Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Interleukin-10 (IL-10) inhibits Borrelia burgdorferi-induced IL-17 production and attenuates IL-17-mediated Lyme arthritis.

Previous studies have shown that cells and cytokines associated with interleukin-17 (IL-17)-driven inflammation are involved in the arthritic response to Borrelia burgdorferi infection. Here, we report that IL-17 is a contributing factor in the development of Lyme arthritis and show that its production and histopathological effects are regulated by interleukin-10 (IL-10). Spleen cells obtained from B. burgdorferi-infected, "arthritis-resistant" wild-type C57BL/6 mice produced low levels of IL-17 following stimulation with the spirochete. In contrast, spleen cells obtained from infected, IL-10-deficient C57BL/6 mice produced a significant amount of IL-17 following stimulation with B. burgdorferi. These mice developed significant arthritis, including erosion of the bones in the ankle joints. We further show that treatment with antibody to IL-17 partially inhibited the significant hind paw swelling and histopathological changes observed in B. burgdorferi-infected, IL-10-deficient mice. Taken together, these findings provide additional evidence of a role for IL-17 in Lyme arthritis and reveal an additional regulatory target of IL-10 following borrelial infection.

High-powered gas-cooled microwave ablation: shaft cooling creates an effective stick function without altering the ablation zone.

OBJECTIVE: The purpose of our study was to validate the ability of a new gas-cooled microwave device to secure antennas into tissue before ablation via shaft cooling and to verify that such cooling does not compromise the intended ablation. MATERIALS AND METHODS: The force required to extract several types of applicators from ex vivo bovine liver before and after ablation was measured. Six groups were compared: cooled needle and multitined radiofrequency electrodes, secured and unsecured cryoprobes, and gas-cooled microwave antennas (n = 6 each). Ablations were next created in in vivo porcine livers for 2 and 10 minutes (n = 6 each) using the gas-cooled microwave system at 140 W. Extraction force was again measured before and after ablation and compared between groups using analysis of variance with post hoc Student t tests. Histologic analysis of the ablation zone was performed to evaluate cellular necrosis along the antenna shaft. RESULTS: Ex vivo, the secured cryoprobe and microwave antenna required significantly more force to remove than unsecured radiofrequency, cryoprobe, and microwave applicators (p < 0.05, all comparisons). The multitined radiofrequency electrode and cooled radiofrequency electrode required significantly more force to remove after ablation than before ablation (p = 0.006 and 0.02, respectively). In vivo, the secured antenna required significantly more force to remove before ablation than after ablation at both 2 (p < 0.0001) and 10 minutes (p < 0.0001). There was no histologic evidence of cell preservation along the antenna shaft. CONCLUSION: The gas cooling used in this microwave device can effectively secure antennas into tissue without altering ablation shape or reducing the intended thermal damage.

Rheumatoid arthritis: Methods for two murine models.

Rheumatoid arthritis is an incurable chronic inflammatory disease for which the pathophysiology is not fully understood, and treatment options are flawed. Thus, animal models are used to dissect disease pathogenesis and to develop improved therapeutics. However, accurately modeling all aspects of human rheumatoid arthritis in mice is not possible, and each model has pros and cons. Two useful murine models of rheumatoid arthritis are collagen induced arthritis and TNF induced arthritis. Both recapitulate the chronic inflammatory, erosive arthritis of human rheumatoid arthritis. Collagen induced arthritis has the added similarity to human rheumatoid arthritis of pathogenic autoantibodies, but can have variable degrees of arthritis severity, a challenge for experiments. In contrast, TNF induced arthritis tends to be uniform, but primarily models the innate arm of the immune response. Here we describe the benefits, limitations, and details for both models to help investigators select and implement an appropriate model to achieve the goals of their experiments.

Peptidylarginine Deiminase 2 in Murine Antiviral and Autoimmune Antibody Responses.

The peptidylarginine deiminases (PADs) and the citrullinated proteins that they generate have key roles in innate immunity and rheumatoid arthritis, an inflammatory arthritis with antibodies that target citrullinated proteins. However, the importance of PADs, particularly PAD2, in the adaptive immune response, both normal and pathogenic, is newly emerging. In this study, we evaluated a requirement for PAD2 in the antibody response in collagen-induced arthritis (CIA), a T and B cell-driven murine model of rheumatoid arthritis, and in the protective antibody response to murine influenza infection. Using PAD2-/- and PAD2+/+ mice on the DBA/1J background, we found that PAD2 is required for maximal anti-collagen antibody levels, but not collagen-specific plasma cell numbers, T cell activation or polarization, or arthritis severity in CIA. Also, we found that PAD2 is required not just for normal levels of persistent hemagglutination inhibiting antibodies but also for full protection from lethal influenza rechallenge. Together, these data provide evidence for a novel modest requirement for PAD2 in a normal antiviral antibody response and in an abnormal autoantibody response in inflammatory arthritis.

