Cyclic peptides

Background Rheumatoid factors (RFs) and antibodies against cyclic citrullinated peptides (CCPs) of IgG, IgA and IgM isotype have been shown to precede disease onset by years. Objective To evaluate serological risk markers in fi rstdegree relatives from multicase families in relation to genetic and environmental risk factors. Methods 51 multicase families consisting of 163 individuals with rheumatoid arthritis (RA) (mean±SD age, 60±14 years; disease duration 21 years; 71.8% female) and with 157 fi rst-degree relatives unaffected by RA (54±17 years; 59.9% female) were recruited. Isotypes of antibodies against CCPs (IgG, IgA and IgM) and RFs (IgM and IgA) were determined using automated enzyme immunoassays. Cut-off levels were established using receiver operating characteristic curves based on values for 100 unrelated healthy controls. Results The concentrations and frequencies of all anti-CCP and RF isotypes were signifi cantly increased in fi rst-degree relatives and patients with RA compared with unrelated healthy controls. The relative distribution of IgA and IgM isotypes was higher than IgG in the relatives, whereas the IgG isotype dominated in patients with RA. The patients carried human leucocyte antigenshared epitope (HLA-SE) signifi cantly more often than the relatives (71.4% vs 53.9%, p=0.01), while the frequency of the PTPN22 T variant was similar. HLA-SE, combined with smoking, was signifi cantly related to all combinations of anti-CCP and RF isotypes in patients with RA. No such relationships were found for the fi rst-degree relatives. Conclusions All anti-CCP and RF isotypes analysed occurred more commonly in unaffected fi rst-degree relatives from multicase families than in controls, but with different isotype distribution from patients with RA.

Various tumours, classically specified as either neuroendocrine or non-neuroendocrine, contain high numbers of somatostatin receptors, which enable in vivo localization of the primary tumour and its metastases by scintigraphy with the radiolabelled somatostatin analogue octreotide. In addition granulomas and autoimmune processes can be visualized because of local accumulation of somatostatin receptor-positive activated mononuclear leucocytes. In many instances a positive scintigram predicts a favourable response to treatment with octreotide. It is tempting to speculate that octreotide labelled with an appropriate radionuclide might be used in cancer therapy. The successful application of radiolabelled octreotide in scintigraphy indicates the possible usefulness of other radiolabelled peptides, either native peptides or derivatives of these, in, for example, nuclear oncology. The small size of these peptides, e.g. bombesin and substance P, is of the utmost importance for a relatively fast blood clearance, thus leading to low background radioactivity. In this way peptides are powerful alternatives to (fragments of) monoclonal antibodies, the application of which to scintigraphic localization of specific cell surface antigen-bearing tumours is plagued by slow blood clearance and, hence, high background levels.

P-glycoprotein (Pgp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance in the treatment of cancers. Substrate promiscuity is a hallmark of Pgp activity, thus a structural description of polyspecific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo-Pgp at 3.8 Å reveals an internal cavity of ∼6,000 Å 3 with a 30 Å separation of the two nucleotide binding domains (NBD). Two additional Pgp structures with cyclic peptide inhibitors demonstrate distinct drug binding sites in the internal cavity capable of stereo-selectivity that is based on hydrophobic and aromatic interactions. Apo-and drug-bound Pgp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

Gene-encoded anti-microbial peptides (AMPs) are widespread in nature, as they are synthesized by microorganisms as well as by multicellular organisms from both the vegetal and the animal kingdoms. These naturally occurring AMPs form a first line of host defense against pathogens and are involved in innate immunity. Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the organism against environmental pathogens. They are classified into three major groups: (i) peptides with an a-helical conformation (insect cecropins, magainins, etc.), (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins, protegrin, etc.), and (iii) peptides with an over-representation of some amino acids (proline rich, histidine rich, etc.). Most AMPs display hydrophobic and cationic properties, have a molecular mass below 25-30 kDa, and adopt an amphipathic structure (a-helix, b-hairpin-like b-sheet, b-sheet, or a-helix/b-sheet mixed structures) that is believed to be essential to their anti-microbial action. Interestingly, in recent years, a series of novel AMPs have been discovered as processed forms of large proteins. Despite the extreme diversity in their primary and secondary structures, all natural AMPs have the in vitro particularity to affect a large number of microorganisms (bacteria, fungi, yeast, virus, etc.) with identical or complementary activity spectra. This review focuses on AMPs forming a-helices, b-hairpin-like b-sheets, bsheets, or a-helix/b-sheet mixed structures from invertebrate and vertebrate origins. These molecules show some promise for therapeutic use.

