Regulation of autoimmune and anti‐tumour T‐cell responses by PTPN22

A number of polymorphisms in immune‐regulatory genes have been identified as risk factors for the development of autoimmune disease. PTPN22 (that encodes a tyrosine phosphatase) has been associated with the development of several autoimmune diseases, including type 1 diabetes, rheumatoid arthritis and systemic lupus erythematosus. PTPN22 regulates the activity and effector functions of multiple important immune cell types, including lymphocytes, granulocytes and myeloid cells. In this review, we describe the role of PTPN22 in regulating T‐cell activation and effector responses. We discuss progress in our understanding of the impact of PTPN22 in autoimmune disease in humans and mouse models, as well as recent evidence suggesting that genetic manipulation of PTPN22 expression might enhance the efficacy of anti‐tumour T‐cell responses.     

Induction of micronuclei and cell cycle arrest by some tri‐ and tetrachlorobiphenyls in mammalian cells deficient in xenobiotic‐metabolizing enzymes

Polychlorinated biphenyls (PCBs) are persistent organic pollutants with continued public health concerns. The lower chlorinated biphenyls are supposed to be mutagenic following metabolic activation. However, in a preliminary study, we recently observed induction of micronuclei by several PCBs in a subclone of Chinese hamster V79 cell line, V79‐Mz, which is deficient in xenobiotic‐metabolizing enzyme activities. In this study, metabolism‐free genotoxicity of PCBs was investigated, using 10 tri‐ and tetrachlorobiphenyls, in V79, V79‐Mz, and human hepatoma (HepG2) cell lines. Among the four tetrachlorobiphenyls, 2,4,4′,5‐ and 2,3′4,4′‐tetrachlorobiphenyl—both having a noncoplanar configuration—induced micronuclei in V79‐Mz cells, while their coplanar analogs 3,4,4′,5‐ and 3,3′,4,4′‐tetrachlorobiphenyl were inactive. Furthermore, 2,3,3′‐ (PCB 20) and 2,3,4′‐trichlorobiphenyl (PCB 22) started to induce micronuclei in V79‐Mz cells at 10 μM and higher concentrations, demonstrating more potent effects than observed with 2,2′,3‐, 2,2′,4‐, 2,2′,5, and 2,4,4′‐trichlorobiphenyl. As representative compounds, PCB 20 and 22 induced micronuclei in relatively high concentrations in HepG2 cells (p53‐proficient), though they did not induce Hprt gene mutations in V79‐Mz cells. PCB 20 and 22 increased mitotic index and induced cell cycle arrest at the G2/M phase, with effects more potent in V79‐Mz than in V79 cells. This study suggests that 2,3,4′‐ and 2,3,3′‐substituted PCBs are micronuclei inducers and G2/M arresters among a number of trichlorobiphenyls in mammalian cell lines, though with potency lower than that observed recently in V79‐derived cells expressing human CYP2E1. Similarly, some noncoplanar tetrachlorobiphenyls possess metabolism‐independent chromosome‐damaging potentials. Environ. Mol. Mutagen. 58:199–208, 2017. © 2017 Wiley Periodicals, Inc.     

Defective antiviral CD8 T‐cell response and viral clearance in the absence of c‐Jun N‐terminal kinases

The c‐Jun N‐terminal kinase (JNK) signalling pathway appears to act as a critical intermediate in the regulation of lymphocyte activation and proliferation. The majority of studies on the importance of JNK are focused on its role in T helper responses, with very few reports addressing the mechanisms of JNK in governing CD8 T‐cell‐mediated immunity. By using a well‐defined mousepox model, we demonstrate that JNK is involved in CD8⁺ T‐cell‐mediated antiviral responses. Deficiency of either JNK1 or JNK2 impaired viral clearance, subsequently resulting in an increased susceptibility to ectromelia virus in resistant mice. The impairment of CD8 responses in JNK‐deficient mice was not directly due to an inhibition of effector T‐cell expansion, as both JNK1 and JNK2 had limited effect on the activation‐induced cell death of CD8⁺ T cells, and only JNK2‐deficient mice exhibited a significant change in CD8⁺ T‐cell proliferation after acute ectromelia virus infection. However, optimal activation of CD8⁺ T cells and their effector functions require signals from both JNK1 and JNK2. Our results suggest that the JNK pathway acts as a critical intermediate in antiviral immunity through regulation of the activation and effector function of CD8⁺ T cells rather than by altering their expansion.     