Discordant influence of Blastomyces dermatitidis yeast-phase-specific gene BYS1 on morphogenesis and virulence.

Blastomyces dermatitidis is a thermally induced dimorphic fungus capable of causing lung and systemic infections in immunocompetent animal hosts. With the publication of genomic sequences from three different strains of B. dermatitidis and the development of RNA interference as a gene-silencing tool, it has become possible to easily ascertain the virulence and morphological effects of knocking down the expression of candidate genes of interest. BYS1 (Blastomyces yeast-phase-specific 1), first identified by Burg and Smith, is expressed at high levels in yeast cells and is undetectable in mold. The deduced protein sequence of BYS1 has a putative signal sequence at its N terminus, opening the possibility that the BYS1-encoded protein is associated with the yeast cell wall. Herein, strains of B. dermatitidis with silenced expression of BYS1 were engineered and tested for morphology and virulence. The silenced strains produced rough-surfaced cultures on agar medium and demonstrated a propensity to form pseudohyphal cells on prolonged culture in vitro and in vivo, as measured in the mouse lung. Tests using a mouse model of blastomycosis with either yeast or spore inocula showed that the bys1-silenced strains were as virulent as control strains. Thus, although silencing of BYS1 alters morphology at 37 degrees C, it does not appear to impair the pathogenicity of B. dermatitidis.

Quantification of hepatic steatosis with 3-T MR imaging: validation in ob/ob mice.

PURPOSE: To validate quantitative imaging techniques used to detect and measure steatosis with magnetic resonance (MR) imaging in an ob/ob mouse model of hepatic steatosis. MATERIALS AND METHODS: The internal research animal and resource center approved this study. Twenty-eight male ob/ob mice in progressively increasing age groups underwent imaging and were subsequently sacrificed. Six ob/+ mice served as control animals. Fat fraction imaging was performed with a chemical shift-based water-fat separation method. The following three methods of conventional fat quantification were compared with imaging: lipid extraction and qualitative and quantitative histologic analysis. Fat fraction images were reconstructed with single- and multiple-peak spectral models of fat and with and without correction for T2* effects. Fat fraction measurements obtained with the different reconstruction methods were compared with the three methods of fat quantification, and linear regression analysis and two-sided and two-sample t tests were performed. RESULTS: Lipid extraction and qualitative and quantitative histologic analysis were highly correlated with the results of fat fraction imaging (r(2) = 0.92, 0.87, 0.82, respectively). No significant differences were found between imaging measurements and lipid extraction (P = .06) or quantitative histologic (P = .07) measurements when multiple peaks of fat and T2* correction were included in image reconstruction. Reconstructions in which T2* correction, accurate spectral modeling, or both were excluded yielded lower agreement when compared with the results yielded by other techniques. Imaging measurements correlated particularly well with histologic grades in mice with low fat fractions (intercept, -1.0% +/-1.2 [standard deviation]). CONCLUSION: MR imaging can be used to accurately quantify fat in vivo in an animal model of hepatic steatosis and may serve as a quantitative biomarker of hepatic steatosis.

Sebaceous cell carcinoma of the eyelid: a rapidly enlarging lesion with massive xanthogranulomatous inflammation.

A 76-year-old man presented atypically with a 4-week history of a rapidly enlarging ulcerated nodular lesion of the left upper eyelid that was found to be sebaceous cell carcinoma. Further investigation showed no metastatic disease, and Mohs surgery was performed to resect the tumor. Histopathologic analysis showed features diagnostic of sebaceous cell carcinoma. However, most of the mass consisted of xanthomatous granulomatous inflammatory reaction vastly out of proportion with the tumor burden. The patient was spared from orbital exenteration, and no evidence of recurrence was present 6 months after resection.

Attenuated Salmonella typhimurium-mediated interleukin-10 delivery is beneficial for dextran sodium sulfate-induced murine colitis.

