bluuebunny/crossref_metadata_2025 huggingface dataset:
C‐reactive protein binds to immune complexes
Authors: Agrawal Alok , Radha Hima B , Cropsey Christopher L , Suresh Madathilparambil V , Singh Sanjay K
Published: June 2021

C‐reactive protein (CRP) has been shown to bind to Fcγ receptor IIa (also known as CD32). CD32 is present on the cells of the immune system and is the receptor for IgG‐containing immune complexes. In this study, we explored CRP‐CD32 interaction in an ELISA‐based solid‐phase binding assay using recombinant CD32‐coated plates. CRP bound to CD32 in a dose‐dependent manner. Surprisingly, when CD32 was blocked with anti‐CD32 IgG antibody, the binding of CRP to CD32 was not inhibited, suggesting a possible interaction between CRP and CD32‐anti‐CD32 complexes. CRP also bound to immobilized IgG when the plates coated with either normal human IgG or anti‐BSA IgG were used in the assays. The inability of the binding of a mutant form of CRP to immobilized IgG further showed the specificity of CRP‐IgG interaction. CRP also bound to immune complexes prepared from BSA and anti‐BSA antibody, indicating that the presence of the antigen did not affect the binding of CRP to IgG. CRP, however, did not bind to immune complexes in the fluid‐phase. These data suggest that CRP may also bind to immune complex‐occupied CD32‐bearing cells and may modulate intracellular signaling triggered by the binding of immune complexes to CD32. Our findings have implications for the functions of CRP in the antibody‐mediated clearance of pathogens and in immune complex‐mediated pathologic conditions.
Th2 cell differentiation first occurs in the T:B zone of the lymph node and is associated with specific innate immune cell populations
Authors: Rozo Cristina T , Liu Qian , Gause William
Published: June 2021

The cell populations and molecules involved in triggering the development of Th2 cells remain uncertain. Our previous studies have shown that in vivo Th2 cell differentiation occurs in the absence of IL‐4 production by non‐T cells. Using in situ cytokine detection methodologies and laser microdissection, transferred DO11.10 Th2 cell differentiation was tracked in IL‐4 KO recipient mice and the T:B zone of the draining lymph node was identified as the microenvironment where in vivo Th2 cell differentiation first occurs as early as 3–4 days after intracutaneous N. Brasiliensis inoculation. To examine in vivo what innate immune cell populations contribute to the development of IL‐4 producing T cells in the T:B zone microenvironment, antigen presenting cells were identified with specific antibodies that detect Ag complexed to MHCII and with DQ‐OVA, which fluoresces following Ag processing. We observed that under Th2 conditions, interstitial DCs present Ag and that DX5+ basophils accumulate as early as 12 hours after immunization, but do not present Ag. Further analysis of basophils is currently being conducted to determine their contribution to the development of the Th2 response.
Modulation of IFN‐γ‐induced gene expression by TLR2 signaling in macrophages is mediated by an NF‐κB‐dependent mechanism
Authors: Benson Sarah , Grabenstein Jens , Ernst Joel
Published: June 2021

Mycobacterium tuberculosis (Mtb) uses multiple mechanisms to avoid destruction by the immune system. We have previously shown that Mtb can inhibit selected macrophage responses to IFN‐γ using TLR2‐dependent and –independent mechanisms. To address the role of TLR2 signaling in mediating this inhibition, we used microarray analysis to identify IFN‐γ‐inducible genes whose expression decreased when macrophages were treated with the specific TLR2/1 ligand Pam3CSK4 (Pam). Four of the genes identified—Ciita, Klrk1, Dnase1l3, and Cxcl11—were further characterized. Ciita and Klrk1 induction by IFN‐γ was rapidly inhibited by Pam while Dnase1l3 and Cxcl11 required prolonged Pam pretreatment prior to IFN‐γ. Histone H4 acetylation at the Ciita and Klrk1 promoters was reduced in macrophages treated simultaneously with Pam and IFN‐γ compared to those treated with IFN‐γ alone. This effect was dependent on histone deacetylase activity. Treatment with the NF‐κB inhibitor parthenolide restored IFN‐γ‐induced gene expression and histone H4 acetylation at the promoters of selected Pam‐inhibited genes including Klrk1 and Cxcl11. These results indicate that TLR2‐dependent inhibition of IFN‐γ‐induced gene expression by Mtb depends on the length of exposure to a TLR2 agonist, is mediated by a mechanism affecting chromatin remodeling, and involves multiple signaling pathways including NF‐κB.

NIH‐R01‐AI46097
Cell‐Based Microfluidic Platform – Showing A Cure For Sepsis?
Authors: Fitzgerald Kate , Lowes Damon , Kashanin Dmitry , O’Dowd Frank , Galley Helen , Williams Vivienne
Published: June 2021

Several studies have recognised the benefit of patients receiving statin therapy in reducing the incidence of sepsis in ICU patients. The potential protective effect of statin therapy reducing the incidence of sepsis is probably due to the immunomodulatory mechanisms of these compounds that are capable of inhibiting the inflammatory pathways. LFA‐1 is an integrin that is critical for T‐cell adhesion, T‐cell priming and cytokine secretion. Statins inhibit LFA‐1 interaction with ICAM by either directly binding to a novel site that is distant from the ICAM binding site (not pravastatin) or inhibit the prenylation of small GTPases. Our results have shown that physiologically relevant concentrations of fluva‐, meva‐, lova‐, simva‐ or pravastatin markedly inhibit T‐cell adhesion to ICAM equally, irrespective of direct LFA‐1 binding. Therefore, the reported clinical outcome of septic patients receiving statin therapy can be at least be partially associated with T‐cell‐ICAM binding activities. In conclusion the Cellix Microfluidic Platform allows for T‐cell adhesion to be accurately determined using physiological shear stress with physiologically relevant concentrations of statins.
Effect of PBMC shipment and storage conditions on ELISpot activity
Authors: Dryga Sergey A , Moskowitz Keith A , Moshkoff Dmitry A , Jeffrey Lisa , Manak Mark
Published: June 2021

INTRODUCTION: Cellular immune response is often evaluated on frozen Peripheral Blood Mononuclear Cells (PBMC). Here, we evaluate the effect of shipping and storage conditions on PBMC viability and functional response using ELISpot assay.

