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Article ; Online: B cell extracellular vesicles contain monomeric IgM that binds antigen and enters target cells.

Gutknecht, Michael F / Holodick, Nichol E / Rothstein, Thomas L

iScience

2023 Volume 26, Issue 9, Page(s) 107526

Abstract: The production and release of small phospholipid membrane vesicles, or extracellular vesicles (EVs), is a trait of most prokaryotic and eukaryotic cells. EVs display heterogeneity in content, size, biogenesis, activity, and function. B cells uniquely ... ...

Abstract	The production and release of small phospholipid membrane vesicles, or extracellular vesicles (EVs), is a trait of most prokaryotic and eukaryotic cells. EVs display heterogeneity in content, size, biogenesis, activity, and function. B cells uniquely express immunoglobulin and produce EVs; however, the relationship between these entities has not been clarified. Here, we used several methodologies to isolate large (11,000 × g) and small (110,000 × g) EVs and evaluate their IgM content, characteristics and activity. We found that B cells from multiple cell lines and primary B cells produce EVs that display monomeric IgM on the surface and contain encapsulated monomeric IgM, which is independent of secreted pentameric IgM. Our data indicate EV IgM can bind antigen specifically, and EV IgM can be incorporated intracellularly into secondary cells. These results suggest immunological activities different from secreted pentameric IgM that may constitute a separate and distinct antibody distribution system.
Language	English
Publishing date	2023-08-03
Publishing country	United States
Document type	Journal Article
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2023.107526
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Article ; Online: Microparticle immunocapture assay for quantitation of protein multimer amount and size.

Gutknecht, Michael F / Kaku, Hiroaki / Rothstein, Thomas L

Cell reports methods

2022 Volume 2, Issue 5, Page(s) 100214

Abstract: Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral ... ...

Abstract	Cellular stress and toxicity are often associated with the formation of protein multimers, or aggregates. Numerous degenerative disorders, including Alzheimer's, Parkinson's, and Huntington's disease, prion-propagated disease, amyotrophic lateral sclerosis, cardiac amyloidosis, and diabetes, are characterized by aggregated protein deposits. Current methods are limited in the ability to assess multimer size along with multimer quantitation and to incorporate one or more ancillary traits, including target specificity, operative simplicity, and process speed. Here, we report development of a microparticle immunocapture assay that combines the advantages inherent to a monoclonal antibody:protein interaction with highly quantitative flow cytometry analysis. Using established reagents to build our platform, and aggregation-prone amyloid beta 1-42 peptide (Aβ42) and alpha-synuclein to demonstrate proof of principle, our results indicate that this assay is a highly adaptable method to measure multimer size and quantity at the same time in a technically streamlined workflow applicable to laboratory and clinical samples.
MeSH term(s)	Humans ; Amyloid beta-Peptides/metabolism ; Amyloidosis/metabolism ; Prion Diseases/metabolism ; Huntington Disease
Chemical Substances	Amyloid beta-Peptides
Language	English
Publishing date	2022-05-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2667-2375
ISSN (online)	2667-2375
DOI	10.1016/j.crmeth.2022.100214
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Article ; Online: nanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro

Griessl, Marion / Gutknecht, Michael / Juranić-Lisnić, Vanda / Cook, Charles H.

Journal of Virological Methods. 2022 Mar., v. 301 p.114436-

2022

Abstract: Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most ... ...

Abstract	Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software. Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency. mCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM. We found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue. For viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.
Keywords	Muromegalovirus ; computer software ; fibroblasts ; gene expression ; genes ; lungs ; mice ; reverse transcriptase polymerase chain reaction ; reverse transcription ; sequence analysis ; transcriptome ; viruses ; Murine cytomegalovirus ; nanoString ; Viral transcription
Language	English
Dates of publication	2022-03
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2021.114436
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Article: Fas Apoptosis Inhibitory Molecule Blocks and Dissolves Pathological Amyloid-β Species.

Kaku, Hiroaki / Ludlow, Alexander V / Gutknecht, Michael F / Rothstein, Thomas L

Frontiers in molecular neuroscience

2021 Volume 14, Page(s) 750578

Abstract: A number of neurodegenerative diseases are associated with the accumulation of misfolded proteins, including Alzheimer's disease (AD). In AD, misfolded proteins such as tau and amyloid-β (Aβ) form pathological insoluble deposits. It is hypothesized that ... ...

