Tumor Necrosis Factor alpha (TNF-α) is a pleiotropic cytokine which plays a primary role in the induction of inflammation in autoimmune diseases. The newest anti-TNF-α agent is adalimumab , a human-derived antibody. This review summarizes the characteristics of adalimumab, highlighting its clinical use in systemic and ocular inflammatory disorders, and the possible therapeutic strategies. Adalimumab has been successfully used for the treatment of rheumatoid arthritis, ankylosing spondylitis, and psoriasis arthritis. More recently, adalimumab has shown promising qualities in controlling intraocular inflammations, even though this has been used prevalently as a rescue therapy for unresponsive cases. This biologic agent was also used in pediatric cases, showing a good safety and efficacy profile. Albeit no direct comparison with other biologics has been done, and adalimumab seems to be equivalent to the other anti-TNF-α, the switching to adalimumab can offer a better uveitic control. Adalimumab is a promising drug for the treatment of uveitis, even though further studies are needed on its application as a primary therapy in uveitis. Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine produced by a variety of cells, comprehending T lymphocytes. TNF-α mediates its effects through two receptors, known as p55 TNF-R1), and p75 TNF-R2)., and p75., and p7134Since noninfectious intermediate, posterior, and panuveitis515Infliximab , a chimeric IgG monoclonal antibody, was the first commercially available drug targeting TNF-α.18et al.In 1998, Dick The newest anti-TNF-α agent is adalimumab , a recombinant human IgG1 monoclonal antibody targeting the TNF-α; adalimumab has been recently introduced for the treatment of RA,In addition, adalimumab exhibited favorable safety and efficacy profiles while demonstrating sustained control of rheumatic diseases activity early in the course of therapy. Moreover, adalimumab has been proven effective as mono-therapy or in combination with other disease-modifying anti-rheumatic drugs (DMARDs).Moreover, the convenient subcutaneous (SQ) dosing and administration schedule is easier for the patients compared to the other anti-TNF-α agents.On the other hand, many questions on its use have remained unanswered and represent a challenge for the treatment strategy. Particularly, it is not clear when to initiate therapy, which agent, at what dosage, and how long the treatment should continue.23In the event of failure of desired response to one biologic therapy, the efficacy of switching from infliximab to adalimumab has been described in other diseases, such as RA,25The efficacy of switching from a certain biologic agent to another has been recently proven effective,In the following review, we will summarize adalimumab characteristics, highlighting the potentialities, and the possible treatment strategies, which could be applied for the therapy of ocular inflammatory diseases.in vitro when it is the presence of complement.27Adalimumab is human-derived antibody, produced by recombinant deoxyribonucleic acid (DNA) technology. Adalimumab tightly binds to human TNF-α, which is a naturally occurring cytokine involved in the acute phase of inflammatory immune responses. The highly specific affinity of adalimumab to TNF-α provides a blocking by interacting with the p55 and p75 cell-surface TNF receptors.TNF-α seems to play a primary role in pathological inflammation and tissues damage: TNF-α is prevalent in all the tissues affected by an active inflammation, such as the synovial fluid of patients with RA or PsA, and the eye during acute uveitis.However, the anti-TNF-α activity of adalimumab is highly specific and it does not inhibit the lymphotoxin, known as TNF-β, a cytokine produced by lymphocytes that affects a variety of cells, albeit TNF-β modulates several biological responses, resulting by the TNF-α stimulation. Specifically, these biological responses significantly influence three adhesion molecules which are responsible for leukocyte migration: endothelial leukocyte adhesion molecule (ELAM-1), vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule (ICAM-1).In healthy adults, 40-mg SQ dose of adalimumab can provide a bioavailability, which is esteemed near 64%. The maximum serum concentration of adalimumab is 4.7 ± 1.6 mcg/mL, occurring within 131 ± 56 h after one 40-mg SQ dose.The mean terminal half-life is reported to be approximately 2 weeks, with a range of 10–20 days across studies.28et al.Adalimumab has been recently introduced for the treatment of RA,Regarding the safety results, no statistically significant differences were observed between the groups. The meta-analysis showed an evident efficacy of adalimumab, even though clinicians should be careful regarding the potential adverse events.Adalimumab has been successfully used in PsA. Adalimumab has been approved for moderate-to-severe plaque-type psoriasis and psoriatic arthritis (PsA). There is evidence concerning the efficacy, clinical effectiveness, safety, and cost-effectiveness of adalimumab in the treatment of psoriasis: Schmitt and WozelIn addition, TNF-α blockers have been shown to be very effective and safe for the treatment of a variety of sequela secondary to systemic diseases, such as RA, PsA, ankylosing spondylitis, juvenile idiopathic arthritis (JIA), and Crohn’s disease.Although adalimumab has been prevalently used as mono-therapy3137et al.TNF-α plays a key role in uveitis. In the model of endotoxin-induced uveitis (EIU) in rats, an early elevation of TNF-α in aqueous humor and serum is detectable.On the basis of this observation, blocking TNF-α seems to be a promising approach in the therapy of uveitis. At this time, three drugs are commercially available to affect TNF-α expression: Etanercept, a recombinant fusion protein, combining extracellular human p75 TNF receptors with the Fc domain of a human IgG1, neutralizing TNF-α before binding to its receptor; infliximab, a mouse-human chimeric IgG1 monoclonal antibody to TNF-α, neutralizes both the soluble and the membrane-bound form of TNF-α.Adalimumab, the newest one, is a recombinant human IgG1 monoclonal antibody, also binding soluble and the membrane-bound form of TNF-α.In addition to the standard use in rheumatic diseases, TNF inhibitory drugs have also shown to be effective in preventing experimental uveitis.et al. used Etanercept in the endotoxin-induced model (EIU).et al.,18Avunduk et al.43Various studies have demonstrated the efficacy of infliximab in Behçet’s disease and other intraocular inflammations. Mushtaq et al.The most relevant publication has been recently published on adalimumab use in uveitis. Rudwaleit et al.Another hot topic in biologics is the switching from a certain anti-TNF-α agent to another. Recently, Takase et al. assessed the efficacy and safety of adalimumab in treating refractory autoimmune uveitis. Nineteen patients were enrolled, and they received a 40-mg SQ injection of adalimumab every other week during 1 year. All patients had an active intraocular inflammation and optic coherence tomography (OCT) proved that 33 eyes (86%) had cystoid macular edema (CME) at baseline. Visual acuity improved by -0.3 log Mar in 12/38 (31%) eyes, and worsened by +0.3 log Mar in 1/38 (2.6%); 12/19 patients (63%) achieved control of their inflammation with adalimumab until the last follow-up. At the end of follow-up, there was a complete resolution of CME in 18/33 eyes (54.54%). All patients were able to reduce at least 50% of the dose of the concomitant immunosuppressive drugs at the end of follow-up. Nevertheless, adalimumab did not show serious adverse events in all patients, and only local minor side effects at the SQ injection site were observed.The same application can also be hypothesized for refractory uveitis.Another potential application of adalimumab in ocular inflammations can be in idiopathic panuveitis associated with CME, unresponsive to the traditional immune suppressive treatment . In our Pediatric uveitis represents another hot topic in ocular inflammations: long lasting, recurrent diseases, and especially chronic AU needs early and aggressive treatment, for resulting in good visual acuity. Because periocular and periarticular steroid therapy is difficult to apply in children, oral corticosteroids remain the first line of treatment. Side effects such as Cushing’s Syndrome and growth retardation are serious and not tolerable for a longer time in children. Therefore, a variety of other immunosuppressive agents are used in paediatric patients: methotrexate, cyclosporin A, azathioprine, cyclophosphamide and mycophenolate mofetil are used as appropriateThe most relevant inflammatory disease in childhood is AU secondary to JIA with or without antinuclear antibodies, even though many other uveitis sub-types can provoke several severe sequela, such as intermediate uveitis and some other posterior uvetis.et al.,Recently, Tynjälä TNF-α is a pleiotropic cytokine produced by a variety of cells that plays a primary role in uveitis pathogenesis. The effect of anti-TNF-α treatment in uveitis is explained by increasing the fraction of peripheral CD4+ T cells expressing IL-10 with a recovery of visual function.TNF-α block causes deviation of the immune response toward the Th2 type. This has been demonstrated in patients who were treated with a recombinant protein generated by fusing the p55 TNF-α receptor with human IgG1. In addition, down regulation of macrophages has been shown to be an important mechanism for TNFα-blocking drugs.51Adalimumab has been recently used for the treatment of several rheumatic diseases and represent the newest biologic agent targeting TNF-α. The application in the field of uveitis is relatively new, stimulating authors to produce an increasing number of publications. Adalimumab has been successfully used in refractory uveitis in adults,The application of adalimumab in certain aggressive types of ocular inflammation, such as Behçet disease, represent a main topic in uveitis treatment.43An important issue for adalimumab is represented by the pediatric use:In conclusion, adalimumab is a promising drug for the treatment of several types of uveitis, even though further studies are needed on its application as primary treatment in ocular inflammatory diseases.


P450 reductase  is known to be important in the reductive activation of the benzotriazene-di-N-oxide tirapazamine (SR 4233). Using a panel of six human breast adenocarcinoma cell lines we have examined the relationship between P450 reductase activity and sensitivity to tirapazamine. The toxicity of tirapazamine was found to correlate strongly with P450 reductase activity following an acute (3 h) exposure under hypoxic conditions, the drug being most toxic in the cell lines with the highest P450 reductase activity. A similar correlation was also observed following a chronic (96 h) exposure to the drug in air but not following acute (3 h) exposure in air. We have also determined the ability of lysates prepared from the cell lines to metabolise tirapazamine to its two-electron reduced product, SR 4317, under hypoxic conditions using NADPH as an electron donor. The rate of SR 4317 formation was found to correlate both with P450 reductase activity and with sensitivity to tirapazamine, the highest rates of SR 4317 formation being associated with the highest levels of P450 reductase activity and the greatest sensitivity to the drug. These findings indicate a major role for P450 reductase in determining the hypoxic toxicity of tirapazamine in breast tumour cell lines.


