes, all three cytokine mixtures markedly downregulated the dopamine D3 receptor, while Th1 and Th2 cytokines downregulated neuropeptide Y receptor 5. An unexpected finding was the large number of changes related to lipid metabolism, including several suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Using QRT-PCR we validated the results for regulation of genes for iNOS, arginase and P glycoprotein/multi-drug resistance protein 1 (MDR1) seen at 6 hours with microarray. Conclusion Each of the three cytokine mixtures differentially regulated gene expression related to metabolism and signaling that may play roles in the pathogenesis of MS, most notably with regard to mitochondrial function and neurotransmitter signaling in glia.

Abstract Background In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells. Methods To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment. Results In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells. Conclusion Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system.

Background: Cytokines secreted by immune cells and activated glia play central roles in both the pathogenesis of and protection from damage to the central nervous system (CNS) in multiple sclerosis (MS).Methods: We have used gene array analysis to identify the initial direct effects of cytokines on CNS glia by comparing changes in early gene expression in CNS glial cultures treated for 6 hours with cytokines typical of those secreted by Th1 and Th2 lymphocytes and monocyte/macrophages (M/M).Results: In two previous papers, we summarized effects of these cytokines on immune-related molecules, and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. In this paper, we present the effects of the cytokines on molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. Many of the changes in gene expression were similar to those seen in ischemic preconditioning and in early inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), related to ion homeostasis, mitochondrial function, neurotransmission, vitamin D metabolism and a variety of transcription factors and signaling pathways. Among the most prominent changes, all three cytokine mixtures markedly downregulated the dopamine D3 receptor, while Th1 and Th2 cytokines downregulated neuropeptide Y receptor 5. An unexpected finding was the large number of changes related to lipid metabolism, including several suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Using QRT-PCR we validated the results for regulation of genes for iNOS, arginase and P glycoprotein/multi-drug resistance protein 1 (MDR1) seen at 6 hours with microarray.Conclusion: Each of the three cytokine mixtures differentially regulated gene expression related to metabolism and signaling that may play roles in the pathogenesis of MS, most notably with regard to mitochondrial function and neurotransmitter signaling in glia. [ABSTRACT FROM AUTHOR]

Background: Definitive surgical care is often delayed in hypertrophic pyloric stenosis (HPS). Our aim is to evaluate the effect modifiable factors in preoperative HPS management have on efficiency of care., Methods: A retrospective review of all patients undergoing pyloromyotomy for HPS at two US children's hospitals between 2008 and 2018 was performed., Results: 406 patients were included in the study. The majority (310, 76 ​%) were adequately resuscitated and ready for surgery upon diagnosis in the ER. However, only 133 patients (43 ​%) had surgery on the day of admission. Patients diagnosed between 12pm and 6pm were more likely to have surgery the next day than those diagnosed before noon (67 ​% vs 33 ​%, p ​< ​.001), which correlated with a longer length of stay (32 vs 47 ​h, p ​< ​.001)., Conclusion: The majority of patients presenting with HPS can safely undergo same day surgery. Delaying surgery due to an afternoon diagnosis is common, and leads to a modifiable increased total length of stay., Competing Interests: Declaration of competing interest All authors declare that they have no pertinent conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