Determination of Homopolymerization Kinetics of 10‐(N‐Methylacrylamido)‐decylphosphonic acid,Its Diethyl ester,and 10‐(Methacryloyloxy)‐decylphosphonic acid,and Their Copolymerization with Methyl methacrylate

Two polymerizable phosphonic acids destined to be constituents of self‐etch adhesives as well as a diethyl phosphonate bearing an N‐methylacrylamide group are examined for their polymerization and copolymerization activity in methanol between 45 and 65 °C. Polymerization proceeds readily through a thermal free radical initiator. The intensity exponents for the monomer and initiator are only slightly over 1 and ≈0.5, respectively, in accordance with the results typically observed for an ideal free radical polymerization with bimolecular termination. The specific feature of this process is the absence of auto‐acceleration during the phosphonic acid monomer polymerization and polymer network formation in case of the phosphonate monomer. The kinetics of copolymerization with methyl methacrylate (MMA) are monitored by online ¹H NMR spectroscopy. Two copolymerization reactions for each pair of comonomers are sufficient to evaluate the kinetic data using the Jaacks method, the Fineman–Ross method, and the nonlinear least squares method. All three methods give similar results for particular monomer/MMA couples.

     

Evaluation of in vivo genotoxicity induced by N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and 4‐nitroquinoline‐1‐oxide in the Pig‐a and gpt assays

The recently developed Pig‐a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor‐deficient red blood cells caused by a forward mutation in the Pig‐a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig‐a assay could be integrated into repeat‐dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig‐a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N‐ethyl‐N‐nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4‐nitroquinoline‐1‐oxide (4NQO, 50 mg/kg) in the Pig‐a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig‐a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7‐week terminal sacrifice. ENU increased both Pig‐a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig‐a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two‐ or threefold above control) were detected in the bone marrow in both the Pig‐a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig‐a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

Retinoic acid promotes the development of Arg1‐expressing dendritic cells for the regulation of T‐cell differentiation

Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in DCs requires retinoic acid. Induction of Arg1 by retinoic acid is directly mediated by retinoic acid‐responsive elements in the 5′ noncoding region of the Arg1 gene. Arg1, produced by DCs in response to retinoic acid, promotes the generation of FoxP3⁺ regulatory T (Treg) cells. Importantly, blocking the retinoic acid receptor makes DCs hypo‐responsive to known inducers of Arg1 such as IL‐4 and GM‐CSF in Arg1 expression. We found that intestinal CD103⁺ DCs that are known to produce retinoic acid highly express Arg1. Our results establish retinoic acid as a key signal in expression of Arg1 in DCs.     

Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4⁺ T cells. A human dominant‐negative mutant class II transactivator (CIITA‐DN) transgene under a CAG promoter with an endothelium‐specific Tie2 enhancer was constructed. CIITA‐DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA‐DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4⁺ T‐cell response to cells from CIITA‐DN and wild‐type (WT) pigs was compared. Lymphocyte subset numbers and T‐cell function in CIITA‐DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen‐presenting cells from CIITA‐DN pigs was significantly reduced (40–50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4⁺ T‐cell response to CIITA‐DN pAECs was significantly weaker than to WT pAECs (60–80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4⁺ cells in the PBMCs from CIITA‐DN (20%) than from WT (30%) pigs (P < 0·01), T‐cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA‐DN pigs should be partially protected from the human cellular immune response.     

Influence of β‐Cyclodextrin on the Free‐Radical Copolymerization of N‐(4‐Methylphenyl)maleimide with N‐Vinylpyrrolidone in Water

Randomly methylated β‐cyclodextrin (me‐β‐CD) is used to include the hydrophobic monomer N‐(4‐methylphenyl)maleimide (MPM) ( 1 ) yielding the corresponding water‐soluble host‐guest structure 1a . Free‐radical copolymerization of 1a with N‐vinylpyrrolidone (NVP) ( 2 ) is performed and the reactivity ratios r₁ and r₂ are determined: 0.24 ± 0.03 (r₂) and 1.10 ± 0.05 (r_1a). This indicates a preferred incorporation of complexed maleimide into the copolymer chain. In contrast to that, the copolymerization of the uncomplexed monomers 1 and 2 is carried out using organic solvent (DMF/H₂O) showing reactivity ratios corresponding to nearly alternating copolymerization (r₂ = 0.09 ± 0.02; r₁ = 0.34 ± 0.03).

