Inhibition of endogenous antagonists with an engineered BMP-2 variant increases BMP-2 efficacy in rat femoral defect healing

Bone morphogenetic proteins (BMP) have been used successfully by orthopedic clinicians to augment bone healing. However, these osteoinductive proteins must be applied at high concentrations to induce bone formation. The limited therapeutic efficacy may be due to the local expression of BMP antagonists such as Noggin that neutralize exogenous and endogenous BMPs. If so, inhibiting BMP antagonists may provide an attractive option to augment BMP induced bone formation. The engineered BMP-2 variant L51P is deficient in BMP receptor type I binding, but maintains its affinity for BMP receptor type II and BMP antagonists including Noggin, Chordin and Gremlin. This modification makes L51P a BMP receptor-inactive inhibitor of BMP antagonists. We implanted β-tricalcium phosphate ceramics loaded with BMP-2 and/or L51P into a critical size defect model in the rat femur to investigate whether the inhibition of BMP antagonist with L51P enhances the therapeutic efficacy of exogenous BMP-2. Our study reveals that L51P reduces the demand of exogenous BMP-2 to induce bone healing markedly, without promoting bone formation directly when applied alone.

Predictors of inflammation in response to anthracycline-based chemotherapy for breast cancer

Although chemotherapy for breast cancer can increase inflammation, few studies have examined predictors of this phenomenon. This study examined potential contributions of demographics, disease characteristics, and treatment regimens to markers of inflammation in response to chemotherapy for breast cancer. Thirty-five women with stage I-III-A breast cancer (mean age 50 years) were studied prior to cycle 1 and prior to cycle 4 of anthracycline-based chemotherapy. Circulating levels of inflammatory markers with high relevance to breast cancer were examined, including C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), Interleukin-1 receptor antagonist (IL1-RA), vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule-1 (sICAM-1), Interleukin- (IL-6), soluble P-selectin (sP-selectin), and von Willebrand factor (vWf). Chemotherapy was associated with elevations in VEGF (p < or = 0.01), sICAM-1 (p < or = 0.01), sP-selectin (p < or = 0.02) and vWf (p < or = 0.05). Multiple regression analysis controlling for age and body mass index (BMI) showed that higher post-chemotherapy levels of inflammation were consistently related to higher pre-chemotherapy levels of inflammation (ps < or =0.05) as well as to certain disease characteristics. Post-chemotherapy IL-6 levels were higher in patients who had larger tumors (p < or = 0.05) while post-chemotherapy VEGF levels were higher in patients who had smaller tumors (p < or = 0.05). Post-chemotherapy sP-selectin levels were highest in women who had received epirubicin, cytoxan, 5-fluorouracil chemotherapy (p < or = 0.01). These findings indicate that chemotherapy treatment can be associated with elevations in certain markers of inflammation, particularly markers of endothelial and platelet activation. Inflammation in response to chemotherapy is most significantly related to inflammation that existed prior to chemotherapy but also potentially to treatment regimen and to certain disease characteristics.

IL1B and DEFB1 Polymorphisms Increase Susceptibility to Invasive Mold Infection After Solid Organ Transplantation.

BACKGROUND  Single nucleotide polymorphisms (SNPs) in immune genes have been associated with susceptibility to invasive mold infection (IMI) among hematopoietic stem cell (HSCT) but not solid organ transplant (SOT) recipients. METHODS  24 SNPs from systematically selected genes were genotyped among 1101 SOT recipients (715 kidneys, 190 liver, 102 lungs, 79 hearts, 15 other) from the Swiss Transplant Cohort Study. Association between SNPs and the endpoint were assessed by log-rank test and Cox regression models. Cytokine production upon Aspergillus stimulation was measured by ELISA in PBMCs from healthy volunteers and correlated with relevant genotypes. RESULTS  Mold colonization (N=45) and proven/probable IMI (N=26) were associated with polymorphisms in interleukin-1 beta (IL1B, rs16944; log-rank test, recessive mode, colonization P=0.001 and IMI P=0.00005), interleukin-1 receptor antagonist (IL1RN, rs419598; P=0.01 and P=0.02) and β-defensin-1 (DEFB1, rs1800972; P=0.001 and P=0.0002, respectively). The associations with IL1B and DEFB1 remained significant in a multivariate regression model (IL1B rs16944 P=0.002; DEFB1 rs1800972 P=0.01). Presence of two copies of the rare allele of rs16944 or rs419598 was associated with reduced Aspergillus-induced IL-1β and TNFα secretion by PBMCs. CONCLUSIONS  Functional polymorphisms in IL1B and DEFB1 influence susceptibility to mold infection in SOT recipients. This observation may contribute to individual risk stratification.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity.