What is the optimal management of spontaneous pneumomediastinum (SPM) and is there a risk of esophageal perforation in patients with SPM? We performed a retrospective case-control study of children through age 21, diagnosed with SPM in one hospital system over 10 years with the primary aim of describing the diagnostic workup, treatment patterns, and clinical outcomes. We hypothesized that SPM is a self-limited disease and is not associated with esophageal injury. Cases were identified using International Classification of Disease codes and excluded for trauma or severe infections. Median age was 16 years, 66% were male ( n = 179). Chest radiography was performed in 97%, chest computed tomography (CT) in 33%, and esophagrams in 26%. Follow-up imaging showed resolution in 83% (mean = 17.2 days). SPM was not associated with esophageal perforation. We recommend avoiding CT scans and esophagrams unless there is discrete esophageal concern. Management of SPM should be guided by symptomatology., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Recent studies have demonstrated that patients undergoing cervical endocrine surgery could be comfortably discharged with minimal opioid analgesia. However, no study to date has examined the efficacy of limiting administration of opioids intraoperatively. We have developed a novel protocol for patients undergoing cervical endocrine surgery that eliminates perioperative opioids. We sought to determine the efficacy of this protocol and its impact on opioid use at discharge., Methods: We conducted a prospective opt-in opioid-limited surgery program study to opioid-naive patients scheduled for cervical endocrine surgery beginning in August 2019. Postoperatively, nonopioid analgesia was encouraged, but patients were also given a low dose prescription for opioids at discharge. Patients were then matched with 2 retrospective control groups, patients from 2014-2016 and 2017-2018, in order to account for increased public awareness of opioid-prescribing patterns. Primary end points included perioperative opioid use. Secondary end points included postoperative pain scores and complications., Results: 218 patients underwent cervical endocrine surgery with our opioid-limited protocol between August 2019 and February 2020. Nine patients received opioids intraoperatively (4%) and 109 (50%) filled their opioid prescriptions at discharge. Compared to retrospective control groups, the average oral morphine equivalents (OME) administered intraoperatively and prescribed postoperatively were significantly lower ( P < .0001). Pain scores and complication rates were similar in all groups ( P = .7247)., Discussion: Our novel opioid-limited surgery protocol used in conjunction with preoperative counseling is an effective approach for pain control in patients undergoing cervical endocrine surgery and limits opioid exposure throughout the perioperative period.

