[Ultrasonographic features of thyroid carcinoma of different sizes: comparison between medullary thyroid carcinomas and papillary thyroid carcinomas].

Objective: To investigate the ultrasonographic features of medullary thyroid carcinomas (MTCs) of different sizes and supply valid information for separating MTCs from papillary thyroid carcinomas (PTCs). Methods: There were 87 patients with MTC and 220 patients with PTC detected by ultrasonography and confirmed by pathology at Tianjin Medical University Cancer Institute and Hospital from June 2018 to March 2022. Nodules were divided into the large nodule group (the maximum diameter of the tumor was＞1 cm) and the small nodule group (the maximum diameter of the tumor was ≤1 cm). There were 97 cases in the small nodule group, including 28 cases of MTC and 69 cases of PTC. There were 210 cases in the large nodule group, including 59 cases of MTC and 151 cases of PTC. After stratification by thyroid nodules, ultrasonographic features of thyroid nodules and metastatic lymph nodes, preoperative serum calcitonin (CT) and carcinoembryonic antigen (CEA) levels were compared between MTC and PTC patients. Results: In the small nodule group, the proportion of MTCs exhibiting hypoecho, smooth margins, and having blood flow signals was higher than that of PTCs, with statistically significant differences (all P＜0.05). In the large nodule group, the proportion of MTCs showing cystic solidity, hypoecho, smooth margins, blood flow, and the type Ⅳvascular distribution was higher than PTCs, and the difference of calcification type between them was also statistically significant (all P＜0.05). In contrast, the differences in the number of lesions and aspect ratio between MTCs and PTCs were not statistically significant regardless of nodule size (all P＞0.05). In the small nodule group,6 metastatic lymph nodes of medullary thyroid carcinoma (LNM-MTC) and 11 metastatic lymph nodes of papillary thyroid carcinoma (LNM-PTC) were correctly diagnosed by ultrasound, respectively. The diagnostic compliance rate of ultrasound was 78.6% (22/28) and 78.3% (54/69), respectively, with no statistically significant difference (P=0.973). In the large nodule group, 28 LNM-MTC and 11 LNM-PTC were correctly diagnosed by ultrasound, respectively. The diagnostic compliance of ultrasound was 88.1% (52/59) and 73.5% (111/151), respectively, which was statistically significant (P=0.022). Among them, 82.1% of LNM-MTC and 56.6% of LNM-PTC showed abnormal blood flow signals, with a statistically significant difference (P=0.016). There were significant differences in preoperative serum CT and CEA levels of different sizes of MTCs (all P＜0.05). Conclusions: Different sizes of MTCs require diverse demonstrative criteria. Abnormal blood flow signal is of great significance in the diagnosis of LNM-MTC. Within the absence of ultrasonic characteristics, preoperative serum CT test can provide confidence for the diagnosis of MTC.

A novel ex vivo culture model for inflammatory bone destruction.

Pathological alterations in the balance of bone metabolism are central to the progression of inflammatory bone diseases such as periodontal disease. We have developed and characterized a novel ex vivo murine mandible model of inflammatory bone destruction. Slices of mandible were cultured for 14 days in the presence or absence of P. gingivalis lipopolysaccharide (LPS) or pro-inflammatory cytokines. Following culture, cell viability and tissue histomorphometry were assessed with quantification of matrix proteins, resident osteoclasts, ligament cells, monocytes, macrophages, and neutrophils. In the absence of inflammatory factors, culture viability, osteoclasts, and matrix components were maintained. LPS or TNFα stimulation demonstrated an increase in cellular proliferation, monocyte cells, osteoclast differentiation, and matrix degradation. Pathophysiological bone metabolism can be induced via exposure to LPS and direct influence of TNFα within the model despite the absence of systemic circulation, providing a model for inflammatory bone destruction and investigation of the effects of novel therapeutics.

Interleukin-18 system plays an important role in keloid pathogenesis via epithelial-mesenchymal interactions.