METHODS: Cryopreserved human PBMC were evaluated after overnight placement on dry ice and subsequent storage at −80oC, −20°C, in LN2 and on dry ice for up to 6 months. Cell count and viability was measured at time of thaw and after 24 hour rest. ELISpot assays were performed using huIFN‐γ ELISpot Kit (SeraCare) after stimulation with PHA, CMV and CEF peptide pools.

RESULTS: Cells stored overnight on dry ice showed no significant decrease in viability at thaw and post rest. Cells stored at −20°C were not suitable for ELISpot assay. Cells stored on dry ice, at −80°C or in LN2 for 6 month demonstrated 92–93% viability upon thaw and 80–86% after 24 hr rest. The control cells remained 90% viable both post thaw and post rest. ELISpot responses of cells stored at −80°C or in LN2 for 6 month, or cells stored on dry ice for 4 weeks were comparable to control.

CONCLUSIONS: The PBMC shipped in dry ice can be stored at −80°C for up to 6 months or on dry ice for 4 weeks with no appreciable loss of viability or functionality. Therefore, the use of PBMC for ELISpot proficiency testing is possible even in laboratories which lack ultra‐cold storage facilities.

SUPPORT: This work was supported by NIAID contract NO1‐AI‐85341.
Associated Factors in diabetes prevalence in Mexican elderly people
Authors: Ruán Ma del Carmen Morales , Levy Teresa Shamah , Hernández Salvador Villalpando , Aguilar Alejandra Jimenez
Published: June 2021

The prevalence of type 2 diabetes (TTD) is very high in Mexico especially in elderly population and its complications represent high expenditures in health care and reduction in their quality of life. The objective of this investigation is to assess some risk factors associated to TTD in a probabilistic sample of Mexican 60 yr. an over from the Mexican National Health and Nutrition Survey 2006 (ENSANUT 2006).

A health questionnaire including information about previous diagnostic of TTD made by a doctor or other health personnel, weight, height, waist circumference, blood pressure measurements and capillary blood cholesterol determinations in a sub sample 1568 individuals were performed. The overall prevalence of TTD by previous diagnostic was 17.8% (women 19.6 %, men 5.4 %). In a logistic regression model the risk for TTD increased with Factors waist circumference > 90 cm (OR 1.6, CI95% 0.9, 2.6), to live in an urban community (OR 3.0, CI95% 2.0, 4.6) and cholesterol ¡Ý 240 mg/dL (OR 2.8 CI95% 1.4, 5.8).

Nutritional intervention to maintain ideal weight, a waist circumference under 90 cm and adequate blood cholesterol concentrations, are essential in this population to guarantee a good quality of life in this elderly population.
Down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) and ABCG1 expression by unsaturated fatty acids in vivo and in vitro
Authors: Park Young‐Ki , Rasmussen Heather E , Ehlers Sarah J , Ku Chai Siah , Jesch Elliot D , Carr Timothy P , Lee Ji‐Young
Published: June 2021

Regulatory roles of fatty acids (FA) in the expression of ATP binding cassette transporter A1 (ABCA1) and ABCG1 were investigated in vivo and in vitro. Male C57BL/6 mice were fed a modified version of the NIH‐07 diet enriched with palm, sheanut, high oleic acid sunflower or safflower oil (15% fat, 1% Cholesterol by wt) for 4 weeks. The hepatic ABCA1 protein was significantly lowered in the animals fed sheanut, sunflower and safflower oil compared with chow‐fed animals with no changes in mRNA level. HepG2 cells and RAW 264.7 macrophages were incubated with 16:0, 18:0, 18:1, 18:2, 18:3 or EPA (100 μM, 2.5:1 of FA to BSA molar ratio) without or with TO901317, a liver X receptor (LXR) agonist. In HepG2 cells, ABCA1 mRNA was not altered by FA without TO901317, but with the agonist, polyunsaturated FA significantly increased ABCA1 mRNA compared with control. In discordance with mRNA, ABCA1 protein was diminished by unsaturated FA both without and with TO901317. In RAW 264.7 macrophages, ABCA1 and ABCG1 mRNA levels were significantly lowered by unsaturated FA without TO901317 whereas the effect of unsaturated FA was abolished in the presence of the agonist. However, unsaturated FA decreased protein levels for ABCA1 and ABCG1 regardless of TO901317. The data suggest that unsaturated FA reduce the expression of ABCA1 and ABCG1 in the liver and macrophages transcriptionally or post‐transcriptionally depending on the site of regulation.
Effect of copper deficiency on intestinal gene expression in beef cattle
Authors: Rilington Valencia D , Hansen Stephanie L , Hicks Julie A , Spears Jerry W , Liu Hsiao‐Ching
Published: June 2021

Copper (Cu) deficiency has been shown to impact iron (Fe) metabolism. We compared global gene expression in the duodenum of Cu‐deficient (‐Cu) and Cu‐adequate (+Cu) calves. Calves were born to cows that received −Cu or +Cu diets for 410 days prior to calving. After weaning calves remained on their respective treatments for 275 days. Liver and plasma Cu concentrations indicated that −Cu calves were severely Cu deficient. An oligonucleotide array containing 8,400 bovine mRNAs was used to determine gene expression changes due to Cu deficiency. Expression changes of genes involved in Fe metabolism were of particular interest, given that Cu and Fe metabolism are closely linked. Total RNA was extracted from duodenum mucosal scrapings of Cu+ and Cu− calves. Cy5 and Cy3 labeled cDNA was synthesized to probe three oligonucleotide arrays. A dye swap was also performed to control for dye bias. Arrays were probed overnight and scanned on a ScanArray 4000. Preliminary t‐test results indicated a differential gene expression between −Cu calves and +Cu calves, including genes involving cellular transport, transcription and translation. Interestingly genes involved in Fe metabolism did not demonstrate statistically significant expression changes. This result suggests that perhaps the major link between Cu and Fe metabolism is not at the gene expression level but rather further downstream in the metabolic pathway.
SBP2 binding affinity is a major determinant in differential selenoprotein mRNA translation and sensitivity to nonsense‐mediated decay
Authors: Squires Jeffrey E
Published: June 2021

