cobalamin , c63h88o14n14pco ( figure 1 ) , participates in only two known mammalian enzymatic reactions . yet , these two cbl - dependent enzymes , cytosolic methionine synthase ( ms ) [ ec 2.1.1.13 ] , requiring methylcobalamin ( mecbl ) , and mitochondrial methylmalonyl - coa mutase ( mu ) [ ec 5.4.99.2 ] , requiring adenosylcobalamin ( adocbl ) [ 1 , 2 ] , are critically involved in key metabolic pathways essential for gene expression and regulation , via formation of s - adenosylmethionine ( sam ) and methylation , and in protein synthesis and catabolism , cellular respiration , and energy .
activation of methionine synthase also ensures key antioxidant defense status , as it triggers concurrent activation of cystathionine -synthase ( cs ) , the pivotal enzyme at the homocysteine junction in the trans - sulfuration pathway to glutathione ( gsh ) .
anaemia , and macrocytic or megaloblastic anaemia , as well as for subacute combined degeneration of the spinal cord .
however , an increasing body of work suggests that cbl may also play a central role in the regulation of immunity and inflammation ( reviewed in ) .
cbl confers significant protection in various animal models of shock , from anaphylaxis to trauma and sepsis [ 57 ] , and has remarkable organ / tissue protective effects when used clinically for the treatment of analogous inflammation in cn poisoning ( reviewed in ) . amongst cbl 's known immunological effects
are an augmentation of the cd8+/cd4 + t - lymphocyte ratio and natural killer cell activity [ 9 , 10 ] , both significantly reduced in inflammatory pathology , with negative consequences in septic patients .
interesting homeostatic links between cbl and pivotal cytokines are also emerging , indicative of complex but still incompletely defined regulatory circuits : mecbl lowers interleukin-6 ( il-6 ) expression in peripheral blood monocytes , whilst cbl deficiency raises circulating il-6 in humans   and cbl physiological status regulates il-6 levels in rat cerebrospinal fluid
. moreover , in both rodents and humans there appears to be an inverse relation between cbl physiological levels and tumour necrosis factor alpha ( tnf- ) serum levels . in vitro ,
a reasonable hypothesis is that such cbl / tnf-/il-6 regulation may be partly effected via cbl indirect regulation of the central immune regulatory transcription factor , nuclear factor kappa b ( nf-b ) .
normal physiological levels of cbl in spinal fluid appear to correlate with nf-b quiescence , at least , in a non - inflammatory / non - immune challenge model .
recently , a kinetic study reported that cob(ii)alamin reacts with superoxide at rates approaching superoxide dismutase .
cncbl protects human aortic endothelial cells , and neuronal cells , in vitro , against superoxide induced injury [ 19 , 20 ] .
given that oxidative stress is a major trigger of nf-b activation , this potential antioxidant effect of cbl could theoretically lead to nf-b inhibition .
it may also be of critical local importance in vivo , as the phagocytic burst includes release of the cbl carrier , haptocorrin ( hc / tc 3 ) , in the immediate vicinity of nadph oxidase [ 8 , 21 ] one of the major biochemical sources of superoxide in immune challenge and inflammation .
hc / tc3 , moreover , is upregulated by il-1 , itself expressed within fifteen minutes of inflammatory challenge . nevertheless ,
though antioxidant effects of cbls have been observed in vitro   and may , indeed , be important in vivo , no systematic analysis of the in vivo mechanisms of cbl conferred protection against inflammation during acute immune challenge has hitherto been done .
we wondered if a more comprehensive explanation for cbl effects on inflammation and immunity , and thence beneficial outcomes in sepsis and other forms of shock , may lie in a potential direct / indirect regulation by cbl of one or more of the several actions of nitric oxide ( no ) as a ubiquitous , cell - signal transduction molecule and second messenger for post - translational modification , whose targets include soluble guanylate cyclase .no is the product of three nitric oxide synthases ( nos ) : two constitutive , nnos ( neuronal nos ; nos i ) and enos ( endothelial nos ; nos iii ) , and one inducible , inos ( nos ii ) , at much higher levels of expression , with the potential to produce 1000-fold higher than normal amounts of no , during gestation , growth , and the immune response .
cobalamins are known to have effects on no [ 2729 ] , but these have hitherto been thought to be a consequence of cbl / no scavenging effects [ 7 , 3035 ] demonstrable chemically and in vitro [ 36 , 37 ] , but biologically unproven in vivo and still controversial [ 3840 ] .
nitrosylcobalamin has not been detected , to date , in vivo or in vitro , amongst naturally occurring intracellular cbls .
nevertheless , if the hypothesis that cbl is involved in nos catalysis has any substance [ 42 , 43 ] , then it is conceivable that , in analogy to previously observed ferric - heme - no complex formation at the conclusion of nos catalysis , nocbl might be transiently formed , just prior to release of free no by the nos .
such a theoretical transience and discrete localisation might account for the failure to detect nocbl in vivo to date .
ubiquitous and continuous cbl scavenging of no , on the other hand , may pose biochemical hazards .
for no has important antioxidant and cell - signalling actions   which might be obstructed by hocbl 's previously proposed , indiscriminate no scavenging , or even just by a recently proposed , cbl structural - based , direct inhibition of the nos tout court and nothing else .
there is some evidence that hocbl can discriminate between exogenous no donors and the natural endogenous donor , s - nitrosoglutathione , gsno , actually prolonging only gsno - induced , gastric fundus relaxations .
moreover , there are also diverse indications that positive cbl status is allied to beneficial no activity : in diabetic rats , high cobalamin levels correlate with high nos protein levels , no activity , and increased erectile function ; cbl supplementation of vegetarians with low cbl status significantly increases enos no release in the brachial artery ; in the digestive tract of endotoxemic rats , the highest expression of inos is in the ileum , precisely where cbl is internalized , and both cbl and no are known to mediate cell protective effects via erk1/2 and akt [ 5054 ] .
these protective effects of no and cbl include induction and regulation of heme oxygenase-1 ( ho-1 ) [ 52 , 5558 ] , which converts biliverdin to the powerful antioxidant bilirubin , and carbon monoxide .