PURPOSE: Interleukin-10 is a potent immunoregulatory agent that appears to play a role in inflammatory bowel disease. We hypothesized that interleukin-10 delivery to the distal gastrointestinal tract using a unique delivery vehicle may serve as a novel therapeutic for the treatment of experimental colitis. METHODS: A murine interleukin-10 cDNA was subcloned and transformed into attenuated Salmonella typhimurium. In vitro interleukin-10 production and biofunction were evaluated. This construct was then used against dextran sodium sulfate-induced murine colitis. RESULTS: A murine interleukin-10 producing S. typhimurium model was constructed. Enzyme linked immunosorbent assay and mast cell bioassay revealed interleukin-10 production. After single oral gavage feeding of 10 bacteria, persistence was noted within mesenteric lymph nodes at 6 weeks. Inoculation with/without the interleukin-10 plasmid (n = 7 per group) was performed before and after dextran sodium sulfate exposure. Postdextran sodium sulfate treatment revealed enhanced weight recovery in the S. typhimurium/interleukin-10 group compared to S. typhimurium/plasmid and phosphate buffered saline controls (P < 0.0001). The mean histology score for S. typhimurium/interleukin-10 was 0.86 compared to 3.14 and 3.17 for the S. typhimurium/plasmid and phosphate buffered saline controls respectively (P = 0.028). CONCLUSIONS: Attenuated S. typhimurium producing interleukin-10 can be successfully delivered to the murine gastrointestinal tract by single oral dosing. This novel delivery method improved recovery of chemically-induced murine colitis.

Microwave ablation with triaxial antennas tuned for lung: results in an in vivo porcine model.

PURPOSE: To prospectively determine in swine the size and shape of coagulation zones created in normal lung tissue by using small-diameter triaxial microwave antennas and to prospectively quantify the effects of bronchial occlusion and multiple antennas on the coagulation zone. MATERIALS AND METHODS: The study was approved by the research animal care and use committee, and all husbandry and experimental studies were compliant with the National Research Council's Guide for the Care and Use of Laboratory Animals. Twenty-four coagulation zones (three per animal) were created at thoracotomy in eight female domestic swine (mean weight, 55 kg) by using a microwave ablation system with 17-gauge lung-tuned triaxial antennas. Ablations were performed for 10 minutes each by using (a) a single antenna, (b) a single antenna with bronchial occlusion, and (c) an array of three antennas powered simultaneously. The animals were sacrificed immediately after ablation. The coagulation zones were excised en bloc and sectioned into approximately 4-mm slices for measurement of size, shape, and circularity. Analysis of variance and two-sample t tests were used to identify differences between the three ablation groups. RESULTS: The overall mean diameters of coagulation achieved with a single antenna and bronchial occlusion (4.11 cm +/- 1.09 [standard deviation]) and with multiple-antenna arrays (4.05 cm +/- 0.69) were significantly greater than the overall mean diameter achieved with a single antenna alone (3.09 cm +/- 0.83) (P = .016 for comparison with multiple antennas, P = .032 for comparison with bronchial occlusion). No significant differences in size were seen between the coagulation zones created with bronchial occlusion and those created with multiple antennas (P = .68). The coagulation zones in all groups were very circular (isoperimetric ratio > 0.80) at cross-sectional analysis. CONCLUSION: A 17-gauge triaxial microwave ablation system tuned for lung tissue yielded large circular zones of coagulation in vivo in porcine lungs. The coagulation zones created with bronchial occlusion and multiple antennas were significantly larger than those created with one antenna.

Role of IL-17, transforming growth factor-beta, and IL-6 in the development of arthritis and production of anti-outer surface protein A borreliacidal antibodies in Borrelia-vaccinated and -challenged mice.

We showed recently that the adaptive immune events leading to the development of arthritis in Borrelia burgdorferi isolate 297-vaccinated and Borrelia bissettii-challenged mice involve IL-17. Here, we show in Borrelia-vaccinated and -challenged mice that two cytokines known to induce the production of IL-17, IL-6 and transforming growth factor (TGF)-beta, are also involved in the development of arthritis. Vaccinated and challenged mice administered either anti-TGF-beta or anti-IL-6 antibodies developed histopathologic changes of the hind paws similar to or greater than untreated control mice. By contrast, simultaneous blockage of these cytokines reduced the severity of arthritis in Borrelia-vaccinated and -challenged mice. Moreover, administration of anti-IL-17 antibodies to these dual-antibody-treated mice completely prevented the development of histopathologic changes of the ankle joints, significantly reduced edema of the hind paws, and prevented the production of anti-outer surface protein A borreliacidal antibodies. These findings demonstrate a role for the combined effects of IL-17, IL-6, and TGF-beta in the adaptive immune events leading to the development of Borrelia-induced arthritis.