P = 0.02), less advanced tumour stage (P = 0.02) or TNF-α and TGF-β positive cancers (P = 0.01 and P = 0.03) showed a favourable prognosis in terms of overall survival. Since our previous studies demonstrated a significant association between NSCLC behaviour, neoangiogenesis and bcl -2 expression, we investigated the putative relation between TNF-α and TGF-β on the one hand, and vascular count (as a measure of tumour angiogenesis) and bcl -2 protein expression, on the other hand. Our results showed a significant direct association between TNF-α and bcl -2 (P = 0.05) and an inverse association between TNF-α and microvessel count (P = 0.03). Moreover, as previously demonstrated, we observed a significant inverse correlation between bcl -2 protein expression and vascular count (P = 0.05), suggesting that the favourable effect of TNF-α on clinical outcome may be related to a bcl -2-mediated low neovascular development. © 2000 Cancer Research CampaignRecent in vivo and in vitro studies have demonstrated a wide spectrum of biologic activities of cytokines in the pathogenesis and progression of malignancy. Tumour necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) have emerged as two of the many host-derived mediators that seem to interfere with both antiproliferative and tumorigenic effects in malignant tumours including lung cancer. However, their association with tumour prognosis or prognostic factors has not yet been completely clarified. In this study, we assessed TNF-α and TGF-β mRNA expression by RT-PCR technique in 61 NSCLC samples, demonstrating the presence of TNF-α and TGF-β mRNA in 55.74% and 45.9% of cases, respectively. We also evaluated the expression of the two distinct transmembrane TNF receptors. TNFR-I and TNFR-II, with a PCR-positive signal in 70.49% and 65.57% of cases, respectively. In 49 of the 61 cases, we evaluated the prognostic impact of the two growth-inhibiting factors using the Kaplan–Meier analysis. In the univariate analysis patients without nodal metastatic involvement (


The pharmacokinetics of the hypoxic radio-sensitizer nimorazole were studied in 19 individuals after single oral doses of between 0.5-3.5 g. HPLC measurements showed, after a rapid absorption, a linear relationship between peak plasma concentration and given dose. Mean elimination half life was 3.1 h. A tendency to a dose-dependent variation in the apparent volume of distribution, total body clearance and elimination half life suggest non-linear pharmacokinetics of nimorazole. Tumour concentrations measured in 5 patients gave tumour/plasma ratios between 0.8-1.3. No toxicity was observed. The results indicate that nimorazole may have potential as a clinically useful hypoxic radiosensitizer.


Marginal posterior genotype probabilities need to be computed for genetic analyses such as geneticcounseling in humans and selective breeding in animal and plant species.i requires recomputing the likelihood for each of the possible genotypes of individual i. This can be done efficiently by storing intermediate results in two types of cutsets called anterior and posterior cutsets and reusing these intermediate results to compute the likelihood.In this paper, we describe a peeling based, deterministic, exact algorithm to compute efficiently genotype probabilities for every member of a pedigree with loops without recourse to junction-tree methods from graph theory. The efficiency in computing the likelihood by peeling comes from storing intermediate results in multidimensional tables called cutsets. Computing marginal genotype probabilities for individual A small example is used to illustrate the theoretical concepts discussed in this paper, and marginal genotype probabilities are computed at a monogenic disease locus for every member in a real cattle pedigree. This sample must be drawn from the marginal posterior genotype probability distribution of i given the modified pedigree, which may still have many loops. Furthermore, marginal posterior genotype probabilities are needed in genetic counseling in humans and selective breeding in domesticated species. An efficient, exact, deterministic algorithm is available to compute the marginal posterior genotype probabilities for every member in a pedigree without loops [For monogenic or oligogenic traits, algorithms for efficient likelihood computations have been described for both pedigrees without loops , and pedut loops . Howeverut loops . Most geut loops  is not sFollowing is a brief outline of the presentation. First we define pedigree loops. Next we discuss the relationship between the likelihood and marginal posterior genotype probabilities of pedigree members. Following this, anterior and posterior cutsets are introduced. Anterior cutsets are used to compute the likelihood by the Elston-Stewart algorithm (peeling), and anterior and posterior cutsets are used to describe an efficient algorithm to calculate marginal probabilities for every member of a pedigree with loops. Next, marginal genotype probabilities are calculated for every member in a cattle pedigree that contains loops. Finally, in the appendix, a small example is used to illustrate in detail the theoretical concepts discussed in this article.Here we define pedigree loops indirectly by providing a simple algorithm to determine if a pedigree contains loops. A pedigree is a set of individuals, each of which can be classified as a founder or a non-founder. A founder is a pedigree member whose parents are not in the pedigree, and a non-founder is a pedigree member with both parents present in the pedigree. A nuclear family consists of a set of parents and all their off spring. A terminal family is a family that has at most one member who belongs to at least one other nuclear family. Terminal members of a pedigree are members of terminal families that do not belong to another family. The algorithm used to determine if a pedigree contains loops relies on identifying and then eliminating terminal members from the pedigree. If a pedigree does not contain any loops, repeated removal of terminal members from the pedigree will result in all members being removed from the pedigree. On the other hand, if a pedigree contains any loops, not all members of the pedigree can be removed by repeated removal of terminal members. See additional file n individuals, and let gi denote the possible genotype and yi the observed phenotype of an arbitrary pedigree member i. Note that both gi and yi can be a function of a single locus or of multiple loci on the chromosome. The likelihood for a genetic model given the observed data can be written asConsider a pedigree with F denotes the joint distribution of all gi (g) and all yi (y) in the pedigree, ρ is the vector of recombination rates between loci, q is the vector of gene frequencies, and θ is the vector of parameters in the genetic model that relates yi and gi [where i and gi . Furthersi, and where h is the conditional probability of the phenotype yi given the genotype gi , Pr(gi| q) is the marginal probability that a founder has genotype gi (founder probability) and Pr(gi| ρ) is the probability that a non-founder has genotype gi given that its mother (mi) has genotype fi) has genotype gi, i in the pedigree, a set si is defined that contains either one or three individuals. For founders, si contains only i, while for non-founders, si contains i, mi and fi. For an arbitrary pedigree member i, marginal genotype probabilities can be written aswhere rference ,12. NoteL is the likelihood defined in 2, and gi fixed at genotype x. Thus, the efficient computation of marginal genotype probabilities using equation 4 requires an efficient algorithm to compute the likelihood. The computation of the likelihood using 2 is not efficient for pedigrees having more than about 20 members. However, the Elston-Stewart algorithm, which is also known as peeling, can be used to efficiently compute the likelihood [N unknown genotypes of individual i requires recomputing the likelihood with gi = x for each of the N values of x. Furthermore, this has to be repeated for all n individuals in the pedigree. In the following section we introduce an algorithm to avoid repeating computations by storing intermediate results in multidimensional tables called anterior and posterior cutsets.where kelihood ,13. Stilg1 of the product of all factors in 2 that contain g1, for each combination of the other genotypes that occur together with g1. Results of these summations are stored in a multidimensional table that has been called a cutset [g1 will be denoted by Computing the likelihood by peeling involves summing over the genotypes of one individual at a time and storing the intermediate results. For convenience, here we assume that individuals are numbered in the order that they are peeled. Peeling the first individual amounts to computing the sum over a cutset . Here weV1 is a set of pedigree members defined as follows. Using the sets si defined earlier for each individual in the pedigree, U1 is defined as the union of all sj that contain individual 1. Then V1 is obtained by removing individual 1 from U1. Further, V1. Note that the product in 5 is over those pedigree members j that contain individual 1 in their sj.where g1, summed over g1, with Replacing in 2 the product of all factors containing g1 = {g2 ... gn} is the set of possible genotypes of the individuals that remain to be peeled, and the product is over those pedigree members r that do not contain individual 1 in their sr. The likelihood expressed as above after peeling g1, will be referred to as LE1, and in general after peeling gi, will be referred as LEi.where g1 has been peeled, the summation in 6 is only over the genotypes of individuals 2 ... n. As described below, and later illustrated through a hypothetical example in the Appendix, as each individual is peeled, an anterior cutset is generated. After peeling the last individual, the final anterior cutset will have only a single value that is equal to the likelihood. Note that for a pedigree with n members, there are n! possible peeling orders. Although any choice of a peeling sequence will lead to the same value for the likelihood, not all choices of the peeling sequence lead to anterior cutsets of the same size. As the amount of memory required does depend on the size of the cutsets, a peeling sequence leading to smaller cutsets is more desirable. However, even for moderately large n, an exhaustive search for an efficient peeling sequence is not feasible. Furthermore, there is no known algorithm to efficiently find the peeling order with the lowest storage requirements [i that remains to be peeled, we compute the size of the anterior cutset that would be generated by peeling i. The individual with the smallest anterior cutset size is chosen to be peeled next [Note that after irements . Howeverled next .V1, we can define a second multidimensional table called a posterior cutsetNow it is convenient to introduce the posterior cutset which will be used to avoid repeating computations in calculating genotype probabilities. By factoring out g1. As a result we can rewrite the likelihood as followswhere i. The first set si has already been described earlier, and it is completely determined by the pedigree. The second set Vi contains the individuals in the cutset that is generated