Purpose: Computed tomography (CT) is currently the standard for evaluation of intra-abdominal injury (IAI) after BAT. Pediatric patients receiving CT scans based on adult clinical protocols are potentially exposed to unnecessary radiation. The purpose of this study is to determine the rate of CT scans before and after implementation of a pediatric BAT decision tool., Methods: We adapted and implemented an evidence-based decision tool for pediatric BAT based on five clinical variables. We reviewed patient charts 18 months pre- and post-implementation. Demographics and outcomes were compared using Chi-square and Fisher's exact test, accordingly., Results: The pre and post-implementation groups were uniform when comparing age, sex, mechanism, and Injury Severity Score. The decision tool was utilized in 85% of patients post-implementation. Fewer CT scans were obtained in the post-implementation group (28 vs. 21%, p = 0.215) with no missed injuries or late diagnoses., Conclusion: Implementation of a pediatric BAT decision tool decreased CT usage and radiation exposure without an obvious compromise to patient care. This experience supports the utilization of these tools for the assessment of IAI after BAT and have resulted in more selective use of CT during pediatric BAT in our program., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: The most common endoleak after endovascular aneurysm repair is type II. Although type II endoleaks (TIIEL) are generally considered benign, there are reports that they can lead to aortic rupture. In this study, we reviewed the effect of TIIEL on sac size change to determine if sac expansion owing to a TIIEL could result in the development of a type IA endoleak (TIAEL)., Methods: After internal review board approval, all aortoiliac endovascular aneurysm repairs performed at a single institution between June 2006 and June 2012 were retrospectively reviewed. Patient demographics, comorbidities, aneurysm diameter, graft type, need for reintervention, and complications were collected. Patients with TIIEL diagnosed on follow-up imaging were categorized as those who underwent intervention for their TIIEL and those who did not. Outcomes were tabulated with attention to sac size change, development of TIAEL, rupture, and survival., Results: Six hundred twenty-seven patients underwent aortoiliac stent graft placement at our institution during this time period. Patients with an operative indication other than nonruptured infrarenal abdominal aortic aneurysm and those without preoperative computed tomography angiography or follow-up data available for review were excluded. The total number of patients included was 389 with an average follow-up of 58.8 months (range, 0-194 months). Follow-up imaging diagnosed 124 patients with TIIEL (32%). Patients with TIIEL were significantly older (P < .0001) and more likely to be hypertensive (P < .05) but less likely to be smokers (P = .01). They had a significantly larger sac size increase than patients without TIIEL (9.50 vs -0.78 mm; P < .0001). Those with TIIEL were significantly more likely to develop a TIAEL than patients who did not have TIIEL (14% vs 5%; P = .004), but the rate of rupture was not significantly different (4% vs 2%; P = .33). In those with a TIIEL, the average sac size increase at which TIAEL developed was 13 mm. Patients in the TIIEL group who underwent intervention for their TIIEL survived significantly longer than patients who did not undergo intervention (140 months vs 100 months; P = .004)., Conclusions: Our data suggest that there is an increased incidence of late TIAEL in patients with TIIEL compared with those without a TIIEL. Our study also demonstrates an increased overall survival in TIIEL patients who underwent intervention. Future studies are necessary to better define the association between TIIEL with enlarging sac and the development of TIAEL. However, it is reasonable to conclude that intervention for TIIEL should be undertaken at or before a cumulative sac size increase of 13 mm., (Copyright © 2020 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Investigating molecular mechanisms involved in the formation of carotid atherosclerotic plaques has been challenging. Isolating high-quality RNA from plaque tissue can be difficult because of acellularity, calcification, and degradation. It is essential that the mRNA isolated from this tissue preserves and reflects the actual relative gene expression. Two common methods for RNA preservation, snap-freezing and stabilizing reagent, were compared using surgically resected human carotid atherosclerotic tissue. In addition, isolation methods were compared for integrity and quantity: column-based extraction, phenol-based extraction, and a combination of the two. We found that using a stabilizing reagent with column filtration resulted in the lowest yield and quality. Phenol-based extraction resulted in higher yields but also increased fragmentation. Snap-frozen tissue coupled with column-based extraction yielded the highest quality. The higher quality and quantity RNA obtained when processing snap-frozen tissue with column-based extraction make it possible to use difficult sample types for molecular downstream applications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inflammatory mediators, including cytokines, contribute to neuronal and axonal dysfunction and cell death. To examine the roles of cytokines in pathogenesis and regeneration in the central nervous system (CNS), we analyzed effects of cytokines on early gene regulation (6h) in neuronal cultures, employing gene arrays. Our hypothesis is that neuronal gene expression is differentially regulated in vitro by cytokine mixtures typical of Th1 and Th2 T cells and monocytes/macrophages (M/M). Th1 and M/M cytokines showed similar patterns for regulation of numerous pathways including cytokine-receptor interactions, MAP kinase, toll like receptors, apoptosis, PPAR signaling, cell adhesion molecules (CAMS), antigen processing, adipocytokine, and JAK-STAT signaling. M/M cytokines uniquely regulated genes in T cell, B cell and ECM receptor signaling pathways. Th2 cytokines had few effects on pathways regulated by Th1 and MM cytokines, but uniquely regulated genes related to neuroactive ligand-receptors and calcium. Th1 and MM cytokines markedly upregulated a wide array of cytokine-related genes. Notably, M/M cytokines uniquely upregulated G-CSF, GM-CSF, CXCL5 and lymphotactin (Xcl1). Th2 cytokines did not upregulate cytokine-related genes, with the exception of CCL11 and FMS-like tyrosine kinase 1, a VEGF receptor. In neuroactive ligand-receptor pathways, Th1 and M/M cytokines upregulated gene expression for tryptophan hydroxylase. Th1 cytokines upregulated gene expression for GABA A receptor, delta, while Th2 cytokines downregulated GABA A receptor, gamma 3. Significant changes occurred in several genes in the wnt and Notch signaling pathways, which are highly conserved and play critical roles in neuronal and glial differentiation. In the ubiquitin-proteasome pathway, proinflammatory cytokine mixtures induced upregulation of several genes, notably ubiquitin D (Ubd/FAT10), ubiquitin ligase and several proteasomal proteins. In agreement with microarray results, QRT-PCR showed marked upregulation of gene expression for Ubd with Th1 and M/M, for transglutaminase 2 with M/M, and for arginase 1 with Th2 cytokines. Expression of Ubd in the nervous system has not been previously reported. Both message and protein for Ubd are expressed in neurons, and upregulated by pro-inflammatory cytokines. Transglutaminase 2 has been implicated in neurodegenerative diseases, and proposed as a therapeutic target. Upregulation of arginase by Th2 cytokines could be potentially neuroprotective by decreasing NO generation and enhancing neurite outgrowth. Our analysis of changes in neuronal gene expression at the time of initial exposure to an abnormal cytokine milieu provides the opportunity to identify early changes that could be reversed to prevent later irreversible neuronal damage and death in multiple sclerosis and other CNS diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) lesion formation is modulated by cytokines secreted within the central nervous system (CNS). Th1 lymphocytes and monocyte/macrophages (MM) likely induce lesion formation, whereas Th2 lymphocytes may inhibit formation. To explore the role of cytokines in MS lesions, we used gene arrays to investigate effects of cytokines representative of Th1 and Th2 cells and M/M on gene expression in cultured CNS glia; at 6 hr, all three increased expression of the interleukin-6 (IL-6) gene and decreased expression of the leptin receptor gene (obr), which mediates IL-6 production and other inflammatory responses. However, expression of a closely related gene, the interleukin-6 signal transducer or gp130 (IL-6st), showed no changes at 6 hr. IL-6st is an essential component of receptor complexes for IL-6 and other cytokines and growth factors that play critical roles in CNS inflammation, protection, and/or regeneration. To analyze expression of IL-6st and leptin receptor over time, we incubated rat CNS glial cultures for 6 hr to 5 days with the cytokines. All three cytokine mixtures down-regulated both IL-6st and leptin receptor mRNA and protein for up to 5 days. Immunocytochemical staining showed expression of both IL-6st and leptin receptor in all three types of glia, with lower IL-6st expression by 3 days. Down-regulation of IL-6st and leptin receptor in glia by cytokines could lead to decreased signaling by the proinflammatory IL-6 and reduced responses to regenerative/protective growth factors such as leukemia inhibitory factor and ciliary neurotrophic factor, potentially affecting the disease course in MS., ((c) 2009 Wiley-Liss, Inc.)

