MH_dev_6

Role of chronic inhibition of dopamine-metabolizing enzymes in the regulation of renal sodium and phosphate excretion in the rat remnant kidney . BACKGROUND / AIMS : The present study examined the effects of chronic selective or combined inhibition of type A monoamine oxidase ( MAO ) and catechol-O-methyltransferase ( P21964 REA ) on daily urinary excretion of dopamine and metabolites and on natriuresis and phosphaturia in 3/4 nephrectomized ( 3/4 nx ) and Sham rats . METHODS : The 3/4 nx and Sham rats were placed in metabolic cages and received the P21397 REA - selective inhibitor Ro - 411049 ( 7.5 mg x kg ( - 1 ) bid ) and / or the P21964 REA - selective inhibitor DB03336 MEN 3-202 ( 30 mg x kg ( - 1 ) bid ) orally for 3 days during high sodium diet . RESULTS : Selective P21964 REA inhibition increased the urinary excretion of the deaminated metabolite ( 3,4- dihydroxyphenylacetic acid , DOPAC ) and decreased the urinary excretion of the methylated ( 3 - methoxytyramine , 3 - MT ) and deaminated plus methylated metabolite ( homovanillic acid , HVA ) in both groups . Selective P21397 REA inhibition increased the urinary excretion of 3 - MT and reduced the urinary excretion of both DOPAC and HVA in either 3/4 nx or Sham rats . Combined inhibition of P21397 REA and P21964 REA did not significantly change the urinary excretion of DOPAC and markedly decreased the urinary excretion of 3 - MT and HVA in both groups . Selective or combined inhibition of P21397 REA and P21964 REA did not alter the daily urinary excretion of dopamine , sodium or phosphate in either 3/4 nx or Sham rats . CONCLUSIONS : Chronic selective or combined inhibition of P21397 REA and P21964 REA is not of major importance in regulating the dopamine-dependent natriuresis and phosphaturia in either 3/4 nx or Sham rats .

Cross-talk between PKA-Cβ and p65 mediates synergistic induction of Q07343 REA by roflumilast and NTHi . Phosphodiesterase 4B ( Q07343 REA ) plays a key role in regulating inflammation . DB01656 MENMAX DB01656 MEN , a phosphodiesterase ( PDE ) 4 - selective inhibitor , has recently been approved for treating severe chronic obstructive pulmonary disease ( P48444 REA ) patients with exacerbation . However , there is also clinical evidence suggesting the development of tachyphylaxis or tolerance on repeated dosing of roflumilast and the possible contribution of Q07343 REA up-regulation , which could be counterproductive for suppressing inflammation . Thus , understanding how Q07343 REA is up-regulated in the context of the complex pathogenesis and medications of P48444 REA may help improve the efficacy and possibly ameliorate the tolerance of roflumilast . Here we show that roflumilast synergizes with nontypeable Haemophilus influenzae ( NTHi ) , a major bacterial cause of P48444 REA exacerbation , to up-regulate PDE 4B2 expression in human airway epithelial cells in vitro and in vivo . Up-regulated PDE 4B2 contributes to the induction of certain important chemokines in both enzymatic activity-dependent and activity-independent manners . We also found that protein kinase A catalytic subunit β ( PKA-Cβ ) and nuclear factor-κB ( NF-κB ) p65 subunit were required for the synergistic induction of PDE 4B2 . PKA-Cβ phosphorylates p65 in a DB02527 - dependent manner . Moreover , Ser 276 of p65 is critical for mediating the PKA-Cβ-induced p65 phosphorylation and the synergistic induction of PDE 4B2 . Collectively , our data unveil a previously unidentified mechanism underlying synergistic up-regulation of PDE 4B2 via a cross-talk between PKA-Cβ and p65 and may help develop new therapeutic strategies to improve the efficacy of DB05876 inhibitor .

