Prepared and screened a modified TNF-alpha molecule as TNF-alpha autovaccine to treat LPS induced endotoxic shock and TNF-alpha induced cachexia in mouse

Overexpression of TNF-alpha in the body is critically involved in many diseases. A strategy to construct TNF-alpha autovaccine by introducing a T cell helper epitope to the protein has been developed and may be an alternative because it is cheaper and highly efficient. However, the induction of high level anti-TNF-alpha neutralizing autoantibodies by TNF-alpha autovaccine is depend on a proper T cell help epitope. In order to evaluate the effect of different T helper cell epitopes on the immunogenicity of mouse TNF-alpha (mTNF-alpha), three T helper cell epitopes, TT (QYIKANSKFIGITEL), HEL (NTDGSTDYGILQINSR), and PADRE (AKFVAAWTLKA), were chosen for this study. The sequence (amino acids 126-140) of mTNF-alpha was replaced with those of the T cell help epitopes, respectively. The three fusion proteins (mTNF-TT, mTNF-HEL, mTNF-PADRE) were expressed in Escherichia coli and purified with a simple strategy. The abilities of the proteins elicited TNF-alpha autoantibodies in BALB/c mice were investigated. The results showed that mTNF-PADRE is the most effective among the three modified TNF-alpha molecules. In the absence of adjuvant, the therapeutic effect of TNF-PADRE on LPS induced endotoxic shock mice and mTNF-alpha induced cachexia mice was observed. This study suggests that mTNF-PADRE may be a better candidate of mTNF-alpha autovaccine.     

Spatially distinct functions of Clb2 in the DNA damage response

In budding yeast four mitotic cyclins (Clb1–4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G₂/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.     

B cells and TCR avidity determine distinct functions of CD4+ T cells in retroviral infection

The T cell-dependent B cell response relies on cognate interaction between B cells and CD4(+) Th cells. However, the consequences of this interaction for CD4(+) T cells are not entirely known. B cells generally promote CD4(+) T cell responses to pathogens, albeit to a variable degree. In contrast, CD4(+) T cell responses to self- or tumor Ags are often suppressed by B cells. In this study, we demonstrated that interaction with B cells dramatically inhibited the function of virus-specific CD4(+) T cells in retroviral infection. We have used Friend virus infection of mice as a model for retroviral infection, in which the behavior of virus-specific CD4(+) T cells was monitored according to their TCR avidity. We report that avidity for Ag and interaction with B cells determine distinct aspects of the primary CD4(+) T cell response to Friend virus infection. Virus-specific CD4(+) T cells followed exclusive Th1 and T follicular helper (Tfh) differentiation. High avidity for Ag facilitated expansion during priming and enhanced the capacity for IFN-γ and IL-21 production. In contrast, Tfh differentiation was not affected by avidity for Ag. By reducing or preventing B cell interaction, we found that B cells promoted Tfh differentiation, induced programmed death 1 expression, and inhibited IFN-γ production by virus-specific CD4(+) T cells. Ultimately, B cells protected hosts from CD4(+) T cell-mediated immune pathology, at the detriment of CD4(+) T cell-mediated protective immunity. Our results suggest that B cell presentation of vaccine Ags could be manipulated to direct the appropriate CD4(+) T cell response.     

Pleiotropic functions of a conserved insect-specific Hox peptide motif

The proteins that regulate developmental processes in animals have generally been well conserved during evolution. A few cases are known where protein activities have functionally evolved. These rare examples raise the issue of how highly conserved regulatory proteins with many roles evolve new functions while maintaining old functions. We have investigated this by analyzing the function of the ;QA' peptide motif of the Hox protein Ultrabithorax (Ubx), a motif that has been conserved throughout insect evolution since its establishment early in the lineage. We precisely deleted the QA motif at the endogenous locus via allelic replacement in Drosophila melanogaster. Although the QA motif was originally characterized as involved in the repression of limb formation, we have found that it is highly pleiotropic. Curiously, deleting the QA motif had strong effects in some tissues while barely affecting others, suggesting that QA function is preferentially required for a subset of Ubx target genes. QA deletion homozygotes had a normal complement of limbs, but, at reduced doses of Ubx and the abdominal-A (abd-A) Hox gene, ectopic limb primordia and adult abdominal limbs formed when the QA motif was absent. These results show that redundancy and the additive contributions of activity-regulating peptide motifs play important roles in moderating the phenotypic consequences of Hox protein evolution, and that pleiotropic peptide motifs that contribute quantitatively to several functions are subject to intense purifying selection.     