Selenoprotein mRNAs are potential targets for degradation via nonsense‐mediated decay due to the presence of in‐frame UGA codons that can be decoded as either selenocysteine or termination codons. When UGA decoding is inefficient, as occurs when selenium is limiting, termination occurs at these positions. Based on predicted exon‐intron structure, fourteen of the 25 human selenoprotein mRNAs are predicted to be sensitive to nonsense‐mediated decay. Among these, sensitivity varies widely, resulting in a hierarchy of preservation or degradation of selenoprotein mRNAs and thus, of selenoprotein synthesis. Potential factors dictating the hierarchy of selenoprotein synthesis are the SECIS RNA‐binding proteins, SBP2 and nucleolin. To investigate the mechanistic basis for this hierarchy and the role of these two proteins, we carried out knockdowns of SBP2 expression and analyzed the effects on selenoprotein mRNA levels. We also investigated in vivo binding of selenoprotein mRNAs by SBP2 and nucleolin, via immunoprecipitation of the proteins and quantitation of bound mRNAs. We report that SBP2 exhibits strong preferential binding to some selenoprotein mRNAs over others, whereas nucleolin exhibits minimal differences in binding. Thus, SBP2 is a major determinant in dictating the hierarchy of selenoprotein synthesis via differential selenoprotein mRNA translation and sensitivity to nonsense‐mediated decay.
Soy Components Genistein And Lunasin Regulate E‐Cadherin And Wnt Signaling In Mammary Epithelial Cells
Authors: Su Ying , de Lumen Ben O , Simmen Rosalia CM
Published: June 2021

Enhanced Wnt/Î²‐catenin signaling and loss of E‐cadherin expression are considered hallmarks of tumorigenesis. We previously showed by microarray gene profiling that dietary intake of soy‐based AIN‐93G diets altered components of Wnt/Î²‐catenin signaling in rat mammary epithelial cells. To further evaluate the molecular effectors, biological consequence, and mechanism(s) underlying dietary soy effects, we utilized the mouse mammary epithelial cell line HC11 treated with the soy isoflavone genistein and the soy peptide lunasin at physiologically relevant doses. Genistein (40 nM) inhibited basal and Wnt1‐activated cell proliferation after 7 days of treatment. Further, genistein increased Î²‐catenin and E‐cadherin protein levels and formation of membrane E‐cadherin/Î²‐catenin complexes. Lunasin (1 Î¼M) induced E‐cadherin gene and protein expression, but had no effect on membrane E‐cadherin/Î²‐catenin complex formation. Cells treated with Wnt1(10ng/ml) had increased nuclear Î²‐catenin localization and expression of its target genes cyclin D1 and c‐myc, relative to the controls. Genistein completely abolished Wnt1‐mediated induction of these genes. Results implicate genistein and lunasin in the regulation of E‐cadherin and Wnt/Î²‐catenin signaling in mammary epithelial cells, and provide a mechanism by which dietary soy protects against mammary tumors.
Anti‐inflammatory activity of extracts of Thai spices and herbs
Authors: Tuntipopipat Siriporn , Failla Mark L.
Published: June 2021

Many spices and herbs contain a variety of compounds with anti‐oxidant and anti‐inflammatory activities. The aim of this study was to assess the anti‐inflammatory activity of spices/herbs that are common ingredients in Thai dishes. Tissues of 13 spices/herbs were processed according to their use in habitual Thai diets. All plant tissues except Roselle and Chili were extracted with ethanol; Chili was extracted with hexane: acetone (2:1), while Roselle was extracted with 70% acetone. Extracts were tested by monitoring inhibition of LPS‐induced nitric oxide synthase (NOS) activity and TNF‐α production in RAW 264.7 cells. Seven extracts inhibited NOS activity and TNF‐α production in a dose‐dependent manner without exerting cytotoxicity. Limnophila aromatica (Pak Kyeng) extract exhibited greatest potency suppressing NOS activity, followed by Dill, Kaffer Lime, Pak Tiew, Sweet Basil, Mint and Devil's Fig, respectively, (range of IC50 = 8.8 to 55.2 ug/mL). Pak Kyeng also showed the greatest inhibition of TNF‐α production (IC50 = 8.1 ug/mL); Mint extract rather than Devil's Fig extract was least potent (range of IC50 =121.5 ug/mL). IC50 values for NOS activity and TNF‐α production in LPS‐activated RAW264.7 cells for these extracts were highly correlated (r = 0.964, p <0.01). These results support the use of NOS activity in RAW264.7 cells as a cost effective tool for screening the anti‐inflammatory activity of extracts of spices/herbs in Thai recipes to identify specific compounds of interest. (Supported by International Nutrition Foundation/Ellison Medical Foundation Post‐Doctoral Fellowship Program and OARDC)
Role of MAPK and NF‐kappaB in the stimulatory effects of American ginseng polysaccharides on murine macrophage (RAW 264.7) cells
Authors: King Mandy L , Ebersole Gregg , Hantak Alison , Murphy Laura L
Published: June 2021