when i is peeled. Thus, Vi is determined by the pedigree and the peeling order. In general, peeling individual i amounts to computing the sum over gi of the product of all factors in LEi-1 that contain gi, for each combination of the other genotypes that occur together with gi. These summations are stored in the anterior cutset for i:In the general description of peeling given below, we make extensive use of two sets defined for each individual j is an individual whose function fj, and Vi = Ui-i. Replacing in LEi-1 the sum over gi of the product of all factors containing gi with LEi:where LEi-1 that were not used in the calculation of LEi-1 that were not used in the calculation of i by removing LEi:where gi. Thus, in general we can write the likelihood as followsNote that m ∈ Vi using anterior and posterior cutsets. As in equation 4, marginal genotype probabilities for m can be written asNow we are ready to explain how to compute genotype probabilities for any individual gm fixed at x. If m is in more than one set of pedigree members Vi, identifying the set Vi with smallest number of members will minimize the required computations. However, if m is not in any Vi, we first write the likelihood 12 as a product of the anterior and posterior cutsets for m. In this expression, however, m has already been peeled. Equation 9, which is used to compute the anterior cutset for an arbitrary individual, contains that individual prior to it being peeled. Thus, by substituting in 12, the expression given in 9 for The denominator of 13 is given by 12, while the numerator is obtained by computing 12 with gm fixed at x.Now the numerator of 13 is obtained by computing 14 with Provided a good peeling sequence is available, computation of the required anterior cutsets and the summation over i: the set of members that contribute to Vi. For example, in computing the numerator of 13 by using 12, the intermediate results from peeling individuals in the first set were stored in n - 1, the likelihood expression LEn-1 becomesThe key principle that we have used to compute marginal posterior probabilities efficiently is that any pedigree member can be assigned into one of three mutually exclusive sets with respect to any individual n remains to be peeled, Vu and Vn-1 contain only n. The likelihood now becomesBecause only individual Further, using 9, L = Note that in 16 and 17 the right-hand sides are identical, and thus i, and thus i is peeled, is used in the calculation of the anterior cutset generated when k = min(Vi) is peeled. The resulting anterior cutset can be written asThe anterior cutset k ∈ sr, and k ∈ Vj in addition to where and by using (19) in (20) we can writeUk = Vk∪ {k}, and thus we can writeRecall that we have defined the set of individuals Note that both (12) and (22) contain the term and using 12 we can writei can be expressed as a function of some anterior cutsets and the posterior cutset for individual k >i. Starting at individual n - 1 all posterior cutsets can be computed in the reverse order of peeling because Thus, the posterior cutset for individual i in a pedigree:In summary, the following procedure can be used to recursively compute the posterior cutset of an arbitrary individual 1. Compute anterior cutsets for all individuals in the pedigree. This step is done only once.2. Identify the anterior cutset 3. Replace 4. If Note that to compute marginal posterior genotype probabilities for an arbitrary member of the pedigree using this algorithm, we need to calculate all anterior cutsets and a subset of all posterior cutsets. Both the anterior and the posterior cutset of a given individual have the same size. The computation of an anterior cutset involves the summation over the genotypes of one individual. The computation of a posterior cutset can involve summations over the genotypes of a variable number individuals. The theoretical concepts introduced in this section are illustrated in detail for a simple example in the Appendix. In the following section we discuss a real data application of the theoretical concepts described above.Consider the pedigree given in the first three columns of Table Next, we augment the genetic information used to calculate posterior genotype probabilities, by including genetic data on two marker loci flanking the hypothesized position of the recessive locus. Each marker locus has three alleles and the two loci are separated by 0.8 cM with the hypothesized position of the recessive locus 0.5 cM from the left marker (M1). The allele scores of the two markers used are given in Table j using equation 13, requires recomputing the likelihood for each of the possible genotypes of individual j. For the last individual in the peeling sequence, this can be done efficiently because intermediate results from peeling individuals 1 through n - 1, for each possible value of gn, have been stored in the anterior cutset m that is in more than one set Vi we identify the smallest Vi containing m. The intermediate results from peeling individuals 1 through i are stored in anterior cutsets, including Vi. Thus, by making use of the intermediate results stored in both Vi (except m) need to be repeated to compute the numerator m that is not in any Vi the expression used to compute genotype probabilities (14) cannot be written as a product of a single anterior and posterior. However, any of the anterior the posterior cutsets used in 14 can be computed efficiently. Thus, this new peeling based algorithm provides an efficient method to compute marginal genotype probabilities for an arbitrary member of a pedigree with loops. The computational cost of obtaining posterior genotype probabilities for all members of a pedigree would approximately be equal to twice that of computing the likelihood because computing the likelihood only requires computing the anterior cutsets while computing all genotype probabilities would require computing the posterior cutsets also. As stated by Jensen and Kong [As stated by Jensen and Kong  current and Kong , a peeliand Kong .k loci, the number of genotypes increases exponentially with k. In such situations, writing the likelihood as a sum over allele state and origin allele variables may lead to more efficient computations [Throughout this paper the likelihood was written as a sum over genotype variables. However, when the genotype of an individual is defined over utations . AlgoritThe authors declare that they have no competing interests.LRT and RLF developed and programmed the algorithm in C++. The analysis of the real cattle pedigree was performed by LRT. KJA contributed to the C++ implementation of the algorithm. The manuscript was prepared by LRT and RLF. All authors have read and approved the final manuscript.The pedigree given in Figure First we show how to use the Elston-Stewart algorithm to compute the likelihood for a genetic model given this pedigree. Next we describe how to calculate marginal posterior genotype probabilities for an arbitrary member of this pedigree using the efficient algorithm described above.As shown in 2, the likelihood given the observed data can be written asf1 is the only function that involves g1. Peeling g1 amounts to computing the sum over g1 of f1, for each combination of the genotypes for individuals 5 and 4, and storing the results of these summations in the anterior cutsetIn the pedigree given in Figure N5 × N4, where N5 and N4 are the number of possible genotypes for individuals 5 and 4. Replacing the sum over g1 of f1 in 25 with LE1:Note that LE1 f2 is the only function that involves g2. Therefore, the anterior cutset for 2 (obtained by peeling g2) isNote that in g2 of f2 in LE1 with LE2:Replacing the sum over LE2 f3 is the only function that involves g3. Therefore, the anterior cutset for 3 (obtained by peeling g3) isNote that in g3 of f3 in LE2 with LE3:Replacing the sum over LE3 not only f4, but also g4. Thus, peeling g4 yields the following anterior cutsetNote that in N7 × N6 × N5, where N7, N6 and N5 are the number of possible genotypes for individuals 7, 6 and 5. LE3 the factors f4, g4. Thus, the likelihood expression LE4 becomesThe resulting anterior cutset LE4 both f5 and g5. Peeling g5 yields the following anterior cutsetNote that in LE4 the factors f5 and g5. Thus, the likelihood expression LE5 becomesThis cutset replaces in LE5 both f6(g6) and g6. Peeling g6 yields the following anterior cutsetIn f6(g6) and g6 with LE5, the likelihood expression LE6 becomesBy replacing g7 yields the numerical valueNote, however, that the anterior cutset obtained by peeling LE7:and thus the likelihood expression Recall that for an arbitrary member of the pedigree  we can calculate marginal genotype probabilities as followsg3 fixed at x. As discussed earlier, using this procedure to compute marginal genotype probabilities for N unknown genotypes of individual 3 requires recomputing the likelihood for the entire pedigree N times. However by writing the likelihood as in 12, these computations can be done efficiently. Consider computing marginal posterior genotype probabilities for individual 3. Recall that, as shown in 26, g3 f3. Using this in 12 we obtainwhere g3 in 32 at x. To complete the calculations, however, we need to compute Note that 32 can be used to calculate the denominator of 31, while the numerator of 31 can be obtained by fixing g4 and g5, the sum over g7 and g6 is computed to obtainStep 1 of the procedure is to compute anterior cutsets for all individuals in the pedigree, and this has already been done. Following step 2, we determine that Following step 4, note that g7, g6 and g5, we obtainFollowing step 2, we determine that Following step 4, note that Following step 2, we determine that g7 and g6 we obtainFollowing step 3, Following step 4, note that Following step 2, we determine that g7 we obtainFollowing step 3, Following step 4, note that A numerical example to illustrate algorithm to detect loops in a pedigree. Algorithm to detect loops in a pedigree.Click here for file


The structural requirements for L-selectin shedding were studied in murine leukocytes. Uponactivation, L-selectin on both lymphocytes and neutrophils undergoes cleavage by a membranemetalloprotease, resulting in the generation of a soluble ectodomain and a membrane-retained 6 kD fragment. Radiochemical sequencing demonstrated a cleavage site in themembrane-proximal region (MPR) between R321 and S322, which is homologous to thehuman site. Although intact neutrophil L-selectin is larger, it is cleaved at the same, or veryclose, site. Analysis of several transfectants expressing L-selectin point mutations and chimericconstructs suggest that, like human shedding, the proteolytic process has relativelyloose sequence specificity for the substrate site. In addition, some constructs are susceptibleto slow constitutive cleavage, but their shedding does not increase upon PMA stimulation,showing that basal and activated shedding are separable processes. Insertion of the 15 aminoacid MPR into murine B7.2 conferred upon this molecule susceptibility to constitutive shedding.PMA stimulation results in little or no acceleration of down regulation of this molecule.These results suggest that recognition of both the membrane-proximal cleavage site and of asite distant from the MPR are required for maximal induction of L-selectin shedding.