Effects of endothelin P25101 REA receptor antagonism on granulocyte and lymphocyte accumulation in LPS-induced inflammation . Endothelin peptides play active roles in different aspects of inflammation . This study investigates the contribution of endogenous endothelins to lipopolysaccharide ( LPS ) pulmonary inflammation by assessing the influence of ET ( A ) receptor antagonism on leukocyte accumulation , granulocyte adhesion molecule expression , and chemokine / cytokine modulation . Local pretreatment with BQ - 123 or A - 127722 ( 150 pmol ) , two selective and chemically unrelated endothelin ET ( A ) receptor antagonists , inhibits neutrophil and eosinophil accumulation in LPS-induced pleurisy at 24 h but not neutrophil migration at 4 h . The effect of endothelin antagonism on neutrophil accumulation at 24 h was concomitant with inhibition of eosinophil and P01730 REA and CD8 T lymphocyte influx . It is surprising that the ET ( A ) receptor blockade did not inhibit the accumulation of gammadelta T lymphocytes , cells that are important for granulocyte recruitment in this model . Blockade of ET ( A ) receptors did not influence the expression of adhesion molecules ( CD11b , CD49d ) on granulocytes but abrogated the increase in tumor necrosis factor alpha levels 4 h after LPS stimulation and also markedly inhibited increases in levels of interleukin - 6 and keratinocyte-derived chemokine / CXC chemokine ligand 1 but not eotaxin / chemokine ligand 11 . Thus , acting via ET ( A ) receptors , endogenous endothelins play an important role in early cytokine / chemokine production and on granulocyte and lymphocyte mobilization in LPS-induced pleurisy .

In vivo treatments with fulvestrant and anastrozole differentially affect gene expression in the rat efferent ductules . Estrogen plays a key role in maintaining the morphology and function of the efferent ductules . We previously demonstrated that the antiestrogen fulvestrant markedly affected gene expression in the rat efferent ductules . The mechanism of fulvestrant action to modulate gene expression may involve not only the blockade of P03372 REA and Q92731 REA estrogen receptors , but also the activation of P03372 REA and Q92731 REA when the receptors are tethered to AP - 1 or SP1 transcription factors , or the activation of the Q99527 REA . We therefore compared the effects of two strategies to interfere with estrogen action in the rat efferent ductules : treatment with fulvestrant or with the aromatase inhibitor anastrozole . Whereas fulvestrant markedly increased Mmp 7 and Q9BX95 , and reduced Nptx 1 mRNA levels , no changes were observed with anastrozole . DB00947 MEN caused changes in epithelial morphology that were not seen with anastrozole . DB00947 MEN shifted P09237 REA immunolocalization in the epithelial cells from the supranuclear to the apical region ; this effect was less pronounced with anastrozole . In vitro studies of ( 35 ) S-methionine incorporation showed that protein release was increased , whereas tissue protein content in the efferent ductules of fulvestrant-treated rats was decreased . Although fulvestrant markedly affected gene expression , no changes were observed on AP - 1 and SP1 DNA-binding activity . The blockade of ESRs seems to be the major reason explaining the differences between both treatments . At least some of the effects of fulvestrant appear to result from compensatory mechanisms activated by the dramatic changes caused by P03372 REA blockade .

Selective use of multiple vitamin D response elements underlies the 1 alpha , 25 - dihydroxyvitamin D3 - mediated negative regulation of the human O15528 REA gene . The human 25 - hydroxyvitamin D3 ( DB00146 ) 1alpha - hydroxylase , which is encoded by the O15528 REA gene , catalyzes the metabolic activation of the DB00146 into 1alpha , 25 - dihydroxyvitamin D3 ( DB00136 MEN ) , the most biologically potent vitamin D3 metabolite . The most important regulator of O15528 REA gene activity is DB00136 MEN itself , which down-regulates the gene . The down-regulation of the O15528 REA gene has been proposed to involve a negative vitamin D response element ( nVDRE ) that is located approximately 500 bp upstream from transcription start site ( TSS ) . In this study , we reveal the existence of two new P11473 REA - binding regions in the distal promoter , 2.6 and 3.2 kb upstream from the TSS , that bind vitamin D receptor-retinoid X receptor complexes . Since the down regulation of the O15528 REA gene is tissue - and cell-type selective , a comparative study was done for the new DB00136 MEN - responsive regions in P29320 REA - 293 human embryonic kidney and MCF - 7 human breast cancer cells that reflect tissues that , respectively , are permissive and non-permissive to the phenomenon of DB00136 MEN - mediated down-regulation of this gene . We found significant differences in the composition of protein complexes associated with these O15528 REA promoter regions in the different cell lines , some of which reflect the capability of transcriptional repression of the O15528 REA gene in these different cells . In addition , chromatin architecture differed with respect to chromatin looping in the two cell lines , as the new distal regions were differentially connected with the proximal promoter . This data explains , in part , why the human O15528 REA gene is repressed in P29320 REA - 293 but not in MCF - 7 cells .