Alternative genetic pathways in colorectal carcinogenesis

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER− subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER− and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-ras mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the p53 gene mutation was less frequently detected, the adenomatous polyposis coli (APC) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of APC and tumor growth factor β receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER− tumors may be more different than previously thought.

Approaches to enhancing the retroviral transduction of human synoviocytes

This report concerns a clinical trial for rheumatoid arthritis (RA), approved by the US National Institutes of Health and the Food and Drug Administration. An amphotropic retrovirus (MFG-IRAP) was used ex vivo to transfer a cDNA encoding human interleukin-1 receptor antagonist (IL-1Ra) to synovium. The protocol required the transduced cells to secrete at least 30 ng IL-1Ra/106 cells per 48 h before reimplantation. Here we have evaluated various protocols for their efficiency in transducing cultures of human rheumatoid synoviocytes. The most reliably efficient methods used high titer retrovirus (approximately 108 infectious particles/ml). Transduction efficiency was increased further by exposing the cells to virus under flow-through conditions. The use of dioctadecylamidoglycylspermine (DOGS) as a polycation instead of Polybrene (hexadimethrine bromide) provided an additional small increment in efficiency. Under normal conditions of static transduction, standard titer, clinical grade retrovirus (approximately 5 × 105 infectious particles/ml) failed to achieve the expression levels required by the clinical trial. However, the shortfall could be remedied by increasing the time of transduction under static conditions, transducing under flow-through conditions, or transducing during centrifugation.

Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway.

Plants can recognize and resist invading pathogens by signaling the induction of rapid defense responses. Often these responses are mediated by single dominant resistance genes (R genes). The products of R genes have been postulated to recognize the pathogen and trigger rapid host defense responses. Here we describe isolation of the classical resistance gene N of tobacco that mediates resistance to the well-characterized pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator (Ac) transposon. We confirmed isolation of the N gene by complementation of the TMV-sensitive phenotype with a genomic DNA fragment. Sequence analysis of the N gene shows that it encodes a protein with an amino-terminal domain similar to that of the cytoplasmic domains of the Drosophila Toll protein and the interleukin 1 receptor in mammals, a putative nucleotide-binding site and 14 imperfect leucine-rich repeats. The presence of these functional domains in the predicted N gene product is consistent with the hypothesis that the N resistance gene functions in a signal transduction pathway. Similarities of N to Toll and the interleukin 1 receptor suggest a similar signaling mechanism leading to rapid gene induction and TMV resistance.

Fine mapping of the tomato I-3 gene for fusarium wilt resistance and elimination of a co-segregating resistance gene analogue as a candidate for I-3

