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1.
Biochemistry (Mosc) ; 89(5): 853-861, 2024 May.
Article in English | MEDLINE | ID: mdl-38880646

ABSTRACT

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.


Subject(s)
Tumor Necrosis Factor-alpha , Animals , Humans , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Mice , Gene Editing/methods , Neoplasms/immunology , Neoplasms/genetics , Neoplasms/therapy
2.
Biochemistry (Mosc) ; 87(7): 590-604, 2022 Jul.
Article in English | MEDLINE | ID: mdl-36154880

ABSTRACT

Peptides are widely used for the diagnostics, prevention, and therapy of certain human diseases. How useful can they be for the disease caused by the SARS-CoV-2 coronavirus? In this review, we discuss the possibility of using synthetic and recombinant peptides and polypeptides for prevention of COVID-19 via blocking the interaction between the virus and its main receptor ACE2, as well as components of antiviral vaccines, in particular, against new emerging virus variants.


Subject(s)
COVID-19 , Angiotensin-Converting Enzyme 2 , Antiviral Agents/therapeutic use , Humans , Peptides/therapeutic use , SARS-CoV-2
3.
Semin Arthritis Rheum ; 49(3S): S39-S42, 2019 12.
Article in English | MEDLINE | ID: mdl-31779851

ABSTRACT

In spite of successful therapeutic neutralization of proinflammatory cytokines in several autoimmune diseases, such therapy is not entirely free of side effects. The main reason relates to the fact that cytokine signaling may have protective components that need to be spared. Several approaches to achieve a less damaging cytokine inhibition are being explored. In our experimental studies we are using bispecific reagents based on VHH-modules from the heavy-chain-only antibodies to limit bioavailability of TNF and IL-6 produced by myeloid cells. After evaluation of their properties in vitro and in vivo we argue that these types of reagents may have an advantage over systemic blockers.


Subject(s)
Autoimmune Diseases/metabolism , Autoimmunity , Cytokines/metabolism , Myeloid Cells/metabolism , Animals , Autoimmune Diseases/immunology , Biological Availability , Humans
4.
Cytokine ; 101: 33-38, 2018 01.
Article in English | MEDLINE | ID: mdl-27624866

ABSTRACT

Cytokines are involved in a wide range of functions shaping the normal immune response, yet inflammatory changes in the immune system due to dysregulated cytokine signaling may lead to the induction of autoimmunity. Cytokine inhibitors have revolutionized the treatment of many autoimmune diseases in recent years. Systemic cytokine ablation, however, is often associated with the development of adverse side effects and some patients simply do not respond to therapy. TNF, IL-1 and IL-6 are the best characterized proinflammatory cytokines considered as the main therapeutic targets for the treatment of several autoimmune and inflammatory diseases. But can anti-cytokine therapy become more selective and thus more efficient? This mini-review discusses several recently emerging paradigms and summarizes current experimental attempts to validate them in mouse studies.


Subject(s)
Autoimmunity/immunology , Cytokines/antagonists & inhibitors , Disease Models, Animal , Immunotherapy , Animals , Autoimmune Diseases/immunology , Autoimmune Diseases/therapy , Cytokines/immunology , Inflammation/therapy , Interleukin-1/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , Mice , Tumor Necrosis Factor-alpha/antagonists & inhibitors
5.
J Leukoc Biol ; 102(3): 783-790, 2017 09.
Article in English | MEDLINE | ID: mdl-28546502

ABSTRACT

Cytokine neutralization is successfully used for treatment of various autoimmune diseases and chronic inflammatory conditions. The complex biology of the two well-characterized proinflammatory cytokines TNF and IL-6 implicates unavoidable consequences when it comes to their global blockade. Because systemic cytokine ablation may result in unwanted side effects, efforts have been made to develop more specific cytokine inhibitors, which would spare the protective immunoregulatory functions of a given cytokine. In this article, we review current research and summarize new strategies for improved anti-TNF and anti-IL-6 biologics, which specifically target only selected parts of the signaling cascades mediated by these ligands.