BACKGROUND: Keloid scarring is a dermal fibroproliferative disorder characterized by increased fibroblast proliferation and excessive production of collagen and extracellular matrix (ECM) components. To date, the role of cytokines in keloid pathogenesis has not been completely unravelled. Interleukin (IL)-18 is a pro-inflammatory cytokine that plays important roles in wound healing, fibrogenesis and carcinogenesis. OBJECTIVES: Our aim was to study the role of the IL-18 system in keloid pathogenesis. MATERIALS AND METHODS: Expression and localization of IL-18 and its receptor (IL-18R) were investigated in normal skin and keloid tissues using Western blot and immunohistochemistry. We further studied the expression of the IL-18 system in normal and keloid-derived cell lines in a coculture model. RESULTS: Results from Western blot and immunohistochemistry revealed that IL-18, IL-18Rα and IL-18Rß expression was elevated in keloid tissue compared with normal skin tissue. Studies on the expression of IL-18 and its antagonist, IL-18 binding protein (IL-18BP), using a coculture model demonstrated severe IL-18/IL-18BP imbalance in keloid keratinocyte/keloid fibroblast (KK/KF) cocultures with significant elevation of bioactive IL-18 whereas IL-18BP levels remained the same. This overproduction of bioactive IL-18 in keloid cocultures could be due to increased caspase-1 and decreased caspase-3 expression in keloid tissue, as well as decreased soluble IL-10 levels observed in keloid cocultures. The important inductive effects of IL-18 on KFs were further underscored by the observation that exposure of KF to IL-18 resulted in increased collagen and ECM component synthesis, and increased secretion of profibrotic cytokines such as IL-6 and IL-8. Finally, the addition of phosphatidylinositol 3-kinase (PI3K), mitogen activation protein kinase (MAPK), specificity protein 1 (Sp1) and mammalian target of rapamycin (mTOR) inhibitors inhibited IL-18 secretion in keloid cocultures. CONCLUSIONS: The present study has proven that the IL-18 system plays an important role in keloid pathogenesis via epithelial-mesenchymal interactions. It also suggests a therapeutic potential of PI3K, MAPK, Sp1 and mTOR inhibitors in the treatment of keloid scarring.

Elevated expression of transmembrane IL-15 in immune cells correlates with the development of murine lupus: a potential target for immunotherapy against SLE.

Presentation in trans by the Interleukin-15 receptor alpha chain (IL-15Ralpha) has been suggested as the main mechanism for IL-15 anchoring to the cell surface, but it is also evident that IL-15 can exist as a transmembrane protein. We herein demonstrate that replacement of the first 41 residues of human IL-15 (hIL-15) with Igkappa chain leader sequence resulted in secretion of most of the recombinant hIL-15 expressed in transfectant cells, thus identifying the transmembrane region of IL-15. A fusion protein (hIL-15Ralpha-Fc) between the extracellular domain of hIL-15Ralpha and the Fc fragment of IgG1 was prepared and shown to be able to bind with transmembrane IL-15 (tmIL-15). The level of tmIL-15 expression in macrophages, activated T cells and B cells from 6-month-old BXSB male mice, an animal model for systemic lupus erythematosus (SLE), was significantly increased compared with that from BXSB females or young males. In addition, hIL-15Ralpha-Fc was able to block the T cell stimulating and anti-apoptotic effect of the tmIL-15-positive BXSB macrophages in vitro. Intravenous administration of hIL-15Ralpha-Fc reduced the titre of autoantibodies against dsDNA and also proteinuria in aged BXSB males, implying that neutralization of IL-15 activity in vivo may be an effective way of treating SLE.

Production and utilization of interleukin-15 in malignant catarrhal fever.