Clinical needs for novel antifungal agents have altered steadily with the rise and fall of AIDS-related mycoses, and the change in spectrum of fatal disseminated fungal infections that has accompanied changes in therapeutic immunosuppressive therapies. The search for new molecular targets for antifungals has generated considerable research using modern genomic approaches, so far without generating new agents for clinical use. Meanwhile, six new antifungal agents have just reached, or are approaching, the clinic. Three are new triazoles, with extremely broad antifungal spectra, and three are echinocandins, which inhibit synthesis of fungal cell wall polysaccharides – a new mode of action. In addition, the sordarins represent a novel class of agents that inhibit fungal protein synthesis. This review describes the targets and mechanisms of action of all classes of antifungal agents in clinical use or with clinical potential.

Patients with persistent fever and neutropenia often receive empirical therapy with conventional or liposomal amphotericin B for the prevention and early treatment of invasive fungal infections. Caspofungin, a member of the new echinocandin class of compounds, may be an effective alternative that is better tolerated than amphotericin B.

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Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms. We also explored effects of preincubation of C. albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic-and biofilm-exposed blastospores to examine drug effects on cell structure. Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays.

Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids. The amino acid sequences of these peptides are not found in the primary sequence of EPO. The signaling pathways activated by these peptides appear to be identical to those induced by the natural ligand. This discovery may form the basis for the design of small molecule mimetics of EPO.

The mammalian suprachiasmatic nucleus (SCN) is a master circadian pacemaker. It is not known which SCN neurons are autonomous pacemakers or how they synchronize their daily firing rhythms to coordinate circadian behavior. Vasoactive intestinal polypeptide (VIP) and the VIP receptor VPAC 2 (encoded by the gene Vipr2) may mediate rhythms in individual SCN neurons, synchrony between neurons, or both. We found that Vip -/and Vipr2 -/mice showed two daily bouts of activity in a skeleton photoperiod and multiple circadian periods in constant darkness. Loss of VIP or VPAC 2 also abolished circadian firing rhythms in approximately half of all SCN neurons and disrupted synchrony between rhythmic neurons. Critically, daily application of a VPAC 2 agonist restored rhythmicity and synchrony to VIP -/-SCN neurons, but not to Vipr2 -/neurons. We conclude that VIP coordinates daily rhythms in the SCN and behavior by synchronizing a small population of pacemaking neurons and maintaining rhythmicity in a larger subset of neurons.

FK228 is a histone deacetylase (HDAC) inhibitor, the molecular mechanism of inhibition of which has been unknown. Here we show that reduction of an intramolecular disulfide bond of FK228 greatly enhanced its inhibitory activity and that the disulfide bond was rapidly reduced in cells by cellular reducing activity involving glutathione. Computer modeling suggests that one of the sulfhydryl groups of the reduced form of FK228 (redFK) interacts with the active-site zinc, preventing the access of the substrate. HDAC1 and HDAC2 were more strongly inhibited by redFK than HDAC4 and HDAC6. redFK was less active than FK228 in inhibiting in vivo HDAC activity, due to rapid inactivation in medium and serum. Thus, FK228 serves as a stable prodrug to inhibit class I enzymes and is activated by reduction after uptake into the cells. The glutathionemediated activation also implicates its clinical usefulness for counteracting glutathione-mediated drug resistance in chemotherapy.

BACKGROUND-Rheumatoid arthritis has a complex mode of inheritance. Although HLA-DRB1 and PTPN22 are well-established susceptibility loci, other genes that confer a modest level of risk have been identified recently. We carried out a genomewide association analysis to identify additional genetic loci associated with an increased risk of rheumatoid arthritis.

Multiple strains of Bacillus spp. were demonstrated to stimulate plant defence responses. However, very little is known about the nature of molecular determinants secreted by these Gram-positive bacteria that are responsible for the elicitation of the induced systemic resistance (ISR) phenomenon. This study shows that the lipopeptides surfactins and fengycins may be involved in this elicitation process. In bean, pure fengycins and surfactins provided a significant ISR-mediated protective effect on bean plants, similar to the one induced by living cells of the producing strain S499. Moreover, experiments conducted on bean and tomato plants showed that overexpression of both surfactin and fengycin biosynthetic genes in the naturally poor producer Bacillus subtilis strain 168 was associated with a significant increase in the potential of the derivatives to induce resistance. In tomato cells, key enzymes of the lipoxygenase pathway appeared to be activated in resistant plants following induction by lipopeptide overproducers. To our knowledge, such lipopeptides constitute a novel class of compounds from non-pathogenic bacteria that can be perceived by plant cells as signals to initiate defence mechanisms.

Invasive candidiasis is an important cause of morbidity and mortality among patients with health care-associated infection. The echinocandins have potent fungicidal activity against most Candida species, but there are few data comparing the safety and efficacy of echinocandins in the treatment of invasive candidiasis.