These findings suggest a possibility that T cell-derived IFN-γ up-regulates macrophages to elaborate TNF-α, which may play a role in amplifying allergic inflammation in the nose through the cytokine network.Tumour necrosis factor-α (TNF-α) was measured by enzyme-linked immunosorbent assay and eosinophil cationic protein (ECP) by radio-immunoassay to evaluate TNF-α in nasal allergy. There was no significant difference either between the mean concentrations of TNF-α in nasal secretions from the patients with perennial nasal allergy and those of normal subjects, or between the TNF-α and ECP concentrations. However, reverse transcription polymerase chain reaction showed a specific increase of TNF-α mRNA and IFN-γ mRNA in allergic nasal mucosa after allergen challenge


Alcoholic liver disease (ALD) develops in approximately 20% of all alcoholics with a higher prevalence in females . The devTNF-alpha is thought to play a particularly critical role in the pathogenesis of ALD. TNF-alpha is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity and apoptosis ,11 In adProduction of TNF-alpha is one of the earliest responses of the liver to injury . CirculaProduction of inflammatory cytokines is a highly regulated process; regulation has been reported at the level of transcription, translation and secretion ,22. TranEthanol disrupts a number of hormone and neurotransmitter dependent signaling pathways , includiMale Wistar rats (150 g) were allowed free access to liquid diet  with 17%Kupffer cells were isolated as previously described  except t6 cells/ml and plated onto 96 well (0.2 ml/well), 6-well (3 ml/well) or 100 mm plates (5 ml/well) culture plates. After 2 h, non-adherent cells were removed by aspiration and fresh media supplied. Assays were initiated after 20–24 h in culture.Isolated Kupffer cells were suspended in CMRL with 10% fetal bovine serum and penicillin-streptomycin at a concentration of 2 – 102 atmosphere. For ethanol treatments, cells were incubated with 25 mM ethanol for 48 h; culture dishes were wrapped in parafilm to minimize the evaporation during culture. Culture dishes with untreated cells were also wrapped in parafilm.The mouse macrophage-like RAW264.7 cell line was obtained from American Type Culture Collection . Cells were cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin at 37 degrees C in a 5% COin vitro transcribed anti-sense riboprobes and ribonuclease protection assays were carried out following manufacturer's instructions. Samples were run on 5% sequencing gels, dried and autoradiographed.After 20 h in culture, Kupffer cell media was removed and replaced with fresh media containing 10% fetal bovine serum. Cells were then stimulated with 0 or 100 ng/ml LPS for 1 h. In some experiments, cells were pre-incubated with PD98059, SB20380 or vehicle (DMSO) for 2 h prior to LPS stimulation, or treated with 5 micrograms/ml actinomycin D 1 h after LPS stimulation and then harvested after 1–2 h. Total RNA was isolated by the TRIzol method . Rat cytokine multiprobe DNA templates  were used to synthesize Kupffer cells isolated from rats fed ethanol for 4 weeks accumulated 2-fold higher concentration of TNF-alpha in their media in response to challenge with LPS, with no difference in TNF-alpha accumulation between the pair- and ethanol-fed groups when cells were not treated with LPS  ,19. Incrin vivo response. RAW264.7 macrophages were cultured with and without 25 mM ethanol for 48 h and then stimulated or not with 100 ng/ml LPS for 2 to 4 h. While LPS increased TNF-alpha production in control cells, secretion of TNF-alpha in response to LPS was increased by 1.7–2.2-fold in cells cultured with ethanol compared to controls , but was not different from pair-fed at higher concentrations of LPS . SimilarTaken together, these data indicate that there is a strong similarity between the effects of ethanol feeding on Kupffer cell responses to LPS and the in vitro response of RAW 264.7 macrophages to culture with chronic ethanol. These results suggest that changes in TNF-alpha production by macrophages observed after long term ethanol exposure in vivo are not due solely to systemic responses, such as increased exposure to endotoxin/LPS ,6,33, buin vivo or during culture, had no net effect on TNF-alpha transcription [While LPS-induced TNF-alpha production is controlled at transcriptional, post-transcriptional and post-translational levels , increascription .1/2 > 100 min), compared to those isolated from pair-fed rats . A similar effect of chronic ethanol was observed on the TNF-alpha mRNA stability in RAW 264.7 macrophages. In control cells, LPS-induced TNF-alpha mRNA decayed with an approximate half-life of 35 min. However, treatment of cells with 25 mM ethanol for 48 h not only increased the accumulation of TNF-alpha mRNA, but also substantially stabilized the TNF-alpha transcript (t1/2 >100 min) [in vivo and in vitro results in a marked stabilization of LPS-induced TNF-alpha mRNA.Since chronic ethanol was not acting to increase transcription of TNF-alpha, we hypothesized that chronic ethanol-induced increases in LPS-stimulated TNF-alpha mRNA accumulation might reflect an increased half-life of the transcripts. To test this hypothesis, mRNA stability experiments were performed in Kupffer cells and in RAW264.7 macrophages. Kupffer cells from ethanol- and pair-fed rats were stimulated with 0 or 100 ng/ml of LPS for 60 min. Cells were further incubated in the presence or absence of actinomycin D for 1–2 h. RNA was isolated and TNF-alpha mRNA expression measured by ribonuclease protection assays . Chronic100 min) . These din vivo and during culture, increases both LPS-stimulated ERK1/2 and p38 activation [Activation of p38 and ERK1/2 MAP kinases has been linked to mRNA stabilization of otherwise short-lived cytokine and other immediate early response genes -39. We htivation ,20,30. Tin vitro during macrophage culture, resulted in a stabilization of the LPS stimulated TNF-alpha mRNA transcripts [Chronic ethanol exposure, either by in vivo feeding to rats or nscripts . Interesnscripts  recentlynscripts . Taken tnscripts . These d


Seteria cervi. However, vaccine potential of GSTs of lymphatic filarial parasites has not been evaluated before.Lymphatic filarial parasites survive within the lymphatic vessels for years despite the complex immune environment surrounding them. Parasites possibly accomplish this by adopting various immunomodulatory strategies, which include release of glutathione-S-transferases (GSTs) that counteract the oxidative free radicals produced by the host. Since GSTs produced by parasites appear to be critical for the survival of parasites in the host, several studies evaluated the potential of parasite GSTs as vaccine candidates especially against schistosomiasis, fascioliasis and GST gene was cloned from the third stage larval (L3) cDNA libraries of Wuchereria bancrofti, and recombinant GST (WbGST) was expressed and purified. Serum samples from individuals living in an endemic area were analyzed for their reactivity with rWbGST. These findings showed that sera from endemic normal individuals (EN) carry significant levels of anti-WbGST IgG antibodies compared to subjects who are microfilaraemic (Mf) or show symptoms of clinical pathology (CP). Isotype analysis of the anti-WbGST IgG antibodies showed a predominance of IgG1 and IgG3 antibodies in EN individuals. Subsequent functional analysis of the rWbGST showed that the rWbGST protein retained the enzymatic activity of GST and the antibodies in EN sera could inhibit this enzymatic activity. Similar results were obtained when anti-rWbGST antibodies raised in mice were used in the neutralization assay. Brugia malayi GST and WbGST show significant sequence similarity. Therefore, to evaluate the vaccine potential of rWbGST, we used B. malayi L3 as challenge parasites. Vaccine potential of rWbGST was initially evaluated by confirming the role of human and mice WbGST antibodies in an antibody dependent cellular cytotoxicity (ADCC) assay. Subsequent vaccination studies in a jird model showed that approximately 61% protection could be achieved against a B. malayi L3 challenge infection in jirds immunized with rWbGST.In the present study, the B. malayi challenge infection in a jird model. The study also showed that the WbGST protein retained the enzymatic activity of GST and this enzymatic activity appears to be critical for the survival of the parasite in the host.Results of this study show that rWbGST is a potential vaccine candidate against lymphatic filariasis. Nearly 61% protection can be achieved against a  Seteria cervi, are also involved in inducing protective immunity in the host. The schistosome 28 kDa GST has been successfully developed into a vaccine and is currently in Phase II clinical trials. Thus, GST appears to be a potential target for vaccine development. Therefore, in the present study, we cloned W. bancrofti GST, and expressed and purified the recombinant protein. Immunization and challenge experiments showed that 61% of protection could be achieved against B. malayi infections in a jird model. In vitro studies confirm that the anti-WbGST antibodies participate in the killing of B. malayi L3 through an ADCC mechanism and enzymatic activity of WbGST appears to be critical for this larvicidal function.Lymphatic parasites survive for years in a complex immune environment by adopting various strategies of immune modulation, which includes counteracting the oxidative free radical damage caused by the host. We now know that the filarial parasites secrete antioxidant enzymes. Among these, the glutathione-S-transferases (GSTs) have the potent ability to effectively neutralize cytotoxic products arising from reactive oxygen species (ROS) that attack cell membranes. Thus, GSTs have the potential to protect the parasite against host oxidative stress. GSTs of several helminthes, including schistosomes, fasciola and the filarial parasite  Wuchereria bancrofti, Brugia malayi or Brugia timori that affects 120 million people in 73 countries and another 1100 million people are at risk Lymphatic filariasis is a mosquito borne infection caused by in vitro and in vivo cytotoxic response to the larvae Especially, antigens of the infective third stage larvae of filarial parasites are of special interest since they represent the first larval stage that enters into the human host. Thus, anti-parasitic mechanisms against these infective larvae can potentially prevent the infection. Previous studies show that both antibodies and effector cells are important in this anti-parasitic mechanism functioning via an antibody dependent cellular cytotoxicity (ADCC) mechanism Lymphatic filarial parasites reside inside the lymphatic system and bathe in lymph that carry immune cells and molecules, yet they survive for years without any major harm and appear to be not damaged by the oxidative free radicals released from the host cells. This is largely possible because of the ability of the parasite to produce and