Concord grape juice attenuates platelet aggregation, serum cholesterol and development of atheroma in hypercholesterolemic rabbits.

Intake of Concord grape juice (CGJ), rich in polyphenolics, inhibits platelet aggregation (PA), a risk factor for cardiovascular disease (CVD), in normocholesterolemic animals and humans. It is unclear whether CGJ can attenuate hypercholesterolemia-enhanced PA. The effects of daily CGJ consumption on hypercholesterolemia-enhanced PA and the development of atherosclerosis were investigated. Two groups of rabbits (Control and Treated; n=10 each) were fed a hypercholesterolemic diet for 48 days. Treated group then received supplemental CGJ (225mL/day) while Control group received supplemental iso-caloric sugar water for 48 days. Collagen-, collagen+epinephrine- and phorbol-12-myristate-13-acetate-induced whole blood PA responses were measured on Days 0, 48 and 96; total serum cholesterol and blood pressure were also measured. The development of aortic atheroma was quantified at the end. Both groups showed significant increases in PA and serum cholesterol at Day 48. However, at Day 96, Treated group showed significantly lower PA and development of atheroma (30.7+/-3.9% lower (p<0.001)) than Control group; Treated group also had significantly lower total serum cholesterol and blood pressure than Control group. In conclusion, daily consumption of CGJ attenuates hypercholesterolemia-enhanced PA, blood pressure, total serum cholesterol and development of atheroma in rabbits.

Susceptibility of gnotobiotic transgenic mice (Tgepsilon26) with combined deficiencies in natural killer cells and T cells to wild-type and hyphal signalling-defective mutants of Candida albicans.

Germfree transgenic epsilon 26 mice (Tgepsilon26), deficient in natural killer cells and T cells, were colonized (alimentary tract) with Candida albicans wild-type or each of two hyphal transcription factor signalling mutant strains (efg1/efg1, efg1/efg1 cph1/cph1). Each Candida strain colonized the alimentary tract, infected keratinized gastric tissues to a similar extent, and induced a granulocyte-dominated inflammatory response in infected tissues. Both wild-type and mutant strains formed hyphae in vivo and were able to elicit an increase in cytokine [tumour necrosis factor alpha, interleukin (IL)-10 and IL-12] and chemokine (KC and macrophage inflammatory protein-2] mRNAs in infected tissues; however, administration of the wild-type strain was lethal for the Tgepsilon26 mice, whereas the mice colonized with the mutant strains survived. Death of the Tgepsilon26-colonized mice appeared to be due to occlusive oesophageal candidiasis, and not to disseminated candidiasis of endogenous origin. In contrast, the mutant strains exhibited a significantly reduced capacity to infect (frequency and severity) oro-oesophageal (tongue and oesophagus) tissues. Therefore, the two hyphal signalling-defective mutants were less able to infect oro-oesophageal tissues and were non-lethal, but retained their ability to colonize the alimentary tract with yeast and hyphae, infect keratinized gastric tissues, and evoke an inflammatory response in orogastric tissues.

A novel murine model for the study of human renal cryoablation.

OBJECTIVE: to develop a reproducible, non-debilitating in vivo murine model of human renal cryoablation using a standard closed argon-delivery system. MATERIALS AND METHODS: Custom engineered 2-mm conical tip cryoprobes for use on the standard argon-based cryoablation unit (Endocare, Inc. Irvine, CA, USA) were used to create small controllable iceballs (−160 °C) in the mouse kidney. The time to create a 4-mm cryolesion was compared using a contact vs puncture technique in 10 mice. To show consistency of the induced-freeze injury, a 4-mm iceball was created in 20 murine renal units and the time to creation and the size of the resultant cryolesion measured. To investigate lesion regression and histological changes, we created a 4-mm renal cryolesion in 28 mice and killed four each at 1, 3, 7, 14, 21, 28, and 35 days. The measured coronal cross-sectional area of the cryoablation site at necroscopy was compared to the initial calculated area as a percentage. To assess renal preservation, blood urea nitrogen (BUN) and creatinine levels at 1 week after cryoablation or sham ablation was compared (10 mononephric mice in each group). RESULTS: The time to create the desired iceball was 1.9 times quicker using the puncture vs the contact technique. The mean (sd) time to forming a 4-mm iceball was 35.3 (4.8) s with a mean maximum length of the resultant post-thaw injury of 5.7 (0.5) mm and a 9% coefficient of variance. Regression analysis of the two-dimensional cross-sectional coronal area of the cryolesion showed a statistically significant linear pattern of regression over time (P = 0.037) and classic histological findings. There was no significant difference in the BUN or creatinine levels in mononephric mice 1 week after cryotherapy compared with the sham-ablated controls. CONCLUSIONS: We describe a reproducible, non-debilitating, easily manipulated murine model for the study of human renal cryoablation.