Aqueous extracts of ginseng polysaccharides have been shown to modulate the immune response by inducing cytokine and nitric oxide (NO) production, however, the exact mechanism(s) of action remains unclear. The aim of the current study was to determine the effect of ginseng polysaccharides (PS) on MAPK and NF‐κB pathways, known mediators of the immune response, using murine macrophage cells (RAW 264.7). Cells were treated with or without PS for 0–60 minutes, harvested at 15 minute increments, and cellular proteins were analyzed for phosphorylated levels of I κB, ERK, JNK, and p38. Time‐dependent increases in phospho‐IκB, phospho‐ERK, and phospho‐JNK were observed in PS treated cells. Further analysis of nuclear extracts by EMSA showed enhanced NF‐κB activity following 24 hours of PS treatment. Cells were then pre‐treated with specific inhibitors of ERK (PD98059), p38 (SB202190), JNK (SP60012), and NF‐κB (PDTC), followed by PS, and supernatants were assessed for NO and tumor necrosis factor‐alpha (TNFα) production. Nitric oxide levels were robustly increased in PS treated cells, and this effect was partially blocked by PD98059 and PDTC. TNFα secretion was also stimulated by PS treatment, however only PDTC was able to attenuate the PS response. These results show that both MAPK and NF‐κB pathways are involved in PS‐induced nitric oxide production, while the effects on TNFα release appear to be mediated by NF‐κB only.
Dietary (−)‐epigallocatechin‐3‐gallate (EGCG) inhibits expression of hepatic lipogenic enzymes in C57BL/6J obese mice
Authors: Lee Mak‐Soon , Kim Yangha
Published: June 2021

Obesity is strongly associated with the development of insulin resistance, type 2 diabetes, metabolic syndrome, and cardiovascular disease. In this study, we examined the effect of dietary EGCG on the expression related with hepatic lipogenic enzymes. Male C57BL/6J mice were fed with high‐fat diet for 8 wks to induce obesity, and subsequently fed with 0.2% or 0.5% EGCG (w/w) supplemented high‐fat diet for 8 wks. The mRNA levels of hepatic lipogenic enzymes were analyzed using real‐time quantitative PCR. Experimental diets supplemented with 0.2% or 0.5% EGCG reduced the body weight by 14% or 20% respectively, compared to control group. EGCG diet also decreased the adipose tissue mass, and lowered the plasma triacylglyceride, free fatty acid and total cholesterol concentration. The mRNA levels of lipogenic sterol regulatory‐element binding protein‐1c (SREBP‐1c), fatty acid synthease (FAS), steroyl‐CoA desaturase 1 (SCD‐1) and 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CoA reductase) gene in the liver were decreased in the EGCG‐fed groups compared with the control group. Our results suggest that EGCG may reduce the risk of obesity through amelioration of lipid metabolism and inhibition of hepatic lipogenesis. “This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10510130005‐07N1013‐00510).”
Subependymal giant cell astrocytoma incidentally found in an elderly patient: Report of an autopsy case
Authors: Takei Hidehiro , Powell Suzanne Z
Published: June 2021

Background: Subependymal giant cell astrocytoma (SEGA) is a benign, slowly growing tumor typically occurring in the first two decades of life. SEGA usually occurs in the setting of tuberous sclerosis complex (TSC), however there are several reported cases in which patients with solitary SEGA had no other stigmata of TSC. These lesions are generally considered to be forme fruste of TSC. We report such a case of SEGA in the oldest patient reported to date.

Case report: The patient was a 75‐year‐old woman who had a history of radical vulvectomy for malignant melanoma and died of widespread systemic metastasis, which was confirmed at autopsy. Postmortem examination of the brain revealed a single intraventricular nodule in the left lateral ventricle, measuring 2.1×1.0×0.8cm. Histologically, the tumor was composed of interlacing bundles of spindle shaped tumor cells with thin delicate processes admixed with relatively large atypical cells with abundant glassy eosinophilic cytoplasm, as seen in SEGA. Ultrastructural study supported the diagnosis. Autopsy study showed no manifestations of TSC systemically or intracranially.

Discussion: This is a very rare case of solitary SEGA incidentally found at autopsy in an elderly patient lacking any other findings of TSC. This is the second reported case of SEGA found in an elderly patient. This autopsy case showed that SEGA can occur outside the setting of TSC.
Myofibroblastoma presenting as a cystic lesion with a mural nodule in the brain parenchyma
Authors: Gaurav Gupta , Meera Hameed , Catrambone Jeffrey , Eun‐Sook Cho
Published: June 2021

An 18‐year‐old female with no past medical history presented with left sided generalized tonic clonic seizures. Imaging showed a large cystic lesion within the right frontal lobe, just superior to the sylvian fissure. It measured 3.3cm × 4.0cm × 4.1cm. Within this large cystic lesion, there was a homogeneously enhancing lobulated nodule measuring 1.5cm × 1.0cm × 0.9cm. There was edema in the surrounding frontal white matter.

At operation the lesion had no attachment to the dura and was completely intraparenchymal. The mural nodule was resected en bloc. Histologically the tumor showed alternating zones of spindle or ovoid cells that were separated by thick collagen bundles. Reticulin fibers are abundant and frequently wrapped around individual cells. Immunocytochemistry showed diffuse positivity for vimentin and focal positivity for bcl 2 and α‐smooth muscle actin. Tumor cells are negative for CD34, desmin, HHF35, S‐100, and EMA. This immunocytochemical profile is consistent with a myofibroblastoma.