secrete molecules such as glutathione-S-transferases (GSTs), SOD, catalase, glutathione peroxidase, peroxiredoxins Seteria cervi have already been established S. mansoniS. bovisS. mansoni 28 GST appears to be due to an inactivation of the GST enzymatic activity Fasciola gigantica GST26 could confer from 77–84% protection against challenge infection in mice F. gigantica GST26 and S. mansoni GST28 show significant sequence similarity such that immunization of mice with recombinant F. gigantica GST26 could confer cross protection against challenge infections with S. mansoni in mice. Thus, GST appears to be a critical protein for the survival of the parasite in the host. Based on homology sequences Nathan et alW. bancrofti GST (WbGST) and predicted sites for docking of GST inhibitors as a new approach to develop drugs against W, bancrofti. These studies suggest that WbGST may be a promising target for vaccine development. Therefore, in this study we have attempted to evaluate the vaccine potential of filarial GST.Neutralizing the effect of these molecules by immunization or vaccination could affect the ability of the parasite to survive in the host. A protective role for immunization with GSTs from several helminth parasites including schistosomes, fasciola B. malayi parasites  used in this study were obtained from NIH/NIAID Filariasis reagent repository center, College of Veterinary Medicine, University of Georgia, Athens, GA.Blood samples were collected from the following clinical groups after obtaining informed consent: 1) Endemic normal (EN) subjects, who are asymptomatic, non-microfilaraemic and reside in villages surrounding Sevagram, Wardha, India with no symptoms of the disease; 2) Symptom-free microfilaraemic carriers (MF) (identified positive by microscopic examination of night blood smear); 3) chronic pathology (CP) patients who are visiting the out-patient department of Kasturba Hospital and were showing clinical symptoms of filariasis. Non-endemic normal (NEN) blood samples were collected from undergraduate medical students coming from non-endemic areas  with no circulating parasites and no evidence of filarial disease who were registered at the time of their admission to Mahatma Gandhi Institute of Medical Sciences, Sevagram. Use of human subjects in this study was approved by the IRB committees at Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, India and at the University of Illinois, Rockford, USA. Sera separated from the blood samples were stored at −20°C freezer until use.WbGST gene was amplified from WbL3 cDNA library using insert specific primers (Forward: 5′CGCGGATCCATGAGTTATAAACTGAC3′ and Reverse: 5′CCGGAATTCTCACTGTTTTCCATTTCC3′). PCR parameters were as follows: 94°C of denaturation for 30 s, 50°C of primer annealing for 30 s, 72°C of primer extension for 30 s, and cycled for 30 cycles; a final extension of 5 min was performed at 72°C. PCR products obtained were digested with Bam HI and Eco RI enzymes and ligated to similarly digested T7 expression vector pRSET A . Insert DNA was sequenced on both strands to ensure authenticity of the cloned nucleotide sequence. The pRSET A-WbGST construct was transformed into E. coli BL21 pLysS to express the recombinant protein.E. coli BL21 containing pRSET A-WbGST construct was grown to A600 ∼0.6 in LB medium containing ampicillin/chloramphenicol at 37°C and the expression of rWbGST was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM IPTG. The culture was further incubated for another 3 h at 37°C. Bacteria were pelleted by centrifugation and resuspended in phosphate-buffered saline . Finally, the cells were lysed by sonication and the debris was precipitated by centrifugation at 12000 rpm at 4°C for 20 min.The Collected supernatant was passed through immobilized cobalt metal affinity column chromatography  to purify the His tagged rWbGST as per manufacturer's instructions. The expression pattern and purity were analyzed by 15% SDS PAGE. Presence of Histidine-tag in the purified protein was detected using penta His-HRP monoclonal antibodies . Concentration of protein was estimated by micro BCA method .−1 cm−1 for CDNB.GST(s) enzyme activity was determined spectrophotometrically at 340 nm by measuring enzymatic conjugation of glutathione with 1-chloro-2, 4-dinitrobenzene (CDNB) 2O2 chromogenic-substrate complex (Thermo fisher Scientific).Sera from different groups of subjects from the endemic and non-endemic regions were screened for the presence of antibodies against recombinant WbGST using both Immunoblot analysis and an indirect ELISA. For immunoblot analysis, rWbGST protein was separated on 15% SDS-PAGE, transferred to nitrocellulose membrane and blocked overnight with 5% skimmed milk. After washing, the membrane strips were incubated individually with diluted (1∶50) pooled sera from MF, CP, EN or NEN individuals. The bound antibodies were probed with HRP labeled mouse anti-human IgG and color was developed with DAB/HAn indirect ELISA was performed to measure the level of IgG antibodies in individual sera sample. A total of 176 sera samples belonging to 45 microfilaraemics (Mf), 45 chronic pathology (CP), 43 endemic normal (EN) and 39 non endemic normal (NEN) individuals were screened for anti WbGST IgG antibodies using rWbGST as antigen in the indirect ELISA. Briefly, wells of an ELISA plates were coated with 1 µg/ml of rWbGST. After blocking the non-specific binding sites human sera samples were added at 1∶100 dilutions. After washing the plates, horse radish peroxidase labeled anti human IgG  was added and color was developed using OPD substrate.Isotype of anti-rWbGST IgG antibodies were also analyzed in the sera of various study groups by indirect ELISA using biotinylated mouse monoclonal antihuman IgG1, IgG2, IgG3 and IgG4 and probed with avidin-horse-radish-peroxidase  as the secondary antibodies.B. malayi L3s in vitro.Use of animals in this study was approved by the IACAUC committee at the UIC College of Medicine, Rockford and by the Institutional Animal Ethics Committee (IAEC) under the guidelines of Committee for the purpose of control and supervision on experiments on Animals (CPCSEA), Wardha, India. Five 2 months old male Balb/c mice were immunized intraperitoneally for antibody generation and seven two-months-old jirds were immunized intraperitoneally with 15 µg of rWbGST per animal in 100 µl of 0.5 M PBS along with equal volume of alum (Thermo fisher Biotech) as adjuvant. Three doses of the antigen were given at 15 days interval followed by one booster dose. Age matched control mice and jirds  received alum alone. One week after the final booster dose, sera were collected and tested for anti-WbGST IgG antibody levels. These sera samples were also used in downstream ADCC assays to determine their ability to kill Total anti WbGST IgG antibody levels in the sera samples of mice and jirds were determined using an indirect ELISA as described above. HRP-labeled goat anti-mouse IgG (ThermoFisher Scientific) was used as the secondary antibody. In addition, we also determined the isotype of anti-WbGST IgG antibodies  in the mice sera using a kit purchased from ThermoFisher Scientific. Color was developed using ABTS  substrate and absorbance was measured at 405 nm in a microplate reader.WbGST gene was PCR amplified from the cDNA libraries of third stage infective larvae (L3), microfilariae (MF) and adult female (AF) of W. bancrofti using gene specific forward and reverse primers. PCR conditions used were an initial denaturation at 94°C for 5 min, 30 cycles  of amplification, and a final extension at 72°C for 5 min. The amplified products were analyzed on a 1% agarose gel.Anti-WbGST antibodies in the sera of immunized mice and in the pooled sera of EN individuals were depleted by passing the sera over rWbGST coupled to Cobalt IMAC resin (Clontech). Briefly, 1 mg of his-tagged rWbGST was coupled to the resin, washed and incubated overnight at 4°C with about 200 µl of neat sera. Supernatant was collected by centrifuging the resin mixture for 2 min at 750 rpm. Depletion of anti-WbGST antibodies were confirmed using an ELISA as described above.Evaluation of the inhibition of GST catalytic activity by anti-WbGST antibodies was determined as described previously using chloro-2, 4-dinitrobenzene as the substrate for GST activity B. malayi L3s were incubated with 14 µM of 1-chloro-2, 4-dinitrobenzene for 2 hrs at 37°C. Following these the cDNB-treated L3s were used in antibody dependent cell cytotoxicity.For neutralization of GST activity in the parasite, we followed the method described by B. malayi L3s, we performed an in vitro ADCC assay as described previously B. malayi to a suspension of 2×105 peritoneal exudates cells (PEC) collected from normal Balb/c mice or PBMC's collected from NEN (WbGST antibody negative) individuals. Approximately, 50 µl of the sera samples were diluted with 50 µl of RPMI 1640 media and added to the suspension to make a final volume of 200 µl. The suspension was then added to wells of a 96 well culture plates (ThermoFisher) and incubated for 48 h at 37°C and 5% CO2. Larval viability was determined under a light microscope 48 h after incubation. Parasites those were limpid and straight with no movements were counted as non-viable and if they were still limpid and straight for the next 8 hours at 37°C, they were counted as dead. The live larvae were still active. We followed the method used by B. malayi L3s to neutralize the GST activity in the parasite and measure ADCC.To determine the cytotoxic effects of anti rWbGST antibodies against B. malayi L3 per animal10 days after the last dose of immunization. We decided on the heterologous challenge for three reasons (1) sequence similarity of WbGST and BmGST (2) lack of an animal model for W. bancrofti and (3) due to the fact that W. bancrofti infections are more prevalent in endemic countries than B. malayi.. 