Insulin resistance may be a key component of the pathogenesis of this disorder. Pioglitazone is a thiazolidinedione derivative that acts by improving insulin resistance via the peroxisome proliferator-activated receptor-γ (PPAR-γ) pathway. Reportedly, pioglitazone improves the anovulation status in patients with PCOS. In the present study, we examined whether pioglitazone directly affects ovarian follicular development and steroidogenesis using in vitro mouse preantral follicle culture was used to test the effects of pioglitazone on the follicle development and steroidogenesis. Tumor necrosis factor-α (TNF-α), which plays a role in insulin resistance, has been reported to inhibit the follicle stimulating hormone (FSH)-induced follicular development and steroidogenesis in an in vitro mouse preantral follicle culture system. Therefore, we examined whether pioglitazone counteracts these effects by TNF-α. We assessed the follicle diameter and follicle survival and antral-like cavity formation rates, the 17β-estradiol (E2) levels in the culture medium, and the ovulation rate using the in vitro preantral follicle culture.An isolated individual P < 0.05), antral-like cavity formation , E2 concentration in the culture medium , and human chorionic gonadotropin-induced ovulation rate .Pioglitazone treatment counteracted the inhibition of TNF-α in FSH-induced follicle development in a dose-dependent manner. Pioglitazone, at a concentration of 5 μM, which was the minimum effective concentration, significantly counteracted the inhibition of TNF-α in FSH-induced follicle survival (29 versus 56%, in vitro mouse preantral follicle culture. The results suggest that pioglitazone may directly affect the follicular development and steroidogenesis.Pioglitazone counteracted the inhibition by TNF-α on FSH-induced follicle development and steroidogenesis in the  Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age, affecting 5–10% of the population . Hyperinin vitro culture [The circulating levels of adipocytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 6, which raise insulin resistance, are higher in obese and nonobese women with PCOS than those in weight matched non-PCOS control women ,8. TNF-α culture .in vitro[Insulin resistance with compensatory hyperinsulinemia is observed in approximately 70% of patients with PCOS, irrespective of obesity ,13. Sevein vitro. Pioglitin vitro mouse preantral follicle culture system to assess the effects of bezafibrate, a lipid-lowering fibrate, on follicular development [We previously established the elopment . As bezaelopment ,25, we delopment . Becausein vitro mouse preantral follicle culture system.The aim of this study was to examine whether pioglitazone affects the ovarian follicular development and steroidogenesis using an This study was performed with permission from the Committee of Animal Experimentation at the Yamagata University Faculty of Medicine. Mature female ICR mice at 6 weeks or older were used in this study. They were housed in a temperature- and light-controlled room at 23–25°C, on a 12-h light:dark cycle  and fed pellet food and water ad libitum.Human pituitary follicle-stimulating hormone (FSH)  and TNF-α , were purchased from Sigma-Aldrich® . Pioglitazone was kindly donated by Takeda Pharmaceutical Company Limited, Osaka, Japan.N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-minimal essential medium  supplemented with 5% fetal bovine serum . After washing with HEPES-MEM 2 times, follicles with the following characteristics were selected: (1) diameter, 120–150 μm; (2) immature oocyte centrally located within the follicle; (3) intact basal membrane; and (4) surrounded by theca cells [Collection of preantral follicles from the mice was reported previously . BrieflyIn vitro preantral follicle culture was reported previously [in vitro follicle culture system. Follicles were individually cultured per well in a 24-well plate  containing 1 mL culture medium at 37°C with 5% CO2 in air atmosphere. Follicles were cultured for 13 days. Subsequently, follicular growth was monitored every 2 days  under an inverted microscope at a 200× magnification. Each follicle image was captured by a cooled charge-coupled device camera system , and follicle diameter was measured using Image J imaging system software version 1.43 . A total of 500 μL conditioned medium was refreshed with preincubated medium every 2 days . The spent media collected on the indicated days were pooled per culture plate and frozen at −80°C for a successive E2 determination. When follicles clearly formed an antral-like cavity on day 12, ovulation and meiotic resumption were induced by refreshing media with culture media supplemented with 5 IU/mL human chorionic gonadotropin . On day 13, follicles were checked for ovulation 16 h after hCG administration, and ovulation was considered when the follicle was visually ruptured and the cumulus oocyte complex (COC) extruded from the follicles. The ovulated COCs were evaluated for cumulus expansion and germinal vesicle breakdown (GVBD), indicating meiotic resumption of oocytes, after removing the surrounding cumulus cells by repeated pipetting in the presence of 300 IU/mL hyaluronidase.eviously . The folin vitro preantral follicle culture. Briefly, the follicular stage is characterized by preservation of the follicle basal membrane. Initially, theca cells grow out to the culture plate and granulosa cell proliferation is limited. The follicles remain in this stage until day 4 of culture. The diffuse stage is characterized by marked granulosa cell proliferation and a large preantral follicle. From day 4 to day 6 of culture, granulosa cells overgrow the theca cell monolayer through the basement membrane, and the follicles gradually enlarge. From day 6 or 8 up to day 12 of culture, granulosa cells differentiate into 2 spatially and functionally distinct populations, cumulus cells and mural granulosa cells, and the follicles form an antral-like cavity, which characterizes the antral stage.Using the method reported by Adriaenssens et al.  and LeniIn this culture system, surviving follicles were defined as those that could retain their oocyte completely embedded within the granulosa cell mass and that exhibited no signs of degeneration ,29. FollMedia cultured with individual mouse follicles were replaced every other day, and the collected media were pooled and stored at −80°C until the measurement of E2 levels was performed. E2 levels in the collected culture media were measured using the estradiol enzyme immunoassay kit (Estradiol EIA Kit)  according to the manufacturer’s protocol. Culture media from non-surviving follicles were excluded from the experiment.in vitro culture period. TNF-α was dissolved in distilled water at 10 μg/mL and was stored at −80°C until use. Pioglitazone was dissolved in dimethyl sulfoxide (DMSO) at 10 mM and was stocked at −80°C until use. The final concentration of DMSO in the medium never exceeded 0.1%. In the preliminary study, we tested the effects of DMSO as a vehicle on follicle development. There were no significant differences of follicle development between the groups treated with and without DMSO (data not shown).In this experiment, the control group consisted of follicles cultured in media without FSH in the presence of the vehicle. All compounds were added to the culture media during the Effects of various treatments on follicle development and steroidogenesis were evaluated as follows: follicle diameter, follicle survival, antral-like cavity formation, and measurement of E2 concentration in culture media. Moreover, we evaluated the effects of various treatments on ovulation and GVBD, indicating resumption of meiosis.in vitro follicle culture system. Experimental scheme shows in Figure We previously demonstrated that TNF-α significantly inhibited FSH-induced follicular development and steroidogenesis in a dose-dependent manner . In the P < 0.05.All experiments consisted of at least 5 independent experimental runs. Differences between means were calculated by one-way ANOVA, followed by a post hoc test, and data expressed as percentages were analyzed by Fisher’s exact probability test using GraphPad Prism version 5.0 for Windows . Values are given as mean ± SEM. Significant differences are defined as Our previous data show that 100 mIU/mL of FSH treatment significantly induced the follicle growth and increase in E2 concentration in the culture medium compared to control samples without FSH treatment . While fin vitro culture, TNF-α significantly inhibited FSH-induced follicular development and steroidogenesis in a dose-dependent manner with a minimal effective dose of 5 ng/mL. Therefore, we used the in vitro preantral follicle culture system with TNF-α as a model to study follicular development of PCOS.TNF-α is a pro-inflammatory cytokine that induces cell death . TNF-α aWe examined whether pioglitazone could counteract the inhibition of FSH-induced follicular development and steroidogenesis by TNF-α. Pioglitazone significantly counteracted the inhibition of FSH-induced follicular development and steroidogenesis by TNF-α in a dose-dependent manner Figure . TNF-α A. TNF-α We examined the effects of pioglitazone on the hCG-induced ovulation and meiotic resumption of oocytes with or without FSH and TNF-α Table . While Tin vitro mouse preantral follicle culture system. While TNF-α inhibited the FSH-induced follicle development, such as follicle survival, antral-like cavity formation, and E2 production, pioglitazone counteracted these inhibitory effects by TNF-α.In the present study, we examined the direct effects of pioglitazone on the follicular development and steroidogenesis by using an In vitro mouse ovarian culture methods provide a useful tool for studying follicle development [in vitro mouse ovarian follicle culture system for up to 12 days to study the effects of various compounds on FSH-induced follicle development and steroidogenesis. The mechanism of TNF-α inhibition of FSH-induced follicle development and steroidogenesis remains unknown. TNF-α is a well-known cytokine that is capable of inducing apoptosis in cells [elopment ,33-35. Win cells . TNF-α iin cells . In the in cells ,10. Morein cells ,37. Sevein cells ,39.in vitro culture system. The precise mechanisms of restoration of TNF-α inhibition of FSH-induced follicle development and steroidogenesis by pioglitazone were unknown. Recently, we examined whether bezafibrate, a ligand for PPARs  rescued the TNF-α inhibition of follicular development and steroidogenesis [In the present study, we showed that pioglitazone counteracted the TNF-α inhibition in FSH-induced follicle development and steroidogenesis in an ogenesis . Our preogenesis .in vivo[It is well known that thiazolidinediones, such as pioglitazone and rosiglitazone, specifically bind to PPAR-γ, which is located on macrophages, adipose tissue or human granulosa lutein cells ,41. Piogin vivo,47. PiogPioglitazone counteracted the inhibition of FSH-induced follicular development and steroidogenesis by TNF-α. These results suggest that pioglitazone may directly act on ovarian functions through PPAR-γ activation in patients with PCOS.N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; MEM: Minimal essential medium; COC: Cumulus oocyte complex; GVBD: Germinal vesicle breakdown; DMSO: Dimethyl sulfoxide.PCOS: Polycystic ovary syndrome; PPAR-γ: Peroxisome proliferator-activated receptor-γ; TNF-α: Tumor necrosis factor-α; FSH: Follicle stimulating hormone; E2: 17β-estradiol; hCG: Human chorionic gonadotropin; HEPES: The authors declare that they have no competing interests.SH carried out the experiment of follicle culture and drafted the manuscript. TT participated in the design of the study and drafted the manuscript. MA carried out the measurement of E2. HI performed the statistical analysis. HK participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


The question of the effect of anti-TNF-alpha in skin carcinogenesis is especially relevant in view of the increased use of these drugs for the treatment of autoinflammatory immune diseases. Since ultraviolet radiation and human papillomavirus are involved in skin carcinogenesis, we wished to investigate the effect of TNF-alpha antagonists on the UVB-induced apoptosis of keratinocytes infected by HPV38. Our results indicate that anti-TNF agent, infliximab, does not contribute to the survival of HPV38-transduced keratinocytes with UVB-induced DNA damages. Numerous studies indicate that TNF-α may influence the fate of HPV-infected cells and suggest that HPV-mediated TNF resistance is a key event in the multistep process leading to cervical cancer [Tumor necrosis factor-alpha  irradiation which is the most important risk factor involved in the development of NMSC, cutaneous beta-HPV infection is also considered as an important cofactor . Similarα agent, infliximab, on the UVB-induced apoptosis of keratinocytes infected by HPV38.Since anti-TNF agents are also associated with an increased risk of viral infections , we wish2) to induce apoptosis. To assess the role of TNF-α, 100 ng/mL of TNF-α  and 20 μg/mL of infliximab  were added after UVB irradiation. Apoptosis was evaluated by flow cytometry (annexin V/propidium iodide labelling), fluorescence microscopy and PARP, and cleaved caspase-3 western blot. Soluble TNF-alpha was measured by ELISA  in the supernatants. HaCaT keratinocytes and HaCaT keratinocytes transduced with empty vector pLXSN and with pLXSN-HPV38 E6/E7 vector [The gene expression of E6 and E7 in HaCaT cell lines was confirmed by RT-PCR analysis . To ensuα in the supernatants from both pLXSN and HPV38 E6/E7-HaCaT cells  restored UVB-induced apoptosis level  restored a similar level of apoptosis in both pLXSN-HaCaT and HPV38 E6/E7-HaCaT cells. This suggests that exogenous TNF-α is able to suppress the inhibitory effect of HPV38 E6 and E7 expression on UVB-induced apoptosis of keratinocytes. Moreover, our results demonstrate that anti-TNF-α agent, infliximab, does not modulate UVB-induced apoptosis of both HaCaT cells and HPV38 E6/E7-infected keratinocytes. In a previous ex vivo study, Gambichler et al. [α receptor fusion protein, had no effect on the number of caspase-3 apoptotic cells present in skin biopsies of psoriasis patients treated with UVB and etanercept. In contrast, Faurschou et al. [α-induced apoptosis. The authors suggested a reverse signalling pathway via membrane-bound TNF-α.Previous studies have already demonstrated that UVB light induces the release of TNF-inocytes  and thataT cells , 10. FuraT cells  demonstrr et al.  observedu et al.  observedμg/mL) was too low to overcome the endogenous TNF-α production. However, the dose tested was still able to neutralize exogenous TNF-α (100 ng/mL) which induced a 2-fold apoptotic rate compared with UVB alone (α by UVB-irradiated keratinocytes was not altered by HPV38 E6/E7 infection. It may be argued that in our study infliximab dose (20 VB alone . It shouα agents in different autoinflammatory immune diseases.Altogether, our results indicate that anti-TNF agent, infliximab, does not contribute to the survival or the elimination of HPV38-transduced keratinocytes with UVB-induced DNA damages. Other TNF-independent pathways may be involved , 11. The


Science 314∶787–790), a high-impact paper that focused on relationships between marine biodiversity and ecosystem services. This paper sparked much controversy through its projection, highlighted in the press release, that all marine fisheries would be collapsed by 2048. Analysis of 664 citing papers revealed that only a small percentage (11%) referred to the 2048 projection, while 39% referred to fisheries collapse in general, and 40% to biodiversity and ecosystem services. The 2048 projection was mentioned more often in papers published soon after the original paper, in low-impact journals, and in journals outside of fields that would be expected to focus on biodiversity. Citing papers also mentioned the 2048 projection more often if they had few authors (28% of single-author papers vs. 2% of papers with 10 or more authors). These factors suggest that the more knowledgeable the authors of citing papers were about the controversy over the 2048 projection, the less likely they were to refer to it. A noteworthy finding was that if the original authors were also involved in the citing papers, they rarely  mentioned the 2048 projection. Thus the original authors have emphasized the broader concerns about biodiversity loss, rather than the 2048 projection, as the key result of their study.Citation patterns were examined for Worm et al. 2006 ( Science and Nature on fisheries research. The first of these two papers, “Faith-based fisheries” by Ray Hilborn Fisheries, argued that a long string of papers published in Science and Nature : from the text in Worm et al. To assess whether the framing of the press release influenced subsequent citations, three word clouds were created using the online software package Wordle  which returns the ratio of male to female babies born with a particular name. Where the citing reference only listed the initials of the first author, a search was made for the full name by looking for other papers by the same author and examining the author’s web page. Using these methods, gender was obtained for all but two citing papers.Of these attributes, gender required some sleuthing. Where gender was not obvious it was inferred from the website Baby Name Guesser  differed from frequencies expected by random chance. P-values were computed using a Monte Carlo simulation test with 100,000 random samples, as implemented by Commonly used words in Worm et al. PLoS ONE and Marine Policy, each accounting for 5% of all citations), spanning many fields. First authors were often male (71%) and resided in 42 countries, most frequently the USA (37%) and Canada (13%). Within the citing papers, Worm et al. was cited most often in the introduction (62%) and discussion (44%), a surprising number of citations occurring in the first paragraph (46%), first sentence (20%), or last paragraph (7%). Citations were seldom situated in the abstract (1%), methods (3%) or results (<1%).Papers citing Worm et al. Relatively few citing papers mentioned the 2048 projection  compared with those that mentioned fisheries collapses , or the relationship between biodiversity and stability ; and 28% did not refer to any of these three topics, instead mentioning other topics in the original paper such as marine protected areas, biodiversity in general, and ecosystem degradation.There were some clear patterns in which types of citing papers mentioned the 2048 projection . The oriCiting papers referring to fisheries collapses did not have many clear explanatory factors ; no statCiting papers referred to the relation between biodiversity and ecosystem services in the opposite manner to those noted for the 2048 projection or fisheries collapse. Biodiversity and ecosystem services were referred to more often by ecology and biology journals and by high-impact journals . In addiWorm et al. The key characteristic of papers that referred to the 2048 projection was related to the familiarity of authors with the controversy over this projection. At the one extreme, coauthors of the original paper, who would have been acutely aware of the controversy, only once mentioned the 2048 projection in their own papers out of 55 papers in which they cited Worm et al. Similarly, papers with many authors were also very unlikely to refer to the 2048 projection (1 of 57 papers with 10 or more authors), perhaps because this increased the probability that one or more of the authors would be aware of the controversy. Additionally, papers published in journals with high impact factors, and presumably reaching a larger and more general audience, were less likely to refer to the 2048 projection–this was mentioned in only 4% of papers with an impact factor greater than 5.0, but 18% of papers with an impact factor less than 2.0. One possibility is that low-impact journals were trying to court controversy to increase their impact factors. Finally, although it would be expected that most citations of Worm et al. Similar characteristics explained patterns in which citing papers referred to biodiversity and ecosystem services, but here the patterns were reversed in a striking mirror-image pattern in These results suggest that press coverage does influence how papers are cited, but despite this influence, only 11% of citing papers referred to the 2048 projection. Papers that mentioned the 2048 projection had characteristics that suggest unfamiliarity with the controversy surrounding this projection, namely papers with few authors, published in journals with low impact factors, in fields far removed from ecology and fisheries, and sharing no coauthors with the Worm et al. paper. For such papers, it is easy to see how the authors, editors, and reviewers could be unaware of the controversy over the 2048 projection. Most interestingly, the original coauthors of Worm et al. only once out of 55 occasions mentioned the 2048 projection in subsequent citations of their own paper.Table S1Complete list of papers examined, citation texts referring to Worm et al., and coded variables used in the analysis.(XLSX)Click here for additional data file.


Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions. Tumor Necrosis Factor alpha (TNF-α) is a cytokine that has a wide variety of functions including immune cell activation and proliferation.et al. reported the isolation and characterization of a transcription factor termed Lipopolysaccharide Induced TNF-Alpha Factor (LITAF), and showed that LITAF plays a role in the regulation of TNF-α gene expression in a human monocyte/macrophage cell line (THP-1).et al. also reported that both LITAF and TNF-α mRNA and protein levels are increased in colonic tissue resected from patients with CD and ulcerative colitis specifically localized to lamina propria macrophages (LPM).TNF-α gene expression is highly regulated by a complex promoter region that contains multiple response elements, including the proinflammatory transcription factor NF-κB.Recently, LITAF mac -/- mice were generated using a Cre/LoxP expression system to specifically target LITAF expression in cells of the monocyte cell lineage.BioChemika, 1 M in H2O) (1.5 mg in 100 µL of 50% Ethanol/water solution) by enema under isoflurane anesthesia. Colonic tissue was dissected at 24–72-hours after TNBS treatment at times associated with acute inflammation in prior studies.Mice were housed in Association for Assessment and Accreditation for Laboratory Animal Care-approved facilities and protocols were approved by the Boston University Medical Center's Institutional Animal Care and Use Committee. Experiments were conducted in adult, gender-matched male and female C57BL/6 mice obtained from Charles River Laboratories . LITAF mac -/- mice were generated on a C57BL/6 mouse background using the Cre-LoxP system as previously described by Tang et. al. Colonic tissue was dissected and fixed in 10% formalin prior to being imbedded in paraffin. The paraffin blocks were then cut into 5 µm specimens and fixed to a slide and then stained with hematoxylin and eosin using an automated staining system Sakura Diversified Stainer DRS-601 . The histological score of each tissue sample was assessed by a blinded observer and assigned a score of 0-4 based on a previously described method Kinematica, Inc., Bohemia, NY) in homogenization buffer . Samples were then centrifuged at 43,000 x g for 30 minutes at 4°C. Cellular pellets were washed with homogenization buffer and centrifuged again, as above. Following this wash, the pellet was resuspended in sonication buffer  to a final concentration of 100 mg/mL and sonicated  on ice for 5, 2-second intervals at full power. Samples were incubated on ice for 20 minutes and then centrifuged for 15 minutes at 12,500 x g at 4°C. To perform the myeloperoxidase (MPO) activity assay, samples and known standards were allowed to react with assay buffer  for exactly 3 minutes before the addition of the stop buffer (0.66 M sulfuric acid). Absorbance was read in samples and standards at 450 nm and milliunits (mU) of MPO were determined.Equal weight colonic tissue sections were completely homogenized on ice using a Polytron PT-2100 (Lamina propria macrophages (LPM) were harvested from freshly dissected colonic tissues.TNF-α levels in the media were assessed using a mouse TNF-α ELISA set  according to the manufacturer's instructions. TNF-α concentrations were determined using a standard and values were normalized to total DNA present in the well using DNA quantification kit .Total RNA was isolated from LPM and colon tissue as previously described using the SV Total RNA Isolation System .LPM were washed with HBSS and lysed with M-PER solution  containing protease inhibitor cocktail (100x). Lysates were sonicated using a Sonic Dismembrator , for 10 seconds on ice. Total protein content in cleared lysates was determined using BIO-RAD Protein assay (BIO-RAD). Samples were separated using Invitrogen gels (4–12% Bis-Tris 120 V for 1.5 hours) transferred to PVDF membranes using 30 V for 1 hour. LITAF protein levels were detected using a custom made LITAF antibody generated against the c-terminal region of mouse LITAF . This antibody was shown previously to be specific for both human and mouse forms of LITAF. Blots were then stripped and reprobed for the housekeeping protein Actin or GAPDH. Optical density of the bands was determined using Scion image . OD for LITAF levels were normalized to the OD of housekeeping proteins.All of the data are expressed as means ± SEM and were analyzed by 1-way or 2-way analysis of variance with the Sigma Stat program . When significant effects (p<0.05) were detected, the differences between specific means were determined by post hoc analysis. In all cases a Dunn test of analysis of variance by ranks followed by the appropriate nonparametric post hoc test was used. Groups were deemed to be significantly different from one another when p<0.05.Following TNBS administration, colonic tissue levels of MPO activity were significantly increased by 24 hours compared to non-inflamed controls indicating a substantial inflammatory response  p<0.001. Based oin vivo TNBS inflammatory stimulus such that the ex vivo response to LPS is significantly greater.The baseline secretion of TNF-α into the media was significantly increased in freshly harvested LPM from inflamed colonic tissue from TNBS-treated mice compared to LPM from non-inflamed colonic tissue from non-treated controls (p<0.001) clearly documenting a change in the properties of the LPM in response to the inflammatory stimulus . After 2ex vivo stimulation with LPS induced a 40% increase (p<0.05) in LITAF protein compared with non-treated LPM harvested from inflamed colonic tissue (LITAF mRNA levels were significantly (p<0.05) increased in LPM harvested from inflamed colonic tissue and stimulated with LPS compared to LPM harvested from non-inflamed colonic tissue . Althougc tissue . The incIn the context of the potential importance of anti-LITAF therapeutic approaches we investigated whether an inhibitor of p38 would reduce the elevated TNF-α response in LPM harvested from inflamed colonic tissue since p38 signaling pathway is known to be involved in LITAF activation The deletion of the LITAF gene in LITAF mac -/- was confirmed by western blot of peritoneal macrophages collected by peritoneal lavage at time of necropsy and compared to peritoneal macrophages harvested from a C57BL/6 wildtype mice . Peritonin vivo. LITAF mac -/- mice lost significantly (P<0.05) less body weight in response to a 24 hours inflammatory challenge with TNBS than did their counterpart wildtype C57BL/6 mice  which leads to activation of Mitogen Activated Protein Kinase (MAPK) pathways, including p38.ex vivo system, inhibitors of NF-κB signaling also caused a reduction in TNF-α secretion in response to LPS stimulation in LPM harvested from inflamed colonic tissue. This is not surprising given that NF-κB is a ubiquitous transcription factor that regulates over 300 genes, including TNF-α, many involved in inflammation and cancer.Additionally, TLR4 is known to activate the NF-κB pathway through the ubiquitination and degradation of IκB. IκB degradation allows for the translocation of the p65 and p50 heterodimeric unit to the nucleus where transcription of inflammatory genes, including TNF-α, is induced.The most direct evidence that LITAF is associated with the capacity of LPM to secrete TNF-α comes from the use of LITAF deficient LPM in these studies. Using LPM harvested from LITAF mac -/- mice following TNBS induced inflammation, the data supports the conclusion that the deletion of the LITAF gene leads to a significant reduction in TNF-α secretion in response to LPS stimulation compared to wildtype controls. Since differences in TLR4 mRNA levels were not found between peritoneal macrophages derived from C57BL/6 and LITAF mac -/- mice (data not shown) it very likely that this process is not dependent on changes in TLR4. In this system, the remaining TNF-α signal that we have observed in LITAF deficient macrophages is likely due to the remaining proinflammatory pathways including NF-κB.LITAF mac -/- mice exhibit reduced bodyweight loss, have lower MPO levels and, on examination of their colonic tissue, express less TNF-α mRNA as compared to C57BL/6 mice. Despite the apparent reduction in the acute TNBS-induced inflammatory response, LITAF mac-/- mice do not exhibit a reduction in the overall histological damage to the colonic tissue following 24 or 48 hours after TNBS administration. These findings suggest that the animals deficient in LITAF have a delayed healing response compared to C57BL/6 mice. This may be due, in part, to the rapid and strong inflammatory stimulus that TNBS administration imparts when administered rectally. Another explanation for this result may be related to a beneficial dysregulation of the inflammatory response in LITAF mac -/- animals.et al. identified an acyclic derivative ((E)-5-biphenyl-4-yl-5-hydroxy-3-methoxy-pent-2-enoic acid methyl ester) and showed this compound to suppress LITAF levels while simultaneously reducing TNF-α secretion from macrophages stimulated with LPS.Interestingly, while making synthetic derivatives of the lactone kavain, Pollastri In summary, endogenous colonic LPMs increased their base line and LPS mediated TNF-α secretion by a LITAF dependant pathway when harvested from acutely inflamed colonic tissue suggesting the usefulness of anti-LITAF drug development for the treatment ulcerative colitis. We propose that LITAF plays a substantial role in TNF-α secretion from LMP isolated from an acutely inflamed colon and represents an alternative pathway to modulation of the TNF-α component of the inflammatory response. A better understanding of the role that LITAF signaling plays in regulating TNF-α gene expression in the LPM of the inflamed colon may provide alternative targets for therapeutic interventions not only in IBD, but other chronic inflammatory diseases exacerbated by elevated TNF-α levels.


The RV144 HIV-1 vaccine trial showed protection from HIV-1 acquisition with vaccine efficacy of 31.2%. Study of the immune correlates demonstrated an inverse association of V1/V2 antibodies with infection risk. A key task for HIV-1 vaccine development is to improve the level of efficacy seen in the RV144 trial with subsequent vaccine designs.E.A244 V1/V2 Env tags contains an N-terminal Ig leader sequence and C-terminal Avi- and His6-tags linked to the V1/V2 domain, was expressed in 293F cells and purified by nickel column. Binding of Tier 1 neutralizing mAb CH58 from RV144 vaccinees, V2 conformational mAb 697D and broadly neutralizing antibodies (bnAb) CH01 and PG9/PG16 to 33 HIV-1 gp140/gp120s and 12 HIV-1 V1/V2 scaffold Envs was tested by ELISA and surface plasmon resonance.Among 45 HIV-1/SIV Envs tested, E.A244 V1/V2 tags and E.A244 gp120Δ11 Env were the only Env antigens recognized by all three types of mAbs: CH58, 697D, and bnAbs CH01, and PG9/PG16. E.A244 V1/V2 tag bound CH58 with a Kd of 0.33 nM and 697D with a Kd of 117 nM. Although PG9 preferentially recognizes trimers, PG9 bound well to both E.A244 gp120Δ11 (Kd = 47.3) and E.A244 V1/V2 tags (Kd = 83.3 nM). BnAb CH01 bound V1/V2 tags as well (Kd = 334 nM). E.A244 V1/V2 Env tags was also recognized by the unmutated ancestor antibodies (UAs) of CH58 with ELISA EC50 = 4.9 nM and CH01 with EC50 = ~1μM. E.A244 V1/V2 tags and AE.gp70 V1/V2 scaffold were the best recombinant Envs for detection of plasma V1/V2 antibodies in RV144 vaccinees.Recombinant E.A244 V1/V2 Env tags Env expresses linear as well as conformational determinants recognized by V1/V2 mAbs and some of their UAs. This V1/V2 construct is a candidate immunogen to target RUAs and intermediate ancestors of V1/V2 antibodies to drive their induction.


To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le Caliciviridae and the major cause of non-bacterial gastroenteritis worldwide Human noroviruses (NoVs) are members of the family To explore the process of viral attachment to cells, we and others have used an experimental system of virus-like particles (VLPs) and the human intestinal cell line, Caco-2. Caco-2 cells were originally isolated from a human colorectal carcinoma b HBGA by α1-3/α1-4 fucosyltransferase, FUT3. Moreover, type H HBGAs are further modified on Gal by a GalNAc or a Gal in α1-3, resulting in type A and B HBGAs, respectively. This step is catalyzed by the A and B enzyme. Hemagglutination assays showed Norwalk VLPs bind to human type O and type A red blood cells specifically Although the ligand(s) for productive NoV infection is unknown, type H HBGAs have been proposed as initial attachment factors in vitro experiments based on enzyme-linked immunosorbent assays (ELISAs) characterized their interactions. In vitro, VLP-carbohydrate binding assays showed that Norwalk VLPs bound to type H1, H2, Leb- and Ley-synthetic saccharides a, and Leb synthetic carbohydrates. VLPs of another genotype, GII.1 (the 485 strain) failed to bind any HBGAs VLP binding to HBGAs can be strain-dependent. Several b HBGAs, depending on the state of cell differentiation NoV VLP-HBGA binding has been well studied; however, the involvement and contribution of HBGAs in NoV and NoV-VLP binding to cells before internalization are not clear. Using confocal laser-scanning microscopy, we showed Ueno 7k VLPs (GII.6) specifically bound to a population of undifferentiated Caco-2 cells that formed clusters b HBGAs was assessed on undifferentiated and differentiated Caco-2 cells, and binding of VLPs was determined by immunofluorescence. Furthermore, the involvement of HBGAs in binding of VLPs was evaluated in tissues from fresh ileal biopsies also by immunofluorescence.In this study, we investigated the co-localization of NoV VLPs and HBGAs to determine if expression of HBGAs has an effect on the binding of VLPs to Caco-2 cells. Expression of type H1, H2 and Le2 at 37°C. Cells were seeded at 50,000 cells/cm2 on type I collagen-coated coverslips  in six-well plates and on type I collagen-coated culture inserts in six-well plates . Cells on coverslips were cultured for 3 days and used as undifferentiated Caco-2 cells. Cells on culture inserts were cultured for 21 days and used as differentiated Caco-2 cells.Caco-2 cells were obtained from the American Type Culture Collection  and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , 1% non-essential amino acids solution (Sigma), 100 U/ml penicillin and 100 µg/ml streptomycin, and grown under humidified 5% COg in an SW32 rotor . VLPs with the native virion size  were purified by CsCl ultracentrifugation. Purified VLPs were applied to a carbon-coated electron microscopy grid, stained with 2% uranyl acetate, and examined by electron microscopy.VLPs of the Ueno 7k and 485 strains were prepared in a baculovirus expression system as described Polyclonal antisera against Ueno 7k and 485 were obtained by immunizing rabbits with VLPs of Ueno 7k and 485, respectively Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described 10 particles) in 200 µl of DMEM at 4°C for 1 hour. After washing with DMEM without supplements three times, cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS and blocking with NETG buffer  for 2 hours at room temperature, cells were incubated with appropriate antibodies in NETG overnight at 4°C. Cells were subsequently incubated with Alexa dye–labeled antibodies in PBS for 1 hour at room temperature. Cell nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI) . After washing with PBS, cells on coverslips or culture inserts were mounted onto glass slides in a drop of Prolong Gold anti-fade regent (Invitrogen). Imaging was performed on a Zeiss Axioplan2 microscope equipped with a LSM5 PASCAL laser-scanning confocal optics .Caco-2 cells cultured on coverslips or culture inserts were incubated with (+) and without (−) 0.5 µg of VLPs . Areas of the red-channel signal corresponding to VLPs were measured by Photoshop software, version CS5 . Statistically significant differences between undifferentiated and differentiated Caco-2 were evaluated by Student's t-test. Error bars represent standard deviations, and statistical significance was defined as p<0.01.Nine microscopic images of Caco-2 cells were taken randomly at ×200 magnification (area of each image = 0.143 cm10 particles) in 500 µl of PBS(-) for 1 hour at 4°C. After washing three times with PBS(-), specimens were fixed with periodate lysine paraformaldehyde  for 4 hours at 4°C. The specimens were dehydrated in a graded series of sucrose/PBS , embedded in OCT compound , and frozen at −80°C until use. Cryostat sections of 6 µm were incubated with primary antibodies and stained with the Alexa dye–conjugated secondary antibodies. Cell nuclei were stained with DAPI. Immunofluorescence images were obtained by confocal laser-scanning microscopy (Carl Zeiss). Pseudo-colors in the images, except for 5B18 detection, were changed by the Zeiss LSM Image Browser software (Carl Zeiss) as follows; the pseudo-color of the NoV VLPs, whose channel was red, was changed to green, and of each HBGA, whose channels were green, were changed to red.The study was approved by the Ethics Committee of the National Defense Medical College, and all biopsy samples were collected from two blood type O individuals . Written informed consent was obtained from all donors. Biopsy specimens from human ileum were immediately cooled and incubated with 1.25 µg of VLPs  was thought to not be in excess. The VLP layers observed in differentiated Caco-2 cells might be due to the structure of the apical cell membrane. After differentiation, Caco-2 cells form microvilli on the apical surface, and the VLPs were assumed to bind the microvilli. The formation of microvilli is a likely reason for the thick VLP layer on Caco-2 cells. During cell differentiation, Caco-2 cells express many proteins on the microvilli surface that are mainly involved in xenobiotic and drug metabolism and lipid metabolism Caco-2 cells change morphological and functional characteristics remarkably during differentiation, and so, we estimated the effect of cell differentiation on the VLP binding. In previous studies of VLP-cell binding with the same cells and VLPs, approximately 5% of VLPs were internalized into cells at 37°C b, but not type H1, HBGAs . These results suggested the Ueno 7k VLPs rely on molecule(s) other than type H1, H2 and Lein vivo in ileum biopsies. Fresh biopsy specimens were incubated with VLPs at 4°C to suppress cellular uptake by phagocytosis, pinocytosis or endocytosis. Furthermore, we suspended the VLPs in PBS(-) to suppress cellular uptake activity, instead of DMEM/FBS which was thought to maintain that activity. The concentrations of VLPs (2.5 µg/ml) added to fresh biopsy samples were similar to those in previous studies (0.4–5 µg/ml) b HBGAs gave different and characteristic staining images  enters RAW264.7 macrophage cells by a non-clathrin, non-caveolae-, dynamin- and cholesterol-dependent pathway Unlike previous studies of VLP-binding to intestinal biopsy et al., the binding patterns of VLPs on the intestinal specimens were similar. Intestinal goblet cells synthesize and secrete high-molecular-weight mucus glycoproteins, mucins, that provide a viscous, presumably protective blanket on the mucosal surface. The synthesized mucins are stored within subcellular secretory vesicles, termed mucin granules, within goblet cells and secreted by exocytosis to the apical side of the cells. In the present study, mucin granule-like signals were clearly observed after staining with the H2- and Leb-antibodies  bound to ileum biopsy  other than HBGAs for cell binding/internalization.We found the Ueno 7k VLPs were likely to bind to type H1 and Lem biopsy . The preTo clarify the involvement of HBGAs in VLP-cell binding/internalization, examination of the mutant VLPs, which are mutated in the HBGA-binding sites will be required. Tests of the GI.3 strains (Desert Shield and VA115), which were reported not to bind to any tested HBGAs, including human saliva samples b HBGAs. To our knowledge, this is the first report to incubate VLPs directly with fresh human intestinal biopsy specimens to examine VLP-cell binding. This result, that VLPs could bind and internalize to epithelial and goblet cells, might provide keys to disclose the mechanism of NoV infection.The results from previous studies that NoVs have the HBGA-binding site Figure S1NoV VLPs colocalized with type H2 HBGA in intestinal biopsy specimens from a different individual. Fresh human ileum biopsy specimens from a single individual  were incubated with 2.5 µg of NoV VLPs in PBS(-) for 1 h at 4°C and subjected to immunofluorescence microscopy. Cryostat sections were incubated with rabbit anti-Ueno 7k VLP serum and mouse anti-type H2 HBGA antibody and stained with Alexa dye–conjugated secondary antibodies. Panels d–f and g–i are high magnification views of areas in boxes in panels a–c, respectively. Green, NoV VLPs; red, type H2 HBGA. Scale bars in panel c = 100 µm, and in panel f and i = 20 µm.(TIF)Click here for additional data file.


Niu-Huang-Jie-Du Pian (NHJD) is a widely used traditional Chinese medicine containing realgar (As4S4). Realgar has been included in many traditional medicines, but is often taken as arsenite for risk assessment. To evaluate true risk of realgar and realgar-containing NHJD, their toxicity was compared with common arsenicals. In cultured cells, the LC50 for NHJD (1200 μM) and realgar (2000 μM) was much higher than arsenite(35 μM), arsenic trioxide (280 μM), and arsenate (400 μM). Acute toxicity in mice showed more severe liver and kidney injury after arsenite or arsenate, but was mild after realgar and NHJD, corresponding to cellular and tissue arsenic accumulation. The expressions of arsenic-sensitive stress gene metallothionein-1 were increased 3–7-folds after arsenite or arsenate, but were unaltered after NHJD and realgar. Thus, realgar and NHJD are much less toxic than arsenite and arsenate. The use of total arsenic to evaluate the safety of realgar and realgar-containing NHJD is inappropriate. NHJD is composed of realgar (6.4%), Niu-Huang , Huang-Qin , Ju-Geng (Platycodon grandiflorum), Gan-Cao , gypsum , and Bing-Pian (Borneol), and it is used for antipyretic, cold, gingivitis and other inflammatory diseases [Niu-Huang-Jie-Du Pian  is a major determinant for its disposition and toxicity. For example, realgar-containing An-Gong-Niu-Huang Wan (安宫牛黄丸) was much less toxic in cultured cells [NHJD, a most popular patent Chinese medicine, in cultured cells and in intact animals, as compared to common arsenicals. The results fortified our prior conclusions that chemical forms of metals are very important in determining the disposition and toxicity of metal-containing traditional medicines.We have recently shown that chemical form of realgar , and realgar (6.4%)-containing NHJD were obtained from Beijing Tong-Ren-Tang Technologies Co., Ltd., Beijing, China; The common arsenicals As2S2, As2O3, NaAsO2, and Na2HAsO4·7H2O were purchased from Sigma Chemical Company , all chemicals were of reagent grade.Realgar  in 10–30 mM and serially diluted in culture media before addition to the cultures at 5-6 different concentrations as indicated. For cytotoxic assay, chemicals were added to 96-wells when initiating the culture, and 48 h later, the cytotoxicity was measured by the MTS assay as described previously [μM) for 30–120 min, and cellular As was determined after thoroughly washing.Human pharyngeal carcinoma FaDu cells were obtained from Shanghai Institute of Biochemistry and Cell Biology  and cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) with penicillin and streptomycin. Cells were cultured at 37°C in a 5% COAdult male and female Kunming mice (22 ± 2 g) were purchased from the Animal Center of the Third Military Medical University . Mice were kept in a regulated environment  with a 12 h : 12 h light:dark cycle. All animal procedures follow the WHO Guidance of Humane Care and Use of Laboratory Animals. NHJD (600 mg/kg), realgar (600 mg/kg), and the equal amount of As doses as sodium arsenite (36 mg/kg) and sodium arsenate (88 mg/kg). The NHJD 600 mg/kg is approximately 2 times of clinical dose . Animals were monitored closely for clinical symptoms after gavage, and tissues were harvested for analysis 8 hrs later. The doses of arsenicals selection were based on our recent publications [Mice were orally administered with ications , 12.NHJD and various arsenicals.Serum was separated from whole blood by standing for 1 hr, and blood biochemistry was determined with an autoanalyzer (GLAMOUR1600). The serum activities of alanine aminotransferase (ALT) and concentrations of blood urea nitrogen (BUN) were quantified to evaluate the hepatotoxicity and nephrotoxicity of animals treated with Total arsenic contents in cells and tissues were analyzed by atomic fluorescence spectrometry (AFS) as described previously . Brieflyβ-actin of the same sample and expressed as percentage of controls.RNA isolation and real-time RT-PCR analysis approximate 50–100 mg tissue was homogenized in 1 mL TRIzol agent , and total RNA was extracted according to manufacturer's instructions, followed by purification with RNeasy columns . The quality of RNA was determined by the 260/280 ratios, and by gel electrophoresis to visualize the integrity of 18S and 28S bands. Total RNA was reverse transcribed with MMLV reverse transcriptase and oligo-dT primers. The PCR primers were designed with Primer Express software  as MT-1 (BC027262), forward: AATGTGCCCAGGGCTGTGT; reverse: GCTGGGTTGGTCCGATACTATT. The Power SYBR Green Mater Mix  was used for real-time RT-PCR analysis. The cycle threshold (Ct) values of the interested genes were first normalized with 50 values were estimated via graphics. For animal studies, means and standard error of 6 mice were calculated. Data were analyzed using a one-way analysis of variance (ANOVA), followed by Duncan's multiple range test. The significant level was set at P < 0.05 in all cases.For cytotoxicity analysis, means and standard error were calculated from 5 separate cultures, and the LCNHJD and arsenicals was examined in this cell line. 50 at 48 h for realgar-containing NHJD was 1200 μM, for realgar was 2000 μM. The most toxic arsenicals was sodium arsenite (35 μM), sodium arsenate (400 μM), arsenic disulfide , and arsenic trioxide .Differential cytotoxicity between realgar-containing An-Gong-Niu-Huang Wan and arsenicals was evident in human nasopharyngeal carcinoma FaDu cells . Thus, tNHJD and arsenicals, all at the 100 μM concentrations for 30 min, and cultures were thoroughly washed and cellular uptake of As were determined as described in Methods. The results .Confluent FaDu cells were exposed to  results  show draNHJD (600 mg/kg), realgar , and the equal amount of As doses of sodium arsenite (36 mg/kg) and sodium arsenate (88 mg/kg). Animals were killed 8 hr later and blood biochemistry was performed. The results , realgar , and the equal amount of As doses of sodium arsenite (36 mg/kg) and sodium arsenate (88 mg/kg). Animals were killed 8 hr later and tissue As accumulation was determined. The results  and arsenate (3320 ng/g liver) produced significant As accumulation. Renal As contents were under detection limits for NHJD and realgar, but reached 3350 ng/g kidney after arsenite and 1500 ng/g kidney after arsenate.Mice were orally administered with  results  show thaNHJD and realgar. As shown in NHJD and realgar did not produce significant elevation in renal MT-1 transcripts.NHJD were much less toxic than sodium arsenite and arsenate in cultured cells and in mice, as evidenced by LC50 values, the elevated serum ALT and BUN concentrations in mice. Toxicokinetically, much less As was accumulated in the cells or in tissues after realgar and NHJD. Furthermore, the expression of stress-related genes, namely, MT-1, was increased 2–7-folds after sodium arsenite and arsenate in the liver and kidneys, but was basically unaltered after realgar and NHJD, consistent with tissue damage. The present study fortified our recent observations in cultured cells [NHJD are different from sodium arsenite and arsenate and clearly demonstrate that the chemical forms of arsenicals underlies their disposition and toxicity potentials. The present study clearly demonstrated that realgar and realgar-containing ed cells  and in ied cells –13, indiNHJD is a most popular realgar-containing traditional Chinese medicine (http://www.wickpedia.org/) available in Chinese grocery stores in the USA and in the Europe and is used for many inflammatory diseases [Arsenic has been used as a remedy and a poison since ancient times. In addition to the use of arsenic compounds in cancer chemotherapy , 17, rea3+, arsenite) and pentavalent  forms and is widely distributed in nature. In general, sodium arsenate (LD50 112–175 mg/kg) is 4-5 times less acutely toxic than sodium arsenite (LD50 15–44 mg/kg), and the pentavalent organic arsenicals MMA, DMA, and TMA are 40–100 times less acutely toxic than arsenite [50 values 100–500-fold above arsenite or arsenate [50 for arsenic trioxide  in mice is 32–39 mg/kg, but the LD50 for realgar is 3.2 g/kg [4S4) is much less acutely toxic than arsenic trioxide (As2O3) and is also much less acutely toxic than sodium arsenite (NaAsO2) and arsenate (Na2HAsO4) in the present study. Arsenic exists in the trivalent , corresponding to 28 mg arsenic per pill, of which only 1 mg arsenic finds its way into the blood stream, that is, only 4% of intake realgar is bioavailable [It is generally assumed that the severity of poisoning is related to the total amount of poison ingested, and assessment of health risk associated with arsenic exposure from human ingestion of traditional medicines has typically taken this tactic –7. Howevvailable . Comparevailable . The biovailable .NHJD, fortifying the observations from blood biochemistry that realgar and NHJD are much less acutely toxic than sodium arsenite and arsenate.Various stresses have been proposed as an important mechanism involved in arsenic toxicity and carcinogenesis. MT is a small, cysteine-rich, metal-binding protein playing an important role in metal toxicity . Inducti六神丸,  As) [安宫牛黄丸,  As+Hg) [朱砂安神丸,  Hg) [NHJD is unique in that it is the most popular traditional medicine available not only in China [Traditional medicines are based on empirical experience and have their own theory and are generally safe at clinical doses. The regulation of metal-containing traditional medicines has been a topic of debate , 23–25. 神丸,  As) , An-Gong  As+Hg) –11, and 神丸,  Hg) . In the NHJD are nontoxic. High dose of realgar for the long-term use did produce toxicity to the liver and kidneys [NHJD was reported to produce hepatotoxicity and nephrotoxicity [NHJD in the treatment of various diseases, the balance of the benefit and risk is important. “Dose makes a poison.” Although realgar and NHJD are less toxic than sodium arsenite and arsenate, high-dose and long-term administration should be avoided to reduce undesired adverse effects and toxicity [Realgar is less toxic than arsenic oxide yet effective in cancer chemotherapy based on the recent literature . However kidneys , similartoxicity . In the toxicity , 18. NHJD are much less toxic to the cultured cells and to the intact animals, as compared to sodium arsenite and arsenate. The chemical forms of arsenicals determine their tissue accumulation and toxicity potentials, and thus the use of the total content of As to evaluate realgar-containing traditional medicines is inappropriate.In summary, the present study clearly demonstrated that realgar and realgar-containing


Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.RP1  is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser The EB1 family proteins encoded by three distinct genes (MAPRE1–3) are involved in microtubule stability and integrity Until now little functional information is available for the second EB1 family member RP1. Regulatory mechanisms governing its cellular function are hitherto unknown. Post transcriptional expression control of RP1 by a viral MicroRNA  has been described Recently, RP1 has been identified in a proteomic screen of pancreatic cell lines that had specifically been selected for increased perineural invasiveness With a comparable molecular weight of 30–37 kDa and a size of 268–327 amino acids, the overall homology among the EB1 protein family members averages between 70% and 77% identity and is specifically higher at their C- and N-terminus. All three family members share a N-terminal calponin-like or actin-binding domain and an EB1-like C-terminal domain. Within these domains conservation is high reaching over 90% in the N-terminal and above 80% in the C-terminal domain, respectively. Dimerization of the EB1 proteins depends on their C-terminal moieties. All EB proteins homodimerize, but only EB1 and EB3 have the ability to heterodimerize The interjacent region of the two homologous domains shows the greatest variability between the three proteins. Within this distinctive zone a serine rich stretch is notable only in the RP1 protein sequence. Using different prediction algorithms, we identified a potential CK2 phosphorylation site that is not present in EB1 or EB3. The serine residue at position 236 displays a classical CK2 (X-S/T-X-X-D) motif and is conserved among various species with an inconsequential variety in the X-residues.CK2 is a pleiotrophic ubiquitous and constitutively active protein kinase with a broad range of targets This study examines the relationship between CK2 and RP1 and a putative role of RP1 phosphorylation in adhesion.The antibodies against RP1 and their usage have been described elsewhere Phalloidin-Rhodamine (Molecular Probes) was used at a final concentration of 0.17 µM. DNAse I  was diluted with PBS to a working concentration of 40 µg/ml. The Actin FACS analysis was carried out as described by Knowles and McCulloch Horseradish peroxidase conjugated secondary antibody goat anti mouse , goat anti rabbit  and donkey anti goat  were used diluted 1∶3000 for Western blotting.The conjugated secondary antibody goat-anti-mouse FITC  was employed 1∶500 both in FACS and IF and the goat-anti-rabbit FITC (BD Transduction Labs) 1∶800 for FACS.et al.The antibodies against CK2 subunits (α-CK2/α-CK2α/α-CK2β) used in this study were characterized and described by Faust et al.EcoR I and Not I restriction sites. In this vector RP1 gained a c-terminal 6xHis-Tag.The RP1 wild type (RP1-wt) DNA sequence, initially described by Juwana 236 residue were performed by using the QuikChange II site-directed mutagenesis kit , according to the manufacturer's instructions. The following primers were used:From the new master clone site-directed mutagenesis of various RP1 constructs concerning the Ser5′-GTGGCTCAGCATCCAAGGCAGATAAAGATTTAG-3′RP1-ALA-forward: 5′-CTAAATCTTTATCTGCCTTGGATGCTGAGCCAC-3′RP1-ALA reverse: 5′-GTGGCTCAGCATCCAAGGATGATAAAGATTTAG-3′RP1-ASP-forward: 5′-CTAAATCTTTATCATCCTTGGATGCTGAGCCAC-3′RP1-ASP-reverse: The mutations were then confirmed by DNA sequence analysis .Human embryonic kidney cells (HEK293) were obtained from the American Type Culture Collection . For the radiolabelling experiments HEK293 cells were cultured in DMEM without sodium pyruvate and phosphate. For generation of stable cell lines HEK293 cell lines were transfected using LipofectAMINE Plus (Invitrogen) with the respective RP1 encoding plasmids or empty vector.Clones outgrowing under puromycine pressure were tested by Western blotting for protein expression levels and by immunofluorescence. To avoid clonal bias three different clones from each construct were pooled and propagated in culture for at least four weeks before the respective experiments.His-tagged RP1 wild type protein and mutants were isolated with Ni-NTA-agarose beads from lysates of HEK293 cells stably expressing the respective RP1 construct. After stringent washing with increasing amount of PBS/Tween (0%–0.3%) lysates were run on 10% SDS PAGE gels and subsequently blotted to nitrocellulose membranes. These membranes were probed with CK2 subunit antibodies as indicated in For analysis, the protein gels were stained by either Pro Q Diamond  or by Sypro Ruby  according to the manufactures’ protocols. Phosphorimaging of stained gels was performed by using either a Storm 860 (at 450 nm) or a Typhoon model 9410 (at 512 nm with 560 V)  and ImageQuant Version 5.1 software (Amersham).59, S72, S236 were identified by the software program Prosite located at www.expasy.ch/prosite and then synthesized as peptides . Three µg of these peptides and an irrelevant control peptide were then tested as CK2 phosphorylation substrates using a 33P based phosphorylation assay described before 236 and RP1-ASP236 (0.5–1 µg) were incubated with recombinant CK2 and kinase buffer  containing 2 µCi 33P-gamma-ATP, for 30 minutes at 37°C. The bead/protein complex was dissolved by 100 mM imidazol and centrifuged. The supernatant was subjected to SDS-PAGE and subsequently blotted to nitrocellulose membranes. For control purposes CK2 was incubated with 2 µCi 33P-gamma-ATP and kinase buffer only. The membranes were examined by phosphorimaging.Potential CK2 kinase sites S4 cells/cm2. On day 3 cultured cells (5×105) were fixed as described below and stained with the respective antibodies. Primary antibodies and secondary antibodies were used as described in the section antibodies above. By FACS  1×105 cells were subjected to analysis in each individual experiment.For flow cytometric analysis cells were seeded into T-25 flasks at 10For measurement of N-cadherin level cells were pretreated with a fixation/intracellular staining buffer Cytofix/Cytoperm Kit (BD Biosciences) and incubated for 30 minutes with a monoclonal antibody directed against the cytoplasmic tail  on ice. After one wash with PBS the corresponding secondary antibody goat-α-mouse FITC  was applied for 15 minutes immediately followed by FACS analysis. For negative controls of the FACS experiments primary antibodies were omitted and cells incubated only with a secondary antibody. Complementary cells from the above experiments were mounted onto slides by cytospin and examined by immunofluorescence with a 40× magnification (data not shown).The G- and F-actin content of constitutively RP1 expressing cells were measured following the method described by Knowles and McMulloch 236, RP1-ASP236 or the empty vector bearing cell line as a control) were grown until reaching cell densities of 75%. Then, cells were trypsinized, washed and resuspended in HBSS/0.1% BSA. 1×105 HEK293 cells were added to the flow chamber and allowed to settle for 3 min as described before 2 was applied by flushing 37°C prewarmed HBSS/0.1% BSA using a precision pump  until no cells were further released. Subsequently, shear stress was increased stepwise every 30 seconds to a maximum of 2.5 dyn/cm2. After each step, cells were photographed using a CCD camera  mounted on an inverted stage microscope . Adherent cells were counted manually in four representative fields for every shear stress condition and values were normalized to the number of initially adherent cells set at 100% Parallel plate flow chambers  were seeded with human umbilical vein endothelial cells . The cells were grown in EGM-2 medium  until reaching confluency. Prior to experiments, HUVECs were stimulated overnight with 10 ng/mL TNF alpha. HEK293 cells stably transfected with RP1 isoforms 3, where a is the radius of orbital rotation (1.1 cm), ρ is the density of the medium (1.0 g/ml), η is the viscosity of the medium (7.5×10−3 dynes·s/cm2) and f is the frequency of rotation (rotations/second). As a control HEK293 cells were incubated only. After five hours cells were lysed and 1mg of protein per treatment group was used for immunoprecipitation following the manurfacturer’s protocol . 15 µg goat anti RP1 antibody (Santa Cruz Biotechnology) was bound to agarose and incubated with each cell lysate. Bound proteins were then eluted from the complex, run on a 10% SDS-Page gel, transferred to nitrocellulose membrane with a pore size of 0.22 µm. The membrane was blocked with Roti-Block  for 2 hrs and incubated with goat anti RP1 antibody. Bound antibody was detected applying peroxidase conjugated donkey anti goat IgG and TMB (GE Lifescience) according to manufacturer’s protocol. As loading control 20 µg of lysate protein from HEK293 (sheared and non sheared) cells was analyzed in a Western blot procedure employing mouse anti α-tubulin antibody. Western blot signals were quantified exerting ImageJ software .Fluid shear stress experiments were performed as described by dela Paz and colleagues 2 for five hours as described above. Thereafter, cells were lysed and 1mg of protein was used for immunoprecipitation. 10 µg monoclonal mouse anti-His antibody  were linked to agarose and used as probe on the cell lysate. Bound proteins were eluted, run on a 10% SDS-Page gel and transferred to nitrocellulose membrane. The blocked membrane was incubated with 2 µg/ml rabbit anti-Phosphoserine Antibody . Bound antibody was detected applying peroxidase conjugated goat anti-rabbit IgG and TMB. As loading control lysates from HEK293 RP1-wt (sheared and non-sheared) cells were detected in a Western blot procedure employing goat anti-RP1 antibody.HEK293 RP1-wt cells were grown and exposed to shear stress of 1 dynes/cm236, RP1-ASP236, empty vector, shRNA or sh-control were grown until reaching cell densities of 70%, detached and 2×106 cells per cell line were seeded in the periphery of 100-mm culture dishes and incubated one hour until initial adherence. Thereupon, shear stress of either 1.5, 4.5 or 8.5 dynes/cm2 by shaking the cells at 60 rpm, 125 rpm or 190 rpm was applied for 5 min. Medium and floating cells were removed, cells were stained with crystal violet solution (0.1%) for 25 min at room temperature and rinsed 15 min in ddH2O. Plates were dried on paper and crystal violet dissolved from cells by 0,5% Triton X-100 while shaking for one hour and measured at 570 nm on an Epoch 96-well plate reader .HEK293 cells stably expressing RP1-ALA5 HEK293 cells per well were seeded in a 12-well-plate and cultivated overnight in a cell incubator (at 37°C and 5% CO2). The next day cells were treated with 4 µg/ml Polybrene (Santa Cruz Biotechnology) for 2 hrs followed by 0.1×106 lentiviral particles of RP1 shRNA (Santa Cruz Biotechnology) or control shRNA lentiviral particles. After 48hrs cells were transferred to a cell culture flask and 24 hrs later growth medium was supplemented with 5 µg/ml Puromycin dihydrochloride (Santa Cruz Biotechnology). Single cells were seeded in 96 well plates, grown and 10 µg of protein lysates from different colonies were run on an 10% SDS page gel. Protein was transferred to a nitrocellulose membrane, which was cut horizontally at a protein marker size of 45 kDa. For RP1 detection the lower membrane part was exposed to goat anti-RP1 antibody. For loading control purposes the upper membrane part was incubated with mouse anti α-tubulin antibody (10antibody .www.graphpad.com). For p value calculation the two-tailed Fisher′s exact test was employed unless otherwise noted.Statistical tests were performed using the GraphPad Prism version 5.03. . In this study we have identified three potential CK2 phosphorylation site motifs (X-S/T-X-X-D) at amino acid position 59, 72, 236 within the RP1 sequence . Whereasin vitro kinase assays were performed. Synthesized peptides of RP1 containing S59, S72 and S236 were used as phosphorylation targets. Only the S236 containing peptide and a positive control (DDDDSDDDDD) were phosphorylated by CK2, whereas peptides S59 and S72 were not or only marginally phosphorylated (236 (ALA) and a Phospho-mimicking RP1-ASP236 (ASP) were used in a kinase assay and incubated with recombinant CK2. Wild type RP1 was considerably phosphorylated, whereas RP1-ALA236 was significantly less phosphorylated  or RP1-wt or RP1-ALA236 or RP1-ASP236 on a surface of endothelial cells (HUVEC) in parallel plate flow chambers at defined shear stresses were assessed. Thereby, the influence of RP1 function on resistance of HEK293 cells to detachment upon increasing levels of shear stress as a measure of the adhesion function could be evaluated. With increasing shear stress adhesion of HEK293 cells was declining to 20–45% at 2.5 dyn/cm2. The differences in adhesion behavior under flow between the parental cell line, RP1-wt or the RP1-ALA236 mutant did not reach statistical significance. In contrast, the RP1-ASP236 bearing cells displayed significantly less adhesion from 1 to 2.5 dyn/cm2 compared to the control cell lines . This inTo corroborate these findings, we investigated the cellular amount of endogenous RP1 under shear stress conditions. After applying shear stress of 1 dynes/cm2 on native HEK293 cells over five hours we found endogenous RP1 expression to be reduced to almost undetectable levels by Western blotting .Next, we examined the RP1 phosphorylation status under fluid shear stress. For this, the RP1-wt containing mutant clone was chosen, because endogenous RP1 had been diminished after shear stress as described above  and woulFinally, we performed the shear stress procedure with different intensity  on HEK293 empty vector and the mutant HEK293 cells. Here, we compared control cells transfected with irrelevant shRNA, RP1 specific shRNA bearing cells and the mutant cell lines RP1-ALA236, RP1-ASP236 . The res236 (ALA), RP1-ASP236 (ASP) cells significantly reduced G-Actin contents were found  by Western blotting, a marked decrease of N-cadherin in cells expressing RP1 constructs compared to parental or empty vector-transfected cells  was notenificant . Furthernificant . This renificant . The meain vitro assays map the CK2 phosphorylation site of RP1 to serine 236 , RP1-wt (middle panels) and RP1 ASP (right panels) were seeded to 10 cm dishes and left for 30 minutes before supernatant and unsettled cells were washed off. 24 hours (upper panels) and 48 hours (lower panels) later, representative overview pictures of the respective cells were taken.(TIF)Click here for additional data file.Figure S2Surface attachment of of RP1 shRNA expressing cells. Pictures show increased attachment behavior of RP1 shRNA regulated cells (right panel) compared to control shRNA containing cells. Cells were seeded and washed off as described in S1. Overview pictures were taken at indicated time points.(TIF)Click here for additional data file.Figure S3RP1 shRNA Downregulation of endogenous RP1 protein in HEK293 cells by a specific lentiviral transduced shRNA (right lane) and a respective control shRNA (left lane) is shown by Western blotting. α-tubulin served as a loading control.(TIF)Click here for additional data file.


Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin.Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166–168 and 178–183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain.These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin. In principle, non-neutralizing antibodies might confer protection by effectively lowering the probability of infection from transmitted virus inocula. Several distinct mechanisms have been proposed by which non-neutralizing antibodies to the HIV-1 envelope protein might have a protective effect. These include inactivation of viruses or virus-infected cells by antibody-dependent cell-mediated virus inhibition + T-cells Recombinant gp120 from the MN strain of HIV-1 (MN-rgp120) has been investigated as a candidate HIV-1 vaccine to elicit protective antibody responses Recently, antibodies that bind to the V2 domain of the HIV-1 envelope protein gp120 have attracted considerable attention in connection with the RV144 trial A collection of thirty-six monoclonal antibodies, produced in the mid-1990's against MN-rgp120 or IIIB-rgp140 To determine whether the V2 MAbs recognized linear or conformation-dependent epitopes, MAb binding to unfolded, reduced and carboxymethylated MN-rgp120 (RCM-rgp120) was examined. It was found  that sixFurther information regarding the specificity of the V2 MAbs was obtained in rgp120 cross-reactivity studies. In these experiments, the MAb binding to recombinant gp120 from seven diverse isolates of HIV-1  was measured. The results obtained  indicateWe found  that six125I]-MN-rgp120 to cell surface CD4 , we considered the possibility that the epitopes were contained entirely in this region. Our initial strategy depended on site-directed mutagenesis, where naturally occurring sequence polymorphisms were incorporated into MN-rgp120 based on comparisons of the sequence of MN-rgp120 with envelope proteins from strains  that faiThe binding of the competition group A antibodies, 1019 and 1027, could be inhibited by the naturally occurring G166R mutation and the double Ala mutation of D167A- K168A . These dWe next examined the competition group B MAbs . We founCompetition group C is represented by MAb 1088. This MAb was unique in that it recognized a conformation-independent epitope, as indicated by its ability to bind RCM-rgp120, and by the fact that it exhibited potent, type-specific, neutralizing activity . In addiThe competition group D MAb, 1025, resembled the competition group A antibodies in that it competed for binding with all of the V2 MAbs in this study . HoweverThe competition group E antibody 6E10, which was isolated from a mouse immunized with IIIB-rgp140 Finally, we examined the effect of combining multiple mutations. We observed that several double and triple mutants appeared to prevent the binding of most of the V2 antibodies in the panel. We found that the E182V-P183S mutant and the E182V-P183V mutant prevented the binding of all of the MAbs in the panel except 1025, whereas other double mutants such as G166R-M169V and D167A-K168A selectively inhibited MAb binding. This result suggested that positions 182 and 183 are unique in that they appear to be critically important for maintaining a unique conformation or structural feature important for the binding of all of the MAbs in this study except for 1025. In view of this observation, it is not surprising that the triple mutant G166R-E182V-P183S similarly prevented the binding of eight of the nine MAbs in this study. As noted above, positions 182 and 183 occur immediately adjacent to the α4β7 recognition sequence LDI spanning position 179–181.2+ and Mn2+ (data not shown) which are essential ions for the binding of α4β7 to its normal receptor, MAdCAM. In these experiments we screened several MAbs raised against α4β7  that were critical for binding of one or more of the MAbs . These cThe structure of a scaffold comprising the V1 and V2 domains of gp120 bound to the broadly neutralizing PG9 MAb has recently been elucidated Recent studies have shown that the central aspartic acid (D180) residue within the α4β7 recognition sequence (LDI/V) also appears to be an essential part of the conformational masking mechanism Neutralizing MAbs targeting the V2 domain have been isolated from mice and rats immunized with recombinant envelope proteins, and from chimpanzees and humans infected with HIV-1  as well Several studies have shown that neutralizing antibodies to the V2 domain are present in sera from HIV-infected humans. Walker et al. An important finding of this study was that four of the epitopes  depended on positions 178, 182, and 183, adjacent to the highly conserved LDV/I sequon of the α4β7 binding site in the C–D connecting loop. The observation that the double mutation E182V-P183S blocked the binding of all of the MAbs described in this study (with the exception of 1025) suggests that these two residues are part of a structural element required for the functional conformation of the V2 domain. This result is in agreement with our previous observation Given the fact that MN-rgp120 is able to stimulate the formation of conformation-dependent antibodies to the V2 domain, it is surprising that we didn't observe greater cross-reactivity and greater neutralizing activity. Several possible explanations may account for this. First, there were several radical amino acid polymorphisms in the MN-rgp120 immunogen that are uncommon in clade B isolates . These iThe results described in this paper add to our understanding of the specificity of the immune response to the V2 domain of gp120. They show that the antigenic structure of the V2 domain is extraordinarily complex, containing at least eight to ten conformation-dependent epitopes including the six described in this paper along with those recognized by the PG9, PG16, 2909, C108g, and 10/76b MAbs GNE) has been described elsewhere . The gp120 MNGNE is two percent different than the original MN gp120 sequence published by Gurgo et al. The standard single letter code is used to designate the amino acids. The numbering of amino acids in gp120 is provided with reference to the standard HXB2 numbering system. The sequence of the MN isolate incorporated in the MN-rgp120 immunogen  −ung− CJ236 E. coli strain and then grown in the presence of MK107 helper phage for the purpose of generating a single stranded DNA template. 33- to 40-mer oligonucleotides incorporating the desired mutation were utilized to insert the mutations into the V2 region. The mutants were transformed into bacteria and the colonies were screened by sequencing  to check for the correct mutations. These plasmids containing the MN gp120 V2 mutants were then transfected into the 293 human embryonic adenocarcinoma cell line using the calcium phosphate method described by Graham and van der Eb Site-directed mutagenesis of regions in the MN-gp120 V2 region was performed using the method of Kunkel et al. Fragments of MN-rgp120 were expressed in mammalian cells as described previously 2SO4. The plates were read in a microtiter plate spectrophotometer at a wavelength of 492 nm.The ability of MAbs to bind to various fragments of MN-rgp120 was determined by ELISA. Three days after transfection, the 293 cell culture supernatants were harvested and the gp120 fragments were captured onto wells of microtiter plates coated with a murine MAb (5B6) specific for the gD-1 flag epitope. The ability of HRPO-conjugated MAbs to bind to the captured fragments was measured by ELISA. The cell culture supernatants containing the MN-rgp120 fragments were then added to the wells and incubated for 2 hr at room temperature. The plates were washed twice with PBT  and then incubated for 2 hr at room temperature with a pre-titrated amount of HRPO-labeled MAb. Plates were washed again in PBT buffer and then developed with o-phenylenediamine tablets  for 15 minutes. The reaction was stopped by the addition of 2.5M H2SO4.A capture ELISA similar to that described previously 125I]-labeled MN-rgp120 (2 nM) was mixed with serially diluted MAbs and then transferred onto the cells. The assay was performed in a 50∶50 mix of F12/DMEM  containing 10% fetal bovine serum, 0.01% NaAzide and 12 mM HEPES. The mixture was allowed to react with the cells for 2 hr, and then washed four times with assay medium. The washed cells were then solubilized in 100 µL of 1.0 N NaOH for 30 min and 90 µL was removed for counting in a gamma counter. The percentage of inhibition was calculated by the equation 100-[100*/(antibody control – background)].The CD4 blocking assay was described elsewhere Δenv expression plasmid were obtained from the NIH AIDS Research & Reference Reagent Program (contributed by Drs. John C. Kappes & Xiaoyun Wu). The MN-3 envelope expression plasmid was a gift from Dr. David Montefiori. For standardization of protocols, we used HIV-1 MN-3 pSGΔenv 293T/17 pseudovirus, a generous gift from Jochen Schmidt and Hagen Von Briesen at the HIV Specimen Cryorepository, Fraunhofer Insitute (IBMT), Sulzbach, Germany.A colorimetric MT-2 syncytia inhibition assay, similar to that described previously +, β1− RPMI-8866 B lymphoma cell line 2 and 1 mM MnCl2). APC-conjugated streptavidin  was used to detect biotinylated gp120 binding to the cells. For blocking experiments, MN-rgp120 (20 µg/ml) was pre-incubated with the V2 MAbs (10 µg/ml) for 1 hr at room temperature and then added to the cells. For blocking experiments with the integrin MAbs, cells were pre-incubated with the integrin MAbs (10 µg/ml) for 30 min at 4°C, washed twice with 0.5% BSA in PBS and then the biotinylated MN-rgp120 was added. Data were collected with a FACSAria  at the UC Santa Cruz Flow Cytometry Facility and analyzed with FlowJo 8.8.6. software (TreeStar).Purified MN-rgp120 protein was biotinylated as described above. The binding of biotinylated MN-rgp120 to α4β7 was carried out using the α4β7P values of <0.05 were considered to be statistically significant.Differences between the ability of MN-rgp120 to bind α4β7 in the absence or presence of V2 antibodies were analyzed with one way ANOVA with Bonferroni correction, using Prism 5.0 (GraphPad Software). Figure S1Inhibition of rgp120 binding to CHO cell lines expressing CD4 by monoclonal antibodies to the V2 domain of gp120. CHO cells expressing membrane-bound human CD4 were plated in 96-well microtiter plates until they were 90% confluent. Serial dilutions of MAbs were pre-incubated with a fixed amount of [125I]-gp120 (2 nM) and then incubated with the cells for 2 hr. The cells were then washed four times and the cells solubilized in 100 µL of 0.1 N NaOH. Specific binding was calculated as described previously (PDF)Click here for additional data file.


TNF is an important inflammatory mediator and a target for intervention. TNF is produced by many cell types and is involved in innate inflammation as well as adaptive immune responses. CD8 T cells produce TNF and can also respond to TNF. Deficiency of TNF or TNFR2 has been shown to affect anti-viral immunity. However, as the complete knockout of TNF or its receptors has effects on multiple cell types as well as on lymphoid architecture, it has been difficult to assess the role of TNF directly on T cells during viral infection. Here we have addressed this issue by analyzing the effect of CD8 T cell intrinsic TNF/TNFR2 interactions during respiratory influenza infection in mice, using an adoptive transfer model in which only the T cells lack TNF or TNFR2. During a mild influenza infection, the capacity of the responding CD8 T cells to produce TNF increases from day 6 through day 12, beyond the time of viral clearance. Although T cell intrinsic TNF is dispensable for initial expansion of CD8 T cells up to day 9 post infection, intrinsic TNF/TNFR2 interactions potentiate contraction of the CD8 T cell response in the lung between day 9 and 12 post infection. On the other hand, TNF or TNFR2-deficient CD8 T cells in the lung express lower levels of IFN-γ and CD107a per cell than their wild type counterparts. Comparison of TNF levels on the TNFR2 positive and negative T cells is consistent with TNF/TNFR2 interactions inducing feedback downregulation of TNF production by T cells, with greater effects in the lung compared to spleen. Thus CD8 T cell intrinsic TNF/TNFR2 interactions fine-tune the response to influenza virus in the lung by modestly enhancing effector functions, but at the same time potentiating the contraction of the CD8 T cell response post-viral clearance. During an infection, the immune system must balance the need for a strong immune response against collateral damage. This is particularly true during respiratory infections where too strong a T cell response in the lung can cause immune pathology, but too weak a response can lead to failure to clear the infection, resulting in virus-mediated damage. Several members of the tumor necrosis factor receptor (TNFR) superfamily control the survival of T cells during viral infections –5. TNF, Listeria or model antigens [The role of TNF in CD8 T cell responses appears to be context dependent. There is evidence that TNF binding to TNFR2 is costimulatory for T cells and can prolong the T cell response to antigens –16. Moreantigens –20.Listeria monocytogenes or Mycobacterium tuberculosis, as well as in organizing lymphoid tissues and in airway inflammation [As the complete absence of TNF leads to lymphoid architecture changes and affects many cell types –23, it hammation –30. HereThe results show that CD8 T cell intrinsic TNF/TNFR2 signaling has a dual role during influenza infection, in enhancing effector function per cell as measured by IFN-γ production and degranulation, as well as in potentiating the contraction of the effector response. This increased functionality but more rapid contraction of the CD8 T cell response may function to limit the damage that could be caused by a prolonged and inefficient effector response.-/- mice with congenically marked CD45.1 OT-I TCR transgenic mice, whose TCR is specific for H-2Kb and OVA257–264 (SIINFEKL). This allowed us to monitor the effect of the absence of TNF on the transferred TCR transgenic T cells in a mouse in which all other cells express genetically normal levels of TNF. To analyze disease course in the mice, we transferred ten thousand purified naïve CD8 T cells from WT CD45.1 OT-I or TNF-/- CD45.1 OT-I mice into separate CD45.2 WT mice and one day later infected the mice intranasally with influenza A/HK-X31-OVA (X31-OVA) . As memory CD8 T cells are more resistant to cell death, this may explain the TNF/TNFR2 independence of this secondary contraction [To ask if TNFR/TNF interactions played a similar role in a secondary response, we transferred an equal number of in vitro generated memory-like OT-I CD8 T cells and infected with X31-OVA. Both WT and TNFR2traction .-/- OT-I cells recovered from influenza-infected mice were restimulated with OVA peptide and stained for surface expression of TNFR2 followed by intracellular staining for TNF. Using TNFR2-/- OT-I cells as a staining control, we found that a fraction of WT OT-I cells that express TNFR2 in the lung and spleen also produce TNF  ,39. Thuslo CD127hi cells had the capacity to produce TNF, compared to KLRG1hi CD127lo cells, with KLRG1lo CD127lo cells producing intermediate levels upon restimulation , consistent with previous results showing that 4-1BB expression is related to recent antigen stimulation [hi CD127lo cells have received the highest level of antigen stimulation, whereas the KLRG1lo CD127hi cells may have seen less antigen. Upon restimulation the KLRG1hi cells produce significantly less TNF than the other effector subsets, consistent with their being more terminally differentiated [A significantly greater proportion of KLRG1mulation ,41 and lmulation . TNFR2 aentiated .-/- and TNFR2-/- CD45.1 OT-I T cells for KLRG1 and CD127 expression in the lung and spleen . Thus intrinsic TNF/TNFR2 interactions increase the level of IFN-γ production per cell as well as the extent of degranulation by lung CD8 T cells.We next asked whether intrinsic TNF/TNFR2 activity was modulating not only the quantity of the CD8 T cells, but also their functionality. Lung and spleen cell suspensions from infected WT, TNF-/- and TNFR2-/- OT-I CD8 T cells to investigate this issue. In this model, only the transferred T cells lack TNF or TNFR2 in the context of an otherwise completely normal immune system. Importantly, our model allowed us to ask about the intrinsic effects of TNF and TNFR2 on T cells under conditions where viral load was not different between mice receiving WT, TNF or TNFR2 deficient T cells. Interestingly, during the response to influenza virus, the CD8 T cells showed increased capacity for TNF production over time, continuing until very late in the response, after virus had been cleared. CD8 T cell-derived TNF appears to enhance effector function of CD8 T cells as evidenced by increased IFN-γ and CD107a MFI on the WT T cells compared to TNF-/- and TNFR2-/- T cells upon restimulation, and at the same time increases the contraction of the CD8 T cell response after viral clearance. The finding that largely similar effects were obtained with TNF-/- and TNFR2-/- OT-I T cells suggests that TNF is acting in an autocrine fashion by binding to TNFR2 on CD8 T cells. Consistent with this, a fraction of the CD8 T cells were found to co-express both TNFR2 and TNF. Thus we have identified a dual role for autocrine signaling by TNF/TNFR2 in CD8 T cells, in enhancing CD8 T cell functionality, but also in increasing their contraction through TNF binding to TNFR2. It is of interest that TNFR2 is preferentially triggered by mTNF, whereas sTNF has little impact on TNFR2 signaling [TNF has been extensively characterized as an important mediator in several inflammatory disorders, but until now, its CD8 T cell intrinsic role during influenza infection has been unclear. In this study we utilized an adoptive transfer model with TNFignaling . Thus mT-/- CD8 T cells to activation induced cell death [The results presented here show that TNF contributes to the contraction of the CD8 T cell response in the lung, implying a role for TNF/ TNFR2 interactions in promoting cell death, while paradoxically enhancing the level of effector function per cell. These opposing roles of TNFR2 in promoting death of the effectors while promoting IFN-γ production and degranulation are likely explained by TNFR2 inducing degradation of the downstream signaling molecule TRAF2. Engagement of TNFR2 by TNF on T cells has been shown to result in the cIAP-1 dependent degradation of TRAF2, potentiating TNF mediated apoptosis . Moreovell death .-/- or TNFR2-/- cells. Activation of the alternative NF-κB pathway is normally restricted in resting cells by a complex between TRAF2/3/cIAP1/2, which act as an E3 ligase complex to degrade the upstream activator NIK [The loss of TRAF2 induced by TNFR2 likely also explains the enhanced production of IFNγ by TNFator NIK ,46. In pator NIK . Moreoveator NIK .The effect of autocrine TNF on contraction of the CD8 T cell response was restricted to the lung compared to the spleen and lymph nodes. This may reflect that effector cell contraction in the spleen and LN reflects migration of effectors to the tissues, whereas in the lung, the T cells persist until they become terminally differentiated. It was recently shown using overexpression of human TNFR2 in a TNFR1/2 deficient mouse embryonic fibroblast line that TNFR2 is internalized with its ligand, leading the authors to speculate that TNFR2 internalization provides a negative feedback loop . ConsistTNF and TNFR2 have been shown to contribute to the contraction of T cell responses in other contexts. For example, TNFR2 deficiency in all cells was found to contribute to the contraction of the CD8 response to LCMV, but only when combined with TNFR1 deficiency . Turner Our study examined the T cell intrinsic role of TNF using an adoptive transfer model in which only the transferred T cells lack TNF. This has the advantage of allowing us to assess the specific role of TNF/TNFR2 in the T cell response, in the context of an otherwise normal host. One disadvantage of this adoptive transfer model is that the T cells that we use have developed in the absence of TNF in other cells and thus one cannot completely rule out an effect of TNF during T cell development. Another feature of the adoptive transfer model is that it suppresses the endogenous T cell response , such thListeria monocytogenes, macrophages and neutrophils were found to be the main source of TNF after bacterial challenge, and this source of TNF was critical to mouse survival, with smaller effects of T cell produced TNF on survival of mice to Listeria challenge [M. tuberculosis infection [Others have used conditional knockout approaches to assess the effects of cell specific deletion of TNF –30. In thallenge . Interesnfection . These snfection ,28 have Listeria infection [In contrast to the results shown here, several studies have shown that TNFR2 provides a costimulatory signal to T cells, contributing to their clonal expansion, in vitro and in vivo –15. Siminfection –15, TNFRnfection –52 TRAF2nfection . On the nfection , which iListeria infection [Evidence in the literature suggests that the contraction of the CD8 T cell response is pre-programmed early after infection and takes place independently of pathogen clearance . Previounfection . In the nfection . Moreovenfection . Thus weA previous study showed that the antigen-inducible TNFR 4-1BB can prolong CD8 T cell effector responses in the lung when virus persists, but that 4-1BB is rapidly downregulated when virus is cleared, thus allowing more rapid contraction of the response . Of inte-/- (B6.129S6-Tnftm1Gkl/J) or TNFR2-/- (B6.129S2-Tnfrsf1btm1Mwm/J) both backcrossed for 10 generations onto C57BL/6 background, were obtained from the Jackson Laboratory (Bar Harbor Maine). The TNF-/- and TNFR2-/- mice were crossed with CD45.1 OT-I TCR transgenic mice  to generate TNF-/- CD45.1 OT-I and TNFR2-/- CD45.1 OT-I mice. Mice were housed under specific pathogen-free conditions in sterile microisolator cages.Male C57BL/6 (B6) mice were obtained from Charles River Laboratories . TNFMouse studies were approved by the University of Toronto animal care committee in accordance with the regulations of the Canadian Council on Animal Care, approved protocol number 20009458.-/- CD45.1 OT-I or TNFR2-/- CD45.1 OT-I mice using a negative selection mouse CD8 T cell enrichment kit . The purified CD8 T cells were injected intravenously (i.v.) at 104 cells/mouse in 200µl into 6-8 week old male B6 mice. One day later, mice were infected with X31-OVA, as described below.CD8 T cells were purified from the spleens of naive male CD45.1 OT-I, TNF-/- CD45.1 OT-I or TNFR2-/- CD45.1 OT-I T cells were infected with 1.25 hemagglutinin units (HAU) influenza A/HK-X31-OVA (X31-OVA) [B6 mice that had received CD45.1 OT-I, TNFLungs were excised from mice at various time points after X31-OVA infection and then homogenized in RPMI 1640 medium (1g lung tissue/10ml). Supernatant was obtained and stored at -70°C. TCID(50) was determined by the MDCK assay with the REED and Muench technique as previously described .L , biotin-4-1BB, and in some cases fixable viability dye . For intracellular TNF and IFN-γ staining, splenocytes and lymphocytes isolated from lungs were restimulated at 37°C with 1µM OVA257-64 using Golgi Stop  for 6 and 5 h, respectively. Cells were then surface stained for CD8 and CD45.1, fixed, and then intracellularly stained for IFN-γ and TNF (BD BioSciences). Five µg/milliliter anti-CD107a (BD Biosciences) was added at the beginning of the restimulation culture for detection of degranulation. Fluorescence minus one (FMO) controls and unstimulated samples (no peptide added) were used as negative controls. Following staining, samples were analyzed using a FACSCalibur or FACSCanto (BD Biosciences) and FlowJo software .Adoptively transferred OT-I cells were surface stained with fluorescently-tagged antibodies against the following: CD8α , CD45.1 , KLRG1, CD127, GITR, secondary streptavidin, eomesodermin (eomes), T-bet , Biotin-TNFR1, Biotin-TNFR2, caspase-3 , Bim , Bcl-xt test . One-way ANOVA was used to compare multiple samples. Statistically significant differences between groups are indicated as *p < 0.05, **p < 0.01, or ***p < 0.001.Each symbol is representative of one mouse per group. Where indicated for comparison of two values, p values were obtained using the Student’s Figure S1Transferred CD45.1 OT-I cells from infected CD45.2 mice were recovered as follows: cell suspensions isolated from lung, spleen and lymph node were gated on live cells with FSC-A x SSC-A, singlets with SSC-W vs SSC-H, negative staining of fixable viability dye, CD8+ cells, followed by CD45.1+ cells. Representative gating of a spleen sample from a WT CD45.1 OT-I recipient mouse, using an uninfected mouse that did not receive OT-I cells as a negative control.(TIF)Click here for additional data file.