Malignant catarrhal fever (MCF) is an often fatal lymphoproliferative disease of ungulates caused by either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). The pathogenesis of MCF is poorly understood, but appears to involve an auto-destructive pathology whereby cytotoxic lymphocytes destroy areas of a variety of tissues. The cytokine interleukin-15 (IL-15) is involved in the development and maintenance of cytotoxic lymphocytes and may therefore have a role in the pathogenesis of MCF. Virus-infected large granular lymphocytes (LGLs) were obtained from the tissues of rabbits infected with AlHV-1 or OvHV-2. These cells exhibited a similar proliferative response to IL-15 and to IL-2 in culture, but their content of the activated cytotoxic enzyme (BLT-esterase) was maintained at higher levels in the presence of IL-15 compared with IL-2. The LGLs did not express IL-15 mRNA or produce IL-15 protein. By contrast, there was abundant expression of IL-15 mRNA and protein in affected tissues. IL-15 production was associated with necrotic lesions of the mesenteric lymph node and appendix of OvHV-2-infected rabbits, but was not found in the same tissues of rabbits infected with AlHV-1 in which there were no necrotic lesions. The cellular source of the IL-15 was predominantly lymphoid cells that did not express B cell or monocyte-macrophage markers. Only a few IL-15+ cells (<10%) co-localized with pan-T cells or CD8+ T cells. The abundance of IL-15 in tissue with lesions of MCF suggests that this cytokine may have a role in the pathogenesis of MCF.

Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption.

Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFkappaB and in NFkappaB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFkappaB in osteoclast precursors.

Nitric oxide mediates intestinal pathology but not immune expulsion during Trichinella spiralis infection in mice.

The relationship between intestinal pathology and immune expulsion of gastrointestinal (GI) nematodes remains controversial. Although immune expulsion of GI helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been identified. We have previously shown that while the intestinal pathology accompanying the expulsion of the GI parasite Trichinella spiralis may be dependent on IL-4 and mediated by TNF, parasite loss is independent of TNF. In contrast, intestinal pathology in other disease models has been attributed to Th1 cytokines, although it closely resembles that seen in helminth infections. Whereas production of inducible NO synthase (iNOS) in the gut is important for both homeostasis of the epithelial layer and in protection against pathogenic microorganisms, overproduction of NO has been implicated in the pathogenesis of a number of inflammatory conditions. We therefore investigated the role of NO in T. spiralis infection using iNOS-deficient mice. iNOS-/- and iNOS-/+ mice were infected with T. spiralis, and parasite expulsion and intestinal pathology were followed. Parasite expulsion proceeded similarly in both groups of animals, but significant intestinal pathology was only observed in the heterozygous mice. Thus it appears that, although the protective effects of Th2 responses in GI helminth infection do not require NO, this mediator contributes substantially to the associated enteropathy. NO may therefore be an important mediator of enteropathy in both Th1- and Th2-inducing conditions.

Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase.

Macrophage activation by cytokines or microbial products such as LPS results in the induction and release of several key immune effector molecules including NO and IL-12. These have been shown to play crucial roles in the development of immunity to intracellular pathogens such as Leishmania. The molecular mechanisms underlying the induction of these effector molecules are not fully understood. We now show that the extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases play differential roles in the regulation of LPS-stimulated inducible NO synthase and IL-12 gene expression. In macrophages, LPS stimulates the simultaneous activation of all three classes of MAP kinases, ERK, c-jun N-terminal kinase, and p38, albeit with differential activation kinetics. However, studies using inhibitors selective for ERK (PD98059) and p38 (SB203580) show that while p38 plays an essential role in the induction of inducible NO synthase, ERK MAP kinases play only a minor role in promoting NO generation. In contrast, while p38 promotes induction of IL-12 (p40) mRNA, ERK activation suppresses LPS-mediated IL-12 transcription. The biological relevance of these regulatory signals is demonstrated by our finding that Leishmania lipophosphoglycans, which promote parasite survival, act by stimulating ERK MAP kinase to inhibit macrophage IL-12 production. Thus, as ERK and p38 MAP kinases differentially regulate the induction of the macrophage effector molecules, inducible NO synthase and IL-12, these kinases are potential targets not only for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation.

A proinflammatory role for IL-18 in rheumatoid arthritis.