120 days after challenge infection, animals were euthanized by intraperitoneal injection of sodium pentabarbitol. Thorax and abdomen were opened to collect adult filarial worms from abdominal cavity, testes, heart, lungs and other organs. Number of adults worms recovered from each animal was recorded.rWbGST immunized and control groups of jirds were challenged intraperitoneally with 100 Statistical analysis was performed using XL STAT software v.7.5.2 . Statistical significance between comparable groups was estimated using appropriate non-parametric tests, with the level of significance set at p<0.05.W. bancrofti  were compared with other filarial parasites such as B. malayi , Onchocerca volvulus  and Dirofilaria immitis . These analyses showed significant sequence identity between the various GSTs . EST database search of W. bancrofti showed the presence of only phi- class of GST and did not show the existence of any other classes of GST. Therefore, based on the sequence data and enzymatic activity we believe that the GST cloned in this study belongs to Phi-class of GST.Multiple sequence alignments of the amino acid sequences of GST family of proteins from ous GSTs . Amino aogeneity . After pHumoral immune responses to rWbGST were determined by western blot analysis and ELISA. Immunoblot analysis confirmed the presence of high levels of anti-WbGST antibodies in pooled EN sera. However, anti-WbGST antibodies were absent in pooled MF and NEN sera . Sero-reLevels of anti-WbGST antibodies were also determined in the sera of mice and jirds by ELISA. Results showed that both mice and jirds immunized with rWbGST developed high antibody titer (1∶10000), whereas, control mice or jirds had no detectable WbGST specific antibodies in their sera. Isotype profile of anti-WbGST IgG antibodies showed that higher levels of both IgG1 (Th2) and IgG2a (Th1) anti-WbGST antibodies are present in immunized animals .GST gene was investigated by PCR using gene-specific forward and reverse primers from the cDNA libraries of the various life stages of the W. bancrofti. This analysis showed that transcripts of the GST gene were present predominantly in the L3 stage of the parasite . The level of inhibition of the GST enzyme activity by EN sera or immune mouse sera was only approximately 50% of NEN or pre-immune sera respectively.To determine whether the GST inhibitory activity in the sera of EN individuals and immune sera was due to anti-WbGST antibodies, we first depleted WbGST specific antibodies from the sera of immunized mice and from pooled sera of EN individuals using rWbGST protein coupled to IMAC column. An ELISA analysis confirmed that all the anti-WbGST antibodies were completely depleted from the sera samples (data not shown). Depletion of anti-WbGST antibodies from the sera samples were also confirmed by western blot analysis (data not shown). The antibody depleted sera were then used in the GST enzymatic assay. These studies showed that depletion of anti-WbGST antibodies from EN sera and from sera of immunized mice resulted in the removal of GST enzyme inhibitory activity from these sera samples. The anti-WbGST antibody depleted sera behaved just like the NEN or pre-immune sera suggesting that the anti-WbGST antibodies were potentially responsible for the GST enzyme neutralizing activity .B. malayi L3 using an in vitro ADCC assay. Results from these studies showed that sera from mice immunized with rWbGST promoted adherence of PEC to L3 larvae  carry antibodies to WbGST in their sera. Majority of the antibodies are either of IgG1 or IgG3 isotype suggesting that cytophilic type of antibodies may have a predominant role in the protection against this infection in human. These individuals also showed significant increases in anti-WbGST IgG4 antibodies similar to CP individuals. In another study also we observed a similar Th2 biased responses following immunization of mice with OvGST-2 of et alF. gigantica infections in mice. Yet studies by Rathaur et alS. cervi GST activity could be inhibited by sera from jirds that are vaccinated with S. cervi GST. Similarly, Bal and Das S.digitata GST activity when incubated with sera from individuals infected with W. bancrofti. Anti-S. mansoni 28 GST IgA antibodies can neutralize the parasite GST activity in human schistosomiasis and can impair in vitro hatching of S. mansoni eggs in vitro ADCC assay. Although this is not a direct measure of protection, it does provide some important information about the potential capabilities of anti-WbGST antibodies. Our in vitro results clearly show that human anti-WbGST antibodies can participate in the killing of infective L3s of B. malayi. Major confirmation of this finding comes from the fact that depletion of anti-WbGST antibodies from the EN sera eliminated the L3 killing effect. Since eliminating L3s are important in preventing the infection, this finding also suggests that anti-WbGST may have a crucial role in protection against this infection in EN individuals. In addition, we also neutralized the activity of BmGST in the parasite by incubating B. malayi L3s with cDNB, a glutathione depleting agent and repeated the ADCC assay using EN sera and PBMCs. Results from these studies show that neutralizing the effect of BmGST in the parasite with cDNB had no inhibitory effect on the ADCC ability of EN sera. Although in this study we did not test whether we neutralized all the parasite GST activity with cDNB, it appears that neutralization of GST activity is not critical for ADCC function. These findings were similar to that reported earlier by Morrison et alOur studies also show that anti-WbGST antibodies in the sera of EN individuals and sera of immunized mice have the capability to neutralize parasite GST function. However, we failed to observe 100% inhibition of GST enzyme activity either with the EN sera or with the immune mice sera. This could be possibly because (1) the antibody titer was not optimized for maximum neutralization of GST activity or (2) possibly the anti-WbGST antibodies could only neutralize approximately 50% of the rWbGST activity. Nevertheless studies by Morrison B. malayi L3s in vitro and depletion of the anti-WbGST antibodies from the mouse sera removed the L3 killing effect from the immune sera. These findings further suggest that GST may be a critical protein for the survival of the B. malayi L3. In our studies we did not identify the effector cells. Given that majority of the anti-WbGST antibodies are of IgG subclass, further studies evaluating the Fcγ bearing cells might help identify the effector cells that participate in the ADCC mechanism.We were able to replicate the ADCC finding using a mouse model as well. The mouse anti-WbGST antibodies were also able to participate in the killing of O. volvulus GST Mouse is not a permissive host in that the worms do not mature into adults. Therefore, we did not conduct any challenge experiments in the mouse model. Instead, we used a jird model where the parasite matures into adult male and female parasites B. malayi that express the GST. Animal studies confirm that immunization with rWbGST can confer protection against a challenge infection. These findings thus suggest WbGST could be potentially developed as a vaccine candidate against lymphatic filarial parasites. Since only L3 stages appear to express the WbGST protein it is likely that this vaccination will be more effective in non-infected individuals as a preventive vaccine.In conclusion, results presented in this study show that putatively immune individuals in an endemic area carry antibodies against WbGST. These antibodies can neutralize the parasite GST activity and kill infective larvae of


Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties.in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[γ] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9–12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A.We performed SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately. Regulated secretion in neurons and endocrine cells is mediated by a specialized version of SNARE-mediated membrane fusion . As shown in To determine the specificity of nucleotide binding by SVOP, we tested the ability of 10-fold excess various nucleotides to compete with 8-azido-ATP labeling. ATP, GTP, TTP, CTP, and NAD all decreased labeling, though ATP, GTP and NAD displayed the strongest inhibition. TTP and CTP produced the least inhibition Figure 3To compare the affinity of SVOP for ATP and NAD, we analyzed the effects of increasing concentrations of these two nucleotides on 8-azido-ATP labeling. As shown in To identify the nucleotide-binding site(s) in SVOP, we first applied chemical cleavage and enzymatic digestion to 8-azido-ATP-labeled SVOP-FLAG. Hydroxylamine cleaves the bond between Asn and Gly, with much less efficient cleavage at Asn-Leu, Asn-Ala, and Asn-Met bonds 1- Val267) or carboxy half (Asp266-Glu548) of SVOP  were assessed for binding to 8-azido-ATP. As with hydroxylamine-cleaved SVOP, apparent molecular sizes of the FLAG tagged SVOP1–267 and SVOP266–548 in SDS-PAGE  differed from the calculated molecular weights of 30.9 and 32.4 kDa, respectively. Our observation that the amino portion of SVOP resolves as a larger protein in SDS-PAGE may reflect incomplete denaturation in SDS. Likewise, the apparent smaller size of carboxy end is consistent with this portion of SVOP being very hydrophobic. As shown in 266–548, bound 8-azido-ATP whereas N-terminal half protein (SVOP1–267 ) showed only minimal binding, confirming that the primary nucleotide binding site in SVOP is located on the carboxy terminal portion of the protein. Given that the N-terminal fragment produced by hydroxylamine cleavage did not show labeling, the very low level labeling in the N-terminal half protein is probably due to the nonspecific binding of 8-azido-ATP.We next assessed binding by recombinant proteins corresponding to the amino and carboxy halves of SVOP. Recombinant FLAG fusion proteins containing the amino (Met1–518) retained the nucleotide binding capability (data not shown). Together these data indicate that the nucleotide-binding site in SVOP is located between a.a. 411–518. We note, however, that the tendency of the carboxy end of SVOP to run smaller in SDS-PAGE suggests that the actual binding site may include residues preceding a.a. 411.Trypsin digestion of photolabeled SVOP further defined the site of 8-azido-ATP attachment to SVOP. As shown in The nucleotide binding sites in SV2 and another MF transporter protein, Glut1, are in cytoplamic domains. To determine if this is also true of SVOP, we mutated residues that are – conserved in SVOP across species - in the loops between membrane domains 8/9 and 10/11. SVOP-E450A, SVOP-Y425A, SVOP-R457A and the double mutant, G398A/R399A, which is analogous to residues required for nucleotide binding to Glut 1 50∼0.25 mM versus ∼0.75 mM for ATP), SV2 shows an equal affinity for both NAD and ATP (∼0.4 mM for both).SV2 and SVOP are transporter-like components of synaptic vesicles that share significant sequence similarity although they constitute separate protein families. SV2 proteins contain features not present in SVOP including the cytoplasmic amino terminus, the cytoplasmic loop between the sixth and seventh transmembrane domains and a luminal glycosylated loop between the seventh and eighth transmembrane domains. In the studies reported here we report that both SVOP and SV2 are nucleotide-binding proteins although both the binding site and nucleotide preference differ between the two proteins. SVOP appears to have a binding site located in a region spanning transmembrane domains 9–12, whereas SV2 has two binding sites in its large cytoplasmic domains preceding transmembrane domains 1 and 7 Nucleotide binding is also a feature of the human glucose transporter (Glut1), another member of the MF transporter family Our studies indicate that nucleotide binding is a shared feature of multiple MF transporters, though this feature appears to have arisen separately in each of the proteins. As more is learned about the molecular actions of SV2 and SVOP, it will be important to compare