Understanding the regulation of cell death pathways is critical for protecting myelin-producing cells and their associated axons during injury resulting from multiple sclerosis and other degenerative diseases. The immortalized N20.1 oligodendroglial cell line provides a useful model for identifying mechanisms that can be exploited to attenuate cell death in myelin-producing cells and their precursors. In our hands, the N20.1 cell line exhibits different characteristics and morphology depending on temperature (permissive or non-permissive) and the presence of cAMP-elevating agents (Studzinski et al. [1998] Neurochem. Res. 23:435-441; Boullerne et al. [1999] J. Neurochem. 72:1050-1060; Studzinski et al. [1999] J. Neurosci. Res. 57:633-642). Our laboratory previously observed that NO donors cause primarily necrotic death in N20.1 cells grown at permissive temperature, but the NO donor SNP switched a portion of cell death to the apoptic pathway. We have continued our study of apoptotic death in these cells by comparing the effects of staurosporine, a known apoptotic agent, on cells grown at the permissive temperature ("undifferentiated") vs. the non-permissive temperature in the presence of forskolin ("differentiated"). Undifferentiated N20.1 cells exhibit maximal cell death after 24 hr of exposure to 50 nM staurosporine, whereas differentiated cells show delayed cell death, with maximal death seen after 48 hr. Pyknotic nuclei were observed in both growth conditions; however, differentiated cells were protected by caspase inhibitors, whereas undifferentiated cells were not. Increased ssDNA staining and DNA laddering were found following 24-hr staurosporine treatment in the differentiated cells only. These results support the conclusion that N20.1 cells can switch from necrotic to apoptotic cell death when cell division is slowed and cyclic AMP is elevated., (Copyright 2001 Wiley-Liss, Inc.)

Regulation of intracellular Ca(2+) (Ca(i)) plays a central role in cell survival, proliferation, and differentiation. We previously reported that immature oligodendroglia (OLs) are less susceptible than mature OLs to cell death following increases in Ca(i) (Benjamins and Nedelkoska [1995] Neurochem. Res. 21:471-479). The N20.1 murine OL cell line provides a model of an intermediate stage of OL maturation in which to study responses to Ca(i) increases with regard to viability, as well as the expression of mRNAs for myelin basic protein (MBP), proteolipid protein (PLP), DM-20, SCIP, and the immediate early genes ZIF268, c-fos, and c-jun. Cells were treated with the calcium ionophore A23187 or thapsigargin for 1, 3, and 18 hr. A23187 at 1.0 microM had no significant effect on cell detachment or death, whereas thapsigargin at 1.0 microM slightly increased both. With both agents, SCIP, MBP, and PLP mRNA levels were unaffected by 3 hr, but markedly reduced after 18 hours. DM-20 mRNA levels remained unchanged at both time points. With both agents, ZIF268, c-fos, and c-jun mRNA levels were unaffected after 1 hr; c-jun mRNA levels showed a significant increase after 3 hr of thapsigargin treatment. Thus, in N20.1 cells, increased calcium affects the IEG c-jun first, SCIP is coordinately decreased with MBP and PLP mRNAs at a later time point, and DM-20 message is under different regulation than PLP. J. Neurosci. Res. 57:633-642., (Copyright 1999 Wiley-Liss, Inc.)