The I-3 gene from the wild tomato species Lycopersicon pennellii confers resistance to race 3 of the devastating vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici. As an initial step in a positional cloning strategy for the isolation of I-3, we converted restriction fragment length polymorphism and conserved orthologue set markers, known genes and a resistance gene analogue (RGA) mapping to the I-3 region into PCR-based sequence characterised amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers. Additional PCR-based markers in the I-3 region were generated using the randomly amplified DNA fingerprinting (RAF) technique. SCAR, CAPS and RAF markers were used for high-resolution mapping around the I-3 locus. The I-3 gene was localised to a 0.3-cM region containing a RAF marker, eO6, and an RGA, RGA332. RGA332 was cloned and found to correspond to a putative pseudogene with at least two loss-of-function mutations. The predicted pseudogene belongs to the Toll interleukin-1 receptor-nucleotide-binding site-leucine-rich-repeat sub-class of plant disease resistance genes. Despite the presence of two RGA332 homologues in L. esculentum, DNA gel blot and PCR analysis suggests that no other homologues are present in lines carrying I-3 that could be alternative candidates for the gene.

A critical role for the parabrachial nucleus in generating central nervous system responses elicited by a systemic immune challenge

Using Fos immunolabelling as a marker of neuronal activation, we investigated the role of the parabrachial nucleus in generating central neuronal responses to the systemic administration of the proinflarnmatory cytokine interleukin-1beta (1 mug/kg, i.a.). Relative to intact animals, parabrachial nucleus lesions significantly reduced the number of Fos-positive cells observed in the central amygdala (CeA), the bed nucleus of the stria terminalis (BNST), and the ventrolateral medulla (VLM) after systemic interleukin-1beta. In a subsequent experiment in which animals received parabrachial-directed deposits of a retrograde tracer, it was found that many neurons located in the nucleus tractus solitarius (NTS) and the VLM neurons were both retrogradely labelled and Fos-positive after interleukin-1beta administration. These results suggest that the parabrachial nucleus plays a critical role in interleukin-1beta-induced Fos expression in CeA, BNST and VLM neurons and that neurons of the NTS and VLM may serve to trigger or at least influence changes in parabrachial nucleus activity that follows systemic interleukin-1beta administration. (C) 2004 Elsevier B.V. All rights reserved.

Plasma cytokine changes in relation to exercise intensity and muscle damage

The purpose of this study was to compare the effects of exercise intensity and exercise-induced muscle damage on changes in anti-inflammatory cytokines and other inflammatory mediators. Nine well-trained male runners completed three different exercise trials on separate occasions: ( 1) level treadmill running at 60% VO2max (moderate-intensity trial) for 60 min; (2) level treadmill running at 85% VO2max (high-intensity trial) for 60 min; (3) downhill treadmill running ( - 10% gradient) at 60% VO2 max (downhill running trial) for 45 min. Blood was sampled before, immediately after and 1 h after exercise. Plasma was analyzed for interleukin-1 receptor antagonist (IL-1ra), IL-4, IL-5, IL-10, IL-12p40, IL-13, monocyte chemotactic protein-1 (MCP-1), prostaglandin E-2, leukotriene B-4 and heat shock protein 70 (HSP70). The plasma concentrations of IL-1ra, IL-12p40, MCP-1 and HSP70 increased significantly (P< 0.05) after all three trials. Plasma prostaglandin E-2 concentration increased significantly after the downhill running and high-intensity trials, while plasma IL-10 concentration increased significantly only after the high-intensity trial. IL-4 and leukotriene B4 did not increase significantly after exercise. Plasma IL-1ra and IL-10 concentrations were significantly higher ( P< 0.05) after the high-intensity trial than after both the moderate-intensity and downhill running trials. Therefore, following exercise up to 1 h duration, exercise intensity appears to have a greater effect on anti-inflammatory cytokine production than exercise-induced muscle damage.