Myofibroblastoma is a benign mesenchymal tumor, which typically arises in the breast, less frequently in the lymph nodes and in other soft tissue. There are three reported cases of intracranial myofibroblastoma, all of which was attached to the meninges. Current case is unique in that it presented as a mural nodule in a large cyst within the brain parenchyme.
Novel variant of cortical dysplasia in two cases of infantile intractable epilepsy
Authors: Khanlou Negar , Mathern Gary W , Mitchell Wendy G , Yong William H , Vinters Harry V
Published: June 2021

Cortical dysplasia (CD) is a frequent cause of intractable epilepsy (IE), especially infantile spasms. CD is defined by neuropathologic features of neuronal disarray, dysmorphism and heterotopia, frequently together with neuronal cytologic abnormalities and “balloon cells”. We report two cases of infantile IE, a 5‐year‐old male and a 17‐year‐old female in whom neuroimaging studies showed findings consistent with CD and histopathologic examination of the corticectomies revealed severe CD with abundant Rosenthal fibers (RF), widely distributed throughout subpial regions and within subcortical white matter. The abundance and location of RF was reminiscent of neuropathologic changes seen in Alexander's disease. Sequencing of the GFAP gene was performed on DNA extracted from resected frozen tissue in one case and although no mutation was found three previously known SNPs were identified. Rare similar cases have been reported (Kupsky & Frosch 1993). The abundant RF seen in association with CD may represent a dysregulation of synthesis/metabolism of alpha B‐crystallin, the main component of the RF, aggregating in association with GFAP, or be secondary to the severity of the CD which produces a dys/upregulation of GFAP to levels sufficient to induce RF and localized leukodystrophy due to astrocytic dysfunction.
The distribution of karyopherin β proteins in Alzheimer's disease
Authors: Sheffield Lynette G , Mirra Suzanne S
Published: June 2021

Electron microscopic studies of Alzheimer's disease (AD) revealed an intimate relationship between paired helical filaments and nuclear pores, structures with a key role in bidirectional nucleocytoplasmic transport. This association led us to explore potential abnormalities in transport that may contribute to tangle formation and other protein aggregates in neurodegenerative disease. Previously, we found that antibodies to several nucleoporins highlighted neuronal nuclear contour and irregularity in AD. Immunohistochemical study of nuclear transport factor 2 (NTF2) revealed increased cytoplasmic label in neurons and RanBP2/nup358 antibody labeled tangle‐bearing neurons and Hirano bodies. In the present study, we examined the distribution of karyopherinβ (Kapβ) in AD hippocampus. This group of proteins is variously involved in docking to the nuclear pore complex and importing ribosomal and other proteins into the nucleus. Using antibodies to Kapβ1, β2, and β3, we labeled sections in 3 AD and 3 control cases. Kapβ1 and Kapβ3 displayed finely granular cytoplasmic immunopositivity, the latter with extensive dendritic label. Kapβ2 antibody revealed striking large cytoplasmic granules in many hippocampal neurons, more prominent in AD than controls. Kapβ2 also was present in some tangle‐bearing cells. Further characterization of transport proteins and their potential role in AD is warranted.
Disseminated Subpial Demyelination Presenting as Respiratory Stridor
Authors: Dulai Mohanpal Singh , Vogel Hannes
Published: June 2021

A 61‐year‐old female with long‐standing bipolar disorder presented with recurrent episodes of laryngeal stridor occurring over 2 years. Her stridor was worse while lying supine, and examination of her vocal cords show paradoxical movement. MRI showed scattered diffuse white matter lesions in the cerebral hemispheres without brainstem involvement. Her final episode of stridor was fatal, and an autopsy was performed revealing no abnormalities of laryngeal structures. Examination of her brain showed diffuse anoxic encephalopathy with acute and remote infarctions in or adjacent to bilateral caudate nuclei. There were numerous chronic demyelinating lesions, demonstrated by LFB/PAS and Bielschowsky stains, located in the optic chiasm, cerebellum, midbrain, pons and medulla. Many of these lesions were localized to the subpial parenchyma, a finding which was most evident in the pons and medulla. Involvement of the dorsal motor nucleus and other fibers contributing to the formation cranial nerve X as it exits the medulla was also noted and may have contributed to her stridor. The pattern of demyelination demonstrated in this case has been referred to as disseminated subpial demyelination, which has only been reported twice in the literature in the absence of classic patterns of demyelination, and it may represent an unusual phenotype of multiple sclerosis.
Morphologic Assessment of Muscle Biopsy Abnormalities in Patients with Congenital Fiber Type Disproportion and Tropomyosin 3 Mutation
Authors: Lawlor Michael William , Roumm Emily , DeChene Elizabeth Taylor , Geggel Amelia , Moghadasadeh Behzad , Beggs Alan H
Published: June 2021

Congenital fiber type disproportion (CFTD) is a rare congenital myopathy characterized by hypotonia and generalized muscle weakness. Pathologic diagnosis of CFTD is based on the presence of type I fiber hypotrophy of at least 12% in the absence of other notable pathological findings. Mutations of the ACTA1 and SEPN1 genes have been identified in a small percentage of CFTD cases. The muscle tropomyosin 3 gene, TPM3, is mutated in rare cases of nemaline myopathy that typically exhibit type 1 fiber hypotrophy with nemaline rods. Using genomic PCR and DNA sequencing, we screened the TPM3 gene in patients with a clinical diagnosis of CFTD, nemaline myopathy, and with undefined congenital myopathies. Mutations in TPM3 were identified in 5 out of 12 patients with CFTD as well as in one case of nemaline myopathy. Review of muscle biopsies from patients with diagnoses of CFTD revealed that patients with TPM3 mutation all displayed marked disproportion of fiber size, without type I fiber predominance. Several mutation‐negative cases exhibited other abnormalities such as central nuclei and central cores. These results support the utility of the CFTD diagnosis in directing the course of genetic testing.
Myonuclear degeneration in LMNA null mice
Authors: Bornemann Antje , Mittelbronn Michel , Sullivan Teresa , Stewart Colin L.
Published: June 2021

Lamins A/C, the major constituent of the nuclear lamina, confer mechanical stability to nuclei. We examined the myonuclei of LMNA null mice at the myotendinous junctions (MTJ), the site of longitudinal force transmission from contractile proteins to extracellular proteins. The right soleus and rectus femoris muscles of 5 null mutants aged 5–7 weeks and 2 wild type animals aged 5 weeks and 6 months were examined by electron microscopy. The myofibers merging into the tendons were assessed for nuclear disintegration and cytoplasmic degeneration. The myofibers of the wild type rectus femoris and soleus muscles revealed 19–27 nuclei per 50 myofibers and 5–8/20, respectively, with no signs of degeneration. The rectus femoris muscle fibers of the null mice contained 75–117 myonuclei/50 myofibers, the soleus muscle, 13–36 nuclei/20 myofibers. 11–21/50 myonuclei of the rectus femoris myonuclei showed chromatin clumping, nuclear fragmentation, nuclear inclusions and invaginations, and intranuclear filaments. The values were 12–19/50 for the soleus myonuclei. Moreover, 5–12/50 rectus femoris myofibers and 5–14/20, of the soleus myofibers showed cytoplasmic degeneration. None of these changes was found distal to the MTJ. These results favor the notion that myonuclei lacking a functional lamina are susceptible to mechanical stress in vivo. These alterations may contribute to the development of early joint contractures, a feature of ADEDMD.
Genetic association between the collagen type III alpha 1 polymorphism and its haplotypes with chronic hepatitis B in Koreans
Authors: Lee Seung Ku , Yi Cheong‐Ho , Kim Min‐Ho , Cheong Jae Youn , Cho Sung Won , Kang Jason Jongho , Yang Song‐Ju , Kwack KyuBum
Published: June 2021

The expression of collagens appears to be related to chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). The authors investigated whether a single nucleotide polymorphism of collagen type III alpha 1 (COL3A1), confers genetic susceptibility to liver diseases. Genotyping was performed on 117 patients with CH and 117 spontaneous recovery (SR) controls. The allele of two SNPs of COL3A1 were highly associated with CH to compared with SR in allele model (P = 0.009 and P = 0.035, respectively). Haplotype and diplotype analysis showed significantly associated with the SR and CH (P = 0.005~0.044). We found that intronic SNP, haplotypes, and diplotypes of COL3A1 were strongly associated with the HBV clearance and CH.
Endogenous Cholinergic Anti‐Inflammatory Pathway in Hepatic Ischemia‐Reperfusion (IR)
Authors: Crockett Elahe T , Dowlatshahi Shadi , Kolamal Nageswari , Galligan James J
Published: June 2021

We recently showed anti‐inflammatory role of cholinergic pathway (Ch.Path) in liver IR by pharmacological stimulation of acetylcholine receptor. Here we tested whether endogenous Ch.Path (e.Ch.Path) activation would exert a similar protective effect, and studied liver gene expression modulation by Ch.Path activation.

Methods

Mice treated with CCK‐8 or nicotine, i.p. prior to ischemia (CCK‐8 stimulates acetylcholine release via Ch.Path). A 2nd group was subjected to bilateral vagotomy prior to CCK‐8, while a 3rd group subjected to electrical vagal stimulation for 20min, followed by liver IR (90min I : 3h R). Plasma ALT and cytokines levels and liver histopathology were assessed.

Results

CCK‐8‐ or nicotine treatment significantly reduced liver injury (87%, and %69 respectively) and cytokine production, as compared to saline‐treated mice. CCK‐8 protective effect was absent in vagotomized mice, indicating e.Ch.Path role. Electrical vagal nerve stimulation confirmed the observation as ALT levels and liver injury were significantly decreased in this group. Gene array data showed significant modulation of several pathways and associated genes.

Conclusions

e.Ch.Path activation via humoral or electrical stimuli provides a protective effect on liver IR injury, which could offer therapeutic means to selectively act on targeted genes for treatment of IR inflammatory diseases. Support: NIH HL075475.
Role of TNF converting enzyme in TNF production, neutrophil activation and liver injury in hepatotoxic interaction of lipopolysaccharide and ranitidine
Authors: Deng Xiaomin , Lu Jingtao , Lehman‐McKeeman Lois , Malle Ernst , Crandall David , Ganey Patricia , Roth Robert
Published: June 2021

In rats, cotreatment with nontoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) causes liver injury. Previous studies showed that RAN augmented serum TNF production and neutrophil (PMN) activation after LPS treatment, and both TNF and neutrophils are crucial for the liver pathogenesis. We tested the hypothesis that TNF converting enzyme (TACE) is necessary for augmentation by RAN of TNF production, PMN activation and subsequent liver injury. LPS/RAN cotreatment elevated serum TNF; production as compared to LPS/Veh treatment, whereas it had no effects on hepatic TNF mRNA level. This suggests that RAN augmented TNF production by a post‐transcriptional mode. Indeed, LPS/RAN treatment increased hepatic TACE activation compared to LPS/Veh treatment. A TACE inhibitor, BMS‐561392, administered just before RAN reduced serum TNF protein. The inhibitor also reduced liver injury caused by LPS/RAN cotreatment. Interestingly, the TACE inhibitor also reduced plasma active PAI‐1, which has been shown to be causally involved in the pathogenesis of LPS/RAN‐induced liver injury. In fact, a PAI‐1 inhibitor, WAY‐140312, reduced hepatocellular injury and hepatic PMN activation. In summary, RAN enhanced TNF production after LPS treatment through TACE activation, which in turns led to PAI‐1 production, PMN activation and subsequent liver injury. (Supported by NIH grant DK061315.)
Murine Model of Occupational Injury Induced by Chronic Exposure to Oak Dust
Authors: McClintock Shannon D. , Barron Adam , Warner Roscoe L. , Johnson Kent J.
Published: June 2021