Pleiotropic functions of EAPII/TTRAP/TDP2

EAPII (also called TTRAP, TDP2), a protein identified a decade ago, has recently been shown to function as an oncogenic factor. This protein was also proven to be the first 5’- tyrosyl-DNA phosphodiesterase. EAPII has been demonstrated to have promiscuous protein associations, broad responsiveness to various extracellular signals, and pleiotropic functions in the development of human diseases including cancer and neurodegenerative disease. Emerging data suggest that EAPII is a multi-functional protein: EAPII repairs enzyme (topoisomerase)-mediated DNA damage by removing phosphotyrosine from DNA adducts; EAPII is involved in multiple signal transduction pathways such as TNF-TNFR, TGFβ and MAPK, and EAPII is responsive to immune defense, inflammatory response, virus infection and DNA toxins (chemo or radiation therapy). This review focuses on the current understanding of EAPII biology and its potential relations to many aspects of cancer development, including chromosome instability, tumorigenesis, tumor metastasis and chemoresistance, suggesting it as a potential target for intervention in cancer and other human diseases.     

Pleiotropic effects of positively charged liposomes on immune functions

Summary Positively charged liposomes have been shown to inhibit the proliferation of lymphocytes induced by various polyclonal activators. We demonstrated that this inhibition is essentially restricted to early phases of activation. B cell proliferation, induction of suppressor cells, and cytotoxic activities are all profoundly inhibited, whereas T4+ cells response to mitogenic stimulation is only moderately affected. The results are discussed in terms of membrane perturbations potentially induced by liposome-lymphocyte interactions. This work has been supported by grants from the National Sciences and Engineering Council of Canada.     

Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-alpha causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-alpha (200 U/ml) for 18 h. Western blot analysis indicated that TNF-alpha decreased the amount of monomeric FN detected under reducing conditions. Analysis of (125)I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P < 0.05). Enhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity (P < 0.05). Calf pulmonary artery endothelial cells pretreated with TNF-alpha also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-alpha-induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF-alpha exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability.     

TNF-alpha production in the cornea in response to Pseudomonas aeruginosa challenge

Pseudomonas aeruginosa can cause ulcerative bacterial keratitis or contact lens-induced acute red eye (CLARE) in humans. The present study used a mouse model of ocular infection and inflammation to examine the relationship between TNF-alpha and inflammation in the cornea in response to challenge with either a strain of P. aeruginosa causing keratitis or a CLARE strain. Constitutive TNF-alpha mRNA was detected in the epithelium, mainly towards the periphery. After infection with the keratitis-inducing strain (6294), TNF-alpha expression was elevated four-fold by 24 h post-challenge. No detectable induction of TNF-alpha mRNA was seen with CLARE strain (Paer1) challenge at any time point. The TNF-alpha protein production detected by ELISA showed a corresponding pattern to the mRNA expression, which also correlated with pathological changes. These results suggest that invasive strains of P. aeruginosa create greater pathological changes as a result of elevated TNF-alpha production, which contributes to inflammation during keratitis in vivo.     

Selective sensitivity to hydrocortisone of regulatory functions that determine the magnitude of the antibody response to type III pneumococcal polysaccharide.

Previous studies on the basis for the immunosuppressive potential of adrenal corticosteroids have stressed that the effects of these agents on immune functions depend on the animal species being considered, as well as the subpopulations of lymphocytes involved in the expression of immune functions examined. In the present work, we have evaluated the effect of a single dose of hydrocortisone on three different immunoregulatory functions that can influence the magnitude of an antibody response to Type III pneumococcal polysaccharide (SSS-III) in mice; these functions include suppressor, amplifier, and helper activity that are dependent upon the presence of distinct subpopulations of thymus-derived (T) cells. The results obtained show that a single injection of a relatively large dose of hydrocortisone, when given at the time of priming with carrier, eliminated all evidence of carrier-specific helper T cell activity; hydrocortisone was also found to eliminate a significant amount of helper T cell activity when given after such activity had been generated. But, under the same experimental conditions, suppressor and amplifier T cell activities were unaffected, even in this steroid-sensitive species. Such selective sensitivity may account for some of the immunosuppressive potency of steroids.     