     

Formation of Macrocycles in the Synthesis of Poly(N‐(2‐ethylhexyl)carbazol‐3,6‐diyl)

A series of polymerizations of 3,6‐dibromo‐9‐(2‐ethylhexyl)carbazole was carried out in different monomer concentrations using standard Yamamoto reaction conditions. It was found that the molecular weight of the resulting poly(N‐(2‐ethylhexyl)carbazol‐3,6‐diyl) strongly depends on the monomer concentration in the reaction mixture. Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) measurements confirmed the formation of low‐molar‐mass cyclic oligomers of the 3,6‐disubstituted carbazole. In this paper we describe, for the first time, the formation of large amounts of a cyclic tetramer and of higher macrocycles in the synthesis of poly(N‐alkyl‐3,6‐carbazoles) by the Yamamoto method. This seems to be a limiting factor in the synthesis of high molecular weight poly(N‐alkyl‐3,6‐carbazole)s. The optical, thermal, and electrochemical properties of poly(N‐(2‐ethylhexyl)carbazol‐3,6‐diyl) have been investigated. Poly(N‐(2‐ethylhexyl)carbazol‐3,6‐diyl) is thermally stable, with 5% weight loss at 460 °C in nitrogen. The polymer exhibits a weak blue fluorescence with a maximum at 425 nm. Poly(N‐(2‐ethylhexyl)carbazol‐3,6‐diyl) is electrochemically stable, its highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels are ?5.0 and ?1.6 eV, respectively.

     

Copolymerization of N‐(prop‐1‐yne‐3‐yl)‐4‐(piperidine‐1‐yl)‐1,8‐naphthalimide with Arylacetylenes into Fluorescent Polyacetylene‐Type Conjugated Polymers

N‐(prop‐1‐yne‐3‐yl)‐4‐(piperidine‐1‐yl)‐1,8‐naphthalimide (PNPr), i.e., the monomer with a terminal ethynyl group and 1,8‐naphthalimide fluorophore, has been successfully copolymerized with a series of monoethynylarenes into well‐soluble high‐molecular‐weight (M_w up to 210 000) linear polyacetylene‐type copolymers containing from 14 to 51 mol% units derived from PNPr. The copolymerization of PNPr with bifunctional 4,4′‐diethynylbiphenyl provides polyacetylene‐type micro/mesoporous fluorescent network containing 8 mol% PNPr units and exhibiting the Brunauer–Emmett–Teller surface of ≈1000 m² g^?1. The copolymerizations (catalyzed with acetylacetonato(norborna‐2,5‐diene)rhodium complex, [Rh(nbd)acac]) proceed smoothly despite the fact that the homopolymerization of PNPr fails. The fluorescence of PNPr (emission at ≈ 510 nm) has been retained after the incorporation of PNPr into the copolymers. The fluorescence of the copolymers can be induced by a direct excitation of PNPr units or via an energy transfer mechanism. In the latter case, the comonomeric units with aromatic hydrocarbon fluorophores (e.g., of the biphenyl‐type) emitting at 380–400 nm (after irradiation with 300 nm UV radiation) serve as energy donors for fluorescent PNPr acceptors. The difference between the wavelengths of the primary absorbed radiation and the finally emitted radiation is 210 nm.

     

Sublinear response in lacZ mutant frequency of Muta™Mouse spermatogonial stem cells after low dose subchronic exposure to N‐ethyl‐N‐nitrosourea

The transgenic rodent mutation assay was used to compare the dose–response relationship of lacZ mutant frequency (MF) in spermatogonial stem cells exposed acutely or subchronically to N‐ethyl‐N‐nitrosourea (ENU). Muta^?Mouse males were exposed orally to 0, 25, 50, or 100 mg/kg ENU for acute exposures and 0, 1, 2, or 5 mg/(kg day) for 28‐day subchronic exposures. LacZ MF was measured in sperm collected 70 days post‐exposure to target spermatogonial stem cells. Dose–response data were fit to linear, quadratic, exponential, or power models. Acute exposure resulted in a dose‐dependent increase in MF that was significant (P < 0.05) at all doses tested and was best described by a quadratic dose–response model that was linear in the low dose range. In contrast, similar total doses fragmented over a 28‐day subchronic exposure only resulted in a significant increase in lacZ MF at the highest dose tested. Therefore, the subchronic no observable genotoxic effect level (NOGEL) was 2 mg/(kg day) (or 56 mg/kg total dose). The subchronic dose–response was best described by the exponential and power models, which were sublinear in the low dose range. Benchmark dose lower confidence limits (BMDLs) for acute and subchronic exposure were 3.0 and 1.0 mg/(kg day) (or 27.4 mg/kg total dose), respectively. These findings are supportive of a saturable DNA repair mechanism as the mutagenic mode of action for ENU in spermatogonia and imply that sufficiently low exposures would not cause appreciable genotoxic effects over background. This may have important implications for the quantitative risk assessment of germ cell mutagens. Environ. Mol. Mutagen. 56:347–355, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.     