IL-18 is a novel cytokine with pleiotropic activities critical to the development of T-helper 1 (Th1) responses. We detected IL-18 mRNA and protein within rheumatoid arthritis (RA) synovial tissues in significantly higher levels than in osteoarthritis controls. Similarly, IL-18 receptor expression was detected on synovial lymphocytes and macrophages. Together with IL-12 or IL-15, IL-18 induced significant IFN-gamma production by synovial tissues in vitro. IL-18 independently promoted GM-CSF and nitric oxide production, and it induced significant TNF-alpha synthesis by CD14(+) macrophages in synovial cultures; the latter effect was potentiated by IL-12 or IL-15. TNF-alpha and IFN-gamma synthesis was suppressed by IL-10 and TGF-beta. IL-18 production in primary synovial cultures and purified synovial fibroblasts was, in turn, upregulated by TNF-alpha and IL-1beta, suggesting that monokine expression can feed back to promote Th1 cell development in synovial membrane. Finally, IL-18 administration to collagen/incomplete Freund's adjuvant-immunized DBA/1 mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 can be proinflammatory in vivo. Together, these data indicate that synergistic combinations of IL-18, IL-12, and IL-15 may be of importance in sustaining both Th1 responses and monokine production in RA.

T-Cell responses during Trypanosoma brucei infections in mice deficient in inducible nitric oxide synthase.

We have investigated the possibility that nitric oxide (NO) synthesis may affect the course of a trypanosome infection via T-cell responses using mice deficient in inducible NO synthase (iNOS). Parasitemia levels increased at the same rate in both iNOS-deficient homozygous and control heterozygous mice, and peak parasitemia values were the same in both groups. However, the heterozygous mice maintained higher parasitemia levels after the peak of an infection than the homozygous mice due to a decrease in the rate of clearance of parasites. In iNOS-deficient mice there was an increase in the numbers of total CD4(+) cells and activated (interleukin-2 receptor-expressing) CD4(+) cells in infected mice compared with the numbers in uninfected mice. Spleen cells from infected iNOS-deficient mice displayed increased proliferative responses and gamma interferon secretion when stimulated in vitro than those of control mice. These data suggest that NO production depresses T-helper 1-like responses generated during Trypanosoma brucei infections, thus promoting the survival of the parasite.

Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12 synthesis, strong Th1 responses, and enhanced nitric oxide production.

MRL/MP-lpr/lpr (MRL/lpr) mice have a single mutation (lpr) of the fas apoptosis gene. The mutant mice developed significantly smaller lesions than the wild-type mice at the earlier stage of infection with the intracellular protozoan parasite Leishmania major. However, while all the wild-type mice achieved complete lesion resolution, the disease in the mutant mice progressed inexorably. The mutant mice had more IL-12 and nitrite/nitrate in the serum than wild-type mice following infection. Lymphoid cells from infected MRL/lpr mice produced more IFN-gamma but less IL-4 and IL-5 than cells from MRL-+/+ mice. Peritoneal macrophages from the mutant mice also produced more IL-12 and NO after stimulation with LPS. Thus, Fas expression is essential for resistance against leishmaniasis, and Fas-mediated apoptosis may form an integral part of the Th1-mediated microbicidal function.

Anti-GBM glomerulonephritis in mice lacking nitric oxide synthase type 2.

Nitric oxide is synthesized in experimental immune complex glomerulonephritis due to local induction of type 2 nitric oxide synthase (NOS2). To determine the role of NOS2, the course of accelerated anti-glomerular basement membrane glomerulonephritis (anti-GBM) was examined in mice homozygous for disruption of the NOS2 gene compared with heterozygous littermates. Disease in the wild type strain (129Sv) was characterized by heavy albuminuria, glomerular neutrophil and macrophage infiltration and glomerular thrombosis. NOS2, interleukin 1B (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) mRNA were induced by 24 hours. The NOS2-deficient mutant mice and the heterozygous mice displayed early (24 hr) and full autologous phase (day 6) injury indistinguishable from the wild-type mice. The equivalent degree of albuminuria and glomerular inflammation indicates that NOS2 does not play an essential role in this form of glomerulonephritis in the mouse.

Cytokines and nitric oxide as effector molecules against parasitic infections.