Relationship of Kaposi sarcoma ( KS ) - associated herpesvirus viremia and KS disease in Zimbabwe . The relationship between Kaposi sarcoma-associated herpesvirus ( KSHV ) viremia and KS disease was investigated in 500 subjects who received treatment in Harare , Zimbabwe . Subjects were grouped by results of human immunodeficiency virus ( HIV ) type 1 serological tests , KS diagnosis , and KS clinical stage . The plasma KSHV DNA concentration was associated with concomitant KS and HIV - 1 infection ( AIDS-KS ; P < . 001 ) and AIDS-KS clinical stage ( P= . 01 ) . Plasma KSHV DNA levels were greater in AIDS-KS than in matched HIV - 1 - seronegative KS ( P= . 04 ) . The plasma KSHV DNA level was not associated with age , sex , systemic symptoms , or P01730 REA + lymphocyte count . Plasma and peripheral blood mononuclear cell KSHV DNA concentrations were linearly related ( r2 = . 44 ; P < . 001 ) , and the nucleotide sequence of the P04264 REA gene highly variable region was identical in both compartments . These findings provide evidence that KSHV viremia is common in advanced AIDS-KS in Zimbabwe and suggest a relationship between KSHV lytic replication and untreated HIV - 1 infection .

Expression of P20839 REA and P12268 REA after transplantation and initiation of immunosuppression . BACKGROUND : DB01024 SUB ( DB00603 ) mediates immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase ( IMPDH ) . Induction of IMPDH activity has been observed in whole blood and erythrocyte samples during immunosuppressive therapy . Information concerning the mechanisms for increased IMPDH activity is limited and the potential implications of induction have been debated . METHODS : Whole blood , P01730 REA + cell , and reticulocyte samples were collected from 30 renal transplant patients pre - and posttransplantation . The expressions of two IMPDH isoforms , type 1 and 2 , were analyzed by real-time reverse-transcription polymerase chain reaction and quantified using a housekeeping gene index . The IMPDH activity was determined by ultraviolet high-performance liquid chromatography . RESULTS : Transplantation and the initiation of immunosuppressive therapy was associated with increased P20839 REA ( 50-88 % , P < 0.0005 ) and decreased P12268 REA ( 42-56 % , P < 0.0005 ) expression . In P01730 REA + cells , however , P12268 REA increased ( 15 % , P= 0.009 ) . These changes are probably related to glucocorticoid effects . Two weeks posttransplant , DB00603 - treated patients displayed elevated P20839 REA and 2 in reticulocytes , suggesting enzyme induction in these cells during prolonged DB00603 therapy . Patients with acute rejection during follow-up demonstrated higher P12268 REA expression in P01730 REA + cells pretransplant than nonrejecting patients ( median expression 1.26 vs . 0.87 respectively , P= 0.017 ) . CONCLUSIONS : Knowledge of changes in P20839 REA and 2 expression after transplantation and initiation of immunosuppression is important considering the action of DB00603 on IMPDH and the potential for pharmacodynamic monitoring of DB00603 by measuring IMPDH activity . The expression of P12268 REA in P01730 REA + cells pretransplant may be an indicator of immune activation .

Contact sensitization to oxazolone : involvement of both interferon-gamma and interleukin - 4 in oxazolone-specific Ig and T-cell responses . The synthesis and role of several lymphokines were examined during contact sensitization to oxazolone ( OX ) . Application of OX to the skin of mice increased the delayed-type hypersensitivity ( DTH ) response to challenge , serum titres of OX-specific IgG 1 and IgG 2a , and draining lymph node cell ( LNC ) numbers . At day 3 , LN contained detectable interleukin - 4 ( P05112 REA ) , interferon-gamma ( P01579 REA ) and granulocyte-macrophage colony-stimulating factor ( GM - P04141 REA ) but not P60568 REA or P08700 REA mRNAs ; P08700 REA and higher levels of P05112 REA , P01579 REA and GM - P04141 REA mRNAs were measured after 24 hr culture with anti-CD 3 antibody in OX-primed but not unprimed LNC . As a result of sensitization , LNC secreted P08700 REA constitutively and produced elevated levels of P60568 REA , P08700 REA , P05112 REA and P01579 REA in response to anti-CD 3 antibody ; a similar but weaker lymphokine response was recalled by OX-protein conjugate . P01730 REA + cells were the major source of the anti-CD 3 - induced lymphokines except P01579 REA , which was derived mainly from CD8 + cells . Since both P05112 REA and P01579 REA were synthesized by OX-primed LNC in vivo and in vitro , their role was investigated by administering anti-lymphokine antibodies at the time of sensitization . Anti - P05112 REA treatment reduced OX-specific serum IgG 1 titres without affecting IgG 2a titres , whereas anti - P01579 REA treatment reduced IgG 2a but not IgG 1 titres . Although neither antibody altered DTH responsiveness , anti - P01579 REA treatment markedly increased P05112 REA production by P01730 REA + LNC and reduced P01579 REA production in vitro , particularly by P01730 REA + cells . We conclude that endogenous P05112 REA and P01579 REA reciprocally influence the isotype of the Ig response to OX and that P01579 REA also affects the relative levels of P05112 REA and P01579 REA synthesis by P01730 REA + LNC .

Endothelin receptor antagonists as disease modifiers in systemic sclerosis . Systemic sclerosis ( SSc ) is a multisystem connective tissue disease of unknown etiology that is characterized by inflammation , vascular dysfunction and fibrosis of the skin and visceral organs . SSc is clinically diverse both in terms of the burden of skin and organ involvement and the rate of progression of the disease . Recent studies indicate that the endothelin system , especially ET - 1 and the P25101 REA and ETB receptors may play a key role in the pathogenesis of SSc . A new class of drugs , endothelin receptor antagonists has been introduced for treatment of patients with pulmonary arterial hypertension ( PAH ) . DB00559 , a dual endothelin receptor antagonist as well as DB06268 and DB06403 MEN , selective blockers of the P25101 REA receptor have proven effective in SSc-PAH . This effect may be mediated through both a vasodilatory and antifibrotic effect , thus making these agents attractive as potential disease modifying agents for SSc .

P05231 REA , P01579 REA and P01375 REA production by liver-associated T cells and acute liver injury in rats administered concanavalin A . The relationship between the development of acute hepatitis and the production of P01375 REA P01579 REA and P05231 REA by liver-associated T lymphocytes following intravenous injection of concanavalin A ( Con A ) was studied in rats . Following a single injection of Con A , there was a dose and time-dependent correlation in the serum levels of serum alanine aminotransferase ( ALT ) , P05231 REA , P01579 REA and P01375 REA . These increases correlated with an increase in the numbers of P01730 REA + , CD8 + and CD25 + T cells in blood and P01730 REA + and CD25 + T cells in the liver perfusate , but not with CD8 + T cells in liver perfusate . Increased levels of P05231 REA , P01579 REA and P01375 REA were constitutively produced by liver-associated P01730 REA + T cells when cultured . In Con A-stimulated cultures , liver-associated P01730 REA + T cells secreted increasing levels of P01375 REA in a time-dependent manner following Con A injection , but P01375 REA production by peripheral blood lymphocytes was transient with peak levels detected at 1 h which then declined over 24 h . Histological examination of the liver revealed fatty change , hepatocyte degeneration and necrosis , with an associated cell infiltrate of neutrophils and P01730 REA + T cells both in the portal areas and around the central veins . These results support the hypothesis that Con A-induced liver damage is mediated by P01730 REA + T cells acting within the liver , at least in part through the secretion of P01375 REA , P01579 REA and P05231 REA .

Chromosomal localization of the genes encoding P37088 REA , P62324 REA , P01579 REA and P21397 REA on chicken chromosome 1 by fluorescence in-situ hybridization .