( for a more comprehensive list of coincidences of cbl's / no 's positive actions , see table 1 and its related discussion in ) .
thus , in these studies we explored an alternative hypothesis to that of cbl as just an no , or , indeed , superoxide , mop .
we posited that the principal mechanism behind cbl 's beneficial , pleiotropic effects in inflammation may involve a biphasic regulation of nos expression and protein translation and the ensuing no synthesis , during the two distinct pro- and anti - inflammatory phases of the immune response .
male c57bl/6 mice , weighing 20 to 25  g , were purchased from harlan , uk , and maintained on a standard chow pellet diet , containing standard amounts of cbl ( 50  g / kg vitamin b12/cncbl ) , with tap water supplied ad libitum .
animals were kept in a 12:00  h light / dark cycle , and all were housed for 7 days prior to experimentation .
all experiments were performed in accordance with uk home office regulations ( guidance on the operation of animals : scientific procedures act , 1986 ) .
the coenzymes , 5-deoxyadenosylcobalamin , and methylcobalamin ; vitamin b12a , cyanocobalamin , and hydroxocobalamin ( cas 78091 - 12 - 0 ) were purchased from sigma - aldrich ( uk ) .
glutathionylcobalamin and n - acetyl - cysteinyl - cobalamin were synthesized and supplied by professor nicola brasch ( kent state university , ohio , usa ) .
all cbls ( and cbl - treated animal samples ) were protected from light during storage and handling , and were 98% to 99.5% pure .
5-deoxyadenosylcobalamin ( adocbl ) , methylcobalamin ( mecbl ) , hydroxocobalamin ( hocbl ) , glutathionylcobalamin ( gscbl ) , and n - acetyl - cysteinyl - cobalamin ( nac - cbl ) were all stored at 20c , and fresh solutions of them were made using sterile , pyrogen - free , phosphate - buffered saline ( pbs ; gibco ) , prior to the experiments . for the in vivo experiments
, cobalamins were diluted at 10  ml / kg prior to treatments ( with pbs used as vehicle ) .
raw 246.7 macrophage cells , stably transfected with nf-b luciferase reporter construct ( stratagene ) , were maintained in dulbecco 's modified eagle 's medium , supplemented with 10% ( v / v ) fetal bovine serum , 2  mm l - glutamine , 1  g / ml geneticin , and 50  g / ml g418 .
cells ( 2  10 cells ) were seeded in 96-well plates and then preincubated for 1  h with increasing concentrations ( 110100  m ) of the five principally occurring , intracellular cbls .
thereafter , at time 0  h , cells were stimulated with e. coli lps ( 0111  :  b4 ; 1  g ) for 4  h and then processed for measurement of luciferase activity in a luminometer ( luminometer td-20/20 ; turner designs instruments ) .
endotoxaemia was induced by the intraperitoneal injection of lps ( 0.1  mg / kg ) , alone ( non - lethal ) or , in the lethal endotoxaemia protocol , in combination with 1  g / kg d - galactosamine ( table 1 ) .
sample collection in non - lethal endotoxaemia was carried out at both 4 and 24  h after lps challenge .
animal survival , in all lethal endotoxaemia experiments , was monitored for a total of 5 days , and all data were analysed using chi - squared or kaplan - meier tests . times shown are in relation to time 0  h , when either lps alone or lps+d - gal was administered by intraperitoneal injection .
individual cobalamins were injected into the peritoneum at the doses and times reported in table 1 .
blood ( 500  l ) was centrifuged ( for 5  min , at 2500  rpm ) , and the plasma then collected for elisa analysis .
500  l of trizol reagent ( invitrogen ) was added to the remaining fraction .
20  l ) was treated with 2  u ( 1  l ) of turbo dnase 1 ( ambion , austin , tx ) , as described by the manufacturer , to remove any contaminating genomic dna . an aliquot of the dna - free rna ( 7.6  l ) was then transferred to a new rnase - free tube and reverse - transcribed into complementary dna ( cdna ) , using superscript iii reverse transcriptase ( invitrogen ) , as described by manufacturer .
the following reagents were used : oligo dt primers ( invitrogen ) ; 1  l , 10  mm dntp ( bioline ) ; 4  l of 5x first - strand buffer ; 1  l , 0.1 m dtt ; 1  l ( 40  u ) rnaseout ; and 1  l ( 200  u ) of superscript iii reverse transcriptase ( invitrogen ) . after synthesis
, cdna was quantified using a nanodrop nd-1000 and diluted ( 80  ng/l ) in molecular biology grade water and then loaded into 384-well plates for real - time pcr .
real - time pcr assays were performed on the various samples in order to evaluate the expression of the following genes : gapdh , rpl32 , il-1 , cox-2 , inos , enos , tnf- , and hmgb1 ( table 2 ) .