Nitric oxide (NO) derived from L-arginine by the catalytic action of inducible NO synthase (iNOS) plays an important role in killing parasites. Many cell types express high levels of iNOS when activated by a number of immunological stimuli which include interferon-gamma (IFN-gamma), tumour necrosis factor alpha, and lipopolysaccharide. IFN-gamma is typically produced by the Th1 subject of CD4+ T cells, whose differentiation depends on interleukin-12 (IL-12) produced by macrophages. Mice with a disrupted iNOS gene were highly susceptible to Leishmania major infection compared with similarly infected control wild-type mice. The mutant mice developed significantly higher levels of TH1-cell response compared with the control mice, suggesting that NO is likely to be the effector molecule in the immunological control of this and other intracellular parasitic infections. To ensure their survival, the Leishmania parasites have evolved effective means to inhibit NO synthesis. The highly conserved major surface glycolipids, glycoinositol-phospholipids and lipophosphoglycan (LPG), of Leishmania are potent inhibitors of NO synthesis. Furthermore, LPG can also inhibit IL-12 synthesis, thereby indirectly blocking the induction of iNOS. The evolutionary and therapeutic implications of these findings are discussed.

Skin allograft rejection in mice lacking inducible nitric oxide synthase.

BACKGROUND: During allograft rejection, up-regulation of cytokine-inducible nitric oxide synthase (iNOS) leads to the production of large amounts of nitric oxide (NO). The net effect of NO on the alloimmune response is, however, difficult to predict because of its diverse biological effects, which include potentially opposing roles as an effector and immunoregulatory molecule. METHODS: In this study, the role of iNOS on the in vitro and in vivo alloimmune response was defined using mutant mice that lack a functional iNOS gene. The ability of spleen cells obtained from iNOS-deficient mutants to proliferate and to produce cytokines in response to irradiated BALB/c stimulator cells was determined, and the rate at which iNOS-deficient mice were able to reject BALB/c skin allografts was observed. RESULTS: Spleen cells from homozygous iNOS-deficient (129xMF1)F1 mice, when compared with cells from heterozygous control mice, showed an increased in vitro proliferative response and produced substantially higher levels of interferon-gamma, and also more interleukin-2 and interleukin-12, in response to allogeneic stimulation. The kinetics of BALB/c skin graft rejection were comparable in heterozygous control animals and iNOS-deficient mice. Moreover, no net effect of iNOS on skin allograft rejection was apparent in mice treated with depleting monoclonal antibodies (mAb) to CD4 or CD8 T cells, either alone or in combination, or in mice treated with both anti-CD8 mAb and a neutralizing anti-tumor necrosis factor mAb. CONCLUSIONS: These results show that iNOS has an immunomodulatory effect on the in vitro alloimmune response but lack of iNOS has no net influence on the kinetics of murine skin allograft rejection in either unmodified recipients or recipients in which the early contribution of T-cell subsets and tumor necrosis factor-alpha to graft rejection has been abrogated.

Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients.

We have demonstrated spontaneous nitric oxide (NO) production by primary synovial cultures from rheumatoid (RA) and osteoarthritis patients. Increased NO production followed addition of staphylococcal enterotoxin B. Immunochemical double staining with specific anti-human inducible NO synthase (iNOS) and nonspecific esterase (NSE), or anti-CD68 (markers for tissue macrophages) showed that although many lining layer cells in RA synovium expressed iNOS, most (approximately 90%) were NSE- and CD68-, with only a minor population (approximately 10%) which were iNOS+, CD68+/NSE+. These data demonstrate the capacity for high output of NO by human synovial tissue and show that, although human macrophages can express high levels of iNOS, the majority of cells expressing iNOS are fibroblasts. We also report that synoviocytes, and macrophage cell lines, cultured with the NO donor, S-nitroso-acetyl penicillamine, produced high concentrations of tumor necrosis factor (TNF)-alpha. These results suggest that NO may mediate pathology in RA through the induction of TNF-alpha production.

[Free radical damage to chondrocytes and protection by selenium, tocopherol and ascorbic acid using serum-free medium method].

Free radical derived from xanthine oxidase damage to rabbit articular chondrocytes cultured in serum-free medium and antioxidant defense factors protecting chondrocytes from free radical were studied. The results showed that free radical mediated an inhibition of DNA as well as proteoglycan synthesis in cultured chondrocytes and selenium, a-tocopherol and L-ascorbic acid failed to protect chondrocytes from free radicals.

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.