Changes in inositol lipids and phosphates after stimulation of the MAS-transfected NG115-401L-C3 cell line by mitogenic and non-mitogenic stimuli

A neuronal cell line (NG115-401L-C3) was stimulated by mitogenic (angiotensin) and non-mitogenic (bradykinin) peptides and examined for the time course of changes in the levels of radiolabelled inositol phosphates and phospholipids. Both peptides stimulated the time-dependent production of Ins(1,4,5)P3 and related metabolites. Bradykinin caused a much larger increase in Ins(1,4,5)P3 than did angiotensin. However, both peptides stimulated similar rises in the levels of Ins(1,3,4)P3 and InsP4. Bradykinin but not angiotensin, caused a rapid (within 2 s) fall in the levels of PtdIns(4,5)P2 and PtdIns(4)P. Serum pretreatment of the cells caused a 2-3-fold potentiation of both the responses to bradykinin and angiotensin. Although significant levels of PtdIns(3)P were detected in resting cells neither mitogenic (angiotensin, insulin-like growth factor I, transforming growth factor beta) nor non-mitogenic (bradykinin, nerve growth factor interleukin-1) receptor activation changed its levels, arguing against regulation of either PtdIns 3-kinase or PtdIns(3)P phosphatase. We conclude that, as judged by the levels of its product. PtdIns(3)P, the enzyme PtdIns 3-kinase is not activated. This questions the significance of this activity in the receptor-mediated initiation of DNA synthesis.

Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection

BACKGROUND Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. RESULTS For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. CONCLUSIONS These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels.

Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for ProMMP-2 generates active MMP-2

The suggested model for pro-matrix metalloproteinase-2 (proMMP-2) activation by membrane type 1 MMP (MT1-MMP) implicates the complex between MT1-MMP and tissue inhibitor of MMP-2 (TIMP-2) as a receptor for proMMP-2. To dissect this model and assess the pathologic significance of MMP-2 activation, an artificial receptor for proMMP-2 was created by replacing the signal sequence of TIMP-2 with cytoplasmic/transmembrane domain of type II transmembrane mosaic serine protease (MSP-T2). Unlike TIMP-2, MSP-T2 served as a receptor for proMMP-2 without inhibiting MT1-MMP, and generated TIMP-2-free active MMP-2 even at a low level of MT1-MMP. Thus, MSP-T2 did not affect direct cleavage of the substrate testican-1 by MT1-MMP, whereas TIMP-2 inhibited it even at the level that stimulates proMMP-2 processing. Expression of MSP-T2 in HT1080 cells enhanced MMP-2 activation by endogenous MT1-MMP and caused intensive hydrolysis of collagen gel. Expression of MSP-T2 in U87 glioma cells, which express a trace level of endogenous MT1-MMP, induced MMP-2 activation and enhanced cell-associated protease activity, activation of extracellular signal-regulated kinase, and metastatic ability into chick embryonic liver and lung. MT1-MMP can exert both maximum MMP-2 activation and direct cleavage of substrates with MSP-T2, which cannot be achieved with TIMP-2. These results suggest that MMP-2 activation by MT1-MMP potentially amplifies protease activity, and combination with direct cleavage of substrate causes effective tissue degradation and enhances tumor invasion and metastasis, which highlights the complex role of TIMP-2. MSP-T2 is a unique tool to analyze physiologic and pathologic roles of MMP-2 and MT1-MMP in comparison with TIMP-2.

Interaction between human interleukin-16 and CD4 receptor of HIV-1

AIM: To study the interaction between human interleukin-16 (IL-16) and the receptor CD4 (T-lymphocyte differentiation antigen) of human immunodeficiency virus type 1 (HIV-1). METHODS: Two structurally con served regions (SCRs) of human IL-16 were built by the SYBYL/Biopolymer module using the corresponding transmembrane (TM) domain of human interleukin-1 (HIL-4) and HIL-2 as the templates. The coordinates for amino-terminal residue sequence, carboxyl-terminal residue sequences, and cytoplasm loops were generated using Biopolymer's LOOP SEARCH algorithm. RESULTS: HIL-16 first formed a homodimer, then contacted with CD4 dimer further forming a dimeric complex. Subsequently, the dimeric complex constructed the tetrameric complex by two disulfide bridges between the cysteines of HIL-16 (Cys31-Cys31). CONCLUSION: The interaction model is useful to propose the action mechanism of HIL-16 and is beneficial for rational designing of novel anti-HIV drugs.