for each gene analyzed , reactions were performed using 1  l of the qiagen quantitect primer assay , added to 5  l power sybr green pcr master mix ( applied biosystems , warrington , uk ) and then diluted with 2  l molecular grade water .
a final volume of 8  l was dispensed into each well and 2  l of diluted cdna ( 160  ng / reaction ) was added .
each sample was tested in triplicate for each gene , and pcr reactions were performed using abi prism 7900 real - time pcr equipment . the thermal profile consisted of 95c for 15  min , then 40 cycles of 94c for 15  s , 55c for 30  s , and 72c for 30  s. this
rest mcs software was utilized for the calculation of the relative difference between the test groups .
liver tissues were harvested from ( n = 5 ) animals , after lps endotoxaemia , with or without hocbl treatment and then homogenized in lysis buffer , which contained a cocktail of protease inhibitors .
protein concentrations prior to loading were determined using the bradford assay ( sigma ) : samples were mixed with 6x laemmli sample buffer , and equal protein amounts ( 100  g ) then underwent electrophoresis on a 10% polyacrylamide gel in running buffer ( 0.3% tris base , 1.44% glycine , and 0.1% sds in distilled water ) .
this was followed by transfer of the proteins onto pvdf membranes in transfer buffer ( using 0.3% tris base , 1.44% glycine , and 20% methanol , in distilled water ) .
membranes were blocked for 1  h with 5% nonfat milk solution in tbs containing 0.1% tween 20 .
inos expression was assessed using a specific monoclonal antibody ( 1  :  1000 ; santa cruz , usa ) .
the signal was amplified with hrp - linked anti - mouse secondary antibody ( 1  :  2000 ) and visualized by ecl ( western blotting detection reagent ; amersham biosciences , usa ) .
densitometric analysis was performed using nih imagej software and normalised to tubulin loading controls in the same sample .
animals ( n = 5 ) were challenged with lps and treated with cbls as described above . at 4  h and 24  h after lps challenge ,
lung and liver tissue samples were harvested , homogenized , and processed for determination of nos activity , as measured by nitrate / nitrite end - products of no .
the ultrasensitive , nos assay used ( oxford biomedical research , oxford , mi , usa : ultrasensitive colorimetric nos assay : cat no .
nb78 ) employs an nadph recycling system  nadp , glucose-6-phosphate , glucose-6-phosphate dehydrogenase and the substrate , l - arginine , but not the cofactor , bh4,to ensure that nos operate linearly for up to 6 hours , as no - derived nitrate and nitrite accumulate .
the assay kit can accurately measure as little as 1  pmol / millil ( ~1  millim ) no produced in aqueous solution . in these studies ,
the enzyme nitrate reductase was used to convert all nitrate to nitrite , then griess reagent employed to quantify nitrite levels , with the generation of a nitrite standard , as recommended by the supplier .
after collection , blood was centrifuged and the plasma separated , under low lighting conditions , then stored at 80c until performance of the analyses .
for determination of circulating tnf- and il-6 levels , using elisa assays , samples were diluted 1  :  10 in the assay diluent , as specified by the manufacturer ( r&d , uk ) .
absorbance was plotted in a standard curve , and data expressed as the content of tnf- ( ng ) or il-6 ( pg ) per ml of plasma . unless otherwise stated , all reagents were purchased from sigma - aldrich , poole , uk .
of 5 animals per group for the analyses in non - lethal endotoxaemia and , initially , 79 per group , then 12 per group , for lethal endotoxaemia survival , series i and ii , respectively .
chi - square and kaplan - meier tests were used for the lethality studies . in all cases , a p < 0.05 was taken as significant .
since cbl has been shown to prevent nf-b activation in a non - immune challenge model , and activation of nf-b leads to inos induction , we first looked at the effects of the five principally occurring , intracellular cbls , ( cncbl , hocbl , gscbl , and the two mammalian enzyme cofactors for mu and ms , respectively , adocbl and mecbl ) , on lps - induced nf-b activation in vitro , using a canonical reporter assay .
although the various incoming forms of cbl are all reduced or dealkylated soon after cell entry , prior to ms / mu cofactor formation , the different incoming forms affect both the rate and ratio of formation of the two known active cofactors , adocbl / mecbl [ 6062 ] .
theoretically , this variability in cbl cofactor formation may impact on the effects of cbls in immune challenge with respect to nf-b activation .
raw 264.7 macrophage cells , stably transfected with nf-b luciferase reporter construct , were preincubated for 1  h with increasing concentrations ( 110100  m ) of cbls . upon lps
stimulation , none of the five cbls significantly affected or inhibited lps - driven nf-b activation at 1  h , with no significant inhibitory effect on nf-b at a later time point ( 24  h ) .
cncbl alone slightly stimulated nf-b activity , but only at the 1  h time point and when tested at the concentration of 1  m ( table 3 ) . in two separate experiments ,
each performed in triplicate , raw246.7 cells , stably transfected with nf-b luciferase reporter construct , were seeded in 96-well plates and preincubated for 1  h with increasing concentrations ( 110100  m ) of individual cbls , followed by stimulation with e. coli lps ( 1  g ) . at 1  h , and 24  h , following lps in the respective experiments , cells were processed for measurement of luciferase activity .
basal values of fluorescence were 2.60   0.38 and 3.77  0.31 for 1  h and 24  h incubation , respectively .
p < 0.05 versus lps alone . as there was no observable difference between the effects of alkyl and non - alkyl cbls on nf-b in vitro , we chose to focus these first investigations in vivo principally on hocbl , as a clinically licensed cbl form , known to be partially converted on cell entry to the two cbl cofactors , mecbl and adocbl , for ms and mcm , respectively . furthermore , at supraphysiological doses of 5  g i.v .