Abstract	A number of neurodegenerative diseases are associated with the accumulation of misfolded proteins, including Alzheimer's disease (AD). In AD, misfolded proteins such as tau and amyloid-β (Aβ) form pathological insoluble deposits. It is hypothesized that molecules capable of dissolving such protein aggregates might reverse disease progression and improve the lives of afflicted AD patients. Here we report new functions of the highly conserved mammalian protein, Fas Apoptosis Inhibitory Molecule (FAIM). We found that FAIM-deficient Neuro 2A cells accumulate Aβ oligomers/fibrils. We further found that recombinant human FAIM prevents the generation of pathologic Aβ oligomers and fibrils in a cell-free system, suggesting that FAIM functions without any additional cellular components. More importantly, recombinant human FAIM disaggregates and solubilizes established Aβ fibrils. Our results identify a previously unknown, completely novel candidate for understanding and treating irremediable, irreversible, and unrelenting neurodegenerative diseases.
Language	English
Publishing date	2021-12-14
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2452967-9
ISSN	1662-5099
ISSN	1662-5099
DOI	10.3389/fnmol.2021.750578
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Article ; Online: nanoString evaluation of murine Cytomegalovirus transcription in vivo and in vitro.

Griessl, Marion / Gutknecht, Michael / Juranić-Lisnić, Vanda / Cook, Charles H

Journal of virological methods

2021 Volume 301, Page(s) 114436

Abstract: Background: Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the ...

Abstract	Background: Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software. Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency. Methods: mCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM. Results: We found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue. Conclusions: For viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.
MeSH term(s)	Animals ; Cytomegalovirus/genetics ; Cytomegalovirus Infections ; Mice ; Mice, Inbred BALB C ; Muromegalovirus/genetics ; Transcriptome
Language	English
Publishing date	2021-12-17
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2021.114436
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Article: FAIM Opposes Aggregation of Mutant SOD1 That Typifies Some Forms of Familial Amyotrophic Lateral Sclerosis.

Kaku, Hiroaki / Ludlow, Alexander V / Gutknecht, Michael F / Rothstein, Thomas L

Frontiers in neuroscience

2020 Volume 14, Page(s) 110

Abstract: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative illness that is unremittingly fatal and for which no effective treatment exists. All forms of ALS are characterized by protein aggregation. In familial forms of ALS, specific and ... ...

Abstract	Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative illness that is unremittingly fatal and for which no effective treatment exists. All forms of ALS are characterized by protein aggregation. In familial forms of ALS, specific and heritable aggregation-prone proteins have been identified, such as mutant superoxide dismutase (SOD1). It has been suggested that a factor capable of preventing mutant SOD1 protein aggregation and/or disassembling mutant SOD1 protein aggregates would ameliorate SOD1-associated forms of familial ALS. Here we identify Fas Apoptosis Inhibitory Molecule (FAIM), a highly evolutionarily conserved 20 kDa protein, as an agent with this activity. We show FAIM counteracts intracellular accumulation of mutant SOD1 protein aggregates, which is increased in the absence of FAIM, as determined by pulse-shape analysis and filter trap assays. In a cell-free system, FAIM inhibits aggregation of mutant SOD1, and further disassembles and solubilizes established mutant SOD1 protein aggregates, as determined by thioflavin T (ThT), filter trap, and sedimentation assays. In sum, we report here a previously unknown activity of FAIM that opposes ALS disease-related protein aggregation and promotes proteostasis of an aggregation-prone ALS protein.
Language	English
Publishing date	2020-02-21
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2411902-7
ISSN	1662-453X ; 1662-4548
ISSN (online)	1662-453X
ISSN	1662-4548
DOI	10.3389/fnins.2020.00110
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Article ; Online: Functional significance of mononuclear phagocyte populations generated through adult hematopoiesis.

Gutknecht, Michael F / Bouton, Amy H

Journal of leukocyte biology

2014 Volume 96, Issue 6, Page(s) 969–980

Abstract: Tissue homeostasis requires a complete repertoire of functional macrophages in peripheral tissues. Recent evidence indicates that many resident tissue macrophages are seeded during embryonic development and persist through adulthood as a consequence of ... ...

Abstract	Tissue homeostasis requires a complete repertoire of functional macrophages in peripheral tissues. Recent evidence indicates that many resident tissue macrophages are seeded during embryonic development and persist through adulthood as a consequence of localized proliferation. Mononuclear phagocytes are also produced during adult hematopoiesis; these cells are then recruited to sites throughout the body, where they function in tissue repair and remodeling, resolution of inflammation, maintenance of homeostasis, and disease progression. The focus of this review is on mononuclear phagocytes that comprise the nonresident monocyte/macrophage populations in the body. Key features of monocyte differentiation are presented, focusing primarily on the developmental hierarchy that is established through this process, the markers used to identify discrete cell populations, and novel, functional attributes of these cells. These features are then explored in the context of the tumor microenvironment, where mononuclear phagocytes exhibit extensive plasticity in phenotype and function.
MeSH term(s)	Animals ; Antigens, Ly/analysis ; CX3C Chemokine Receptor 1 ; Cell Lineage ; Cell Movement ; Cytokines/physiology ; Gene Expression Regulation, Developmental ; Granulocyte-Macrophage Colony-Stimulating Factor/physiology ; Hematopoiesis ; Homeostasis ; Humans ; Immune System/embryology ; Immune System/immunology ; Immunity, Innate ; Inflammation ; Macrophage Colony-Stimulating Factor/physiology ; Macrophages/cytology ; Macrophages/immunology ; Mice ; Models, Biological ; Monocytes/classification ; Monocytes/cytology ; Monocytes/immunology ; Neoplasm Metastasis ; Neoplasms/immunology ; Neoplasms/pathology ; Organ Specificity ; Receptors, Chemokine/physiology ; Transcription Factors/physiology ; Tumor Microenvironment
Chemical Substances	Antigens, Ly ; CX3C Chemokine Receptor 1 ; Cx3cr1 protein, mouse ; Cytokines ; Ly-6C antigen, mouse ; Receptors, Chemokine ; Transcription Factors ; Macrophage Colony-Stimulating Factor (81627-83-0) ; Granulocyte-Macrophage Colony-Stimulating Factor (83869-56-1)
Language	English
Publishing date	2014-09-15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	605722-6
ISSN	1938-3673 ; 0741-5400
ISSN (online)	1938-3673
ISSN	0741-5400
DOI	10.1189/jlb.1RI0414-195R
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Article ; Online: Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro.