Subject(s)
Autoimmune Diseases/drug therapy , Autoimmune Diseases/immunology , Interleukin-6/antagonists & inhibitors , Signal Transduction/drug effects , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Humans
6.
AIDS Res Hum Retroviruses ; 33(1): 57-60, 2017 01.
Article in English | MEDLINE | ID: mdl-27649790

ABSTRACT

HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.


Subject(s)
Aorta/pathology , Lipids/analysis , Liver/pathology , nef Gene Products, Human Immunodeficiency Virus/biosynthesis , Animals , Histocytochemistry , Mice, Inbred C57BL , Mice, Transgenic , Staining and Labeling , nef Gene Products, Human Immunodeficiency Virus/genetics
7.
Proc Natl Acad Sci U S A ; 113(11): 3006-11, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26936954

ABSTRACT

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)--a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.


Subject(s)
Antibodies, Bispecific/immunology , Antigens, Differentiation/immunology , Chemical and Drug Induced Liver Injury/therapy , Macrophages, Peritoneal/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Amino Acid Substitution , Animals , Antibodies, Bispecific/genetics , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibody Specificity , Antigen-Antibody Reactions , Antigens, Differentiation/genetics , Antigens, Surface/immunology , Camelus/immunology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/prevention & control , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Galactosamine/toxicity , Genes, Synthetic , Humans , L Cells , Macrophages, Peritoneal/immunology , Mice , Mutation , Random Allocation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Tumor Necrosis Factor-alpha/metabolism
8.
Nat Immunol ; 16(8): 819-828, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26147686

ABSTRACT

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.


Subject(s)
Inflammation/immunology , Intra-Abdominal Fat/immunology , Lymphocytes/immunology , Lymphoid Tissue/immunology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Chemokine CXCL13/genetics , Chemokine CXCL13/immunology , Chemokine CXCL13/metabolism , Flow Cytometry , Gene Expression/immunology , Inflammation/genetics , Inflammation/metabolism , Intra-Abdominal Fat/metabolism , Lymphocytes/metabolism , Lymphoid Tissue/cytology , Lymphoid Tissue/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Myeloid Cells/immunology , Myeloid Cells/metabolism , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/immunology , Receptors, Tumor Necrosis Factor/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/immunology , Stromal Cells/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
9.
Methods Mol Biol ; 1155: 151-62, 2014.
Article in English | MEDLINE | ID: mdl-24788180

ABSTRACT

This chapter provides protocols for in vitro and in vivo analysis of TNF-producing cells from a novel TNF reporter mouse. In these transgenic mice, genetic sequence encoding far-red reporter protein Katyushka (FRFPK) was placed under control of the same regulatory elements as TNF, thus providing the basis for detection, isolation, and visualization of TNF-producing cells.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/pathology , Genes, Reporter , Green Fluorescent Proteins/metabolism , Luminescent Proteins/metabolism , Macrophages/pathology , Tumor Necrosis Factor-alpha/physiology , Animals , Encephalomyelitis, Autoimmune, Experimental/metabolism , Flow Cytometry , Green Fluorescent Proteins/genetics , Image Processing, Computer-Assisted , Indicators and Reagents/metabolism , Luminescent Proteins/genetics , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic/genetics , Recombination, Genetic , Thy-1 Antigens/genetics , Red Fluorescent Protein
10.
PLoS One ; 8(4): e62055, 2013.
Article in English | MEDLINE | ID: mdl-23630623