, hocbl , as a clinical cyanide antidote , has shown remarkable protection against corollary inflammation ( analogous to the inflammation seen in sirs , sepsis , and septic shock ) , that goes beyond merely acting as a magnet for cn .
we therefore next decided to see if the lethality survival protection also conferred by hocbl in a sepsis / endotoxaemia mouse model   was reproducible in a different strain and in a more acute endotoxaemia model .
some groups of animals were alternatively treated with the relatively novel , intracellular cbl , glutathionylcobalamin ( gscbl ) [ 61 , 64 ] whose clinical effects are untested in sepsis , or with n - acetyl - cysteinyl - cobalamin ( nac - cbl ) , a synthetic cobalamin , used as a non - endogenously occurring , thiol cbl comparison . to gain some information on potential clinical dosage
, all cbls were tested in two distinct , high dosing regimes , with or without prophylactic pretreatment . in a severe sepsis protocol ( lps+d - gal ) , using c57bl/6 mice , we administered a relatively low dose of cbls ( 0.2  mg / kg i.p . ) , equivalent to a maximal concentration of approximately 1  m ( considering a total blood volume of 2.5  ml in the mouse , this concentration being well within the range tested in vitro ) .
h prior to lps+d - gal and then given in repeated doses at + 1 , + 2 , + 6 , and + 22  h after lps+d - gal .
alternatively , a high dose cbl protocol ( 40  mg / kg i.p . ) was administered only twice , at + 2 and + 22  h after lps+d - gal , to assess its potential as a rescue regimen .
the urine of all cbl - treated animals was red , within 1  h of administration , an indicator of rapid , high , systemic cbl saturation ( data not shown ) .
lps+d - gal mice rapidly reached 88.9% mortality by 8  h. this did not change further up to 24  h. animals treated with the relatively low - dose regimen of gscbl or nac - cbl , were protected in the early , 48  h time frame ( figure 2(a ) ) . during this period
all cbl - treated animals also exhibited less huddling and pilo - erection ( data not shown ) . at 8  h after lps+d - gal , all relatively low - dose cbl treatments afforded 25% survival ,
p < 0.05 versus lps+d - gal alone ( figure 2(a ) ) . however , only low - dose hocbl treatment maintained this level of protection up to 24  h ( figure 2(a ) ) .
( indeed , as regards the long - term outcomes , 8  h seemed to be a watershed time point at which the outcome was determined for all groups . ) paradoxically , in view of its early protective effects at the lower dose , the high - dose gscbl regimen was less protective within the first 8  h. high - dose nac - cbl , which again provided some degree of protection in the first 6  h , was not significantly different from controls at 24  h. in contrast , high - dose gscbl and hocbl , despite the lesser protection of the former in the first hours , offered a consistent 28.60% survival up to 24  h , p < 0.01 versus lps+d - gal alone ( figure 2(b ) ) .
later , at 72  h following lps+d - gal , in the gscbl high - dose group , mortality was equal to that observed in the nac - cbl high dose group , 85.72% , close to that of lps+d - gal control animals , though this increase in mortality was a late event : with 28.60% survival to 54  h in this group , perhaps indicative of the general cbl protective trend .
nonetheless , the 25% and 28.60% , respectively , of mice that were alive at 24  h , in each of the two distinct , low- or high - dose , hocbl - treated groups , exhibited continued survival up to 72  h , /p < 0.05 versus lps+d - gal alone / p < 0.01 versus lps+d - gal alone ( figures 2(a ) and 2(b ) ) and beyond ( data not shown ) . 
 ii . since these initial endotoxaemia studies might be considered underpowered , we repeated the lethality survival experiments using larger groups of mice ( n = 12 ) and focussing on hocbl alone , as having previously shown the most consistent protective effects .
this time , given the trend towards improved survival seen at the higher hocbl dose , two distinct ultra - high doses of hocbl ( 40  mg / kg and 80  mg / kg ) were tested , with a more concise dose / time frame , + 2  h and + 4  h only for the 40  mg / kg , and , in the case of the 80  mg / kg dose , a single bolus administration at + 2  h. the significant survival advantage of hocbl treatment results demonstrated over 5 days , in figures 3(a ) and 3(b ) , not only shows that our hocbl data is consistently reproducible , but also that increasing the dose of hocbl significantly increases survival , from 25% up to 33.333% : p < 0.01 for hocbl ( 80  mg / kg ) by using the kaplan - meyer test . by comparison , at 24  h in the lps - only group , there was 90% mortality . to gain information about the mechanisms behind the consistent protection afforded by hocbl , and to observe any potential impact on the nos ,
then the expression of inflammatory mediator genes in liver and lung was analysed , in both the pro- and anti - inflammatory phases of the immune response , at the 4  h and 24  h time points .
the early effects of hocbl treatment on enos mrna appeared organ dependent , with significant promotion of enos mrna in the lung and attenuation in the liver ( figures 4(a ) and 4(b ) ) .
for enos , in lps - only animals we observed a decrease in the lung of 2.9  0.1 , whereas there was an increase of 2.1  0.1-fold change in lps+hocbl - treated animals ( figure 4(a ) ) .
paradoxically , in the liver of lps - only treated animals , there was an increase of enos expression of up to ~15-fold compared to up to ~4-fold change only in lps+hocbl  treated animals ( figure 4(b ) ) .