Hsp 27 regulates epithelial mesenchymal transition , metastasis , and circulating tumor cells in prostate cancer . Defining the mechanisms underlying metastatic progression of prostate cancer may lead to insights into how to decrease morbidity and mortality in this disease . An important determinant of metastasis is epithelial-to-mesenchymal transition ( EMT ) , and the mechanisms that control the process of EMT in cancer cells are still emerging . Here , we report that the molecular chaperone Hsp 27 ( P04792 REA ) drives EMT in prostate cancer , whereas its attenuation reverses EMT and decreases cell migration , invasion , and matrix metalloproteinase activity . Mechanistically , silencing Hsp 27 decreased P05231 REA - dependent P40763 REA phosphorylation , nuclear translocation , and P40763 REA binding to the Twist promoter , suggesting that Hsp 27 is required for P05231 REA - mediated EMT via modulation of P40763 REA / Twist signaling . We observed a correlation between Hsp 27 and Twist in patients with prostate cancer , with Hsp 27 and Twist expression each elevated in high-grade prostate cancer tumors . Hsp 27 inhibition by DB06094 MEN , an antisense therapy currently in phase II trials , reduced tumor metastasis in a murine model of prostate cancer . More importantly , DB06094 MEN treatment decreased the number of circulating tumor cells in patients with metastatic castration-resistant prostate cancer in a phase I clinical trial . Overall , this study defines Hsp 27 as a critical regulator of P05231 REA - dependent and P05231 REA - independent EMT , validating this chaperone as a therapeutic target to treat metastatic prostate cancer .

Serial changes in serum vitamin P04264 REA , triglyceride , cholesterol , osteocalcin and 25 - hydroxyvitamin D3 in patients after hip replacement for fractured neck of femur or osteoarthritis . Serum vitamin P04264 REA concentrations were measured at presentation ( just before surgery ) and then at weekly intervals for 3 weeks in two groups of elderly patients requiring either hemiarthroplasty for fractured neck of femur ( FON , n = 13 ) or total hip replacement for osteoarthritis of the hip ( OA , n = 16 ) . In comparison with healthy elderly volunteers ( n = 25 ) , serum vitamin P04264 REA concentrations were significantly lower in both groups at presentation , and fell significantly within 24 h after surgery to concentrations approaching non-detectable , subsequently returning to pre-operative values within 3 weeks . Serum vitamin P04264 REA tended to be lower in the fracture group both before and after operation , although calculation of a vitamin P04264 REA - triglyceride ratio reduced the apparent difference as triglyceride concentrations were lower in the fracture group . P02818 REA concentrations were similar and fell significantly after operation in both groups , returning to pre-operative levels within 7 days . No differences in the two forms of osteocalcin ( carboxylated and undercarboxylated ) were observed either before or after operation in either group . DB00146 concentrations were not significantly different between the two groups at any time . DB01022 MEN status may be lower than desirable in certain groups of the elderly population , and supplementation should be considered as prophylactic therapy .

Favorable coagulation profile with fondaparinux after hip surgery in elderly patients . Twenty-three patients with fondaparinux prophylaxis over 75 years of age who underwent hip fracture surgery were enrolled in the study . DB00569 ( 2.5 mg ) was administered subcutaneously 6 h postoperatively and then every 24 h for 28 days . Coagulation and inflammatory parameters were measured preoperatively , then 10 h , 2 , 7 , and 28 days postoperatively . Increased D-dimers , positive acute phase proteins , and P05231 REA , and decreased negative acute phase proteins were observed preoperatively ( P < 0.05 ) . Maximum values were reached 10 h postoperatively for P05231 REA and D-dimer , and on postoperative days 2 and 7 for positive acute phase proteins ( P < 0.05 ) . P02787 REA , prealbumin and antithrombin levels were lowest 10 h postoperatively and on postoperative day 2 ( P < 0.05 ) . Increased D-dimers , P05231 REA , and positive acute phase proteins , and decreased negative acute phase proteins persisted until postoperative day 28 ( P < 0.05 ) . P00734 REA fragments ( F1 + 2 ) reached peak levels preoperatively and decreased gradually until postoperative day 28 . Fondaparinux promoted the inhibition of thrombin generation , as documented by negative correlation between F1 + 2 and FXa inhibition ( r = -0.46 ; P < 0.001 ) . Fondaparinux-induced FXa inhibition increased gradually until postoperative day 28 . This increase correlated positively with antithrombin activity ( r = 0.4 ; P < 0.05 ) . Fondaparinux prophylaxis counteracted pro-thrombogenic effect associated with hip fracture and subsequent surgery without severe bleeding complications .

DB00174 MEN Synthetase Deficiency : New Inborn Errors of Metabolism . BACKGROUND : DB00174 MEN synthetase deficiency ( P51689 REA ) is a newly identified neurometabolic disorder characterized by severe congenital microcephaly , severe global developmental delay , intractable seizure disorder , and spastic quadriplegia . Brain Q9BWK5 showed brain atrophy , delayed myelination , and simplified gyriform pattern . METHODS : We report P51689 REA deficiency in a 2 - and 4 - year-old sibling . On them , we described clinical , biochemical , and molecular findings , and we compared our results with previously reported cases . RESULTS : We identified a homozygous novel missense mutation in P08243 REA gene in both probands and we demonstrated low P04141 REA and plasma asparagine in both patients . CONCLUSIONS : Clinicians should suspect P51689 REA deficiency in any newborn presented with severe congenital microcephaly followed by severe epileptic encephalopathy and global developmental delay . P04141 REA asparagine level is low in this disorder while plasma may be low .

Lack of pharmacokinetic interactions between argatroban and warfarin . The potential for pharmacokinetic interactions between argatroban and warfarin was studied . In a randomized , crossover study , healthy volunteers participated in three treatment periods , each separated by a nine-day washout interval . Drug regimens consisted of a single oral 7.5- mg dose of warfarin , intravenous argatroban infused at a rate of 1.25 micrograms / kg / min for 100 hours , or both . Blood samples were collected at intervals up to 104 hours to determine clearance ( CL ) and the apparent first-order elimination rate constant ( kel ) for argatroban and the area under the concentration-versus-time curve ( AUC ) and maximum concentration ( Cmax ) for R - and S-warfarin . An interaction was defined as a > 25 % difference in the magnitude of the pharmacokinetic values between administration of one drug alone and coadministration with the other agent . Twelve adult subjects were enrolled . The mean CL and lel for argatroban administered alone differed by < 7 % from the mean values when the two drugs were coadministered . When warfarin was administered alone , the mean Cmax and AUC of R - and S-warfarin differed from the mean values when the two drugs were coadministered by < 10 % . P00734 REA time was prolonged comparably when argatroban was administered alone and with warfarin . No deaths or serious adverse events were reported . No significant pharmacokinetic interactions were detected between i . v . argatroban 1.25 micrograms / kg / min and a single 7.5- mg oral dose of warfarin . DB00278 MEN was well tolerated when administered alone or with warfarin .

P09237 REA facilitates immune access to the CNS in experimental autoimmune encephalomyelitis . BACKGROUND : Metalloproteinase inhibitors can protect mice against experimental autoimmune encephalomyelitis ( EAE ) , an animal model for multiple sclerosis ( MS ) . P14780 REA ( P14780 REA ) has been implicated , but it is not clear if other MMPs are also involved , including matrilysin / P09237 REA - an enzyme capable of cleaving proteins that are essential for blood brain barrier integrity and immune suppression . RESULTS : Here we report that P09237 REA - deficient ( mmp 7 - / - ) mice on the C57Bl / 6 background are resistant to EAE induced by myelin oligodendrocyte glycoprotein ( Q16653 ) . Brain sections from Q16653 - primed mmp 7 - / - mice did not show signs of immune cell infiltration of the CNS , but Q16653 - primed wild-type mice showed extensive vascular cuffing and mononuclear cell infiltration 15 days after vaccination . At the peak of EAE wild-type mice had P09237 REA immuno-reactive cells in vascular cuffs that also expressed the macrophage markers Iba - 1 and Gr - 1 , as well as tomato lectin . Q16653 - specific proliferation of splenocytes , lymphocytes , P01730 REA + and CD8 + cells were reduced in cells isolated from Q16653 - primed mmp 7 - / - mice , compared with Q16653 - primed wild-type mice . However , the adoptive transfer of splenocytes and lymphocytes from Q16653 - primed mmp 7 - / - mice induced EAE in naïve wild-type recipients , but not naïve mmp 7 - / - recipients . Finally , we found that recombinant P09237 REA increased permeability between endothelial cells in an in vitro blood-brain barrier model . CONCLUSION : Our findings suggest that P09237 REA may facilitate immune cell access or re-stimulation in perivascular areas , which are critical events in EAE and multiple sclerosis , and provide a new therapeutic target to treat this disorder .