ABSTRACT

Dendritic cells from mesenteric lymph nodes (MLN) can convert retinal to retinoic acid (RA), which promotes induction of the gut-specific homing receptor α4ß7. In contrast, priming within peripheral lymph nodes leads to upregulation of E- and P-selectin ligands (E- and P-lig). Apart from its α4ß7 promoting effect, RA was shown to suppress E- and P-lig induction in vitro. However, enhanced frequencies of P-lig(+) CD4(+) T cells were reported during intestinal inflammation. To understand this contradiction, we first determined whether location of intestinal inflammation, that is, ileitis or colitis, affects P-lig induction. Both conditions promoted P-lig expression on CD4(+) T cells; however, P-lig expressed on T cells facilitated Th1 cell recruitment only into the inflamed colon but not into inflamed small intestine induced by oral Toxoplasma gondii infection. A majority of P-lig(+)CD4(+) T cells found within MLN during intestinal inflammation co-expressed α4ß7 confirming their activation in the presence of RA. Mesenteric P-lig(+)CD4(+) cells co-expressed the 130 kDa isoform of CD43 which requires activity of core 2 (beta)1,6-N-acetyl-glycosaminyltransferase-I (C2GlcNAcT-I) suggesting that C2GlcNAcT-I contributes to P-lig expression under these conditions. To test whether inflammatory mediators can indeed overrule the inhibitory effect of RA on P-lig expression we stimulated CD4(+) T cells either polyclonal in the presence of IL-12 and IFNγ or by LPS-activated MLN-derived dendritic cells. Both conditions promoted P-lig induction even in the presence of RA. While RA impeded the induction of fucosyltransferase-VII it did not affect IL-12-dependent C2GlcNAcT-I induction suggesting that C2GlcNAcT-I can support P-lig expression even if fucosyltransferase-VII mRNA upregulation is dampened.


Subject(s)
Colitis/immunology , Intestine, Small/immunology , Membrane Glycoproteins/physiology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Movement , Colitis/metabolism , Colitis/pathology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Female , Gastroenteritis/immunology , Gastroenteritis/metabolism , Gastroenteritis/pathology , Integrins/metabolism , Intestine, Small/metabolism , Intestine, Small/pathology , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Th1 Cells/physiology , Tretinoin/physiology
12.
Cell Host Microbe ; 10(1): 44-53, 2011 Jul 21.
Article in English | MEDLINE | ID: mdl-21767811

ABSTRACT

Innate lymphoid cells (ILCs) have emerged as important players, regulating the balance between protective immunity and immunopathology at mucosal surfaces. However, mechanisms that regulate ILCs' effector functions during mucosal pathogenic challenge are poorly defined. Using mice infected with the natural mouse enteric pathogen Citrobacter rodentium, we demonstrate that lymphotoxin (LT) is essential for IL-22 production by intestinal ILCs. Blocking of LTßR signaling dramatically reduced intestinal IL-22 production after C. rodentium infection. Conversely, stimulating LTßR signaling induced an IL-22 protection pathway in LT-deficient mice. Furthermore, exogenous IL-22 expression rescued LTßR-deficient mice. IL-22-producing ILCs were predominantly located in lymphoid follicles in the colon and interacted closely with dendritic cells (DCs). We find that an LT-driven positive feedback loop controls IL-22 production by RORγt(+) ILCs via LTßR signaling in DCs. Taken together, our data show that LTßR signaling in gut lymphoid follicles regulates IL-22 production by ILCs in response to mucosal pathogen challenge.


Subject(s)
Citrobacter rodentium/pathogenicity , Interleukins/metabolism , Intestinal Mucosa/microbiology , Lymphocytes/immunology , Lymphotoxin-alpha/metabolism , Animals , Colon/metabolism , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Enterobacteriaceae Infections/immunology , Enterobacteriaceae Infections/metabolism , Immunity, Innate , Interleukins/immunology , Intestinal Mucosa/immunology , Lymphocytes/metabolism , Lymphotoxin beta Receptor/genetics , Lymphotoxin beta Receptor/immunology , Lymphotoxin beta Receptor/metabolism , Lymphotoxin-alpha/immunology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Signal Transduction , Interleukin-22
13.
Plant Biotechnol J ; 9(1): 22-31, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20444206