The spleen modulates the febrile response of guinea pigs to LPS

The febrile responses of splenectomized (Splex) or sham-operated (Sham) guinea pigs challenged intravenously or intraperitoneally with lipopolysaccharide (LPS) 7 and 30 days after surgery were evaluated. FITC-LPS uptake by Kupffer cells (KC) was additionally assessed 15, 30, and 60 min after injection. LPS at 0.05 microg/kg iv did not evoke fever in Sham animals but caused a 1.2 degrees C core temperature (T(c)) rise in the Splex animals. LPS at 2 microg/kg iv induced a 1.8 degrees C greater T(c) rise of the Splex animals than of their controls. LPS at 2 and 8 microg/kg ip 7 days postsurgery induced 1.4 and 1.8 degrees C higher fevers, respectively, in the Splex than Sham animals. LPS at 2 and 8 microg/kg ip 30 days postsurgery also increased the febrile responses of the asplenic animals by 1.6 and 1.8 degrees C, respectively. FITC-LPS at 7 days was detected in the controls within KC 15 min after its administration; the label density was reduced at 30 min and almost 0 at 60 min. In the Splex group, in contrast, the labeling was significantly denser and remained unchanged through all three time points; this effect was still present 30 days after surgery. Similar results were obtained at 60 min after FITC-LPS intraperitoneal injection. Gadolinium chloride pretreatment (-3 days) of the Splex group significantly reduced both their febrile responses to LPS (8 microg/kg ip) and their KC uptake of FITC-LPS 7 days postsurgery. Thus splenectomy increases the magnitude of the febrile response of guinea pigs and the uptake of systemically administered LPS.     

Divergent response to LPS and bacteria in CD14-deficient murine macrophages

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.     

Metyrapone restores the febrile response to Escherichia coli LPS in pregnant rats

Fever, an important component of the host's defense response to immune challenge, is absent or attenuated in rats near the term of pregnancy. The present experiments were carried out to determine the role of endogenous glucocorticoids in mediating the altered core temperature (Tc) response to exogenous pyrogen (i.e., Escherichia coli LPS). For the experiments, metyrapone-a glucocorticoid synthesis inhibitor-was administered to near-term pregnant rats prior to an EC(100) dose of E. coli LPS. Administration of LPS following vehicle elicited a significant corticosterone response and resulted in a decrease in Tc (i.e., hypothermia). Prior administration of metyrapone, however, which abolished the corticosterone response and altered the pyrogenic/cryogenic cytokine response to LPS, eliminated hypothermia and restored the febrile response. Our results provide evidence that endogenous glucocorticoids play a role in mediating the altered febrile response to immune stimuli observed in rats near the term of pregnancy.     

Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions

Macrophages display remarkable plasticity, allowing these cells to adapt to changing microenvironments and perform functions as diverse as tissue development and homeostasis, inflammation, pathogen clearance and wound healing. Macrophage activation can be triggered by Th1 cytokines and pathogen-associated or endogenous danger signals, leading to the formation of classically activated or M1 macrophages. On the other hand, anti-inflammatory mediators, including IL-4, IL-10, TGF-β and M-CSF, induce diverse anti-inflammatory types of macrophages, known under the generic term M2. In human breast carcinomas, tumor-associated macrophage (TAM) density correlates with poor prognosis. In mouse models of breast cancer, eliminating macrophages from the tumor site, either via genetic or therapeutic means, results in retarded tumor progression. Over the years, multiple signals from the mammary tumor microenvironment have been reported to influence the TAM phenotype and TAM have been propagated as anti-inflammatory M2-like cells. Recent developments point to the existence of at least two distinct TAM subpopulations in mammary tumors, based on a differential expression of markers such as CD206 or MHC II and different in vivo behaviour: perivascular, migratory TAM which are less M2-like, and sessile TAM found at tumor-stroma borders and/or hypoxic regions that resemble more M2-like or "trophic" macrophages. Hence, a further refinement of the molecular and functional heterogeneity of TAM is an avenue for further research, with a potential impact on the usefulness of these cells as therapeutic targets.     

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.