the effects of nucleotides on these actions to the effects of nucleotides reported for Glut1. The fact that both SV2 and SVOP demonstrate a high affinity for NAD suggests their action may be influenced by the rate of synaptic glycolysis, a process that consumes NAD, or by synaptic redox potential. It will be especially interesting to determine whether the apparent convergent evolution of nucleotide binding in MF transporters has rendered them sensitive to the same cellular conditions.cDNA encoding rat SVOP with the FLAG epitope (DYKDDDK) fused to its C-terminus was subcloned into the mammalian expression vectors pIRES2–EGFP . Constructs encoding N-terminal and C-terminal halves of SVOP protein were generated by PCR amplification of rat SVOP cDNA and subcloned into FLAG-pIRES2–EGFP vector. The QuikChange Site-Directed mutagenesis Kit (Stratagene) was used to generate mutant SVOP constructs. These include single point mutations, E450A, Y452A, R457A, and a double mutation G398A/R399A.GCG GTG TAT CCA ACG GCG ACG AGG-3′5′- TAC ACG CCT , E450A r CGC AGG CGT GTA AAC GTA GGC TGC TTG-3′5′- TGG ATA CAC , Y452A f GCG CCA ACG GCG ACG AGG GCG-3′5′- CCT GAG GTG , Y452A r CGC CAC CTC AGG CGT GTA AAC-3′5′- CGC CGT TGG , R457A f GCG GCG CTG GGC CTG GGC ACC TG-3′5′- A ACG GCG ACG , R457A r CGC CGT CGC CGT TGG ATA CAC CTC5′- GCC CAG CGC , G398A/R399A f GCC GCC AAG AAG ACC ATG GCT CTG-3′5′- GAC CGC CTG , and G398A/R399A r GGC GGC CAG GCG GTC GAT GAC CCA C-3′5′- GGT CTT CTT . f and r denote forward and reverse primers. Underlined letters indicate mismatches. DNA sequencing was performed to confirm that no undesired mutation was introduced by PCR.The primers used include: E450A f HEK293 cell culture and transfection of the cells with the Lipofectamine™ 2000 reagent were performed as previously described SVOP-FLAG fusion protein and its mutants were generated as described previously Dried 8-azido-ATP[γ] biotin  was dissolved in the buffer ). Appropriate amount of 8-azido-ATP[γ] biotin as indicated was mixed with SVOP preparation and incubated on ice in the dark for 2 min. Generally, about 5 µg of SVOP-FLAG protein preparation was used for each labeling reaction and the final volume was 50 µl. After incubation, the samples were irradiated with a hand-held UV lamp at 254 nm  for 2 min. The UV-irradiated samples were immediately diluted with SDS-PAGE sample buffer containing β-mercaptoethanol, and subjected to SDS-PAGE and western blot analysis as described below. For hydroxylamine cleavage or proteolytic digestion experiments, the labeled samples were added with DTT to a final concentration of 40 mM after labeling. For the substrate specificity and competition studies, competitive nucleotides were added into the affinity labeling reactions.SDS-PAGE was performed using either precast 4%–15% gradient gels  or 10% gels. Proteins were electro-transferred onto polyvinylidene difluoride membranes (PVDF)  and subjected to western blot analysis. The primary antibodies used include a monoclonal anti-FLAG M2 antibody  and a polyclonal rabbit anti-SVOP antibody made against N-terminally derived peptides of SVOP (LRQLPVVKFRRTGES). Membranes were then incubated with the appropriate horseradish peroxidase (HRP) conjugated secondary antibody, washed and immune complexes were visualized using an enhanced chemiluminescence detection kit . The 8-azido-ATP[γ] biotin labeled samples were probed with ExtrAvidin-HRP  and visualized by chemiluminescence. The net intensity of the regions of interest was quantified using a Kodak Image Station 440, and only the nonsaturated images were used for quantification analysis. The image of 8-azido-ATP labeling blot with trypsin digested SVOP-FLAG  and incubated at 45°C for 4 h. The cleavage reaction samples were then desalted by passing through Zeba desalt spin columns . Aliquots of non-cleaved and cleaved samples were resolved by SDS-PAGE and subjected to western blot analysis with anti-FLAG, anti-SVOP and ExtrAvidin-HRP as described above.8-azido-ATP[γ] biotin labeled SVOP- FLAG protein was digested at 37°C in the presence of sequencing grade trypsin (30∶1 protein: enzyme by weight) . At the time periods indicated, an aliquot was withdrawn and the digestion terminated by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1 mM. The samples were subjected to SDS-PAGE and western blot analysis.


LMP-420 is a boronic acid-containing purine nucleoside analogue that transcriptionally inhibits TNF production but is non-cytotoxic to TNF-producing cells.This study investigated the efficacy of LMP-420 as an anti-inflammatory agent in acute and chronic colitis induced by oral administration of dextran sulfate sodium (DSS) to mice and in chronic colitis following piroxicam administration to IL-10-deficient mice. The severity of colon inflammation was assessed histologically. TNF levels were measured by enzyme immunoassay.-/- mice deficient in T and B cells also developed severe ongoing colitis in response to 3 cycles of DSS, but showed marked differences vs. wild type mice in stool TNF and TNF-RII in response to DSS exposure. Systemic and oral LMP-420 treatment for 16 days decreased colonic TNF levels in IL-10-deficient mice with chronic colitis, with a trend to decreased histologic inflammation for oral LMP-420.Administration of DSS for 7 days resulted in severe acute colitis that was associated with a marked increase in stool and colon tissue TNF levels. Initiation of therapy with intraperitoneal (i.p.) LMP-420 on day 4 of DSS exposure decreased colonic TNF to near normal levels on day 7. However, neither i.p. nor oral treatment with LMP-420 affected the development or severity of acute DSS colitis. Initiation of LMP-420 therapy after 3 cycles of DSS administration to establish chronic colitis also had no effect on the severity of chronic colitis. Analysis of colonic TNF combined with longitudinal analysis of TNF and TNF receptor (TNF-RII) levels in stool during the development of chronic DSS colitis demonstrated that the initially elevated colonic TNF levels returned to normal despite intense on-going inflammation in mice with chronic colitis. RAG-2These studies demonstrate that short-term treatment with a transcriptional inhibitor of TNF production can decrease systemic and local colonic levels of TNF but may not decrease the histologic severity of colitis. Longer term studies using colitis models that are more dependent on TNF elevation should be performed to more accurately assess the potential of LMP-420 for therapy of inflammatory bowel disease. Inflammatory bowel diseases such as Crohn's disease (CD) and ulcerative colitis (UC) are hypothesized to result from abnormal immune responses to antigens derived from intestinal microbiota  that in vitro in the presence or absence of complement . Using Stool for cytokine analysis was collected before DSS exposure began (day 0), at the end of each 5 day cycle of DSS administration . Freshly obtained stool was kept on ice until it was homogenized at 100 mg stool/ml buffer in PBS containing 1% bovine serum albumin, 0.1% Kathon , and Protease Inhibitor Cocktail . Insoluble material was removed by centrifugation at 4°C at 15,000 × g in a microfuge for 10 min. Stool extracts were filtered at 0.2 μm then stored at -20°C until assayed. Fresh colon samples were homogenized at 100 mg tissue/ml buffer using a PowerGen 125 tissue grinder  and the BioPlex Cell Lysis Kit  according to the manufacturer's instructions. Colon tissue extracts were then frozen at -20°C until analysis. TNF and TNF-RII were quantitated in tissue culture supernatants, stool, and colon tissue extracts by enzyme immunoassay using Duo-Set reagents . Results were expressed as pg/ml for culture supernatants or as pg/100 mg tissue or stool.Statistical comparison of groups was performed using Student's t test or analysis of variance (ANOVA). A value of p ≤ 0.05 was considered to be significant.in vitro to macrophages and lymphocytes, cell types that are present in the colonic lamina propria  by LPS-stimulated murine monocytes in vitro is ~500 nM . The molecular basis for the ~10-fold increased sensitivity of human vs. murine cells to LMP-420 is unclear, but suggests that LMP-420 is likely to have greater anti-inflammatory activity in humans than in mice.LMP-420 very effectively inhibits production of TNF mRNA and protein when applied topically in vitro profile, LMP-420 was hypothesized to be an efficient inhibitor of TNF in vivo. The degree of systemic and colonic TNF blockade that resulted from different LMP-420 in vivo dosing protocols was first assessed in wild type mice using a bacterial lipopolysaccharide (LPS) challenge model. Mice treated with 2 mg (100 mg/kg) LMP-420 given intraperitoneally (i.p.) experienced transient behavioral depression consistent with hypotension, but recovered to normal behavior within 20 minutes after injection. TNF was below the limit of detection (< 10 pg/ml) in the serum of mice not challenged with LPS. As expected, serum TNF levels were markedly increased to 1109 ± 87 pg/ml  upon LPS challenge. However, pre-treatment with i.p. LMP-420 decreased LPS-induced serum TNF levels by 42%  and total colonic TNF levels  compared to mice who were not exposed to DSS  (p = 0.03). DSS-exposed mice that were treated with i.p. LMP-420 demonstrated a significant (85%) decrease in colonic TNF levels , that represented near normalization of their colonic levels of TNF compared with control mice that were not exposed to DSS. TNF was not detected in the serum of either vehicle- or LMP-420-treated DSS-exposed or control mice at this time point.The LPS challenge experiments demonstrated that both i.p. and orally-administered LMP-420 significantly inhibited Despite the efficacy of i.p. LMP-420 in decreasing colonic levels of TNF induced by DSS exposure, there was no difference in clinical or histologic severity of colitis observed on day 7 in mice treated i.p. with vehicle- vs. LMP-420 (data not shown). However, the 3 day treatment period used was likely too short to allow healing. Longer treatments were not possible in the acute DSS model due to severe weight loss that required euthanasia for humane reasons. To determine if LMP-420 treatment could prevent the development of acute DSS colitis, LMP-420 was administered i.p. for 5 days prior to as well as throughout the 7 day exposure to DSS. Oral administration of LMP-420 was not used for these studies, since mice rapidly decrease their food consumption when acute colitis develops and it was not possible to maintain a consistent oral dose using drug mixed with food. Systemic (i.p.) treatment with LMP-420 given prior to and during DSS exposure did not alter the clinical or histologic severity of colitis Figure . The sevNext, we determined whether LMP-420 treatment would influence the severity of chronic colitis initiated by multiple cycles of DSS exposure. Treatment with a dose range of LMP-420 was begun immediately upon discontinuation of the 3d DSS cycle when all mice had severe chronic colitis. The LMP-420 dose