liver and lung inos and cox-2 gene expression levels were increased in lps - only treated animals when compared to that of pbs - only injected mice , whose value was set as 1 . however , as for enos , the effects of hocbl on inos expression were once more organ selective , failing to inhibit the rise in inos mrna in the lung , but attenuating it in the liver ( figures 4(c ) and 4(d ) ) . strikingly , in spite of hocbl 's failure to completely inhibit inos expression , hocbl was a consistent inhibitor of cox-2 mrna in both liver and lung , bringing its degree of expression back to and below that of pbs - injected mice ( figures 4(e ) and 4(f ) ) .
hocbl treatment also had a consistent regulatory effect on il-1 expression , which was moderately and significantly decreased in lung and completely inhibited in liver ( figures 4(g ) and 4(h ) ) .
to determine efficiency of translation of the post - lps increased nos mrna , we assessed enos and inos protein expression by western blot in 4  h liver samples .
as predicted by our hypothesis that sepsis may involve a failure in translation of the nos , this revealed that whilst in the lps - only challenged group there was a significant depression of enos protein translation , that was at odds with its high mrna expression , hocbl significantly promoted enos protein translation , above the levels of both pbs control and lps - only treatment groups ( figure 5(a ) ) .
a similar paradoxical pattern was observed in hepatic inos protein translation , with significant depression of inos protein translation in the lps - only challenged group , and promotion of inos protein in the hocbl+lps treated group ( figure 5(b ) ) .
we confirmed that these effects of hocbl on nos protein promotion were not random or artifactual , but were specific to cbl , by repeating the lps non - lethal endotoxaemia experiment using either gscbl or nac - cbl treatment and performing western blots for enos / inos protein .
once again , we observed a significant early promotion of enos / inos protein by these other cbls , when compared to lps only ( figures 5(a ) and 5(b ) ) .
however , when nos activity was measured ( using a nitrite production assay ) both in the early post - lps challenge , pro - inflammatory phase and in the late anti - inflammatory , resolution phase , a further paradoxical result emerged , suggesting that hocbl may exert some post - translational modification of nos activity .
levels of nitrite at 4  h showed an inverse relation to levels of nos protein , with significantly higher levels of nitrite in the lps - only enos / inos - depressed group and significantly lower levels of nitrite being generated in the hocbl / enos / inos - promoted group ( figure 6(a ) ) .
( that this was a general , reproducible cbl effect was confirmed , as stated previously , by also doing the western blot with 4  h gscbl / nac - cbl - treated liver samples and also running the nos activity assay with both thiol - cbl treated samples . ) here we again observed a correlation between gscbl / nac - cbl promoted high nos protein in the western blots and decreased nitrite in the nos activity assay ( figure 7 ) . at 24  h following lps
, levels of nos - derived nitrite , as measured in tissue samples , were even higher than at 4  h in both the lps - only and hocbl - treated groups .
nevertheless , hocbl consistently showed relatively less nitrite production than lps only , in both lung and liver tissue samples ( figures 6(b ) and 6(c ) ) . since high inos expression / protein and no activity in the sepsis literature
are associated with high tnf- , il-6 , and ensuing toxicity , we evaluated how hocbl might impact upon systemic levels of tnf- and il-6 triggered by lps .
levels of il-6 protein were not significantly lower in the hocbl - treated group ( figure 8(a ) ) however , hocbl treatment significantly ( p < 0.05 ) attenuated the post - lps - induced increase in circulating plasma tnf- , as measured at the 4  h time point , ( ~50% reduction : figure 8(b ) ) .
consistent with its effects on plasma tnf- , hocbl also showed some protection from the inhibitory effects of lps on tnf- mrna in the lung and significant attenuation of tnf- mrna in liver ( figures 8(c ) and 8(d ) ) . in the resolution phase of the immune response to lps , the effects of hocbl treatment on nos expression displayed a degree of organ selectivity , though most notable with respect to inos expression . whilst hocbl did not change lps - induced enos mrna inhibition in the lung
, it significantly attenuated its inhibition in the liver , from 75- to 58 fold - change , respectively , for lps only and lps+hocbl ( figures 9(a ) and 9(b ) ) .
hocbl effects on inos mrna in the lung were more distinctive , with ~80% inhibition compared to lps - only ( vehicle group ) . in the liver , hocbl treatment attenuated the lps - induced inhibition of inos mrna by ~40% ( figures 9(c ) and 9(d ) ) .
the consistent hocbl tissue inhibition of cox-2 mrna , seen at the early pro - inflammatory phase time point of 4  h , persisted at 24  h , showing a significantly greater degree of inhibition than lps - only : 7- versus 2.5-fold for lps - only in the lung ; 115- versus 50-fold for lps - only in liver ( figures 9(e ) and 9(f ) ) .
given the early regulatory impact of hocbl on nos / no activity , and cox-2 , il-1 , and tnf- , we expected to see related downstream beneficial effects on expression of the late ( 18  h ) effector of endotoxaemia , high mobility group box 1 ( hmgb1 ) .
this prediction was confirmed . where lps - only presented an inconsistent picture , hocbl treatment consistently inhibited hmgb1 mrna : in the lung , from an increase of 2.5 in lps - only to a near threefold decrease ( setting levels of expression even lower than those observed in the control group , taken as a value of 1 ) ; and in the liver , to a more significant degree , even beyond the remarkable inhibitory effect of lps - only ( figures 9(g ) and 9(h ) ) .