Arthritis is inhibited in Borrelia-primed and infected interleukin-17A-deficient mice after administration of anti-gamma-interferon, anti-tumor necrosis factor alpha and anti-interleukin-6 antibodies.

The role that cytokines play in the induction of Lyme arthritis is gradually being delineated. We showed previously that severe arthritis developed in a T-cell-driven murine model, even in mice lacking interleukin-17A (IL-17A) and administered anti-gamma-interferon (IFN-γ) antibody. Increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two pro-inflammatory cytokines, were detected in cultures of popliteal lymph node cells obtained from these mice. We hypothesized that concomitantly administered anti-IL-6, anti-TNF-α and anti-IFN-γ antibodies would inhibit the development of arthritis in IL-17A-deficient mice. Our results showed that swelling of the hind paws and histopathological changes consistent with arthritis were significantly reduced in IL-17A-deficient mice that administered the three anti-cytokine antibodies. These results suggest that treatment with multiple anti-cytokine antibodies can abrogate the induction of Lyme arthritis in mice.

Borrelia-primed and -infected mice deficient of interleukin-17 develop arthritis after neutralization of gamma-interferon.

The immune mechanisms responsible for development of Lyme arthritis are partially understood with interleukin-17 (IL-17) and gamma-interferon (IFN-γ) playing a generally accepted role. Elevated levels of IL-17 and/or IFN-γ have been reported in samples from human Lyme arthritis patients and experimental mice. In addition, IL-17 and IFN-γ have been implicated in the onset of arthritis in Borrelia-primed and -infected C57BL/6 mice. Recently, we showed that IL-17-deficient mice developed swelling and histopathological changes consistent with arthritis in the presence of high levels of IFN-γ. We hypothesized that neutralization of IFN-γ in IL-17-deficient mice would inhibit Borrelia-induced arthritis. Our results, however, showed that swelling of the hind paws and histopathological changes of arthritis did not differ between Borrelia-primed and -infected IL-17-deficient and wild-type mice with or without neutralization of IFN-γ. We also found higher levels of tumor necrosis factor alpha (TNF-α) and IL-6 in the popliteal lymph node cells of Borrelia-primed and -infected IL-17-deficient mice after neutralization of IFN-γ. These results suggest that multiple cytokines interact in the development of Borrelia-induced arthritis.

Elastographic measurements of in-vivo radiofrequency ablation lesions of the kidney.

BACKGROUND AND PURPOSE: Elastography may prove useful for monitoring radiofrequency ablative (RFA) therapy because heat-ablated tissues are more elastic than untreated tissues. Herein, we report our initial evaluations of the reliability of elastography for delineating thermal-lesion boundaries at the time of RFA of porcine kidneys. MATERIALS AND METHODS: In-vivo RFA was performed on 20 kidneys from 10 40-kg female pigs. Elastography was performed at the time of surgery and after 48 hours. The imaging plane was perpendicular to the axis of the RF electrode so that the ablated region was around the center of the plane. Measurements of the sections representing the same image plane used for elastography were taken at pathologic examination and compared with the measurements obtained from the elastograms. RESULTS: We found a statistically significant correlation between elastography and pathology measurements with respect to the area and volume estimates (r = 0.9302 and r = 0.953, respectively). Overall, elastography slightly underestimated the lesion size, as judged by the digitalized pathologic images, a finding consistent with previous reports. CONCLUSION: We found a correlation between the area and volume estimates of thermal lesions that were based on elastographic images and the measurements from gross pathologic dimensions. A significant limitation of renal RFA is the inaccuracy of current imaging modalities to provide real-time monitoring, and elastography may prove to be reliable for delineating the resulting thermal lesions.

Arthritis is developed in Borrelia-primed and -infected mice deficient of interleukin-17.