The N20.1 immortalized cell line has several characteristics of differentiating oligodendrocytes (OLs), including expression of the glycolipids galactocerebroside (GalC) and sulfatide, and the myelin proteins CNPase and myelin basic protein (MBP) (1,2). Addition of 1-100 microM forskolin to elevate cyclic AMP (cAMP) levels changed cell morphology from irregular and flattened to a more rounded birefringent cell with multiple branched processes. GalC and sulfatide were detected immunocytochemically after permeabilization in the untreated cells and levels appeared to increase slightly following exposure to forskolin. Further analysis showed that most of the glycolipid was internal, with virtually no detectable levels on the cell surface in untreated cells and a very slight change following treatment with forskolin. Synthesis of the two lipids as measured by [H3]galactose incorporation doubled within 24 hours of treatment with forskolin. Levels of message for UDP-galactose: ceramide galactosyl transferase (CGT), a key enzyme in the synthesis of GalC and sulfatide, were compared with those of MBP and proteolipid protein (PLP), before and after elevation of cAMP. No changes were observed in levels of mRNA for CGT and PLP after 24 hours, with a possible increase by 48 hours. In contrast, levels of MBP message dropped precipitously by 24 hours; this was accompanied by an increase in levels of message for suppressed cAMP-inducible POU (SCIP). Thus CGT transcription is regulated independently of MBP and SCIP in N20.1 cells. Analysis of MBP levels by immunocytochemistry and Western blot showed little or no change in protein levels at 24 and 48 hours, in contrast to the sharp decrease in message levels by 24 hours, indicating a relatively long half life for MBP in this cell line. Thus, the N20.1 cells are an informative model for examining regulation of expression of myelinotypic proteins and GalC, as well as the transport of this lipid to the plasma membrane.

Membrane fractions and chloroform-methanol (C-M) extracts of jimpy (jp) and normal CNS at 17-20 days were examined by immunoblot and sequence analysis to determine whether myelin proteolipid protein (FLP) or DM-20 could be detected in jp CNS. No reactivity was detected in jp samples with several PLP antibodies (Abs) except with one Ab to amino acids 109-128 of normal PLP. Proteins in the immunoreactive bands approximately 26 M(r) comigrating with PLP were sequenced for the first 10-12 residues. A sequence corresponding to PLP was found in normal CNS, as expected, but not in the band from jp CNS. Our results provide no evidence for an aberrant form of PLP in jp CNS at 17-20 days. This and other studies suggest that the abnormalities in jp brain are not due to toxicity of the mutant jp PLP/DM-20 proteins. Interestingly, a sequence identical to the amino terminus of the mature proton channel subunit 9 of mitochondrial F0 ATPase was detected in the immunoreactive bands approximately 26 M(r) in both normal and jp samples. This identification was supported by reactivity with an Ab to the F0 subunit and by labeling with dicyclohexylcarbodiimide (DCCD). In contrast to PLP isolated from whole CNS, PLP isolated from myelin was devoid of F0 subunit 9 based on sequence analysis and lack of reactivity with an Ab to the F0 subunit, yet still reacted with DCCD. This finding rules out the possibility that contaminating F0 ATPase gives rise to the DCCD binding exhibited by PLP and confirms the possibility that PLP has proton channel activity, as suggested by Lin and Lees (1,2).