to conclude the 24  h gene expression analyses , tissue levels of il-1 and tnf- mrna were also quantified using rt - pcr . in resolution , hocbl treatment significantly increased inhibition of hepatic il-1 ( in line with its inhibitory effect at 4  h ) and inhibition of tnf- in the lung , whilst also , paradoxically , decreasing lps inhibition of tnf- in the liver ( table 4 ) .
of note was the fact that the late effects of hocbl on tnf- mirrored the degree of late inos expression , in both lung and liver , as , indeed , did lps - only ( figures 9(c ) and 9(d ) and table 4 ) .
our studies present a picture of complex and far - reaching homeostatic regulation of the activation , expression , and translation of nos , no synthesis , and inflammatory mediators by hocbl during the immune response .
we propose that this regulation accounts for the noted survivals in rodent endotoxaemia , both in our more acute , septic shock models i and ii ( a modest but significant 25%/28.60% , and 25%/33.333% , survival ) and in a previous sub - acute , sepsis model ( performed with cncbl / hocbl 
the regulation we show here may also explain the observed organ / tissue - protective effects of hocbl in the clinical treatment of cn poisoning and the ensuing shock , which appear to go beyond what may be expected from the cbl binding of cn alone .
furthermore , given the supraphysiological , saturating doses of cbl used in our studies , if cbl had been acting just as an no scavenger , or even as a nos inhibitor , it seems unlikely that it would have permitted the increasing rise in nos activity ( as indexed by nitrite ) , both early and late , or that its effects would be so subtle , complicated , and ultimately beneficial .
the thiol cbls , on the other hand , present some mysteries , about which one can only give speculative answers at this point . the survival advantage exhibited in the early hours by comparatively low dose gscbl / nac - cbl treated animals , but not with hocbl treatment , may be due to the known higher reduction potential / lability of thiol cbls , resulting in more rapidly available intracellular cbl .
cbl is likely to be , at least in part , functionally deficient in sepsis , as indexed by the down - regulation of the cbl receptors , megalin and cubilin , in the kidneys of endotoxaemic mice , the kidney being known , as a cbl homeostatic regulator , to reduce its cbl uptake in states of cbl deficiency .
pertinently , megalin is down - regulated via the lps - induced erk1/2 signaling pathway , through which , as noted earlier , both cbl and no achieve their coincidentally regulatory , beneficial effects . although the beneficial supply of some extra gsh , as a consequence of thiol cbl lability , might be proposed as an alternative explanation for early survival protection , it is noteworthy that , at the higher dose , gscbl showed no early hours protection .
moreover , in a new series of endotoxaemia lethality , in vivo survival studies that compared protective effects of all endogenous cbls against those of gscbl and nac - cbl , we observed that though gscbl / nac - cbl still consistently conferred the same early / pre-8-hour protection compared to the 4 other cbls , paradoxically , both thiol cbls repeatedly produced poor long - term survival outcomes , equivalent to or worse than lps - only , whereas hocbl / cncbl / mecbl / adocbl all consistently showed significant protection , with cncbl / mecbl in particular , producing even better survival results than the current hocbl - centred study ( brancaleone , dalli et al .
gscbl is certainly known to produce a more rapid early increase in ms activity ( together with fourfold greater formation of adocbl ) , when compared to hocbl .
such an increase in ms activity , normally rapidly deactivated by oxidative stress [ 6870 ] , would increase synthesis of the methyl donor , s - adenosylmethionine ( sam ) , which inhibits lps - induced gene expression by modulating histone methylation . whilst this may have initial short - term benefits , as observed ,
there are also negative long - term consequences from excessive inhibition of the necessary , pro - inflammatory gene expression at this stage
. this may be why ms expression and activity are transiently decreased by 25% and 30% early on in the normal pro - inflammatory phase of the immune response to lps , as well as to allow for gsh synthesis modulation .
the consequent , equally paradoxical failure of the other thiol cbl , nac - cbl , to significantly increase survival beyond 8  h , at both high- and low - dose protocols , may also be attributable to the fact that nac is particularly unstable as a cbl ligand and may therefore have acted independently of cbl as nac alone .
further , though nac can ( 1 ) act as an antioxidant by increasing gsh levels ( 2 ) it can equally act as a pro - oxidant , increasing disulfides , gssg ,   and ( 3 ) is counter - indicated in sepsis , since , whilst nac enhances phagocytosis , it also suppresses the bactericidal respiratory burst in icu patients , with potential negative outcomes .
indeed , consistent with this independent , paradoxical observation , additional data from our in vivo endotoxaemia model shows that in the early 4  h phase of the immune response nac - cbl , in contrast to hocbl / gscbl , significantly increases circulating pmn , specifically granulocytes , yet decreases the intensity of cd11b expression ( see supplementary data available online at http://dx.doi.org/10.1155/2013/741804 ( and researchgate ) ) .
the adhesion molecule / complement receptor , cd11b , is a marker of neutrophil activation , and its high expression normally correlates with a strong respiratory burst .
it is conceivable then that such nac - promoted suppressive effects ultimately outweighed the very early benefits in survival protection conferred by nac - cbl .
the initial in vitro observation that all major endogenous cbls do not inhibit nf-b activation , even at 24  h , may appear surprising , particularly in view of the cbl beneficial outcomes in vivo .
however , it has previously been shown in vivo that early inhibition of nf-b in immune challenge increases and prolongs inflammation , and that persisting late nf-b activation ( 24/48  h ) permits its resolution . moreover ,
this failure to inhibit nf-b by cbl during inflammation , with positive outcomes , has now also been observed by a group who were aware of our findings   and , independently , in a cbl cancer model by marguerite et al .