Chronic exposure to airborne particulates is well described as the primary cause of occupational pulmonary injury and increases the risk of asthma, chronic bronchitis, allergic rhinitis and hypersensitivity pneumonitis. Sources of both inorganic and organic particulates are derived of bacterial, fungal, chemical, plant, or animal origin. While inhalation of wood dust of several particulate sizes has long been thought of as an occupational hazard for those in the woodworking industry it has not been directly implicated as the causative agent for pulmonary injury. Herein we present a mouse model of chronic exposure to wood dust resulting in a pneumonitis type injury. Intratracheal administration of an oak dust suspension (80 micro‐L) was performed three times per week for four weeks. Histological examination demonstrated an influx of inflammatory cells peri‐airway and a resident macrophage population swollen to several times their normal size, due in part to the phagocytosis of particulates. Increased interstitial thickening of the smaller airways and alveolar compartments were noted in the lungs of injured mice relative to uninjured controls. Results from these experiments demonstrate that repeated exposure to oak dust induces lung inflammation and a pneumonitis type injury and may offer a model to better understanding and subsequently treat occupational pulmonary injury. Work supported by NIH RO1 HL07097
Alveolar retention of bone marrow‐derived mesenchymal stem cells in lipopolysaccharide‐treated lungs
Authors: Islam Mohammad Naimul , Otsu Keishi , Lindert Jens , Bhattacharya Jahar
Published: June 2021

Lamellar bodies (LB) of alveolar type 2 cells consist of an aqueous phase that surrounds a phase containing surfactant protein B (SPB) in complex with surfactant phospholipids. Surfactant secretion entails exocytosis of the aqueous and the SPB‐phospholipid phases. To determine the extent to which these phases are secreted differently, we imaged by live confocal microscopy, single alveoli of isolated, blood‐perfused rat lungs held at alveolar pressure (Palv) of 5 cmH2O. Then, by alveolar micropuncture, we co‐loaded type 2 cells with purified, fluorescent SPB (BODIPY‐SPB) and the LB‐localizing marker, LysoTracker Red (LTR) to mark respectively, the phospholipid and the aqueous phases in LB. In single alveoli, type 2 cells were revealed by the high fluorescence of both BODIPY‐SPB and LTR. Thirty minutes after a single 15‐s inflation to Palv of 15 cmH2O, type 2 cell fluorescence decreased for BODIPY‐SPB by 46.6 2.9% of initial (mean SE; n=4), but markedly more for LTR, namely by 72.5 4.5% of initial (p<0.05). Thus, the fluorescence decay rate for BODIPY‐SPB was half that for LTR (P<.05). We interpret that during surfactant secretion, phospholipid versus aqueous secretion rates differ markedly. The difference might be attributable to differences in phase mobility resulting from the geometry of the LB exocytosis pathway (support: HL64896).
Antagonism of the substitution of ethanol, pentobarbital, and midazolam for 1.0 and 2.0 g/kg ethanol by Ro15‐4513 and Ro15‐1788 in cynomolgus monkeys
Authors: Helms Christa Marie , Rogers Laura SM , Grant Kathleen A
Published: June 2021

The GABAA receptors mediating the discriminative stimulus effects of ethanol (EtOH) were studied by comparing the potency of Ro15‐4513 and Ro15‐1788 to antagonize EtOH, pentobarbital (PB) and midazolam (MDZ) substitution for EtOH. Ro15‐4513, a GABAA partial inverse agonist, has high affinity for receptors containing α4/6 and α5 subunits and lower affinity for α1, α2 and α3 subunits. Ro15‐1788 is a structurally‐similar but non‐selective benzodiazepine site GABAA antagonist. Male (n = 9) and female (n = 8) monkeys were trained to discriminate EtOH (1.0 or 2.0 g/kg, i.g., 30‐min pre‐treatment) from water. EtOH, PB and MDZ dose‐dependently substituted for EtOH (≥ 80% EtOH‐appropriate responding). Ro15‐4513 (0.003‐0.56 mg/kg, i.m., 5‐min pre‐treatment) shifted the EtOH, PB and MDZ dose‐response functions rightward in 13/14, 16/16 and 11/11 monkeys tested. Ro15‐1788 (0.30‐10.0 mg/kg, i.m., 5‐min pre‐treatment) shifted the EtOH, PB and MDZ dose‐response functions rightward in 9/14, 8/10 and 7/8 monkeys, respectively. EtOH and PB substitution were more potently antagonized by Ro15‐4513 (mean ± SEM apparent pKB, 6.39 ± 0.07) compared to Ro15‐1788 (4.84 ± 0.09). MDZ substitution was antagonized with similar potency (Ro15‐4513, 7.05 ± 0.08; Ro15‐ 1788, 6.72 ± 0.14). GABAA receptors with high‐affinity for Ro15‐4513 may specifically mediate the discriminative stimulus effects of EtOH. NIH/NIAAA AA10009 and AA13860
Contributions of extracellular and intracellular calcium in the biosynthesis of endocannabinoids
Authors: Placzek Ekaterina A , Barker Eric L
Published: June 2021

The actions of a plant‐derived cannabinoid delta9‐tetrahydrocannabinol (δ9‐THC) are mimicked by the endogenous cannabinoids anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG). The mechanisms that underlie AEA and 2‐AG biosynthesis are not completely understood. It is known that endocannabinoid biosynthesis is a calcium‐dependent process, however the physiological source of calcium is not known. We performed a series of biochemical experiments examining the contributions of extracellular and intracellular calcium in the biosynthetic process. We discovered that extracellular calcium is necessary for AEA and 2‐AG biosynthesis, whereas simply releasing intracellular calcium stores does not have an effect on endocannabinoid biosynthesis. The model for endocannabinoid biosynthesis was extended into MES 23.5 cells, a neuronal cell line. We determined that AEA biosynthesis in MES 23.5 cells may occur as a result of the activation of TRPC 3/6/7‐type channels.
A human anti‐cocaine monoclonal antibody antagonizes the cocaine‐induced reinstatement of self‐administration in rats
Authors: Norman Andrew B. , Norman Mantana K. , Buesing William R. , Tabet Michael R. , Tsibulsky Vladimir L. , Ball William J.
Published: June 2021