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis in experimental mice. For example, neutralization of IL-17 with antibodies inhibits induction of arthritis in Borrelia-primed and -infected C57BL/6 wild-type mice. We hypothesized that mice lacking IL-17 would fail to develop Borrelia-induced arthritis. IL-17-deficient and wild-type C57BL/6 mice were primed with heat-inactivated Borrelia and then infected with viable spirochetes 3 weeks later. No swelling or major histopathological changes of the hind paws were detected in IL-17-deficient or wild-type mice that were primed with Borrelia or infected with viable spirochetes. By contrast, IL-17-deficient and wild-type mice that were primed and subsequently infected with heterologous Borrelia developed severe swelling and histopathological changes of the hind paws. In addition, Borrelia-primed and -infected IL-17-deficient mice exhibited elevated gamma-interferon (IFN-γ) levels in sera and increased frequencies of IFN-γ-expressing lymphocytes in popliteal lymph nodes compared to Borrelia-primed and -infected wild-type mice. These results demonstrate that IL-17 is not required for development of severe pathology in response to infection with Borrelia burgdorferi, but may contribute to disease through an interaction with IFN-γ.

Tumor necrosis factor alpha, citrullination, and peptidylarginine deiminase 4 in lung and joint inflammation.

BACKGROUND: The relationship between lung and joint inflammation in rheumatoid arthritis is poorly understood. Lung inflammation with resultant protein citrullination may trigger anti-citrullinated protein antibodies, inflammation, and arthritis. Alternatively, lung and joint inflammation may be two manifestations of a single underlying pathology. The lung has increased citrullination and TNF-α levels are high in rheumatoid arthritis; however, it is unknown if TNF-α can induce lung protein citrullination. The citrullinating enzyme peptidylarginine deiminase 4 (PAD4) exacerbates TNF-α-induced arthritis, but a role for PAD4 in lung citrullination and TNF-α-induced lung inflammation has not been explored. Our aim was to use TNF-α-overexpressing mice to clarify the intersection of TNF-α, citrullination, PAD4, arthritis, and lung inflammation. METHODS: Lung protein citrullination in wild-type mice, mice that overexpress TNF-α systemically (TNF(+)), TNF(+)PAD4(+/+), and TNF(+)PAD4(-/-) mice was quantified by both gel electrophoresis using a citrulline probe and western blot. Hematoxylin and eosin (H&E)-stained lung sections from TNF(+)PAD4(+/+) and TNF(+)PAD4(-/-) mice were scored for lung inflammation. H&E-stained ankle joint sections from mice that overexpress TNF-α only in the lungs were assessed for arthritis. RESULTS: TNF(+) mice have increased lung protein citrullination. TNF(+)PAD4(-/-) mice do not have significantly reduced lung protein citrullination, but do have decreased lung inflammation compared to TNF(+)PAD4(+/+) mice. Mice that overexpress TNF-α only in the lungs do not develop arthritis. CONCLUSIONS: PAD4 exacerbates lung inflammation downstream of TNF-α without having a major role in generalized protein citrullination in inflamed lungs. Also, TNF-α-induced lung inflammation is not sufficient to drive murine arthritis.

Localization of transfected B7-1 (CD80) DNA in human melanoma cells after particle-mediated gene transfer.

The purpose of this study was to evaluate stable DNA transfection of M-21 human melanoma cells with particle-mediated gene transfer (PMGT) with B7-1 cDNA and to identify sites of gene integration. Stable B7-1 transfectants (M-21-B7) were obtained with PMGT using a plasmid vector containing cDNA for both B7-1 and neomycin phosphotransferase, with subsequent selection with G418. The transfected cells were flow sorted by B7-1 expression into two populations, bright and dim. The bright population had 85%-90% of cells expressing B7-1; the dim population had less than 50% of cells with B7-1 expression. Chromosome analysis with fluorescence in situ hybridization (FISH) and G-banding showed that 70% of bright cells had two main integration sites, with extensive amplification of the transgene. The dim population had random signal distribution, with little or no amplification, despite G418 selection. Because B7-1 has been mapped to 3q21, FISH was performed using a chromosome 3 painting probe (WCP) together with a probe for B7-1. In transfected bright M-21 cells, amplified genes that hybridized with the B7-1 construct were localized to chromosome 3 material inserted into marker chromosomes. These data suggest that B7-1 insertion may involve homologous recombination, but maintenance of integration and amplification required selection.