ABSTRACT

Tumour necrosis factor (TNF) is a major pro-inflammatory cytokine involved in multiple inflammatory diseases. The detrimental activity of TNF can be blocked by various antagonists, and commercial therapeutics based upon this principle have been approved for treatment of diseases including rheumatoid arthritis, Crohn's disease and psoriasis. In a search for new, improved anti-inflammatory therapeutics we have designed a single-domain monoclonal antibody (V(H) H), which recognizes TNF. The antibody component (TNF-V(H) H) is based upon an anti-human TNF Camelidae heavy-chain monoclonal antibody, which was linked to an elastin-like polypeptide (ELP). We demonstrate that ELP fusion to the TNF-V(H) H enhances accumulation of the fusion protein during biomanufacturing in transgenic tobacco plants. With this study, we show for the first time that this plant-derived anti-human TNF-V(H) H antibody was biologically active in vivo. Therefore, therapeutic application of TNF-V(H) H-ELP fusion protein was tested in humanized TNF mice and was shown to be effective in preventing death caused by septic shock. The in vivo persistence of the ELPylated antibody was ∼24 fold longer than that of non-ELPylated TNF-V(H) H.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Shock, Septic/prevention & control , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Disease Models, Animal , Elastin , Escherichia coli , Galactose , Gene Expression , Humans , L Cells , Lipopolysaccharides , Mice , Peptides , Plants, Genetically Modified , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Shock, Septic/chemically induced , Shock, Septic/immunology , Nicotiana/genetics , Tumor Necrosis Factor-alpha/immunology
14.
Blood ; 116(18): 3456-64, 2010 Nov 04.
Article in English | MEDLINE | ID: mdl-20634375

ABSTRACT

Secondary lymphoid organs provide a unique microenvironment for generation of immune responses. Using a cell type-specific conditional knockout approach, we have dissected contributions of tumor necrosis factor (TNF) produced by B cells (B-TNF) or T cells (T-TNF) to the genesis and homeostatic organization of secondary lymphoid organs. In spleen, lymph nodes and Peyer patches, the cellular source of TNF, and its molecular form (soluble versus membrane-bound) appeared distinct. In spleen, in addition to major B-TNF signal, a complementary T-TNF signal contributed to the microstructure. In contrast, B-TNF predominantly controlled the development of follicular dendritic cells and B-cell follicles in Peyer patches. In lymph nodes, cooperation between TNF expressed by B and T cells was necessary for the maintenance of microarchitecture and for generation of an efficient humoral immune response. Unexpectedly, soluble but not membrane TNF expressed by B cells was essential for the organization of the secondary lymphoid organs. Thus, the maintenance of each type of secondary lymphoid organ is orchestrated by distinct contributions of membrane-bound and soluble TNF produced by B and T lymphocytes.


Subject(s)
B-Lymphocytes/immunology , Lymph Nodes/immunology , Peyer's Patches/immunology , Spleen/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Gene Expression Regulation , Immunity, Humoral , Lymph Nodes/cytology , Lymph Nodes/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Peyer's Patches/cytology , Spleen/cytology , Spleen/ultrastructure , Tumor Necrosis Factor-alpha/genetics
15.
Immunity ; 32(3): 403-13, 2010 Mar 26.
Article in English | MEDLINE | ID: mdl-20226692

ABSTRACT

Epithelial cells provide the first line of defense against mucosal pathogens; however, their coordination with innate and adaptive immune cells is not well understood. Using mice with conditional gene deficiencies, we found that lymphotoxin (LT) from innate cells expressing transcription factor RORgammat, but not from adaptive T and B cells, was essential for the control of mucosal C. rodentium infection. We demonstrate that the LTbetaR signaling was required for the regulation of the early innate response against infection. Furthermore, we have revealed that LTbetaR signals in gut epithelial cells and hematopoietic-derived cells coordinate to protect the host from infection. We further determined that LTbetaR signaling in intestinal epithelial cells was required for recruitment of neutrophils to the infection site early during infection via production of CXCL1 and CXCL2 chemokines. These results support a model wherein LT from RORgammat(+) cells orchestrates the innate immune response against mucosal microbial infection.


Subject(s)
Citrobacter rodentium/immunology , Enterobacteriaceae Infections/immunology , Epithelial Cells/immunology , Immunity, Innate , Intestinal Mucosa/immunology , Lymphotoxin beta Receptor/immunology , Signal Transduction , Adaptive Immunity , Animals , Bone Marrow Cells/immunology , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Lymphotoxin beta Receptor/deficiency , Lymphotoxin beta Receptor/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout
16.
Brain Pathol ; 20(4): 771-9, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20070303

ABSTRACT

Systemic or brain-selective hypothermia is a well-established method for neuroprotection after brain trauma. There is increasing evidence that hypothermia exerts beneficial effects on the brain and may also support regenerative responses after brain damage. Here, we have investigated whether hypothermia influences neurite outgrowth in vitro via modulation of the post-injury cytokine milieu. Organotypic brain slices were incubated: deep hypothermia (2 h at 17 degrees C), rewarming (2 h up to 37 degrees C), normothermia (20 h at 37 degrees C). Neurite density and cytokine release (IL 1beta, IL-6, IL-10, and TNF-alpha) were investigated after 24 h. For functional analysis mice deficient in NT-3/NT-4 and TNF-alpha as well as the TNF-alpha inhibitor etanercept were used. Hypothermia led to a significant increase of neurite outgrowth, which was independent of neurotrophin signaling. In contrast to other cytokines investigated, TNF-alpha secretion by organotypic brain slices was significantly increased after deep hypothermia. Moreover, hypothermia-induced neurite extension was abolished after administration of the TNF-alpha inhibitor and in TNF-alpha knockout mice. We demonstrate that TNF-alpha is responsible for inducing neurite outgrowth in the context of deep hypothermia and rewarming. These data suggest that hypothermia not only exerts protective effects in the CNS but may also support neurite outgrowth as a potential mechanism of regeneration.


Subject(s)
Cold Temperature , Entorhinal Cortex/metabolism , Neurites/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Entorhinal Cortex/drug effects , Enzyme-Linked Immunosorbent Assay , Etanercept , Immunoglobulin G/pharmacology , Immunohistochemistry , Mice , Mice, Knockout , Nerve Growth Factors/metabolism , Neurites/drug effects , Neurotrophin 3/metabolism , Organ Culture Techniques , Receptors, Tumor Necrosis Factor , Rewarming , Statistics, Nonparametric , Tumor Necrosis Factor-alpha/antagonists & inhibitors
17.
Eur J Immunol ; 39(10): 2906-15, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19735075

ABSTRACT

TNF, lymphotoxin (LT)-alpha, LT-beta and LIGHT are members of a larger superfamily of TNF-related cytokines that can cross-utilize several receptors. Although LIGHT has been implicated in thymic development and function, the role of TNF and LT remains incompletely defined. To address this, we created a model of modest homeostatic overexpression of TNF/LT cytokines using the genomic human TNF/LT locus as a low copy number Tg. Strikingly, expression of Tg TNF/LT gene products led to profound early thymic atrophy characterized by decreased numbers of thymocytes and cortical thymic epithelial cells, partial block of thymocyte proliferation at double negative (DN) 1 stage, increased apoptosis of DN2 thymocytes and severe decline of T-cell numbers in the periphery. Results of backcrossing to TNFR1-, LTbetaR- or TNF/LT-deficient backgrounds and of reciprocal bone marrow transfers implicated both LT-alpha/LT-beta to LTbetaR and TNF/LT-alpha to TNFR1 signaling in accelerated thymus degeneration. We hypothesize that chronic infections can promote thymic atrophy by upregulating LT and TNF production.