Enhancement of antibody responses to Bacillus anthracis protective antigen domain IV by use of calreticulin as a chimeric molecular adjuvant . The generation of protective humoral immune responses against the receptor-binding domain ( domain IV ) of protective antigen [ PA ( dIV ) ] of Bacillus anthracis represents a plausible approach against anthrax toxin . In the current study , we have developed a naked DNA vaccine encoding calreticulin ( CRT ) linked to PA ( dIV ) of Bacillus anthracis [ CRT / PA ( dIV ) ] . We transfected a human embryonic kidney cell line ( P29320 REA 293 ) with CRT / PA ( dIV ) DNA and performed Western blotting and confocal microscopy analysis . We found that linkage of CRT to PA ( dIV ) targets PA ( dIV ) to the endoplasmic reticulum , resulting in secretion of the chimeric CRT / PA ( dIV ) protein . We then evaluated the ability of CRT / PA ( dIV ) DNA to generate PA ( dIV ) - specific antibody responses and protective immunity against lethal anthrax toxin ( PA plus lethal factor ) challenge . We found that mice immunized with CRT / PA ( dIV ) DNA were capable of rapidly inducing significantly higher PA ( dIV ) - specific antibody responses than mice immunized with PA ( dIV ) DNA alone . Furthermore , we observed that this enhanced antibody response generated by CRT / PA ( dIV ) DNA was P01730 REA dependent , since P01730 REA knockout mice demonstrated a significant reduction in antibody responses . In addition , analysis of the titers and avidity maturation of the induced PA-specific antibodies revealed that vaccination with CRT / PA ( dIV ) DNA vaccine accelerated the avidity maturation of antibodies to PA ( dIV ) compared to vaccination with PA ( dIV ) DNA . Importantly , the enhanced antibody responses correlated to protective immunity against lethal anthrax toxin challenge . Thus , DNA vaccines encoding CRT linked to PA ( dIV ) may dramatically enhance PA-specific protective antibody responses . Our results have significant clinical applications for biodefense against anthrax toxin .

Differential expression and function of phosphodiesterase 4 ( DB05876 ) subtypes in human primary P01730 REA + T cells : predominant role of Q08499 REA . Type 4 phosphodiesterases ( DB05876 ) are critical regulators in TCR signaling by attenuating the negative constraint of DB02527 . In this study , we show that anti-CD 3 / P10747 REA stimulation of human primary P01730 REA ( + ) T cells increases the expression of the DB05876 subtypes P27815 REA , Q07343 REA , and Q08499 REA in a specific and time-dependent manner . P27815 REA and Q08499 REA mRNAs as well as enzyme activities were up-regulated within 5 days , Q07343 REA showed a transient up-regulation with highest levels after 24 h . The induction was shown to be independent of different stimulation conditions and was similar in naive and memory T cell subpopulations . To elucidate the functional impact of individual DB05876 subtypes on T cell function , we used DB05876 subtype-specific short-interfering RNAs ( siRNAs ) . Knockdown of either Q07343 REA or Q08499 REA inhibited P60568 REA release 24 h after stimulation ( time point of maximal P60568 REA concentrations ) to an extent similar to that observed with the panPDE 4 inhibitor RP73401 ( piclamilast ) . Substantial amounts of P01579 REA or P05113 REA were measured only at later time points . siRNA targeting Q08499 REA showed a predominant inhibitory effect on these cytokines measured after 72 h . However , the inhibition of all cytokines was most effective when DB05876 siRNAs were applied in combination . Although the effect of DB05876 inhibition on T cell proliferation is small , the Q08499 REA - targeting siRNA alone was as effective as the panPDE 4 inhibitor , whereas P27815 REA or Q07343 REA siRNAs had hardly an effect . In summary , individual DB05876 subtypes have overall nonredundant , but complementary , time-dependent roles in propagating various T cell functions and Q08499 REA is the form likely playing a predominant role .