range was 0, 5, 15, 45 mg/kg/day for i.p. administration and 0, 26, 63, and 145 mg/kg/day for oral administration. Mice were euthanized after 16 days of treatment to determine colonic TNF content and the histologic severity of colitis. Although gross inflammation  was subjectively decreased in LMP-420-treated mice, there was no difference in histologic scores between treated and untreated mice Figure . DespiteThe effects of LMP-420 on TNF production was profound in acute DSS colitis, but LMP-420 therapy apparently had no effect on inflammation severity in either acute or established chronic DSS colitis. Furthermore, colonic TNF levels were not elevated in untreated control mice with chronic DSS colitis, despite the presence of very severe colonic inflammation . To begin to understand these observations, a longitudinal study of the levels of TNF in the stool was performed. Stool samples were obtained prior to the initial DSS exposure (day 0), at the end of each 5 day cycle of DSS administration , and after 16 days of healing prior to beginning the next cycle of DSS administration . Because levels of soluble TNF receptors increase during inflammation due to receptor shedding after binding TNF and/or increased alternative splicing that generates the soluble form, the levels of TNF-RII  in stool were also measured. In wild type C57BL/6 mice, TNF levels in stool were significantly increased compared to baseline levels at the end of each cycle of DSS administration. Stool TNF then spontaneously decreased to normal levels by the end of each 16 day period of recovery Figure . Stool l-/- mice that lack both T and B cells were exposed to the same regimen of 3 cycles of DSS that induced severe chronic colitis in wild type mice, then euthanized 16 days following the 3rd administration of DSS. All RAG-2-/- mice had severe colitis after exposure to 3 cycles of DSS, with histologic scores of 53 ± 1 . These scores were statistically similar to those observed in wild type mice exposed to a similar regimen of 3 DSS cycles, however the histologic picture was very different. In contrast to the marked mucosal hyperplasia seen in wild type mice, RAG-2-/- mice exhibited minimal mucosal hyperplasia. Mucosal histologic sub-scores were high nonetheless, due to marked squamous metaplasia in the rectum and architectural distortion throughout the colon, manifested primarily by crypt branching and crypt dropout. Inflammatory infiltrates in the RAG-2-/- mice consisted of a small number of mononuclear cells and a moderate to large number of neutrophils, and these infiltrates were more uniformly spread through the tissues. Frank ulcerations and crypt abcesses were present, but less common compared with the wild type mice.Acute DSS colitis has been shown to occur in the absence of T and B lymphocytes , however-/- mice initially developed higher mean levels of TNF in their stool following DSS exposure, compared to wild type mice . The TNF levels in colon tissue measured in a subset of these mice (n = 5) was 72 ± 3 pg/100 mg tissue, which is similar to that seen in control wild type mice without colitis  or oral LMP-420  for 16 days. Effects on colonic TNF and histologic severity of colitis were determined. Colonic levels of TNF were elevated in untreated IL-10-/- mice with colitis compared with wild type mice without colitis  and colonic TNF responses (-67%) to challenge with a lethal dose of LPS. LMP-420 treatment also reduced (-85%) TNF elevations associated with acute DSS colitis to near baseline levels. However, despite its efficacy in reducing TNF levels in vivo, LMP-420 had no effect on the histologic severity of colonic inflammation in response to either acute or chronic DSS administration. A trend toward decreased inflammation (p = 0.06) was observed in IL-10-/- mice treated orally with LMP-420, which correlated with decreased colonic TNF levels. Histologic healing of colonic inflammatory lesions is known to lag behind clinical remission and endoscopic healing. Thus, it is possible that LMP-420 treatments of longer durations might result in beneficial clinical effects that were not observed in this pilot study using short treatment periods.The novel small molecule drug LMP-420 is highly effective in inhibiting TNF production both in vivo. Spohn et al. recently showed that anti-TNF antibodies that bound to both membrane-bound and soluble forms of TNF had a greater anti-inflammatory effect in a murine model of rheumatoid arthritis than antibodies reactive only with soluble TNF [The availability of LMP-420, a small-molecule, orally-active inhibitor of TNF production, provided us an attractive opportunity to define the potential role of TNF in the murine model of DSS-induced colitis, a commonly-used model of inflammatory bowel disease. Although we were able to demonstrate significant inhibition of colon TNF by LMP-420 in this model, we had no effect on the pathological inflammation. However, in contrast to the biological TNF antagonists currently used in humans which are capable of "neutralizing" essentially all circulating TNF, LMP-420 allowed ~20% of colon TNF to be produced in our model. Thus, while our data might be interpreted to suggest that TNF does not play a significant role in this model, we cannot at this time rule out the possibility that the small amount of TNF produced was sufficient to induce pathogenesis. Alternatively, prevention of TNF synthesis without toxicity to TNF-producing cells may not be sufficient to stop the inflammatory cascade uble TNF . Furtheruble TNF . Thus, iuble TNF  despite et al, who previously reported that TNF was not detectable in colon tissue or plasma of CBA/J mice with acute DSS colitis, and that a polyclonal anti-TNF antiserum had no effect on disease severity [et al showed that TNF-deficient mice have increased intestinal inflammation in response to DSS compared with wild type mice [Levels of TNF excreted in the stool correlated well with levels of TNF measured in colon tissues harvested at the time of euthanasia, providing a method to follow colonic TNF levels non-invasively. Longitudinal measurements of TNF in the stool of mice subjected to multiple cycles of DSS demonstrated that, although TNF levels are elevated in acute DSS colitis, these levels decrease spontaneously and eventually return to baseline despite ongoing severe inflammation. Taken together with the lack of efficacy of LMP-420 in acute or chronic DSS colitis despite its ability to significantly lower colonic TNF levels, these data suggest that elevated levels of TNF are not required for the initiation and maintenance of colonic inflammation in this murine model. Our data thus is similar to that of Olson severity . Howeverype mice . These c-/- mice, in contrast to the markedly higher TNF-RII levels observed in stool of wild type mice subjected to multiple cycles of DSS. The manufacturer reports that the presence of TNF-RII does not affect the ability of its TNF ELISA assay to quantitiate TNF. Thus additional mechanisms beyond simple shedding of receptor that has bound TNF likely account for the marked and sustained increase in stool TNF-RII during chronic DSS colitis in wild type mice. The induction of severe chronic colitis following repeated DSS administration to RAG-2-/- mice that lack T and B cells suggests that innate immunity is sufficient to drive the development of chronic colitis in the DSS model. Use of other models that are TNF-dependent or driven by induced T cell responses will be necessary to determine if LMP-420 may have efficacy in maintaining remission of chronic colitis driven by TNF or T cells.Our studies showed that levels of TNF and TNF-RII in the stool are highly correlated for RAG-2-/- mice. The presence of squamous mucosa is typically limited to the anus in both humans and mice. The simple columnar epithelium that is normally present in the terminal colon and rectum functions to absorb fluid from stool as well as to secrete mucus to lubricate its passage. These functions would be missing from metaplastic squamous epithelium. By analogy with other organs, it is possible that mice with extensive squamous metaplasia of the colon might be at increased long-term risk for development of squamous carcinomas of the colon rather than the adenocarcinomas that typically develop at this site. Longer term studies will be needed to address this possibility.The induction of extensive squamous metaplasia in the terminal colon and rectum by multiple cycles of DSS administration is an interesting pathologic observation that is of uncertain clinical significance. Metaplasia has been observed in a range of organs and is thought to occur in response to chronic irritation . T and B-/- model of murine colitis. As we saw for acute DSS colitis, LMP-420 treatment  significantly decreased colonic TNF levels in a setting where TNF was elevated in the colons of mice that did not receive this drug. However, again we saw no statistically significant decreases in severity of colitis as measured histologically, although there was a trend to decreased histologic inflammation for mice that received 41 mg/kg oral LMP-420. Increased potential effect of oral as compared with systemically administered drug suggests that LMP-420 may exhibit local anti-inflammatory activity as it passes through the gastrointestinal tract. Lack of TNF lowering in the IL-10-/- model at higher LMP-420 doses indicates a complex dose-response profile that may reflect dual activity of LMP-420 in competing inflammatory/anti-inflammatory pathways.The effect of LMP-420 was additionally studied in the IL-10-/- mice. A detailed dose-response study and longer treatment durations using other colitis models that are more dependent on TNF elevation should be performed to more accurately assess the potential of LMP-420 for therapy of inflammatory bowel disease.Taken together, these studies demonstrate that short-term treatment with a transcriptional inhibitor of TNF production does not decrease the severity of acute and chronic DSS colitis or piroxicam-accelerated colitis in IL-10BSA: bovine serum albumin; CD: Crohn's disease; DSS: dextran sulfate sodium; FBS: fetal bovine serum; i.p.: intraperitoneal; IBD: inflammatory bowel disease; IFN: interferon; IL: interleukin; LPS: lipopolysaccharide; MCP: monocyte chemoattractant protein; PBS: phosphate buffered saline; SD: standard deviation; SEM: standard error of the mean; TNF: tumor necrosis factor; TNF-RII: tumor necrosis factor receptor, type II; UC: ulcerative colitis.LPH has no competing interests. GJC also has no competing interests, but discloses that he was a co-discoverer of LMP-420 and was associated with LeukoMed Inc (the company that holds the license for LMP-420) until October 2005.LPH conceived of and designed the studies, obtained funding, performed the pathologic and data analyses, prepared figures, and drafted the manuscript. GJC assisted in study design, reviewed data, prepared figures, and helped to draft the manuscript. All authors read and approved the final manuscript.