further , since activation of nf-b is linked to induction of inos , and since inadequately low levels of no  and , indeed , inos gene knockout in mice and nos inhibitors in clinical trials [ 79 , 80 ] have been implicated in sepsis morbidity and mortality , we adopted the hypothesis that nos translation may , in contradiction to the common view , actually be depressed in sepsis and nos catalytic activity   uncoupled  or malfunctioning .
previously observed high no in sepsis is believed to comprise a greater ratio of the more toxic no species , such as peroxynitrite , onoo- , which inhibits enos , as opposed to more antioxidant / cytotoxic no forms , such as s - nitrosothiols / gsno [ 84 , 85 ] or no itself ; although overhigh levels of gsno also have negative effects in sepsis - like inflammation .
since cbl is known to promote gsh [ 3 , 8688 ] , whose synthesis is induced simultaneously with that of inos , it should theoretically alter the ratio of gsno / no to onoo- and related species , so that the more positive actions of no predominate   ( figure 10 ) .
therefore , we predicted that cbl would not inhibit inos expression and translation early on .
this proved correct , with significant hocbl ( and gscbl / nac - cbl ) early promotion of both inos and enos proteins and significantly lower lps - only inos / enos protein .
since enos is known to be depressed in sepsis , with adverse cardiovascular consequences , this early effect of cbl may have positive clinical implications .
there is an apparent contradiction in the data showing relatively low inos / enos mrna leading to strikingly high protein translation in the lps+hocbl treated animals , in direct contrast to the lps - only group , where strikingly high inos / enos mrna yielded much lower levels of inos / enos protein . that this is not an artefact , but a specific cbl effect , also observed by others , is seen by the comparable inverse results achieved with the two , thiol cbls .
( these collective cbl results were also mirrored by decreasing nitrite / nos activity , in inverse proportion to the ascending levels of hocbl / gscbl / nac - cbl inos protein . )
we propose that this paradox may actually be an index of cbl / nos regulation in endotoxaemia and that the high mrna levels in lps - only animals may be due to the observed phenomenon of  relaxed control of rna synthesis  when sam / methyl groups are deficient , as in folate or cbl deficiency , thus consequential on the functional cbl deficiency of endotoxaemia / sepsis , and more permanent ms inactivation by lps , discussed earlier .
furthermore , it is theoretically possible that abnormal cell function in sepsis may result in much of the mrna produced by the lps - only group being  masked  and unavailable for efficient translation .
it is possible also that the translated protein may be unstable and degrade at a faster rate .
this is certainly known to be the case with enos mrna in hypoxia   and in the presence of high tnf- , both characteristic of sepsis .
in contrast , given cbl 's impact on the two coenzymes , ms and mu , upon cbl treatment , the septic cell should afford a degree of metabolic normality and thus economic efficiency in transcription / translation .
a remarkable study , over half a century ago , demonstrated that cbl is capable of reactivating a diversity of key enzymes after acute oxidant stress , most of them also negatively affected in sepsis , including glucose-6-phosphate - dehydrogenase , lactate dehydrogenase , lysine , ornithine , and glutamic decarboxylase .
this last is critical for the supply of alpha - ketoglutarate in the krebs cycle , which is depressed in sepsis , with consequent lower atp production that is associated clinically with increased mortality . in turn
, supply of alpha - ketoglutarate determines the availability of l - arginine , glutamate , and glutamine , all of which also decrease in sepsis , with adverse consequences , in the case of l - arginine especially for the nos .
observed low levels of l - arginine in sepsis   are associated with increased reactive nitrogen species , including high onoo- , and reactive oxygen species production during nos catalysis , interfering with the normal , more beneficial no cell signalling , necessary for the efficient resolution of the pro - inflammatory phase of the immune response .
inos - derived no is known to have a direct regulatory correlation to levels of tnf- [ 100 , 101 ] . this inos / no / tnf- regulation seems operative here with hocbl treatment , not lps - only .
since hocbl permitted a moderate rise in no ( at least , as measured by nos nitrite end - products ) , in tandem with moderate levels of tnf- mrna and protein , and since cbl status has an inverse relation to tnf- levels , it seems reasonable to conclude that such cbl / tnf- regulation occurs downstream of hocbl / nos / no regulation .
additional evidence for this in our studies may be seen even in the resolution phase of the immune response , where hocbl - related levels of inos expression showed a direct correlation to those of tnf- , even to the degree of inhibition , in both lung and liver tissues ( figures 9(c ) and 9(d)/table 3 ) .
importantly , however , in the early pro - inflammatory phase hocbl inos / no regulation does not completely inhibit tnf- : 50% reduction being observed in our experiments .
this is a critical point as anti - tnf- mab treatment increases sepsis mortality in the clinic , since some degree of tnf- production and consequent early pro - inflammatory signalling is essential for an effective immune response . also noteworthy , and crucially consistent with such hocbl / inos / tnf- regulation ,
il-6 is essential for induction of acute phase proteins , whilst simultaneously also decreasing pro - inflammatory cytokines and increasing anti - inflammatory factors .
il-6 regulation of pro - inflammatory factors includes regulation of tnf- and il-1 , expression of the latter also determining that of cox-2 , involved in arachidonic acid - derived prostaglandin and leukotriene synthesis .
il-1 is rapidly expressed ( ~15  min post lps ) , whereas inos , which may be induced by il-1 , is not fully expressed until 6  h after lps .