Immunotherapy may represent a viable alternative to pharmacotherapy for cocaine abuse. The predominantly human sequence anti‐cocaine monoclonal antibody, 2E2, has high affinity (Kd = 4 nM) and specificity for cocaine. In mice, 2E2 sequesters cocaine in the peripheral circulation, thereby decreasing brain cocaine concentrations. Therefore, it was hypothesized that 2E2 would antagonize cocaine‐induced reinstatement (priming) of self‐administration, a partial model of relapse that depends on the brain cocaine concentration. The level of cocaine required to reinstate self‐administration (priming threshold) in trained rats was calculated during daily sessions run 6 days a week. After 2 weeks of baseline measurements, rats were infused with 2E2 (120 mg/kg i.v.) or an equivalent dose of human polyclonal IgG as a control and daily sessions resumed. 2E2 produced a significant almost 3‐fold increase in priming threshold that gradually declined towards baseline values. The control IgG had no effect. The terminal elimination half‐life of 2E2 was 10 days. 2E2 also produced an approximately 20–40% increase in the rate of maintained cocaine self‐administration, which declined over days. This is less than the increase typically produced by competitive dopamine receptor antagonists. 2E2 may represent a therapeutic agent for decreasing the probability of relapse in cocaine abusers. Supported by PHS grant DA018538.
Prenatal and Postnatal Depletion of Tyrosine Hydroxylase by MPTP as a “Sensitization and Precipitation” Model for Parkinson's disease
Authors: Muthian Gladson , Mackey Veronica , King Jennifer , Charlton Clivel
Published: June 2021

Parkinson's disease (PD) is due to the degeneration of dopamine (DA) neurons and loss of DA and tyrosine hydroxylase (TH) in the nigrostriatal (NS) system. In this project we test the hypothesis that timed exposure of fetuses to toxins sensitizes the NS DA neurons and that later challenges further harm the vulnerable neurons, resulting in PD. Thus, a sensitization stage and a precipitating stage underlie idiopathic PD. For the 1st sensitization stage we exposed C57 black mouse fetuses to 10 mg/kg/day 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐ tetrahydropyridine (MPTP) during gestation day 8–12 to target the emerging NS DA neurons. For the 2nd precipitating stage the adult offsprings were challenged with 10, 20 or 30 mg/kg/day MPTP for 7 days. On the 6th day post‐MPTP Western blot analysis of brain TH and histology were performed. The results show that prenatal MPTP affects the NS in a long lasting manner, since in the 3 months old adult, striatal and midbrain TH expressions were still reduced by over 26%. Postnatal MPTP is additive and further decreases TH in a dose‐dependent manner. Prenatal PBS or MPTP, plus postnatal MPTP at 10, 20, 30 mg/kg, reduce striatal TH by 0, 52, 79% or 55, 72 and 91%, respectively. The outcome demonstrates that idiopathic PD may have a fetal basis and that chemical and or functional stress and age‐related changes precipitate PD symptoms by further damaging the NS DA neurons that were made susceptible earlier in life. Supported by NIH RO1NS041674 and NIH R21NS049623
Alpha synuclein and vesicular monoamine transporter2 expression modulate the vulnerability of nigral neurons to dopaminergic neurotoxicants
Authors: Shepherd Kennie Raviie , Wang Min Z. , Dean E. Danielle , Miller Gary W.
Published: June 2021

The vesicular monoamine transporter2 (VMAT2) and α‐synuclein are two abundant proteins in the nigrostriatal system, which has been suggested to be responsible for proper maintenance and homeostasis of the dopaminergic system. VMAT2 is responsible for the packaging of dopamine (DA), preventing its degradation and subsequent production of ROS. α‐synuclein has been shown to be a key regulator of VMAT2, and mutations in α‐synuclein have been shown to cause a rare form of Parkinson's disease (PD). Primary nigra neurons isolated from VMAT2 low expresser mice have significant production of ROS, caspase‐3 activation, and undergo retrograde degeneration as demonstrated by a 33.4% loss of tyrosine hydroxylase (TH) positive neurons and processes (66.5%) after the exposure to DA (10μM). In addition, primary cultures generated from mice with differential expression of α‐synuclein and VMAT2 were treated with rotenone (20nM), and MPP+ (5μM), in the presence of 10μM DA, to determine the role of multiple gene interaction in altering the vulnerability of nigra neurons. Nigral neuron vulnerability was assessed by counted TH positive neurons/processes, DCF staining, and caspase‐3 activation after neurotoxicant exposure. The results show that α‐synuclein and VMAT2 modulate the toxicity of these toxicants to varying degrees. Supported by U54ES‐012068; 2T32NS007480; and the American Parkinson's Disease Association.
Synaptic versus extrasynaptic NMDA receptors: Role in PCP‐induced neurotoxicity and development of locomotor sensitization
Authors: Anastasio Noelle , Xia Yan , Wang Cheng Z , Johnson Kenneth M
Published: June 2021

The relationship between PCP‐induced neurotoxicity in perinatal rats and the development of behavioral deficits is unclear as is the role of specific NMDAR subunits in both. This lab has reported that activation of synaptic NMDAR can prevent PCP‐induced death implying that NR1/NR2A, thought to be synaptic, and NR1/NR2B, thought to be extrasynaptic, are differentially involved in the behavioral and neurotoxic effects of PCP. Using cortical organotypic slices as a model, we found that the NR2A antagonist, NVPAAM007, is neurotoxic, while ifenprodil, NR2B antagonist, is not. Further, rat pups treated with either PCP or PEAQX (NR2A antagonist) showed marked elevation of caspase 3 activity in the cortex, while ifenprodil showed no effect. Rat pups were treated with saline or PCP on PN7, 9 and 11, challenged with PCP between PN28‐35 and locomotor activity measured. This regimen caused a sensitized locomotor response to PCP challenge. A similar response to PCP challenge was observed following treatment with ifenprodil on PN7, 9 and 11. These preliminary data suggest that sensitization to PCP involves changes in NR2B receptors and may not be the result of the neurotoxic effects of PCP. Future experiments with PEAQX will be performed in order to distinguish the role of NR2 subunits in aberrant behaviors and the link between neurotoxicity and locomotor sensitization. Supported by F31DA022824 & DA02073.