Immunocytochemistry and thymidine autoradiography were combined to determine the time elapsed between cell division and the expression of proteolipid protein (PLP) in individual oligodendrocytes in normal mouse brain. In jimpy (jp) brains, autoradiography was used to determine the time elapsed between cell division in an individual oligodendrocyte and evidence of cell death. Oligodendrocytes in normal mouse brain do not express PLP until 72 h after a single injection of [3H]-thymidine. In contrast, oligodendrocytes in jp brains begin to die within 9-11 h after an injection of thymidine. The jp mouse is one of several X-linked, hypomyelinated mutants in which a defect has been demonstrated in the gene coding for PLP. It has been presumed that the lack of this protein in the myelin sheath is responsible for the jp phenotype. However, the present study shows that individual jp oligodendrocytes begin to die long before they would normally have synthesized detectable levels of PLP. Therefore, it seems unlikely that the death of jp oligodendrocytes is due to the absence of PLP in myelin sheaths. Oligodendrocyte death and other early jp abnormalities may be due to the presence of abnormal PLP message which may interfere with glial differentiation. Alternatively, the PLP message may code for another protein which is important for normal development of neuroglia.

We have measured levels and synthesis of proteolipid protein (PLP) and its transport into myelin in female mice heterozygous for the jimpy gene and in their normal female littermates. In both cord and cerebrum, jimpy carriers show deficits in PLP during development followed by compensation in adulthood. Recovery of PLP occurs earlier in cord than in brain. At 13 days levels of PLP in carriers compared to controls are reduced to 0.60 and 0.44, respectively, in cord and cerebrum. By 100 days, normal levels of PLP are attained in cord (1.13) whereas levels of PLP in cerebrum are only 0.78 of control. By 200 days full recovery occurs in cerebrum, with a ratio of 1.21, suggesting a possible over-compensation. The yield of myelin from cerebrum was reduced to 0.78 in carriers compared to controls at 17 days. In brain slices, incorporation of [3H]leucine into homogenate PLP from carriers is the same as in controls, whereas [3H]leucine incorporation into myelin PLP is reduced to 0.68 of control. These results indicate that synthesis of PLP in the carriers is normal at 17 days, but transport of PLP into myelin is reduced. Similarly, acylation of homogenate PLP is normal, whereas acylation of myelin PLP is reduced, as measured by incorporation of [3H]palmitic acid. Transport of PLP into myelin was compared to transport of MBP; transport of both proteins was equally decreased as indicated by the similar ratio of labeled PLP to MBP in myelin from carriers compared to noncarriers.(ABSTRACT TRUNCATED AT 250 WORDS)

High titer antibodies primarily of the IgG class were produced against galactocerebroside (GalC) by including keyhole limpet hemocyanin (KLH) and supplemental M. tuberculosis in the adjuvant mixture used for immunization of rabbits. Antibody titers were determined by an ELISA in which microtiter wells were coated with liposomes containing lecithin, cholesterol and GalC. The antibodies showed reactivity with GalC and psychosine, but not glucocerebroside, sulfatide, mixed gangliosides or asialo GM1. Specificity was further demonstrated by absorption of antibodies with GalC. Binding was inhibited by galactose, but only at high concentrations. Further, the antibodies did not bind to any brain proteins on immunoblots, indicating lack of reactivity with glycoproteins which might contain a terminal galactose. Antibodies to GalC are directed against different determinants than those reacting with peanut agglutinin since the lectin will not react with GalC, and the antibodies will not react with asialo GM1. The antibodies raised to GalC by this method show specific staining for oligodendroglia in culture. Peanut agglutinin binds intensely to process-bearing GalC+ oligodendroglia, but very poorly to the membrane sheets elaborated by oligodendroglia after longer times in culture. Other process-bearing GalC-, GFAP- cells were also stained with peanut agglutinin; these cells may represent glial precursors.

Brain and spinal cord of female mice heterozygous for the jimpy gene were analyzed during development for activity of ceramide galactosyl transferase (CGT) and for levels of myelin basic protein (MBP). CGT activity was low at 13-14 days in brains of heterozygous jimpy females but showed normal levels by 31-36 days, in agreement with our earlier study of this enzyme. In cord, CGT activity was normal or slightly above normal at all ages studied, from 13-14 days into adulthood. In both brain and cord, decreased levels of MBP were observed at 13 days; by 100 days, amounts of MBP approached normal levels. Proven female carriers of the jimpy gene also showed normal levels of CGT activity, MBP, and isolated myelin at 200-250 days of age in both brain and cord. These biochemical findings agree with previous morphologic measurements in cord demonstrating deficits in myelin at early ages but compensation by 100 days. Our results show that compensation occurs earlier in cord than in brain and that levels of MBP show a closer correlation than CGT activity with amounts of myelin, as measured by either morphometric analysis or direct isolation.