Analysis of cellular response to isocyanate using N‐succinimidyl N‐methylcarbamate exposure in cultured mammalian cells

Isocyanates (R? N?C?O), one of the highly reactive industrial intermediates, possess the capability to modulate the bio‐molecules by forming toxic metabolites and adducts which may cause adverse health effects. Some of their toxic degradations have previously been unknown and overlooked; of which, molecular repercussions underlying their genetic hazards upon occupational/accidental exposures still remain as an intricate issue and are hitherto unknown. To assess the genotoxic potential of methyl isocyanate in cultured mammalian cells after in vitro exposure, we performed a study in three different normal cell lines MM55.K (mouse kidney epithelial), B/CMBA.Ov (mouse ovarian epithelial), and NIH/3T3 (primary mouse embryonic fibroblast). Cellular DNA damage response was studied for qualitative phosphorylation states of ATM, γH2AX proteins and quantitative state of p53 phosphorylation; DNA cell cycle analysis and measure of cellular apoptotic index before and after treatment were also investigated. Our results demonstrate that methyl isocyanate by negatively regulating the DNA damage response pathway, might promote cell cycle arrest, and apoptosis in cultured mammalian cells suggestive of causing genetic alterations. We anticipate that these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates. We also predict that increasing knowledge on DNA damage‐triggered signaling leading to cell death could provide new strategies for investigating the effects of DNA repair disorders and decreased repair capacity on the toxicity and carcinogenic properties of environmental toxins. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

T‐cell prolymphocytic leukemia (T‐PLL) with overlapping cytomorphological features with T‐CLL and T‐ALL: A Case Initially Diagnosed by Fine‐Needle Aspiration Cytology and Immunocytochemistry

T‐cell prolymphocytic leukemia (T‐PLL) is a very unusual form of chronic lymphoproliferative disorder, which has rarely been diagnosed by fine needle aspiration (FNA) cytology. We report one such case with some overlapping cytomorphological features with chronic lymphocytic leukemia and acute lymphoblastic leukemia. A 91‐year‐old man presented with generalized lymphadenopathy, pleural effusion, ascites, and an ulcerated growth in rectum. FNA smears from the left cervical lymph node showed a monotonous population of small lymphoid cells having small but distinct nucleoli that was initially diagnosed as chronic lymphocytic leukemia (CLL). Smears from the left axillary lymph node contained both small and medium‐sized lymphoid cells with frequent hand‐mirror cell appearance, which has been described in acute lymphoblatic leukemia (ALL). Immunocyto/histochemical stainings on smears and cell block preparations of the aspirate showed the following immunophenotype: CD3+, CD4+, CD5+, CD7+, CD8‐, CD20‐, CD23‐, and Tdt‐. Total peripheral blood leukocyte count was 26.4 × 10⁹/L and total lymphocyte count, 8.3 × 10⁹/L with predominance of small lymphocytes. T‐cell nature of the neoplasm was confirmed by biopsies from the cervical lymph node (T‐cell lymphoma), bone marrow (T‐cell lymphoid neoplasm/chronic lymphocytic leukemia), and the ulcerated rectal lesion (atypical T‐cell lymphoproliferative disorder). The patient developed deep vein thrombosis, heparin‐induced thrombocytopenia and bleeding from duodenal ulcer. By the time the reports of all the investigations were ready, the patient succumbed to bronchopneumonia. To the best of our knowledge, this T‐CLL/T‐PLL which was diagnosed initially by FNA cytology with immunocytochemical support is first of its kind to be reported. Diagn. Cytopathol. 2013;41:360–365. © 2011 Wiley Periodicals, Inc.     

Multifunctional Electrospun Nanofibers Prepared from Poly((N‐isopropylacrylamide)‐co‐(N‐hydroxymethylacrylamide)) and Their Blends with 1,2‐Diaminoanthraquinone for NO Gas Detection

Multifunctional electrospun (ES) nanofibers are successfully prepared from blends of poly((N‐isopropylacrylamide)‐co‐(N‐hydroxymethylacrylamide)) (poly(NIPAAm‐co‐NMA)) with NO_(g)‐responsive 1,2‐diaminoanthraquinone (DAQ). Different compositions of poly(NIPAAm‐co‐NMA) are synthesized by free radical polymerization. The NMA content significantly affects the stability of the ES nanofibers in water and the NO_(g) detection due to a change of the hydrophilic/hydrophobic characteristics. In addition, the fibers exhibit a significant volume (or hydrophilic/hydrophobic) change during the heating and cooling cycle between 25 and 50 °C, attributed to the lower critical solution temperature (LCST) characteristic of the thermoresponsive NIPAAm moiety. On the other hand, a distinct on/off switching of the optical absorption spectra and high color contrast on detecting the NO_(g) are observed using the ES nanofibers of poly(NIPAAm‐co‐NMA)/DAQ blends. The high surface/volume of ES fibers enhance the sensitivity and responsive speed, compared with that of drop‐cast film. The above studies suggest that the prepared multifunctional ES nanofibers have potential applications in environmentally sensing devices.