Griessl, Marion / Gutknecht, Michael / Cook, Charles H

Journal of virological methods

2017 Volume 248, Page(s) 100–106

Abstract: Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly ... ...

Abstract	Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for in vitro infection studies. For in vivo studies the most suitable reference genes were highly tissue and cell type dependent. Comparing infected and uninfected groups revealed viral influence on transcription of some genes. We provide reference gene guidelines for investigations of gene expression for mCMV Smith strain infection of Balb/cJ mice or NIH-3T3 cells. These results also suggest careful consideration of reference genes for different host tissues evaluated.
MeSH term(s)	Algorithms ; Animals ; Gene Expression ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics ; Herpesviridae Infections/genetics ; Herpesviridae Infections/virology ; Hypoxanthine Phosphoribosyltransferase/genetics ; Liver/virology ; Lung/virology ; Mice ; Mice, Inbred BALB C ; Muromegalovirus/genetics ; Muromegalovirus/physiology ; NIH 3T3 Cells ; Real-Time Polymerase Chain Reaction/methods
Chemical Substances	Glyceraldehyde-3-Phosphate Dehydrogenases (EC 1.2.1.-) ; Hypoxanthine Phosphoribosyltransferase (EC 2.4.2.8)
Keywords	covid19
Language	English
Publishing date	2017-06-26
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2017.06.012
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Article: Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro

Griessl, Marion / Gutknecht, Michael / Cook, Charles H

Journal of virological methods. 2017,

2017

Abstract	Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for in vitro infection studies. For in vivo studies the most suitable reference genes were highly tissue and cell type dependent. Comparing infected and uninfected groups revealed viral influence on transcription of some genes. We provide reference gene guidelines for investigations of gene expression for mCMV Smith strain infection of Balb/cJ mice or NIH-3T3 cells. These results also suggest careful consideration of reference genes for different host tissues evaluated.
Keywords	Muromegalovirus ; endothelial cells ; gene expression ; genes ; guidelines ; in vivo studies ; kidneys ; liver ; mice ; quantitative polymerase chain reaction ; reverse transcriptase polymerase chain reaction ; salivary glands ; spleen ; tissues ; covid19
Language	English
Publishing place	Elsevier B.V.
Document type	Article
Note	Pre-press version
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2017.06.012
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: The nonreceptor protein tyrosine kinase Pyk2 promotes the turnover of monocytes at steady state.

Llewellyn, Ryan A / Thomas, Keena S / Gutknecht, Michael F / Bouton, Amy H

Journal of leukocyte biology

2017 Volume 102, Issue 4, Page(s) 1069–1080

Abstract: Monocytes are short-lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline-rich ... ...

Abstract	Monocytes are short-lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase that regulates numerous immune cell functions, but its role in monocytes is currently unknown. In this study, we sought to characterize the expression and function of Pyk2 in lineage-committed monocyte populations. Here, we report that Pyk2 protein expression is increased in the Ly6C
MeSH term(s)	Animals ; Apoptosis/genetics ; Apoptosis/immunology ; Bone Marrow Cells/cytology ; Bone Marrow Cells/immunology ; Bone Marrow Transplantation ; Focal Adhesion Kinase 2/genetics ; Focal Adhesion Kinase 2/immunology ; Mice ; Mice, Knockout ; Monocytes/cytology ; Monocytes/immunology ; Transplantation Chimera
Chemical Substances	Focal Adhesion Kinase 2 (EC 2.7.10.2) ; Ptk2b protein, mouse (EC 2.7.10.2)
Language	English
Publishing date	2017-07-28
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	605722-6
ISSN	1938-3673 ; 0741-5400
ISSN (online)	1938-3673
ISSN	0741-5400
DOI	10.1189/jlb.1A0217-063R
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