we have observed that treatment with high doses of endogenous cbls ( hocbl / gscbl ) promotes inos mrna expression as early as 2  h following lps ( unpublished data ) , possibly fast forwarding the immune response .
this , together with the hocbl - promoted high nos protein , controlled rise in no synthesis , and thence a moderate production of tnf-/il-6 may form a feedback loop accounting for the tight hocbl regulation of il-1 , and consequently also cox-2 , as seen at 4  h after lps ( figure 10 scheme ) .
cobalamin - promoted nos / no early regulation of tnf-/il-6/il-1/cox-2 seems also to be consistent with , and accounts for , the later inhibition of hmgb1 mrna .
if expressed at > 18  h and then released extracellularly , hmgb1 can trigger further late release of tnf- , il-1 , and inflammatory products from cox-2 , inos , and excessive ros and rni species , leading to pathology .
it is known that the nervous system can modulate circulating tnf- levels via release of acetylcholine by the vagus nerve .
but our studies show that cbl  essential for acetylcholine synthesis is the first known endogenous inhibitor of late hmgb1 mrna expression .
both nicotine and ethyl - pyruvate have been used to block extracellular release of hmgb1   but , in addition to the fact that hocbl appears to impact on hmgb1 much further upstream , at least in tissues , neither drug is endowed with the safety profile of cbl   and , more pertinently , neither is known to exert such a central , endogenous regulation of the immune response .
but , on the evidence of the general anti - inflammatory regulation observed in our studies , we predict that extracellular release of hmgb1 , from macrophages and pmn , should be negligible with hocbl treatment .
the theory that cbl may impact on the nos indirectly , through the contribution of its two known mammalian coenzymatic functions to nos substrate and cofactor assembly and , indeed , to assembly of the nos protein itself , may further explain our findings , including the cbl - promoted high nos protein ( figure 10 scheme ) .
furthermore , a deficiency of any of the nos substrates and cofactors ( the likely result of cbl functional deficiency in endotoxaemia ) is known to result in less tightly  coupled  nos activity and increased free radical generation [ 112 , 113 ] , with a corollary increase in inflammatory mediators and prolonged period of nos activity , indexed by our observed higher lps - only nos nitrite levels .
( in a forthcoming study we will also analyse more exactly how cbl may shift the ratio of no / gsno / onoo and related species ) .
it may also be that cbl , as adocbl and its radical , takes a direct , active part in nos catalysis , as a third mammalian cbl cofactor . from this perspective
, the high nos protein seen with high cbl administration may be a classical instance of the cofactor promoting coenzyme assembly .
such a central , direct cbl / nos , catalytic interaction would further reduce excess production of toxic forms of no , as well as superoxide and other related ros and rni species . the consequent , more precise , pro- and anti - inflammatory signalling should again result in a shorter , more effective period of nos activity ,
thus lower detectable nitrite levels ( as seen ) with the beneficial signalling and antioxidant effects of no predominant . however , direct cbl scavenger interactions , in discrete intracellular compartments , with primarily toxic rnis , such as onoo-/onooh / no2 , with which cbl interacts ex vivo , ,
may also play a part and can not be ruled out as contributing to a more complex picture behind our observed results ( figure 10 ) .
these novel observations on the mechanism behind cobalamin protection in endotoxaemia suggest that we may be looking at the ideal natural , selective and collective regulator of the nos , and thence of cytokines and other pivotal factors , in immune challenge and sepsis .
in fact , it is now accepted that anti - inflammatory therapies ( based on blocking a specific mediator ) fail toutcourt in sepsis and that a more modulatory approach , which regulates the homeostatic inflammatory response , ( in itself beneficial ) , could be successful .
thus , our findings may have significant clinical implications , not only for the treatment of sepsis , but also for other analogous inflammation - driven conditions , such as cancer and malaria , where nos / no deregulation , and consequent loss of control over key inflammatory mediators , are equally pathogenic [ 115 , 116 ] .
 
 background . 
 nos/no inhibitors are potential therapeutics for sepsis , yet they increase clinical mortality . 
 however , there has been no in vivo investigation of the ( in vitro ) no scavenger , cobalamin 's ( cbl ) endogenous effects on nos/no / inflammatory mediators during the immune response to sepsis . 
 methods . 
 we used quantitative polymerase chain reaction ( qpcr ) , elisa , western blot , and nos griess assays , in a c57bl/6 mouse , acute endotoxaemia model . 
 results . during the immune response , 
 pro - inflammatory phase , parenteral hydroxocobalamin ( hocbl ) treatment partially inhibits hepatic , but not lung , inos mrna and promotes lung enos mrna , but attenuates the lps hepatic rise in enos mrna , whilst paradoxically promoting high inos / enos protein translation , but relatively moderate no production . 
 hocbl / nos/no regulation is reciprocally associated with lower 4  h expression of tnf- , il-1 , cox-2 , and lower circulating tnf- , but not il-6 . in resolution , 24  h after lps , hocbl completely abrogates a major late mediator of sepsis mortality , high mobility group box 1 ( hmgb1 ) mrna , inhibits inos mrna , and attenuates lps - induced hepatic inhibition of enos mrna , whilst showing increased , but still moderate , nos activity , relative to lps only . 
 experiments ( lps+d - galactosamine ) hocbl afforded significant , dose - dependent protection in 
 mice conclusions . 
 hocbl produces a complex , time- and organ - dependent , selective regulation of nos/no during endotoxaemia , corollary regulation of downstream inflammatory mediators , and increased survival . this merits clinical evaluation 
 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions