Ae. albopictus as a principal vector. Previously we have demonstrated that a single mutation E1-A226V significantly changed the ability of the virus to infect and be transmitted by this vector when expressed in the background of well characterized CHIKV strains LR2006 OPY1 and 37997. However, in the current study we demonstrate that introduction of the E1-A226V mutation into the background of an infectious clone derived from the Ag41855 strain (isolated in Uganda in 1982) does not significantly increase infectivity for Ae. albopictus. In order to elucidate the genetic determinants that affect CHIKV sensitivity to the E1-A226V mutation in Ae. albopictus, the genomes of the LR2006 OPY1 and Ag41855 strains were used for construction of chimeric viruses and viruses with a specific combination of point mutations at selected positions. Based upon the midgut infection rates of the derived viruses in Ae. albopictus and Ae. aegypti mosquitoes, a critical role of the mutations at positions E2-60 and E2-211 on vector infection was revealed. The E2-G60D mutation was an important determinant of CHIKV infectivity for both Ae. albopictus and Ae. aegypti, but only moderately modulated the effect of the E1-A226V mutation in Ae. albopictus. However, the effect of the E2-I211T mutation with respect to mosquito infections was much more specific, strongly modifying the effect of the E1-A226V mutation in Ae. albopictus. In contrast, CHIKV infectivity for Ae. aegypti was not influenced by the E2-1211T mutation. The occurrence of the E2-60G and E2-211I residues among CHIKV isolates was analyzed, revealing a high prevalence of E2-211I among strains belonging to the Eastern/Central/South African (ECSA) clade. This suggests that the E2-211I might be important for adaptation of CHIKV to some particular conditions prevalent in areas occupied by ECSA stains. These newly described determinants of CHIKV mosquito infectivity for Ae. albopictus and Ae. aegypti are of particular importance for studies aimed at the investigation of the detailed mechanisms of CHIKV adaptations to its vector species.Between 2005 and 2007 Chikungunya virus (CHIKV) caused its largest outbreak/epidemic in documented history. An unusual feature of this epidemic is the involvement of  Alphavirus genus in family Togaviridae, is transmitted to humans by Aedes (Stegomyia) spp mosquitoes, primarily Ae. aegypti. However, transmission by a previously unrecognized vector species, Ae. albopictus, has been a critical contributor facilitating recent epidemics The recent massive epidemics of Chikungunya virus (CHIKV) in Africa, the Indian Ocean islands, India, and the small outbreak in Europe have elevated this arthropod-borne virus (arbovirus) to the status of a major global health problem Ae. albopictus-human transmission cycles have identified the independent acquisition of a common mutation, namely E1-A226V, in strains isolated from different geographic regions Ae. albopictus mosquitoes. Recently we demonstrated that the E1-A226V mutation significantly increases the ability of CHIKV to infect and be transmitted by a laboratory colony of Ae. albopictus mosquitoes when expressed in the background of the well-characterized La Reunion LR2006 OPY1 and West-African 37997 CHIKV strains Ae. albopictus mosquitoes collected from La Reunion Island and Mayotte, as compared with CHIKV isolates bearing alanine at this position Ae. albopictus, and provides a plausible explanation for how this mutant CHIKV caused an epidemic in a region lacking the more typical urban vector, Ae. aegypti.Phylogenetic analysis of CHIKV strains obtained during outbreaks circulating in Alphaviruses are enveloped single-stranded positive-sense RNA viruses. Genomic RNA, of ∼12,000 nt, consists of two open reading frames (ORF): the first, translated from genomic RNA, encodes the four non-structural proteins (nsP1–4), whilst the second ORF is translated from sub-genomic RNA to produce the three main structural proteins . The E2 and E1 envelope glycoproteins form heterodimers on the viral surface, where E2 lies mainly on top of E1 and is believed to interact with cellular receptors Ae. albopictus mosquitoes are native to Southeast Asia, but have recently spread globally due to the advent of modern shipment, with the current geographic range including Europe, Africa, the Middle East, North and South America and the Caribbean Ae. albopictus transmission cycle was established for the first time in Europe, with an estimated 254 human cases in Italy Ae. albopictus has been demonstrated to be susceptible to infection by several clinically important arboviruses including; eastern Ae. albopictus suggested that the epidemiologic success of CHIKV with the E1-A226V mutation was most likely due to enhanced midgut infectivity Ae. albopictus was also compared. When intrathoracically injected into the mosquito hemocoel, CHIKV does not need to infect midgut cells and can directly infect secondary organs including the salivary glands. Since in intrathoracically infected Ae. albopictus the E1-A226V mutation did not enhance dissemination/transmission rates (Higgs lab unpublished data), it was thus concluded that the effect of this mutation occurs before virus is released from the midgut into the hemocoel. These data, together with previous findings Ae. albopictus midgut infectivity resulting from the A226V mutation plays a primary role in enhanced viral transmission.Previous investigations of the effects of the E1-A226V mutation on CHIKV infection of midguts, dissemination into salivary glands, and transmission to a vertebrate host by Ae. albopictus midgut cells might provide an advantage for fusion to CHIKV with the E1-A226V mutation Ae. albopictus CHIKV midgut infectivity based on the presence of E1-226A or E1-226V residues. Based on our data we conclude that a mutation at position E2-60 influences CHIKV infectivity for Ae. albopictus, regardless of the mutations in position E1-226, and also modulates CHIKV infectivity for Ae. aegypti. Furthermore, substitutions at E2-211 are crucial for CHIKV sensitivity to the E1-226V mutation in Ae. albopictus mosquitoes, but have no effect on CHIKV infectivity for Ae. aegypti, and are widely dispersed among CHIKV isolates. These findings provide greater insight into the complexity of the molecular mechanisms involved in adaptation of CHIKV to a new vector.Little is known about the molecular mechanisms responsible for the selective advantage associated with the E1-A226V mutation. Earlier we demonstrated that the E1-A226V mutation was responsible for modulation of the CHIKV cholesterol dependence for replication in C6/36 cells, suggesting that specific lipid composition of the endosomal compartments of Ae. albopictus mosquitoes Ae. albopictus, the E1-A226V mutation was introduced into an enhanced green fluorescent protein (eGFP) expressing infectious clones (i.c.) of the Ag41855 strain of CHIKV (designated as p41855-GFP-226A). The resultant clone was designated p41855-GFP-226V , indicating that introduction of the E1-A226V mutation into the Ag41855 strain therefore does not attenuate this virus in BHK-21 cells . This demonstrates that for the Ag41855 strain, the E1-A226V mutation does not affect Ae. albopictus midgut infectivity  indicating that virus produced from the full-length and eGFP-expressing i.c. retained the infection phenotype of the parental virus in Ae. albopictus. This also indicates that the lower infectivity observed for 41855-GFP-226A and 41855-GFP-226V for Ae. albopictus, as compared with viruses produced from the i.c. of the LR2006 OPY1 and 37997 strains, is attributed to the specific mutation(s) in the genome of Ag41855 strain.The sequence of 41855-GFP-226A was identical to that of the virus stock used for i.c. construction and we previously showed that introduction of the eGFP gene into backbone of LR2006 OPY1 and 37997 strains of CHIKV does not significantly affect infectivity for A clones . The OIDAe. albopictus, and to determine the sites affecting sensitivity to the E1-A226V mutation in Ae. albopictus.Several recent phylogenetic analyses of CHIKV have grouped the Ag41855 and LR2006 OPY1 strains into the East/Central/South African phylogroup Sequence comparison of the Ag41855 and LR2006 OPY1 strains revealed a total of 202 nucleotide differences (1.7%), encoding 31 amino acid substitutions: 18 in the non-structural and 13 in the structural coding sequence . The nucAe. albopictus. Since numerous previous studies identified the E2 protein as a major determinant of mosquito infectivity for different alphaviruses including Sindbis virus (SINV) and VEEV Ae. albopictus.A comparison of amino acid sequences for strains Ag41855, LR2006 OPY1 and 37997  identifiAe. albopictus as compared to Ag41855-GFP-226V virus (OID50 = 3.78) , indicating that this region encodes the major determinant(s) for Ae. albopictus midgut infectivity in the Ag41855 strain. Additionally, these data indicate that mutations in the nsP3 and E1 genes of Ag41855  from the LR2006 OPY1 i.c. containing the E2 60D, 162A, and 211T mutations, was introduced into 41855-GFP-226V  . The rev Ag41855  probably50 for the resultant chimeric virus 41855/LR-GFP-226A was 5.21 Log10TCID50/ml . The OID50 of 41855-GFP-226V with the E2-V162A mutation was not significantly different from the OID50 for the 41855-GFP-226V virus   . The Ae.50 values for LR-GFP-226V with individual mutations E2-D60G and E2-T211I were indistinguishable as compared to OID50 values of the 41855-GFP-226V virus with E2-G60D and E2-I211T mutations expressed individually or in combination with E2-A162V, but were significantly higher as compared with LR-GFP-226V. The OID50 value for LR-GFP-226V with both the E2-D60G and E2-T211I mutations was indistinguishable from the OID50 values of 41855-GFP-226V or chimeric virus LR/41855-GFP-226V (p>0.1). These data suggest that the specific phenotype(s) associated with the E2 mutations introduced into strain Ag41855 would be retained if these mutations were expressed in other CHIKV strains.To further evaluate the relationships between these different mutations, four additional viruses were constructed in which single and double substitutions at positions E2-60, E2-161 and E2-211 from the Ag41855 strain were substituted into the backbone of LR-GFP-226V . The OIDAe. albopictus midgut infectivity for the 41855-GFP-226A virus. In this regard it was important to investigate the individual roles of these particular mutations in the 41855-GFP-226A virus, and to determine how these roles correlated with the effects of these mutations in the 41855-GFP-226V virus. In contrast to the 41855-GFP-226V virus, the introduction of the single mutation E2-G60D into 41855-GFP-226A almost completely restored viral infectivity phenotype to the relatively high levels observed for 41855/LR-GFP-226A and LR-GFP-226A (p>0.1)  , 1. HoweAe. aegypti was the principal vector responsible for most urban epidemics of chikungunya Ae. albopictus mosquitoes, the E1-A226V mutation does not increase infectivity for Ae. aegypti when expressed in the backbones of the LR2006 OPY1 and 37997 strains of CHIKV Ae. albopictus mosquitoes led us to investigate the mosquito infectivity phenotype of this strain in Ae. aegypti.Prior to the 2006–2007 outbreaks, 50 values of 41855-GFP-226A in Ae. aegypti were 6.92 and 7.24 Log10TCID50/ml, which are significantly higher than the OID50 values determined previously for LR-GFP-226A and 37997-GFP-226A viruses (p<0.01) . This finding corroborated our previous results showing that CHIKV with the E1-226A residue is slightly more infectious for Ae. aegypti mosquitoes The OID(p<0.01) . MosquitAe. aegypti, the chimeric viruses 41855/LR-GFP-226A and 41855/LR-GFP-226V as described above, were tested. These viruses were significantly more infectious for Ae. aegypti than 41855-GFP-226A and 41855-GFP-226V (p<0.01), with OID50 values similar to those of CHIKV possessing either an alanine or a valine at position E1-226 when expressed in the backbone of strains LR2006 OPY1 and 37997 (p>0.1) . In contrast, introduction of the E2-A162V and E2-I211T mutations individually or in combination resulted in viruses almost indistinguishable from 41855-GFP-226A and 41855-GFP-226V with respect to their ability to infect Ae. aegypti midguts, indicating that these two positions do not play important roles in CHIKV transmitted by Ae. aegypti.To determine the genomic regions of Ag41855 that are responsible for attenuation of Ag41855 in  (p>0.1) . These dn E1-226 , 6. ExprAe. albopictus and Ae. aegypti that we identified in previous experiments raised an important question regarding the origin of these particular mutations in the genome of the Ag41855 strain, and what evolutionary advantages might be associated with them. To address these questions, we analyzed the distribution of E2-60G and E2-226I mutations among known CHIKV isolates  was necessary and sufficient to elevate the Ae. albopictus infectivity of the Ag41855 strain with an alanine at E1-226. When considered together, these data provide new and important insights into the roles played by E2-G60D and E2-211 mutations in determining CHIKV infectivity for Ae. albopictus mosquitoes.In this work we performed a detailed investigation of the genetic factors responsible for the relatively low infectivity of the Ag41855 strain for Ae. albopictus when expressed with either alanine or valine at E1-226. This indicates that residue E2-60D is important for vector infectivity of CHIKV, but it does not specifically affect the previously observed phenotypes reported for the E1-A226V mutation. This conclusion was further supported by the results obtained from Ae. aegypti infectivity experiments. In this mosquito, the E1-A226V mutation does not increase CHIKV infectivity Ae. aegypti, expression of the E2-60D alone was necessary and sufficient to increase Ag41855 infectivity to that of the LR2006 OPY1 and 37997 levels. Interestingly, the E2-60G residue was found only in three other CHIKV strains, none of which share close phylogenetic relationships. More importantly, the IND-00 CHIKV strain, which is almost 100% identical to the Ag41855 strain based on both nucleotide and amino acid sequences, has a glutamic acid residue at E2-60. This suggests that acquisition of E2-D60G occurs sporadically, possibly during propagation of the virus under laboratory conditions. Altogether, accumulated data suggests that the E2-60 residue affects some basic mechanism used by CHIKV to infect its vector. Disruption of this mechanism exerts a strong inhibitory effect on CHIKV infectivity to both Ae. albopictus and Ae. aegypti, and therefore this mutation should be quickly eliminated from naturally transmitted strains.The mutation E2-G60D significantly increases Ag41855 infectivity for Ae. aegypti, both independently and in combination Flaviviridae) mediates adaptation of TBEV to BHK-21 cells via increasing virus binding to HS To our knowledge, this is the first report incriminating alphavirus position E2-60 as a major determinant of mosquito infectivity. Interestingly, a recent study demonstrated that introduction of two mutations; E2-H55Q and E2-K70E from the TR339 strain into the TE/5'2J of SINV increased infectivity for Ae. albopictus only when expressed together with the E1-226V mutation. If CHIKV has the pre-2005 E1-226A, then the substitution E2-211T has almost no effect on infectivity for Ae. albopictus compared with the E2-211I variant. This indicates that the E2-I211T mutation is responsible for specific modulation of CHIKV infectivity for Ae. albopictus. The results of CHIKV infectivity for Ae. aegypti further support this conclusion. In Ae. aegypti, the E1-A226V mutation does not increase CHIKV infectivity Ae. albopictus. Such viruses would probably not be selected in nature. This conclusion is in agreement with our phylogenetic analyses. It has been shown that the E1-A226V mutation appeared independently at least three times in strains of CHIKV transmitted by Ae. albopictusIn contrast to the E2-G60D, the mutation E2-I211T significantly increases infectivity of the Ag41855 strain for Aedes spp. mosquitoes. Based on isolation frequencies, the main sylvatic vectors of CHIKV are probably Ae. furcifer-taylori, Ae. africanus and Ae. luteocephalusAe. furcifer-taylori more important in southern and western Africa Ae. africanus more important in central regions Ae. africanus. The involvement of the particular species of non-human primates as vertebrate hosts for CHIKV in central Africa is yet to be analyzed.Phylogenetic analysis of the distribution of the E2-211I mutation revealed substantial differences compared to the E2-60G mutation. E2-211I was present in the majority of pre-2005 CHIKV isolates in the ECSA complex, suggesting that it might play an important role for maintenance of CHIKV in the enzootic African cycle involving wild non-human primates and forest-dwelling Ae. aegypti mosquitoes were the main vector in this initial epidemic We cannot exclude the possibility that the observed predominance of E2-211I among CHIKV strains within the ECSA complex arose not due to some selective advantage but rather due to a founder effect. It is possible that the ancestral progenitor of these viruses had an isoleucine at E2-211 and the absence of the selective pressure at this position led to fixation of this mutation in the viral population. It is also possible that CHIKV strains with T and I at E2-211 coexist in nature, and changes at this position occur due to some, as of yet unidentified, conditions. In this case, the apparent prevalence of E2-211I among ECSA strains isolated before 2005 could be an artifact resulting from the limited numbers of CHIKV strains available for analysis. This hypothesis is supported by recent studies of CHIKV evolution on Comoros and Reunion islands Ochlerotatus (Aedes) taeniorhynchus mosquitoes Ae. aegyptiPosition E2-211 is located within, or in close proximity to, the sites that have previously been shown to harbor genetic determinants of host specificity for several alphaviruses. Substitution E2-S218N was responsible for increased VEEV subtype IE infectivity to Ae. albopictus mosquitoes is that this substitution disrupts the ability of CHIKV to interact with a particular receptor on the midgut epithelial cells of Ae. albopictus. This receptor might be responsible for targeting of CHIKV to the specific endosomal compartments/domains with a unique lipid composition that favors fusion of CHIKV possessing valine at E1-226, as compared to alanine. If this pathway is blocked by the E2-211I mutation then CHIKV may infect Ae. albopictus mosquitoes using an alternative receptor(s) that targets virus into different endosomes, in which the presence of the E1-A226V does not result in differential infectivity. It is also tempting to suggest that, in Ae. aegypti mosquitoes, only the second  pathway is available for CHIKV infection, thereby making this species insensitive to the E1-A226V mutation. In agreement with this hypothesis is the fact that mutations at position E1-226 could be responsible for modulation of the lipid requirement for growth of CHIKV Considering the evidence for the involvement of mutations in the region around position E2-211 in receptor binding and in adaptation to new host species, we believe that the simplest explanation for the specific effects of the E2-211I mutation on CHIKV infectivity for Alternatively, mutations at E2-211 might affect the stability of the E2-E1 heterodimers, which would differentially affect fusion properties of the CHIKV with E1-226A or E1-226V. It has been shown that the mutation E2-D216G can rescue a PE2 cleavage mutant of SINV by disrupting the E2-E1 heterodimer stability under acidic condition Ae. albopictusAdditional studies are required to investigate the precise molecular mechanisms responsible for the observed, unique roles of the substitutions at position E2-60 and E2-211 of CHIKV. The widespread and increasing distribution of The plasmid encoding eGFP-expressing full-length cDNA clone derived from CHIKV LR2006 OPY1 strain LR-GFP-226V  has been previously described All plasmids were constructed and propagated using conventional cloning methods NotI and in vitro transcribed from the minimal SP6 promoter using the mMESSAGE mMACHINE kit  following the manufacturer's instructions. The yield and integrity of synthesized RNA were analyzed by agarose gel electrophoresis in the presence of 0.25 µg/ml of ethidium bromide. RNA (∼10 µg) was electroporated into 1×107 BHK-21 (baby hamster kidney) cells as previously described and cells were then transferred to 75 cm2 tissue culture flasks with 15 ml of Leibovitz L-15 (L-15) medium. Supernatants were collected at 24 and 48 h post-electroporation and stored at −80°C. Electroporation efficiency was estimated using an infectious centers assay as previously described 5 electroporated BHK-21 cells were serially 10-fold diluted and seeded in six-well plates containing 106 Vero (green monkey kidney) cells per well in MEM media. Following an incubation for 2 h at 37°C, cells were overlaid with 2 mL of 0.5% agarose containing MEM supplemented with 3.3% FBS. Cells were incubated for 2 d at 37°C until plaques developed and were stained with crystal violet.All plasmids were linearized with Ae. aegypti (white-eyed Higgs variant of the Rexville D strain) and Ae. albopictus  were reared at 27°C and 80% relative humidity under a 16 h light: 8 h dark photoperiod, as previously described http://research.utmb.edu/iacuc/) approved protocols.BHK-21 cells were maintained at 37°C in L-15 medium supplemented with 10% fetal bovine serum (FBS), 100 U penicillin, and 100 µg/ml streptomycin. 10TCID50/ml) as previously described Viral titers from tissue culture supernatants were determined by titration on Vero cells and expressed as tissue culture infectious dose 50 percent endpoint titers , frozen stocks of viruses were thawed at 37°C and four to five 10-fold serial dilutions of virus were made in L-15 medium followed by mixing the samples with an equal volume of defibrinated sheep blood. Each viral dilution was presented to 50 4–5-day post-eclosion Ae. aegypti or Ae. albopictus female mosquitoes (starved for 24 h) using a Hemotek membrane feeding system  fitted with a murine skin membrane. Mosquitoes were permitted to feed for 1 h, after which engorged mosquitoes [stage>3b Most of the studies of oral infectivity of CHIKV in stage>3b  returned10TCID50 infection units (limit of detection). The experiments were performed once or twice for each virus. OID50 values and confidence intervals were calculated using PriProbit program (version 1.63). The SAS equivalent method was used to calculate the fiducial limits , assuming normal function distribution and an “all or nothing” response parameter. The difference between two OID50 values was considered statistically significant if 95% fiducial limits did not overlap.At 7 days post-infection (dpi) mosquitoes were dissected and eGFP expression in infected midguts was analyzed by fluorescence microscopy. A mosquito was considered infected if at least one focus of eGFP-expressing cells was present in the midgut. To compare oral infectivity of non-eGFP-expressing viruses 16 to 24 mosquitoes from each viral dilution were collected on day 7 post-infection, individually triturated in 1 ml of L-15 media and titrated as described Ae. albopictus cells, concentrated using polyethylene glycol (7% W/V) and NaCl (2.3% (W/V), and RNA was extracted using TRIzol  according to the manufacturer's protocol. Overlapping PCR amplicons were amplified from viral RNA using the Titan One Tube RT-PCR system  according to the manufacturer's protocol. Primer sequences and specific PCR protocols are available from the authors upon request. Agarose gel-purified amplicons were sequenced directly using BigDye Terminator v3.1 cycle sequencing kit  designed from a genomic alignment of strains 37997, LR2006 OPY1, 15561, TSI-GSD-218, Ross, and RSU1 sequence. Sequencing was performed in an ABI PRISM model 3100 Genetic Analyzer , and sequences were edited and assembled in VectorNTI v10.3 . Newly-generated CHIKV sequences as well as those available from the GenBank library, along with that of o'nyong-nyong virus  were aligned with ClustalW in MacVector v9.0  or BioEdit v7.0.5.3 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html); gap introductions in the nucleotide alignment were refined using amino acid alignments to preserve codon homology. The phylogeny was produced using neighbor-joining and maximum parsimony methods available in the PAUP* v4.0b 10 package . Bootstrap analysis Entire E2-K6-E1 genome region of the selected CHIKV strains were sequenced or obtained from GenBank. Viruses whose genomes were not available in GenBank were obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch, Galveston, TX. Viruses were passaged in C6/36 Figure S1Schematic representation of the viruses used in this study.(1.20 MB TIF)Click here for additional data file.Table S1Recovery of the viruses with mutations in E2 protein after electroporation of in vitro transcribed RNA. a - amino acids at position of E1-226. b - amino acids at position of E2: 60, 162, 211. c - Specific infectivity of in vitro transcribed RNA. 10(7) BHK-21 cells were transfected with 10 µg of RNA. Electroporated BHK-21 cells were ten fold serially diluted, seeded in 6 well tissue culture plates containing 1×10(6) Vero cells per well in MEM media. Following an incubation for 2 h at 37°C, cells were overlaid with 2 mL of 0.5% agarose containing 3.3% FBS in MEM. Plaques were scored and measured on day 2 post transfection. d - Supernatants of electroporated BHK-21 cells were collected on days 1 and 2. Virus titers were determined by titration on Vero cells and expressed as Log10TCID50/ml. e - Plaque size of infectious centers expressed in millimeters±standard deviation. h - hours post-infection. Blue color corresponds to authentic amino acids at indicated positions of strain Ag41855, red color corresponds to authentic amino acids at indicated positions of strain LR2006 OPY1.(0.06 MB DOC)Click here for additional data file.Table S2Summary of virus strains used in phylogenetic analysis. Genotype: ECSA - Eastern/Central/South African; W. Afr - West African. Passage history: SM: suckling mouse; C6/36: Ae. albopictus cell line; Vero: African green monkey cell line; RMK: Rhesus monkey kidney cell line; MRC-5: human lung epithelium; AP61: Ae. pseudoscutellaris cell line. GenBank Acc - GenBank accession number. ? - information is unavailable to the authors(0.10 MB DOC)Click here for additional data file.


Knock-in mice (gp130F759) with a Y759F point mutation in gp130, a signal transducing receptor subunit shared by members of the IL-6 cytokine family, show sustained activation of STAT3, enhanced acute-phase or immune responses, and autoimmune arthritis. We conducted a detailed analysis of collagen-induced arthritis (CIA) in gp130F759 with a DBA/1J background (D/J.gp130F759).We backcrossed gp130F759 to C57BL/6 and DBA/1J, and compared the pathologic changes, including occurrence of arthritis, in the two distinct genetic backgrounds. We analyzed CIA in D/J.gp130F759 and investigated the effects of methotrexate (MTX) on CIA.C57BL/6 background gp130F759 mice, but not D/J.gp130F759, spontaneously developed polyarthritis and glomerulonephritis. On the other hand, keratitis of the eyes only developed in D/J.gp130F759, indicating the influence of genetic background on disease development in gp130F759 mice. Resistance of the DBA/1J background against spontaneous arthritis urged us to examine CIA in D/J.gp130F759. CIA in D/J.gp130F759 was more severe, with greater bone destruction, than the control mice. After collagen immunization, splenomegaly and serum levels of rheumatoid factor and anti-DNA antibody were augmented in D/J.gp130F759. Bio-Plex analysis of serum cytokines revealed increased IL-12p40 and PDGF-BB before immunization, and increased levels of IFN-γ, IL-17, TNF-α, IL-9, and MIP-1β 8 days after the booster dose. IL-6 and PDGF-BB in D/J.gp130F759 showed distinct kinetics from the other cytokines; higher levels were observed after arthritis development. MTX partially attenuated the development of arthritis and inhibited bone destruction in D/J.gp130F759, with reduction of anti-type II collagen antibody levels, suggesting that MTX mainly affects antigen-specific immune responses in CIA.The Tyr-759 point mutation of the IL-6 family cytokine receptor subunit, gp130, caused autoimmune disease, and this was also influenced by the genetic background. CIA in D/J.gp130F759 is useful for evaluating drugs in a relatively short period because sustained activation of STAT3 may enhance the disease symptoms. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by progressive chronic inflammation of multiple joints, resulting in joint destruction. Several characteristics of RA, such as hyper-γ-globulinemia, autoantibody production, genetic linkage with the HLA-DR locus, and infiltration of T-cells and plasma cells into the synovium, have suggested that immunologic disorders have crucial roles in the pathogenesis of this disease . RA is aIL-6 is a pleiotropic cytokine that regulates various biologic functions, such as the development of the nervous and hematopoietic systems, acute-phase responses, inflammation, and immune responses . A causaThe IL-6 receptor comprises two molecules, IL-6 receptor α-chain and gp130, which is shared among the receptors for the IL-6 cytokine family. Ligand binding of gp130 activates two major signal-transduction pathways  in a manner dependent on the YXXQ motif and tyrosine (Y) 759 of gp130, respectively ,11.in vivo, we generated a series of knock-in mouse lines in which the gp130-mediated STAT3 or SHP-2 signals were selectively disrupted. This was achieved by mutating the tyrosine residues of all the YXXQ motifs or Y759 to phenylalanine . Analyses of these mice indicated that SHP-2-mediated or Y759-dependent signals negatively regulate the biological responses elicited by the STAT3-mediated signals in vivo, and that the balance of positive and negative signals generated through gp130 is skewed or shifted to positive STAT3 signaling in gp130F759 [To clarify the roles of SHP-2- and STAT3-mediated signal-transduction pathways p130F759 . Importap130F759 . HoweverHere, we report the histopathologic abnormalities in two genetic backgrounds of gp130F759, and analyze collagen-induced arthritis (CIA) in gp130F759 with a DBA/1J background for the development of a model for drug evaluation.Experimental procedures were conducted with the approval of the Santen Animal Experimental Ethics Committee.gp130F759/F759 knock-in mice were backcrossed to C57BL/6 or DBA/1J eight times , hereafter abbreviated B6.gp130F759 and D/J.gp130F759, respectively. In all studies, we used wild-type mice (Japan SLC Inc.) as controls. Mice were housed in a specific pathogen-free animal facility at the Research and Development Center of Santen Pharmaceutical Company Limited. A 12-h light/dark cycle was maintained in a room at 23 ± 1°C. Mice had free access to food and water.Mice were sacrificed at 12 months for the comparative study of mice with two genetic backgrounds. Eyes, limbs, spleen, thymus, mesenteric lymph nodes, lungs, and kidneys were histologically-examined. The weights of the spleen and thymus were recorded. For the study of CIA, mice were sacrificed at the end of the experimental period (day 56) by exsanguination. After measuring the weights of the spleen and thymus, the organs were fixed in 10% neutral buffered formalin solution. After fixation, the joints were decalcified in 10% ethylenediaminetetraacetate solution for 3 weeks. Paraffin-embedded samples were sectioned and stained with hematoxylin and eosin. Sections were examined under light microscopy.CIA was induced in D/J.gp130F759 and wild-type mice according to the method described previously . We prevFor drug evaluations, methotrexate  at 0.2 mg/kg was administered p.o. once daily from the first immunization until day 42, and the activity of clinical arthritis was evaluated. At the end of the experimental period (42 days), the joints were processed for histologic examination. Proliferation of synovial cells, joint destruction, and lymphocyte infiltration into the synovial lining were blindly evaluated.-5 M 2-mercaptoethanol, 100 units/mL penicillin, and 100 μg/mL streptomycin). Lymph node cells (4 × 106 cells/mL) with 200 μL of complete medium were cultured in the presence or absence of 5 μg/mL of bovine type II collagen or 1 μg/mL of concanavalin-A for 2 days. Thereafter, supernatants were collected for measurement of IL-2 production.Mice were sacrificed 14 days after the first immunization. The inguinal lymph nodes were removed, and single-cell suspensions were prepared in complete medium . Mouse IL-7, rheumatoid factor (RF) for mouse immunoglobulin (Ig) of IgM classes, and anti-ssDNA antibodies were measured using a commercial ELISA kit . Levels of anti-type II collagen antibody were measured by ELISA using a mouse IgG anti-type II collagen antibody assay kit . COMP levels were measured using a commercial ELISA kit .t-test .Results were evaluated by Student's Abnormalities in gp130F759 mice with the C57BL/6 or DBA/1J background are shown in Tables We attempted to induce CIA in DBA/1J background knock-in mice to determine if the point mutation affected the prevalence or course of CIA because D/J.gp130F759 mice did not develop spontaneous arthritis up to 4 months of observation (data not shown). The severity of polyarthritis in D/J.gp130F759 mice was higher than that in wild-type mice  and platelet-derived growth factor-BB  partially attenuated it Fig. . MTX alsMany abnormalities were shown in B6.gp130F759 mice at 12 months of age. Characteristic abnormalities in C57BL/6 background knock-in mice were not only spontaneous polyarthritis, but also glomerulonephritis . Spontaneous arthritis did not develop in the DBA/1J background, indicating that the genetic background of DBA/1J is resistant to spontaneous arthritis caused by the Y759F mutation of gp130. On the other hand, keratitis was characteristic in D/J.gp130F759 mice. These different abnormalities between mice with two backgrounds may have been caused by differences in their H-2 haplotype . Thymic hyperplasia was observed in both backgrounds, but splenic hyperplasia only occurred in the C57BL/6 background. This abnormality may be an important requisite for the development of spontaneous arthritis.We tried to induce CIA in DBA/1J background knock-in mice resistant to spontaneous polyarthritis. The severity of polyarthritis in gp130F759 mice was greater in wild-type mice when the 2-week interval immunization protocol was followed, which was similar to our previous study results involving a 3-week interval immunization protocol . In the In gp130F759 mice with CIA, the serum levels of TNF-α, IL-9, IL-17, IFN-γ, and MIP-1β were elevated 8 days after the second immunization. TNF-α has an important role in RA , particuElevation of IL-17 in gp130F759 mice  may be p+ T-cells, which is due to elevated production of IL-7 by non-hematopoietic cells as a result of IL-6 family cytokine-gp130-STAT3 signaling [+CXCR3+ T-cells to the inflamed synovium [MIP-1β is a member of the CC chemokine subfamily, and has been shown to signal mainly through CCR5 . It has ignaling . The higsynovium . Bao et synovium  reportedRF and anti-DNA antibody levels were higher in gp130F759 mice, but anti-type II collagen antibody level was slightly lower. IL-2 production in lymph node cells of collagen-immunized D/J.gp130F759 mice stimulated by collagen was slightly higher than that of the control mice. These results suggest that immune reactions in gp130F759 mice and the control mice against heterologous collagen have slightly different properties. However, antigen-specific immune responses were not as highly augmented in gp130F759 from the present data. Therefore, the augmentation of arthritis in gp130F759 mice may be due to the increased response of inflammation or innate immunity. Further studies to clarify the relation between the augmentation of arthritis in gp130F759 mice and the humoral or cellular immunity need to be conducted in the future.We investigated the effects of MTX, which is widely used for RA treatment, on this model. It has been reported that MTX ameliorates T-cell-dependent autoimmune arthritis (including CIA), but not antibody- or fibroblast-induced arthritis . In CIA The point mutation of Tyr-759 of the IL-6 family cytokine receptor subunit, gp130, causes autoimmune disease, and this is also influenced by the genetic background. CIA in D/J.gp130F759 is useful for evaluating drugs in a relatively short period because sustained activation of STAT3 may enhance the disease symptoms.B6.gp130F759: C57BL/6 background gp130F759; CCR5: chemokine (C-C motif) receptor 5; CIA: collagen-induced arthritis; COMP: cartilage oligomeric matrix protein; D/J.gp130F759: DBA/1J background gp130F759; ELISA: enzyme-linked immunosorbent assay; FCA: Freund's complete adjuvant; Gab: GRB2-associated binding protein; IFN: interferon; IL: interleukin; MAPK: mitogen-activated protein kinase; MIP-1β: macrophage inflammatory protein-1 beta; MTX: methotrexate; PDGF: platelet-derived growth factor; RA: rheumatoid arthritis; RF: rheumatoid factor; SHP-2: Src homology 2-conteining tyrosine phosphatase; STAT3: signal transducer and activator of transcription 3; Th: T helper; TH17: IL-17-producing helper T cells; TNF-α: tumor necrosis factor alphaThe authors declare that they have no competing interests.FT carried out animal experiments, cell culture experiments, and cytokine and antibody detection. FT also performed the statistical analysis, and wrote the manuscript. MY and OK carried out the histologic studies. MT performed animal experiments. KI gave valuable advice to FT. HA supervised the study design. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core (OC) do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp), an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.Many pathogens are equipped with factors providing resistance against the bactericidal action of complement.  Yersinia enterocolitica can survive in human serum because of two bacterial surface proteins, YadA and Ail. These proteins have been shown to bind a complement alternative pathway regulator, factor H. Here, we show that both proteins also bind the classical and lectin pathway inhibitor, C4b-binding protein. These results together explain the serum resistance of Y. enterocolitica. The ability to evade complement attack is apparently important for the pathogenicity of Yersinia enterocolitica.To cause disease in humans, pathogenic bacteria have to evade the versatile immune system of the host. An important part of innate immunity is the complement system that is composed of over 30 proteins on host cells and in blood able to detect and destroy foreign material. To survive, bacteria can bind complement regulator proteins onto their surfaces and thus inhibit the activation of complement. Previously, it has been shown that food-borne diarrhoea-causing  Yersinia enterocolitica is a food-borne enteric pathogen of humans and animals. The invasive strains predominantly belong to serotypes O:3, O:9, O:5,27 and O:8. Successful colonization of the intestinal tract is the prerequisite for Y. enterocolitica infection. Bacteria pass through the acidic content of the stomach and reach the small intestine. Soon after invading the M cells, bacteria enter the lamina propria and encounter innate and adaptive immune responses Y. enterocolitica can cause enterocolitis, mesenteric lymphadenitis and, as a post-infectious complication, reactive arthritis Y. enterocolitica can evade the immune system killing and why it causes reactive arthritis are not fully understood.The complement system is a crucial constituent of the innate immunity. Its activation via the classical (CP), lectin (LP) or alternative pathway (AP) may lead to the killing of microbes by direct lysis or by complement opsonin-enhanced phagocytosis Streptococcus pyogenes, S. pneumoniae, Neisseria gonorrhoeae, N. meningitidis, Borrelia burgdorferi, Escherichia coli K1, Moraxella catarrhalis, Bordetella pertussis, Haemophilus influenzae and Candida albicansDistinct pathogenic microorganisms have been demonstrated to be able to bind host complement regulators, such as factor H (FH) or C4bp, to exploit their protective properties and prevent complement activation on the microbial surfaces. These pathogens include e.g. Y. enterocolitica resists efficiently complement-mediated killing Y. enterocolitica serotype O:3, the O-ag homopolymer and the OC hexasaccharide are linked to the inner core forming a branched structure. Though both O-ag and OC are needed for colonization of the gut Ail is a 17 kDa protein encoded chromosomally. It is predicted to comprise eight membrane spanning β-strands and four extracellular loops located close to the cell membrane Y. enterocolitica binds the AP inhibitor FH Y. enterocolitica serotype O:3 bacteria in fact use both YadA and Ail to bind the AP regulator FH  but no studies on possible regulation of the CP or C4bp binding to the bacterium have been reported. Since Y. enterocolitica efficiently escapes all the complement activation pathways we examined in this study whether Y. enterocolitica also interacts with the major CP and LP regulator, C4bp. We show that C4bp binding to Y. enterocolitica is also mediated by YadA and Ail. Since the proteins are located at different layers on the bacterial surface, Ail is masked by O-ag and OC while YadA is well surface-exposed. In consequence, Ail binds C4bp when not blocked by O-ag and OC, while YadA-mediated C4bp-binding occurs regardless of the LPS expression status. As an end result the Y. enterocolitica –bound C4bp retains its function thereby modulating CP and LP activation on the bacterial surface.Similar to other pathogens also Yersinia antibodies were collected from healthy human donors and stored at −70°C. Serum was heat-inactivated (HIS) by incubation for 30 min at 56°C. C4bp with protein S was purified from pooled human plasma as described previously 125I  using the Iodogen method 125I-C4bp binding assays.Human serum samples devoid of anti-The bacterial strains and plasmids used in this study are listed in Y. enterocolitica O:8 the yadA -gene of pYV8081 was cloned in a 4.4 kb XbaI-PvuI fragment  between the EcoRV and XbaI sites of pTM100 to generate plasmid pYMS3221x. The kanamycin resistance GenBlock (KmGB) removed by AccI digestion from pUC4K was cloned into the ClaI site within the yadA gene of pYMS3221x to obtain pYMS3223. The expression of YadA was abolished by the KmGB-insertion (data not shown). pYMS3223 was transformed into E. coli S17-1 and mobilized into the wild type Y. enterocolitica O:8 strain 8081 and recombinants, which had lost the vector plasmid due to double crossing-over between pYV8081 and pYMS3223, were screened for by looking for KmR ClmS strains and one such strain was named as YeO8-116. YeO8-116 did not express YadA as verified by SDS-PAGE and autoagglutination tests (data not shown). Restriction digestions and Southern hybridizations of the isolated virulence plasmid of YeO8-116 showed that it carried the KmGB and an inactivated yadA -gene. In addition, YeO8-116 was calcium dependent, and produced the Yop proteins as released proteins identical to the wild type strain (data not shown). The strain Ye08-116 has been used also in earlier studies E. coli JM109 strain. Triparental conjugation was used to mobilize the plasmids pTM100 and pTM100-ail (from JM109/pTM100 and JM109/pTM100-ail) with a help of E. coli HB101/pRK2013 to YeO3-c-Ail-OCR. The matings were performed as described elsewhere To generate a YadA-negative strain of 8) aliquot of the bacterial suspension was incubated with 50 µl of radiolabeled C4bp  for 30 minutes at 37°C. The assays using BSA (0–300 nM/assay), unlabeled C4bp (0–300 nM/assay) or factor H (0–300 nM/assay) as competitors were performed with 40 µl of bacteria (3×107) in reaction mixtures containing 125I-C4bp, or 125I-BSA as a control. The effects of heparin (0–1000 µg/ml) and NaCl (50–650 mM) on binding were assayed in reaction mixtures containing 125I-C4bp and 40 µl of bacteria (3×108). After incubation the mixtures were centrifuged through 20% (w/v) sucrose in 1/3 GVBS to pellet the bacteria with the bound radiolabeled protein. Tubes were frozen, the bottoms of the tubes were cut out and radioactivities in the pellets and supernatants were measured with a γ-counter . The ratios of bound to total activities were calculated.Bacteria grown to mid-logarithmic phase were suspended in hypotonic 1/3 GVBS. A 50 µl (3×108) were incubated in 5% heat-inactivated serum at 37°C for 30 min. Thereafter, the bacteria were washed 5–6 times with 400 µl of PBS or 1/3 PBS and bound proteins were eluted with 0.1 M glycine-HCl (pH 2.7) or PBS, respectively. Supernatants were collected and those acidified due to elution with glycine were additionally neutralized with 1 M Tris-HCl (pH 7.5). Samples of the last wash and elution fractions were subjected to a non-reducing 8% SDS-PAGE gel electrophoresis and subsequently transferred onto nitrocellulose membranes. The membranes were blocked and incubated with 1∶7,500 diluted sheep anti-human C4bp antiserum  and further with 1∶10,000 diluted peroxidase-conjugated donkey anti-sheep antiserum (Jackson Immunoresearch). The proteins were detected by enhanced chemiluminescence.Bacteria (3×109) were incubated with C4bp  in 40 µl of PBS for 30 min at 37°C with shaking. After washing for four times with PBS, bacteria (109) were pelleted and resuspended in 30 µl of PBS containing FI  and C4b . The reactions were incubated for 45 min at 37°C with shaking. A reaction where C4b and FI were incubated for 45 min at 37°C with 50 µg/ml of C4bp was used a positive control. In addition, a negative control comprising of C4b and FI incubated without C4bp, was included. After incubation the samples were centrifuged, supernatants were collected, mixed with Laemmli buffer and subjected to 12.5% SDS-PAGE and immunoblotting using rabbit anti-human C4c antiserum . The bound anti-human C4c antibodies were detected using HRP-conjugated swine anti-rabbit IgG . Supernatants were also analyzed by immunoblotting with sheep anti-human C4bp antiserum to exclude unbound C4bp.The cofactor assay was performed to analyze the effect of C4bp on FI-mediated cleavage of C4b. Bacteria (4×10E. coli JM103/pYMS4450), Tx-Ail (from E. coli JM109/pTM100-ail) and vector control extracts from E. coli strains JM103/pL2.1 and JM109/pTM100 were prepared as described previously g, 15 min) and incubated on ice for 1 h in 20 ml of lysis buffer . Triton X-114, prepared as described previously g, 10 min) to clear the phases. The Tx-114 phase was recovered and stored at 4°C.The extracts Tx-YadA (from E. coli JM109/pTM100-ail and JM109/pTM100 were prepared using OG as described elsewhere The OG-Ail and vector control extracts from 6 cpm/assay) or 5% NHS. Binding of the protein was detected by autoradiography or immunoblotting with rabbit anti-human C4bp (5 µg/ml) as described above. YadA on the membrane was detected by immunoblotting with the monoclonal antibody 3G12 Samples from Tx-YadA, Tx-Ail and OG-Ail as well as from the three control extracts (see above) were run into a 7.5–17.5% SDS-PAGE gel. Proteins were electrotransferred onto a nitrocellulose membrane. Membranes were blocked with 5% skimmed milk and incubated with the radioactively labeled C4bp (10http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the gene sequence of Ye O:8 YadA is X13881. For the protein sequences the accession numbers are CAE53849 for Ail (Ye serotype O:3) and CAA32086 for YadA (Ye 6471/76 serotype O:3).The GenBank  bound 125I-C4bp significantly less or not at all. The binding percentages by strains that did not bind C4bp (including E. coli K12 C600) ranged between 1–8% under the conditions used (not shown).In order to study whether Y. enterocolitica–bound C4bp, serotype O:3 wild type bacteria and Ail-expressing YeO3-028-OCR strain were incubated with purified C4bp as described in Experimental procedures. A clinical isolate, less virulent Ye biotype 1A strain was included as a control because the strain bound C4bp clearly less than the other two strains used. Unbound C4bp was removed by extensive washing with PBS. The bacteria with surface-bound C4bp were subsequently incubated with FI and C4b. Following the incubation, the C4bp-cofactor activity was verified by immunodetection of C4b-cleavage products in the supernatants using anti-C4c antibodies. As a polyclonal antibody the anti-C4c antibody detects the α′-chain, the β-chain and cleavage fragments of the α′-chain. As shown in Y. enterocolitica-bound C4bp displayed cofactor activity for FI-mediated cleavage of C4b with both strains YeO3 and Ye-028-OCR. This is indicated by the appearance of the C4b α′-chain cleavage fragments (Y. enterocolitica-bound C4bp corresponds to the fragment generated in the absence of bacteria by FI incubated with C4bp and C4b (To examine cofactor activity of the ragments . The molY. enterocolitica depends greatly on the expression of the pYV-encoded YadA protein Y. enterocolitica factor(s) in C4bp-binding.Serum resistance of Y. enterocolitica O:3 surface a set of 12 strains expressing YadA, Ail, LPS O-ag and OC in different combinations was tested for the ability to bind 125I-C4bp in 1/3 GVBS buffer containing 50 mM NaCl. YadA was indispensable for the maximal C4bp binding. Both pYV- and YadA-negative strains  displayed equally low levels of bound C4bp suggesting that the main factor responsible for the binding is pYV-encoded YadA  with pTM100-ail carrying the cloned ail gene. The resulting strain YeO3-c-Ail-OCR/pTM100-ail restored C4bp-binding ability, while the vector control YeO3-c-Ail-OCR/pTM100 strain was unable to bind the CP-regulator  displayed about four-fold higher C4bp binding capacity (80%) than the YeO3-c-OCR strain expressing Ail from the chromosomally-located ail gene (To identify C4bp receptors on ded YadA . Almost egulator . C4bp bidetected . In addiail gene  and 5. TNeither O-ag nor OC contributed to C4bp binding since YadA- Ail- negative strains expressing full LPS (YeO3-c-Ail) or its rough (YeO3-c-Ail-R) or OC-less (YeO3-c-Ail-OC) derivatives bound only negligible amounts of C4bp .To see a direct interaction of YadA and Ail with C4bp we tested the binding of serum C4bp to TritonX-114 extracted YadA and Ail, Tx-YadA and Tx-Ail, respectively, in affinity blotting. C4bp binding to YadA trimer was observed . Bindingtrans. Bacteria were incubated with 125I-C4bp in 1/3 GVBS alone or supplemented with NaCl to create a salt-concentration gradient ranging from 50 to 650 mM. After centrifugation through 20% sucrose bound 125I-C4bp was measured using a gamma-counter. In general, at low salt concentrations both YadA- and Ail-mediated C4bp-binding was the highest and showed a tendency to decrease with increasing salt concentrations . As shown in 125I-C4bp binding to Ail, while significant reduction of 125I-C4bp binding to YadA could only be observed at the highest heparin concentration of 1000 µg/ml. Delayed response to heparin was observed with the strain over-expressing Ail (YeO3-c-Ail-OCR/pTM100-ail).Heparin binds to the N-termini of C4bp α-chains, i.e. to CCP1-3 125I-C4bp to Ail-expressing strain Ye-028-OCR could be dose-dependently reduced to 55% by adding 0–300 nM factor H. C4bp binding to YeO8 wt strain was not affected by the addition of 0–300 nM factor H (data not shown).The relative binding of 125I-C4bp-binding was specific, wild type and YeO3-028-OCR strains were incubated with 125I-C4bp in the presence of unlabeled C4bp or BSA (0–300 nM). In this assay, ten-fold less bacteria (3×107) were used when compared to the heparin and salt inhibition experiments. Under these conditions 125I-C4bp binding to both, YadA (wild type) and Ail (YeO3-028-OCR and YeO3-c-Ail-OCR/pTM100-ail), was significantly inhibited in the presence of unlabeled C4bp . Since this strain lacks O-ag and OC, C4bp binding to Ail was strongly favored  seems to be solely responsible for the serum resistance property in Y. pestisY. pestis Ail protein shares about 70% sequence identity with Y. enterocolitica Ail. As a result of several gene mutations, however, Y. pestis does not express YadA or O-ag Y. enterocolitica strain, removing the Ail-masking O-ag and OC greatly enhances the binding of C4bp . Consequently, the bacteria can avoid complement-mediated lysis and increase their chances to survive in the human host. It is also remarkable that Y. enterocolitica uses both YadA and Ail to recruit both the AP and CP regulators FH and C4bp, respectively. This way the pathogen can ascertain that it will be protected from complement activation during different phases of infection.In summary, this study provides the first evidence that ag or OC . Y. ente


Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters.Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between  Tobacco mosaic virus (TMV) both in vivo and in vitro. When coexpressd with MP, ANK promoted intercellular transport of MP and, by implication, gated PD for its movement and facilitated the viral spread. Conversely, RNAi-based knock-down of the ANK gene expression resulted in reduced MP movement and attenuated viral infection. Coexpression of ANK with MP resulted in reduction of callose deposits at PD, which are known to function as PD channel sphincters. Interestingly, expressing ANK alone did not affect callose deposits. Thus, ANK most likely represents a cellular factor that recognizes MP and acts synergistically with it to gate PD and mediate MP transport through these channels.During infection, plant viruses utilize their cell-to-cell movement proteins (MPs) to gate plant intercellular connections, the plasmodesmata (PD), and spread between the host cells. The mechanism by which MPs facilitate their cell-to-cell translocation remains elusive. We have identified a tobacco ankyrin repeat-containing protein, ANK, that interacts with MP of  Plasmodesma (PD) is a cell-wall spanning channel that interconnects plant cells and is unique for plants. This structure is characterized by a neck-like constriction at its each end, is lined by the plasma membrane, and is traversed by a strand of appressed endoplasmic reticulum, providing endomembrane and cytoplasmic connections to adjacent cells. PD mediate direct macromolecular exchange between the connected cells, and this transport is highly regulated  MP, the archetype of many viral MPs, presumably associates with the viral genomic RNA to form a movement ribonucleocomplex PD are also utilized by plant viruses for infection. After initial inoculation and replication in the infected cell, plant viruses spread to the neighboring cells and throughout the entire plant. For this transport, viruses utilize their cell-to-cell movement proteins, MPs Cucumber mosaic virus and TMV MP may sever the actin filaments to aid their cell-to-cell transport To date, two cellular factors, actin filaments and callose have been implicated in control of transport through PD Potato virus X (PVX) in vitroTo date, several and very diverse host proteins have been shown to bind viral MPs. For example, cytoskeletal elements, calreticulin, pectin methylesterases, and DnaJ chaperones have all been shown to interact with TMV MP Nicotiana tabacum cv. Turk), which lacks the N-gene and, therefore, does not produce a hypersensitive cell death response to TMV To evaluate TMV spread within infected plant tissues, it is useful to employ a plant host that does not develop necrosis upon infection with this virus. Thus, we chose the cultivar of tobacco  and MAGNESIUM PROTOPORPHYRIN IX (MgPP), which are unrelated to ANK, but contain a short, 17-bp nucleotide sequence that is also found in the ANK sequence used for the RNAi suppression , which move between cells presumably due to membrane diffusion through the PD-spanning ER MP is known to associate with the host cell ER ANK under the control of a constitutive promoter. We then selected two of these lines, ANK1 and ANK2, which exhibited moderate levels of the ANK transcript overexpression  in the place of its coat protein. The local movement of this virus was detected by the appearance and spread of the DsRed signal. To gain insight into molecular mechanism of the ANK function in MP movement, we first examined the subcellular localizations of both proteins. When MP tagged with YFP and ANK tagged with CFP were transiently coexpressed in tobacco leaves following agroinfiltration, MP displayed the typical punctate pattern of PD localization , whereasin vivo. To this end, we utilized the bimolecular fluorescence complementation (BiFC) assay in plantaAlthough the expression pattern of ANK is different from that of MP, MP is known to possesses cytoplasmic domain Arabidopsis cytoplasmic NADH kinase  in vivo interaction between MP and ANK at PD was specific.The specificity of the MP-ANK interaction was verified in negative control experiments, for which we chose two unrelated cytoplasmic proteins similar in size to ANK (37 kDa), the in vivo and in vitro.This BiFC data were confirmed by an independent approach, using a renatured gel blot overlay assay for protein interaction Arabidopsis ANK2 is involved in ROS scavenging through its interaction with ascorbate peroxidase Another possibility is that ANK is involved in regulation of callose sphincters of PD. Callose deposits are known to affect viral movement through PD t-test , and its cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Fermentas) with the oligo-dT primer. The full-length ANK cDNA was obtained by PCR using the primer pair 5′GCGAATTCTATGTCTGAGGGAGAGAAAGTTTTGCC3′/5′TCCCCCGGGTCACAGAAACACATCTTTCTCGAGCAG3′ and the total cDNA as template.Total RNA was extracted from the ANK cDNA was inserted into the EcoRI-SmaI sites of pSAT4A-35SP-MCS-35ST ACTIN intron 11 sequence from pSK-int ANK gene specifically, we designed RNAi constructs that target the unique ANK cDNA sequences located between positions 1 and 406. These sequences of ANK were obtained from pANK by digesting it with BamHI-HindIII and BamHI-EcoRI and cloned into the BglII-HindIII and EcoRI-BamHI sites, respectively, of pRNAi. The resulting pRNAi-ANK construct carried the inverted repeat of the first 406 base pairs of the ANK cDNA, flanked by the ACTIN intron 11 sequences.For pANK, the PCR-amplified The entire expression cassettes from pRNAi-ANK and pANK were then cloned as I-SceI fragments into the binary vector pRCS2-nptII, which contains a kanamycin resistance selection marker 5′AAGAATTCATGGCTCTAGTTGTTAAAGGAAAAGTG3′/5′TGCATCCCGGGTTAAAACGAATCCGATTCGGCGACAGT3′ and pTMV004 For pTMV MP, the MP coding sequence was PCR-amplified using the primer pair ANK cDNA was digested with EcoRI and SmaI, and the resulting fragment cloned into the same sites of pSAT4-ECFP-C1 and pSAT4-nEYFP-C1 To construct pCFP-ANK and pnYFP-ANK, the PCR-amplified 5′AAGAATTCATGGCTCTAGTTGTTAAAGGAAAAGTG3′/5′GGCCGGTACCGAAACGAATCCGATTCGGCGACAGT3′ and pTMV004 as template. The amplified fragment was digested with EcoRI and KpnI, inserted into the EcoRI-XbaI sites of pSAT4A-nosP-MCS-nosT by double-ligation with the YFP and cYFP coding sequences excised with KpnI-XbaI from pSAT4-EYFP-N1 and pSAT4-cEYFP-N1 For pTMV MP-YFP and pTMV MP-cYFP, the TMV MP-encoding sequence was first PCR-amplified using the primer pair NADK3 cDNA sequence was obtained by PCR using primers 5′GCGAATTCATGGCGATTAGGAAGCTTTTGCTTCTTTTG3′/5′CATCCCGGGCTAGTACCTTGATCTGATCTGAG3′ and Arabidopsis cDNA library NADK3 cDNA was digested with EcoRI and SmaI, and the resulting fragment was cloned into the same sites of pSAT4-nEYFP-C1 For pnYFP-NADK3, Then, the expression cassette from pnYFP-NADK3 was cloned as an I-SceI fragment into pRCS2-nptII, resulting in pRCS-nYFP-NADK3.CNX and ACA2 cDNAs were amplified from the Arabidopsis cDNA library 5′GCGAATTCTATGAGACAACGGCAACTATTTTCCG3′/5′GGCCGGTACCATTATCACGTCTCGGTTGCCTTTTGC3′, and 5′GCGAATTCTATGGAGAGTTACCTAAACGAGAAT3′/5′GGCCGGTACCAACGGGAATCGTCTTCAGTCCAGCG3′. The amplified products were digested with EcoRI and KpnI, and cloned into the same sites of pSAT4A-YFP-N1.For pCNX-YFP and pACA2-YFP, the The PDCB-mCherry expression construct was kindly provided by Dr. Maule GCGAATTCTATGTCTGAGGGAGAGAAAGTTTTGCC 3′/5′GCGGCCGCTTATTTTTCAAACTGCGGATGGCTCCAGGTACCCAGAAACACATCTTTCTCGAGCAG3′ and pANK as template. The StrepII coding sequence was also included in the reverse primer of the primer pair 5′GCGAATTCATGGCGATTAGGAAGCTTTTGCTTCTTTTG3′/5′GCGGCCGCTTATTTTTCAAACTGCGGATGGCTCCAGTACCTTGATCTGATCTGAGA3′ and used to amplify the NADK3 cDNA from the pnYFP-NADK3. The amplified products were digested with EcoRI and EagI and inserted into the same sites of pET28c(+) (Novagen), producing pET28-ANK-StrepII and pET28-NADK3-StrepII, respectively.The StrepII coding sequence To produce GST-MP, the MP coding sequence was excised from pTMV MP by digesting it with EcoRI and SmaI inserted into the same sites of pGEX-5X-1 , producing pGEX-TMV MP.Pfu Turbo DNA polymerase (Stratagene), and their products were verified by DNA sequencing using an Applied Biosystems 3730 Genetic Analyzer.All PCR reactions were performed using a high-fidelity proofreading Agrobacterium strain EHA105, which was then used to transform N. tabacum cv. Turk as described ANK in the ANK and RNAi ANK lines, respectively, was monitored by reverse transcription (RT) followed by quantitative (q) PCR. Control, wild-type tobacco plants were grown as described pRCS-RNAi-ANK and pRCS-ANK were introduced into the disarmed ANK (5′AGGCTGCACTAACTGCTGGT3′/5′TTACAGCGGCTCCATTCTCT3′), MP (5′AAAGATTTCAGTTCAAGGTCGTTCC 3′/5′TCCGTCTCTCACGTTTGTAATCTTC3′), ACTIN (5′TCACTGAAGCACCTCTTAACC3′/5′CAGCTTCCATTCCAATCATTG3′), and TUBLIN(5′TACACAGGGGAAGGAATGG/CTCGAAACCAACGCTTATC3′).Total RNA was extracted from tissue samples using Tri-Reagent (Ambion), treated with RNase-free DNase I (Fermentas), and 0.5-µg samples of the resulting preparations were reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Fermentas), according to the manufacturer's instructions. RT reaction products were amplified by qPCR in a 7300 Real-Time PCR System (Applied Biosystems) with Maxima™ SYBR Green qPCR Master Mix (Fermentas) using primer pairs specific for ANK or MP mRNA-specific products was normalized to the amount of the product specific for ACTIN and TUBLIN, respectively, which represented an internal control of a constitutively expressed gene. The absence of potential residual genomic DNA contamination was confirmed by control qPCR reactions performed without RT.Relative abundance of the Agrobacterium EHA105 strain as described N. tabacum leaves as described For agroinfiltration, binary vectors were introduced into the t-test. Differences between sets of measurements with p-values less than 0.001, corresponding to the statistical probability of greater than 99.9%, were considered statistically significant.Leaves with the length of 18 cm, excluding petiole, from 4–5 week-old plants were selected for all experiments. Constructs expressing YFP-tagged tested proteins, i.e., pTMV MP-YFP, pCNX-YFP and pACA2-YFP, were microbombarded into the lower epidermis at the equivalent locations on each leaf. When movement of two proteins was compared, their expression constructs were introduced at the symmetrical locations relatively to the mid-rib of the leaf. At least 120 YFP-expressing clusters in each microbombarded tissue were observed under a Zeiss LSM 5 Pascal confocal microscope. After 24 h, all expression clusters were represented by single cells and considered to indicate the absence of movement. After 48 h, the number of fluorescent cells in each expression cluster varied due to cell-to-cell movement. At this time, the number of cells in each expression cluster was recorded, followed by statistical evaluation of resulting data by the unpaired two-tailed Student's t-test. Differences between sets of measurements with P-values <0.001, corresponding to the statistical probability of greater than 99.9%, were considered statistically significant.pTRBO-DsRed  was agroinfiltrated into 18-cm, excluding petiole, leaves from 4–5 week-old plants. Four to 14 days after inoculation, at least 40 DsRed-expressing clusters in each infiltrated tissue were analyzed by confocal microscopy, followed by statistical evaluation of the DsRed-expressing surface area measurements by the unpaired two-tailed Student's 2, 5 mM MES, pH 5.5). The protoplasts were collected by centrifugation at 600 x g for 5 min, resuspended in the MMC buffer  at 2×107 cells/ml. For transformation with the viral infectious clone, 5 ml of 30% PEG 8000 in the MMC buffer and 4 µg of the pTRBO-DsRed DNA were added to 2 ml of protoplast suspension and mixed gently. After 15-min incubation at room temperature in the dark, 40 ml of the culture medium  was added to the protoplast suspension. The incubation was continued for an additional 45 min, after which the protoplasts were collected by centrifugation, resuspended in 35 ml of the culture medium, and incubated further to allow replication of the virus. Protoplast samples (5 ml) were harvested 16, 28, 40, 52, and 64 h after transformation, and their total RNA was extracted by the TRIzol (Invitrogen) method according to the manufacturer's instructions.Protoplasts were prepared from the 3–8-cm, excluding petiole, leaves from 4–5 week old plants. The leaves were sterilized in 50% bleach with 0.1% SDS for 5 min, followed by 5 rinses in distilled water and overnight incubation at room temperature in the protoplasting enzyme mixture (40 mg/ml cellulase Onozuka R-10 (Phytotechnology Laboratories) and 1.5 mg/ml macerozyme  in 500 mM D-sorbitol, 1 mM CaCl600 = 0.0015. For negative controls, bacterial cultures at OD600 of up to 0.6 were used to confirm the absence of reconstructed YFP signal even at very high inocula.For subcellular localization, pTMV MP-YFP and pCFP-ANK were expressed in tobacco leaves following microbombardment, and the subcellular fluorescence pattern was analyzed by confocal microscopy. For BiFC, pRCS-nYFP-ANK and pRCS-TMV MP-cYFP, or pRCS-nYFP-ANK and pRCS-nYFP-NADK3 binary constructs were expressed in tobacco leaves following agroinfiltration, and BiFC was detected by confocal microscopy as described Escherichia coli strain (Novagen) from the pGEX-TMV MP, pGEX-5X-1, pET28-ANK-StrepII and pET28-NADK3-StrepII vectors. The identity of these proteins was confirmed by western blot analysis using anti-GST (Santa Cruz) and anti-StrepII (Genscript) polyclonal antibodies, and the amounts of these proteins in the total bacterial extracts were estimated by scanning densitometry of the corresponding bands on SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R-250, using the known amounts of BSA as standard. Protein extracts containing 1 µg of GST-MP or unfused GST were resolved on 15% SDS-polyacrylamide gels, followed by eletrotransfer to a nitrocellulose membrane. The membrane-immobilized proteins were incubated with 0.5 µg/ml of ANK-StrepII or NADK3-StrepII and processed as described Recombinant GST-MP, unfused GST, ANK-StrepII, and NADK3-StrepII were produced as described FAST 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich). DAB was vacuum-infiltrated into the tissues for 20 min, followed by incubation of the leaf disks in the DAB staining solution for 4 h in the light and 16 h in the dark. After four brief washes in double-distilled water, the stained leaf disks were transferred to 95% ethanol and heated at 55°C to remove chlorophyll. The tissue was rehydrated, and imaged using an EPSON Perfection 4490 photo scanner.Three days after agroinfiltration with binary constructs expressing the tested proteins, i.e., pRCS-ANK and/or pRCS-TMV MP, leaf disks from the agroinfiltrated areas were excised and transferred to the 10 mM MES (pH 5.6) containing SIGMA2, pH 6.9) followed by overnight incubation at 25°C with gentle agitation. The samples were then washed twice in double-distilled water and depleted of chlorophyll by incubation for 2 h at 25°C with gentle agitation in a graded series of ethanol . For permeabilization, the dehydrated samples were air-dried, transferred to the PIPES buffer, and freeze-shattered as described t-test as described for analyses of the cell-to-cell movement data.Three days after agroinfiltration with pRCS-ANK and/or pRCS-TMV MP, tissue samples (<2×5 mm) from the agroinfiltrated areas were fixed by vacuum infiltration with 4% paraformaldehyde in the PIPES buffer  and MAGNESIUM PROTOPORPHYRIN IX (MgPP) genes in wild-type, RNAi ANK1 and RNAi ANK2 plants. AThe shown values were normalized to the amounts of ACTIN transcript in the same samples. BWT, wild-type. CStandard deviations are indicated. DP-values for the sets of data obtained from the WT and RNAi ANK1 or RNAi ANK2 plants were calculated using the Student t-test. Note that they show no statistically significant differences between expression levels of the tested genes between the RNAi transgenic lines and the wild-type plants.Relative expression levels of (0.03 MB DOC)Click here for additional data file.Figure S1N. tabacum genes, aspartate aminotransferase (AB126259.1) and magnesium protoporphyrin IX (AF014052.1). The sequences for primers used for qRT-PCR are also indicated. As these primers are designed to detect the sequence specifically conserved in the ANK family, but not found in other ankyrin repeat-containing proteins, qRT-PCR using this primer set allows specific quantification of all close homologs of ANK.ANK cDNA sequence. The nucleotide sequence encoding the ankyrin repeat-containing domain (positions 670–1032) is shadowed, and the sequence used for the pRNAi-ANK construct is underlined. The segment used for pRNAi-ANK is unique to ANK, except for the 17-bp region highlighted in black (positions 19–45), which is found in two (1.10 MB TIF)Click here for additional data file.Figure S2ANK expression. (A) Quantification of the levels of ANK transcripts with indicated standard deviations. Plants with ANK. gene expression levels reduced to 5% and 15–40% of the wild-type expression level were designated severe and moderate suppressors, respectively. Plants were analyzed four weeks after their transfer to growth chamber from tissue culture. (B) Chlorotic leaf phenotypes in RNAi ANK3, but not in RNAi ANK1 or wild-type (WT) plants.RNAi transgenic plants with severe and moderate suppression of (1.21 MB TIF)Click here for additional data file.Figure S3ANK expression levels do not affect MP-YFP targeting to PD or protein expression. (A–F) PD localization of MP-YFP at 16 h (A–C) and 24 h (D–F) after bombardment in wild type plants , RNAi ANK2 , and ANK1 . Bars  = 20 µm. Plastid autofluorescence is in white. All images are single confocal sections. (G) MP-YFP expression levels in different plant lines. Lane1, wild type; lane 2, ANK1; lane 3, ANK2; lane 4, RNAi ANK1; lane 5, RNAi ANK2. Arrow indicates the position of MP-YFP.Altered (4.60 MB TIF)Click here for additional data file.Figure S4ANK-StrepII in extracts prepared from the independent transgenic lines ANK-StrepII 1 to 6. (B) qRT-PCR analysis of the ANK-StrepII mRNA levels in the same transgenic lines. The shown values were normalized to the amounts of ACTIN transcript in the same samples.Positive correlation between ANK-StrepII transcripts and protein levels. (A) Western blot analysis of (1.53 MB TIF)Click here for additional data file.Figure S5Histochemical detection of ROS. The leaf discs were excised from the indicated plant lines and treated with DAB. No significant difference in ROS levels were observed between plant lines with different expression levels of ANK. WT; wild type. RNAi1 and 2; RNAi ANK1 and RNAi ANK2, respectively.(2.62 MB TIF)Click here for additional data file.File S1Supplemental Methods(0.02 MB DOC)Click here for additional data file.


Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease. We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694 -N -methylamino]-2 - thenoyl)-L-glutamic acid), a drug currently in phase III clinical trial. The degree of resistance was > 20,000 for the human lymphoblastoid cell line W1L2:R and approximately 14 for the ovarian carcinoma cell line CH1:R. In both cases resistance was associated with increased TS activity. The W1L2:R cell line had an approximately 100-fold increase in TS gene copy number and mRNA levels and a 500- to 1000-fold increase in enzyme levels determined using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern and Western blotting. The CH1:R cell line had an approximately 2- to 2.5-fold increase in TS gene copy number, mRNA and protein levels. In both cell lines the fold resistance determined was significantly higher than the fold increase in target enzyme DNA, mRNA or protein levels. Small changes in TS levels may therefore translate to clinically significant alterations in drug sensitivity.


Sir,Myelosuppression is frequently observed during treatment with the multi tyrosine kinase inhibitors (TKIs)  in these patients. As a consequence, a larger decrease in thrombocytes is necessary to develop thrombocytopenia.In our study, only 29% of the patients developed thrombocytopenia in the first treatment cycle as compared with 46% of the patients who developed leukopenia scored according to the criteria of Common Toxicity Criteria for Adverse Events version 3.0. An explanation for this difference might be the high baseline thrombocyte counts (mean: 319 × 10n=59) vs C-allele carriers (CT/CC) (n=134) since a protective effect was observed in C-allele carriers in developing leukopenia in our earlier analysis (vs carriers of the flt-3 738 CT/CC genotype were 0.54 vs 0.65 (P=0.024)  were univariately tested for an association with the flt-3 polymorphism 738T/C. The flt-3 genotype was divided into two groups wild type (TT) (P=0.024) .We conclude that the flt-3 738C allele has a protective effect against sunitinib-induced thrombocytopenia. Combined with our earlier finding with regard to leukopenia it seems that the flt-3 738C>T polymorphism has a role in the variability of sunitinib-induced bone marrow toxicities.


P = 0.012) was found to be significantly high in Kangri cancer cases compared to control (0.736) with a Relative Risk of 2.45 fold. Conversely, the allelic frequency of Hsp70-2 A/A allele in homozygous condition was significantly low in Kangri cancer cases and worked out to be 0.084 (Vs 0.252 in control) with P is equal to 0.001, implicating it as a protective allele against Kangri cancer in subjects with this genotype. Similarly, significantly high frequency of 0.50 (Vs 0.29 in control) of Hsp70-homC/C allele was found in homozygous condition in Kangri cancer cases suggestive of a positive relative risk associated with this genotype  . The overall allele frequency data analysis of Hsp70-2 and Hsp70-hom genes was significant . The study also reveals considerable association of high frequency alleles of HSP70 genes, especially of Hsp70-2 A/G or G/G in Kangri tumors with clinico-pathological features of poor prognosis. These results indicate that the relative risk of Kangri cancer associated with Hsp70-2 and Hsp70- hom gene polymorphisms is confined to Hsp70-2 A/G or G/G and Hsp70homC/C haplotype in our population. The study, therefore, suggests Hsp70-2 A/G or G/G and Hsp70homC/C genotypes as potential susceptibility markers and independent prognostic indicators in Kangri carcinoma patients in Kashmiri population.Kangri cancer is a unique thermally-induced squamous cell carcinoma (SCC) of skin that develops due to persistent use of Kangri (a brazier), used by Kashmiri people, to combat the chilling cold during winter months. We designed a large scale case-control study to characterize the frequency of two polymorphisms within the MHC class III-linked HSP70genes, Hsp70-2 and Hsp70-hom, in order to find any association of these genotypic variants for predisposition to and clinical outcome of Kangri cancer patients from Kashmir valley in North India. Polymerase Chain Reaction and restriction enzymes were utilized to characterize the frequency of two polymorphisms with in Hsp70-2 and Hsp70-hom genes in 118 Kangri carcinoma cases and 95 healthy controls from the same population of Kashmir. Association of high frequency allelic variants of Hsp70genes with various clinicopathological features of prognostic significance was assessed by Chi-square test using SPSS software. In this study, allelic frequency of Hsp70-2 A/G heterozygote (0.87) ( TP53 (wild type TP53 act as repressor of HSP70gene)  of the Hsp70-2gene and a polymorphic Nco1 site at position 2437(C greater than G)  of the Hsp70-hom gene. The position 1267 of the Hsp70-2gene lies in the coding region, but corresponds to a silent mutation. The polymorphic nucleotide 2437 of the Hsp70-hom gene corresponds to a Met greater than Thr aminoacid substitution. The coding sequence of this Hsp70-2 and Hsp70-hom genes were amplified from g-DNA using sequence specific oligo-nucleotide primers. For Hsp70-2, the 5' primer: 5' ACC CTG GAG CCC GTG GAG AA was used in combination with the 3' primer: 5' CAC CCG CCC GCC CCG TAG G. For Hsp70-hom, the 5' primer: GGA CAA GTC TGA GAA GGT ACA G-3' was used in combination with the 3' primer: 5' GTA ACT TAG ATT CAG GTC TGG 3'. The PCR mixture contained to a final concentration of 500 nano-grams of g-DNA, 200 μM of dNTP's, 1.5 mM MgCl1 Vs N0) and histopathological Grade [Grade I Vs (Grade II)].[P less than/equalto 0.05.All statistical analysis was performed using S-PLUS soft-ware. Chi-square test was used to test for a significant association between Kangri cancer and Hsp70-2 and Hsp70-hom genotypes . Relative risk associated with a particular genotype was estimated by the odds ratio formula. Chi-squaade II)]. The leveP less than 0.05). An increase in frequency of Hsp70-2 A/G heterozygote was observed in Kangri cancer cases (0.87) compared to control group (0.736).The allelic frequency of Hsp70-2 A/G heterozygote was significantly more with a relative risk 2.45 fold in Kangri cancer cases (P less than 0.05) compared to control. Conversely the allelic frequency of Hsp70-2 G/G allele was 0.042 in Kangri cancer cases (Vs 0.010 in control) with RR is equal to 4.15. The overall frequency of Hsp70-2 G/G allele in homozygous or heterozygous condition was 0.477 in Kangri cancer cases (Vs 0.378 in control). These results indicate that the RR of Kangri cancer associated with Hsp70-2 polymorphism is confined to Hsp70-2 G allele in homozygous or heterozygous state while as Hsp70-2 A allele in homozygous condition is rather a protective allele for Kangri cancer in our population.The genotype frequencies of Hsp70-2 in patients with Kangri cancer and the control group from the population of Kashmir is shown in P ≤ 0.05). Conversely, the significantly low frequency of Hsp-homGG genotype in homozygous (0.016) or heterozygous condition (0.474) in Kangri cancer cases compared to control (0.073) and 0.631 respectively) suggest it rather a protective allele for Kangri cancer with negative relative risk .These results indicate the relative risk of Kangri cancer associated with Hsp70-hom polymorphism is confined to Hsp70hom CC genotype. The overall Chi-square test used for comparative evaluation of Hsp70-2 and Hsp70-hom gene polymorphic analysis reveals that the data is quite significant .The allelic frequency of Hsp70-hom genotypes in Kangri cancer patients and control group given in P ≤ 0.05) and histopathological Grade II (P ≤ 0.05), features that reflect poor prognosis [P ≤ 0.05) and histopathological grade I (P ≤ 0.05) [The presence of high frequency allelic variants of HSP70's (Hsp70-2 A/G or G/G and Hsp70-hom CC in our population) when compared with various clinico-pathological attributes of Kangri cancer patients showed statistically significant association of Hsp70-2 A/G or G/G genotype with advanced Clinical Tumor Stage [T2 ] (rognosis . However ≤ 0.05) , which sSeveral studies have shown statistical evidence of association between specific human leukocyte antigen (HLA) alleles and risk for or protection against various cancers.17 In theP ≤ 0.05). Conversely, a high relative risk was found in Kangri cancer cases that carry Hsp70-2 G allele in heterozygous or homozygous state [P = 0.0001[P is = 0.0001).[P = 0.0001),[P = 0.006) and in diseases other than cancer.[Comparison of Hsp70-2 allele frequencies in patients with Kangri cancer and control subjects from same population of Kashmir indicated a significant decrease of Hsp70-2 A/A allele in Kangri cancer cases, suggesting it rather a protective allele in homozygous state (us state . Our fin = 0.0001 and 0.28 0.0001). The Hsp7 0.0001), nasopharn cancer.‐22P ≤ 0.05) and the risk decreases significantly as the allelic frequency of Hsp70-homG allele increases in homozygous or heterozygous state .This is contrary to what has been reported from Tunisia where allelic frequency of Hsp70-hom G allele in homozygous or heterozygous is more in breast cancer cases (0.13) compared to controls (0.05) .[Similarly, a striking difference in the frequency of Hsp70-hom genotypes was found in Kangri cancer patients of Kashmir when compared with controls . Hsp70-h = 0.01). More intComparison between the Hsp70-2 and Hsp70-hom haplotype frequencies in patients and in control subjects indicate almost double frequency of Kangri cancer cases (48/114) (0.42) harbour together Hsp70-2 A/G or GG and Hsp70-hom C/C haplotype when compared with controls (18/90) (0.20). These results suggest that Hsp70-2A/G or G/G and Hsp70-homC/C haplotype may represent a specific risk factor for Kangri cancer to our population of Kashmir.The polymorphic Pst1 site in Hsp70-2, although a synonymous variation, has functional significance as per recent reports which show the influence of synonymous gene variation on the expression levels and enzyme activity, possibly, by affecting the secondary structure of mRNA, its stability and the timing of co-translational folding that alters the substrate or inhibitor binding sites.24 NcoI HTP53 mutations in 32% of the sporadic Kangri cancer cases, therefore, an increase in expression of HSP genes is very likely, which further facilitates the various steps involved in tumor development.The high frequency allelic variants of Hsp70s' in Kangri cancer, like Hsp70-homC/C and Hsp70-2 A/G or G/G haplotypes in our population, might express well, possibly by increased stability of variant mRNA or by altering the timing of co-translational folding that produce protein with altered enzymatic activity, so as to facilitate anti-apoptosis and delay senescence, and hence support tumor development. In our previous study, we reported rd-4th class  that suggests low assurance of reproducibility.[The presence of high frequency genotypes of HSP70genes when compared with various clinical parameters of prognostic significance substantiates the role of Hsp70-2A/G or G/G genotype as risk imposing as evident from its presence in Kangri cancer cases bearing poor prognostic features. However, according to Manley's (2005) BADGE (Better Association for Disease and Gene) classification, the Hsp70-2gene-disease association is of 3cibility. In summa


The 1.8-kb mRNA was reported as one of the oncogenesis-related genes of Marek's disease virus (MDV). In this study, the bacterial artificial chromosome (BAC) clone of a MDV field strain GX0101 was used as the platform to generate mutant MDV to examine the functional roles of 1.8-kb mRNA.Based on the BAC clone of GX0101, the 1.8-kb mRNA deletion mutant GX0101Δ(A+C) was constructed. The present experiments indicated that GX0101Δ(A+C) retained a low level of oncogenicity, and it showed a decreased replication capacity in vitro and in vivo when compared with its parent virus, GX0101. Further studies in vitro demonstrated that deletion of 1.8-kb mRNA significantly decreased the transcriptional activity of the bi-directional promoter between 1.8-kb mRNA and pp38 genes of MDV.These results suggested that the 1.8-kb mRNA did not directly influence the oncogenesis but related to the replication ability of MDV. Marek's disease (MD) is a contagious lymphoproliferative disease of poultry caused by the highly oncogenic alphaherpesvirus, MDV, which is characteristic by mononuclear infiltration of peripheral nerves, irises, skin and other visceral tissues ,2. AmongThe 1.8-kb mRNA is unique to MDV and it has no homology with other groups of herpesviruses, and it received attention as a pathogenic determinant following demonstration of the expansion of the 132-bp tandem repeats in the 1.8-kb mRNA region during attenuation of MDV. However, deletion of the two copies of the 132-bp repeat region in a pathogenic MDV demonstrated that the virus was still pathogenic . The traRecent progresses in BAC cloning and mutagenesis technology make it possible to identify specific genes important for MDV replication and oncogenesis. In earlier studies we cloned the full length genome of a virulent MDV strain, GX0101, into a bacterial artificial chromosome (BAC) and reconstituted the infectious virus, bac-GX0101. Studies in specific-pathogen-free (SPF) chickens showed that the virulence of bac-GX0101 was higher than virulent MDV (vMDV) GA strain but lower than very virulent MDV (vvMDV) strain Md5, and there was no difference in growth ability and pathogenicity to birds when compared with its parental virus, GX0101 -16. In tThe deletion of the ORF(A+C) was confirmed by PCR with purified GX0101Δ1(A+C)-BAC and GX0101Δ(A+C)-BAC as templates . As showTo determine whether the deletion of the 1.8-kb mRNA had any effect on GX0101Δ(A+C) growth replication in vitro, the growth rate of GX0101Δ(A+C) virus was compared with that of GX0101. As shown in Figure The viremia levels in 10 birds from each group were determined on days 7, 14, 21 and 28 p.i. As indicated in Table To determinate the activity of the bi-directional promoter, plasmids pP(pp38)-CAT and pP(1.8-kb)-CAT were used to transfect CEF monolayers. The results showed that the CAT expression level in uninfected CEF was very low, nearly 0. The CAT activity in GX0101-CEF was higher than that in GX0101Δ(A+C)-CEF (P < 0.05) Table . The resTo compare the pathogenicity of mutant virus with its parental virus, we examined the growth rates of infected birds. Both viruses strongly inhibited the growth rates of infected birds. As shown in Table During 120 days after challenged with the two viruses, 50% and 40% mortality were observed in groups inoculated with GX0101 or GX0101Δ(A+C), respectively. Furthermore, 22.5% and 12.5% of birds exhibited visceral tumors conformed by histopathologic changes in different tissues  in groups infected with GX0101 or GX0101Δ(A+C), respectively , we found that the replication ability of GX0101Δ(A+C) was decreased compared to that of GX0101 in vitro and in vivo. MDV replication origin locates between pp38 and 1.8-kb mRNA in the long inverted repeat region of the viral genome, therefore, we speculate that 1.8-kb mRNA might affect the MDV replication origin as its effect to the bi-directional promoter. However, further studies are required to confirm this hypothesis and to understand the mechanism of 1.8-kb mRNA.Although GX0101Δ(A+C) was severely impaired for in vivo replication, the virus retained a low level of oncogenicity and thus demonstrated that 1.8-kb mRNA was dispensable for tumor induction. Meanwhile, HI antibody titers to AIV-H9, AIV-H5 and NDV in birds infected with GX0101 were lower than those infected with GX0101Δ(A+C) (P < 0.05). The differences in tumor induction or immunosuppression effects between GX0101Δ(A+C) and GX0101 may be due to the poor replication ability in vivo of GX0101Δ(A+C).These results suggested that the 1.8-kb mRNA gene family did not directly influence the oncogenesis of MDV but related to its replication ability. And our future studies will concentrate on the identification of the 1.8-kb mRNA product and its mechanism.A field virulent MDV strain, named GX0101, was isolated from a layer farm in Guangxi province in China . In our The recombinant plasmids expressing chloramphenicol acetyltransferase (CAT) gene under the control of the bi-directional promoter were constructed in our laboratory . In the R gene was carried out by a procedure of homologous recombination [Escherichia coli EL250 (harboring the GX0101-BAC) grown at 30°C in Luria-Bertani (LB) medium containing chloramphenicol (25 mg/ml) to an optical density (OD600) of 0.6. The expression of recE, recT, and λ gam was induced by 42°C for 15 min. KanR cassette flanked by FRT sites was amplified using primers 1.8-kb mRNA (A+C)-kanR-F and 1.8-kb mRNA(A+C)-kanR-R  copy, the entire procedure was repeated. Once both copies were deleted, an recombinant virus, named GX0101Δ(A+C), was reconstituted by transfecting CEF cultures with purified BAC DNA. And the recombinant virus, in which one copy ORF(A+C) was deleted, named as GX0101Δ1(A+C).Replacement of the ORF (A+C) with the Kanbination . Electroensity OD00 of 0.6The deletion of the ORF(A+C) from the GX0101 BAC DNA were analyzed by PCR using primers, which cross the 1.8-kb mRNA ORF(A+C), as follows: forward primer, 5'-GGCTAGCATTCGATAAGC-3'; reverse primer, 5'-GGAGGTGTAATATAAGG G-3'. GX0101Δ(A+C) was reconstituted by transfecting CEF cultures with purified BAC DNA as previously described . When CEAfter transfection of the mutant viruses into CEF cultures, we compared the plaques of GX0101Δ(A+C) with those of GX0101 by immunofluorescence analysis (IFA) with monoclonal antibody H19 at 96 h after infected in fresh CEF cells as previously described [6 CEFs and incubated at 37°C, 5% CO2, respectively. At hours 0, 24, 48, 72, 96, 120 and 144 p.i., the infected cells were trypsinized and serial 10-fold dilutions were added onto six-well plates of CEFs, visible viral plaques were counted on days 5 p.i. by IFA.The rates of growth in vitro of GX0101Δ(A+C) were measured as follows, briefly, 100 PFU GX0101 and GX0101Δ(A+C) (from stock virus in liquid nitrogen) were inoculated onto six-well plates seeded with 2×106cells/ml) collected after centrifugation were used to inoculate two duplicate 35-mm plates with CEF monolayer. To determine viremia levels, visible viral plaques were counted on days 5 p.i. by IFA.In vivo replication of GX0101Δ(A+C) were measured as follows. In brief, blood samples in anticoagulants were collected from 10 birds of each group on days 7, 14, 21 and 28 p.i., and 1 ml blood from each bird was mixed with 9 ml DMEM. Blood suspensions were centrifuged for 5 min at 500 g to separate white blood cells. And 500 μl white blood cells  infected CEF, plasmids pP(pp38)-CAT and pP(1.8-kb)-CAT were used to transfect GX0101-CEF or GX0101Δ(A+C)-CEF, respectively. Construction of recombinant plasmids expressing CAT gene under the control of the bi-directional promoter was performed as previously described . Brieflyncubator . The expOne-day-old male SPF chickens were randomly divided into three groups and kept in three isolators under positive filtered air. In the experiment, 40 birds were inoculated intra-abdominally with 1000 PFU of GX0101 or GX0101Δ(A+C). A control group of 13 birds was inoculated with uninfected CEF. During 120 days after challenge with the two viruses, all chickens were examined for gross MD lesions. Body weight measurements of the birds in different groups were made on weeks 3, 4, 5, 6 and 8 p.i., in order to evaluate the effect of the two viruses on chicken growth rates.In order to evaluate the immunosuppressive effects of the two viruses, on one-day-old, chickens were inoculated intra-abdominally with 1,000 PFU of GX0101 or GX0101Δ(A+C), while control chickens were inoculated with uninfected CEF cultures. On days 9 p.i., all chickens from each treatment were vaccinated with inactive Newcastle disease virus (NDV), H5 avian influenza viruses (AIV) and H9 AIV. On days 28 p.i., the birds' serums were collected to measure the HI antibody titers to NDV, AIV-H5 and AIV-H9.The authors declare that they have no competing interests.AJS and YPL carried out most of the experiments and wrote the manuscript. JBD and ZZC carried out study design, and revised the manuscript. JYW, SS and HJC helped in vivo experiments, participated data organization and statistical analysis. HFZ helped in revision of the manuscript. All authors read and approved the final manuscript.