Subject(s)
Atrophy/genetics , Gene Expression/genetics , Lymphotoxin-alpha/genetics , Lymphotoxin-beta/genetics , Thymus Gland/pathology , Tumor Necrosis Factor-alpha/genetics , Animals , Apoptosis/genetics , Apoptosis/immunology , Atrophy/pathology , Bone Marrow Transplantation , Cell Count , Cell Proliferation , Epithelial Cells/pathology , Gene Dosage/genetics , Humans , Keratin-8/metabolism , Lymphotoxin beta Receptor/genetics , Lymphotoxin-alpha/metabolism , Lymphotoxin-beta/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, Tumor Necrosis Factor, Type I/genetics , Stem Cells/pathology , T-Lymphocyte Subsets/pathology , T-Lymphocytes/pathology , Thymus Gland/metabolism , Tumor Necrosis Factor-alpha/metabolism
18.
J Biotechnol ; 142(2): 170-8, 2009 Jun 15.
Article in English | MEDLINE | ID: mdl-19439388

ABSTRACT

Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn's disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (V(H)H) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF-V(H)H does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF-V(H)H molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF-V(H)H and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF-V(H)H-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF-V(H)H was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF-V(H)H proteins were much more active than the monomeric hTNF-V(H)H. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF-V(H)H proteins is caused by avidity effects.


Subject(s)
Immunoglobulin Heavy Chains/metabolism , Protein Multimerization , Recombinant Fusion Proteins/metabolism , Transglutaminases/metabolism , Tumor Necrosis Factor-alpha , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Cytotoxicity Tests, Immunologic , Escherichia coli/genetics , Etanercept , Humans , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains/genetics , Protein Binding , Receptors, Tumor Necrosis Factor/metabolism , Ruminants/immunology , Spectrometry, Fluorescence , Streptomyces/enzymology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/immunology
19.
Cytokine Growth Factor Rev ; 19(3-4): 231-44, 2008.
Article in English | MEDLINE | ID: mdl-18502680

ABSTRACT

TNF is an exciting cytokine which has helped to establish many paradigms in immunology. Although TNF itself has found only very limited use in the clinic, anti-cytokine therapy, which targets this single molecule, has enjoyed astounding success in treatment of a growing number of human diseases. However, since TNF mediates unique physiologic functions, in particular those related to host defense, TNF blockade may result in unwanted consequences. Much of our understanding about TNF intrinsic functions in the body, as well as about consequences of its overexpression and ablation, is based on studying phenotypes of various genetically engineered mice. Here we review mouse studies aimed at understanding TNF physiologic functions using transgenic and knockout models, and we discuss additional mouse models that may be helpful in the future.


Subject(s)
Tumor Necrosis Factor-alpha/physiology , Animals , Autoimmune Diseases/metabolism , Gene Targeting , Mice , Mice, Knockout , Mice, Transgenic , Models, Animal , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics
20.
Eur J Immunol ; 35(5): 1592-600, 2005 May.
Article in English | MEDLINE | ID: mdl-15832287

ABSTRACT

We generated a novel tumor necrosis factor (TNF) null mutation using Cre-loxP technology. Mice homozygous for this mutation differ from their "conventional" counterparts; in particular, they completely lack Peyer's patches (PP) but retain all lymph nodes. Our analysis of these novel TNF-knockout mice supports the previously disputed notion of the involvement of TNF-TNFR1 signaling in PP organogenesis. Availability of TNF-knockout strains both with and without PP enables more definitive studies concerning the roles of TNF and PP in various immune functions and disease conditions. Here, we report that systemic ablation of TNF, but not the presence of PP per se, is critical for protection against intestinal Listeria infection in mice.


Subject(s)
Mice, Knockout/immunology , Peyer's Patches/immunology , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/genetics , Animals , Flow Cytometry , Hematopoiesis/immunology , Immunohistochemistry , Listeriosis/immunology , Mice , Mice, Knockout/genetics , Mutation , Peyer's Patches/pathology , Tumor Necrosis Factor-alpha/immunology
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