Background. Invasive aspergillosis (IA) is an important cause of morbidity and mortality among immunocompromised patients. Echinocandins are novel antifungal molecules with in vitro and in vivo activity against Aspergillus species. Methods. We investigated the efficacy and safety of caspofungin in the treatment of IA. Ninety patients with IA who were refractory to or intolerant of amphotericin B, lipid formulations of amphotericin B, or triazoles were enrolled to receive caspofungin. Results. Efficacy was assessed for 83 patients who had infection consistent with definitions of IA and who received у1 dose of study drug. Common underlying conditions included hematologic malignancy (48% of patients), allogeneic blood and marrow transplantation (25% of patients), and solid-organ transplantation (11% of patients). Seventy-one patients (86%) were refractory to and 12 patients (14%) were intolerant of previous therapy. A favorable response to caspofungin therapy was observed in 37 (45%) of 83 patients, including 32 (50%) of 64 with pulmonary aspergillosis and 3 (23%) of 13 with disseminated aspergillosis. Two patients discontinued caspofungin therapy because of drug-related adverse events. Drug-related nephrotoxicity and hepatotoxicity occurred infrequently. Conclusion. Caspofungin demonstrated usefulness in the salvage treatment of IA.

Biomedical researchers have become increasingly aware of the limitations of time-honored conventional 2D tissue cell cultures where most tissue cell studies have been carried out. They are now searching for 3D cell culture systems, something between a petri dish and a mouse. It has become apparent that 3D cell culture offers a more realistic micro-and local-environment where the functional properties of cells can be observed and manipulated that is not possible in animals. A newly designer self-assembling peptide scaffolds may provide an ideally alternative system. The important implications of 3D tissue cell cultures for basic cell biology, tumor biology, high-content drug screening, and regenerative medicine and beyond could be profound.

Antibodies against citrullinated proteins are specific and predictive markers for rheumatoid arthritis although the pathologic relevance of these antibodies remains unclear. To investigate the significance of these autoantibodies, collagen-induced arthritis (CIA) in mice was used to establish an animal model of antibody reactivity to citrullinated proteins. DBA/1J mice were immunized with bovine type II collagen (CII) at days 0 and 21, and serum was collected every 7 days for analysis. Antibodies against both CII and cyclic citrullinated peptide, one such citrullinated antigen, appeared early after immunization, before joint swelling was observed. Further, these antibodies demonstrated specific binding to citrullinated filaggrin in rat esophagus by indirect immunofluorescence and citrullinated fibrinogen by Western blot. To evaluate the role of immune responses to citrullinated proteins in CIA, mice were tolerized with a citrulline-containing peptide, followed by antigen challenge with CII. Tolerized mice demonstrated significantly reduced disease severity and incidence compared with controls. We also identified novel murine monoclonal antibodies specific to citrullinated fibrinogen that enhanced arthritis when coadministered with a submaximal dose of anti-CII antibodies and bound targets within the inflamed synovium of mice with CIA. These results demonstrate that antibodies against citrullinated proteins are centrally involved in the pathogenesis of autoimmune arthritis. Nonstandard abbreviations used: ABTS, 2,2′-azinobis(3-ethylbenzthiazoline-6sulfonic acid)

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

In protein structure prediction, it is often the case that a protein segment must be adjusted to connect two fixed segments. This occurs during loop structure prediction in homology modeling as well as in ab initio structure prediction. Several algorithms for this purpose are based on the inverse Jacobian of the distance constraints with respect to dihedral angle degrees of freedom. These algorithms are sometimes unstable and fail to converge. We present an algorithm developed originally for inverse kinematics applications in robotics. In robotics, an end effector in the form of a robot hand must reach for an object in space by altering adjustable joint angles and arm lengths. In loop prediction, dihedral angles must be adjusted to move the C-terminal residue of a segment to superimpose on a fixed anchor residue in the protein structure. The algorithm, referred to as cyclic coordinate descent or CCD, involves adjusting one dihedral angle at a time to minimize the sum of the squared distances between three backbone atoms of the moving C-terminal anchor and the corresponding atoms in the fixed C-terminal anchor. The result is an equation in one variable for the proposed change in each dihedral. The algorithm proceeds iteratively through all of the adjustable dihedral angles from the N-terminal to the C-terminal end of the loop. CCD is suitable as a component of loop prediction methods that generate large numbers of trial structures. It succeeds in closing loops in a large test set 99.79% of the time, and fails occasionally only for short, highly extended loops. It is very fast, closing loops of length 8 in 0.037 sec on average.

A novel fungal metabolite, apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2- amino-8-oxodecanoyl)], that exhibits potent, broad spectrum antiprotozoal activity in vitro against Apicomplexan parasites has been identified. It is also orally and parenterally active in vivo against Plasmodium berghei malaria in mice. Many Apicomplexan parasites cause serious, life-threatening human and animal diseases, such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and new therapeutic agents are urgently needed. Apicidin's antiparasitic activity appears to be due to low nanomolar inhibition of Apicomplexan histone deacetylase (HDA), which induces hyperacetylation of histones in treated parasites. The acetylation-deacetylation of histones is a thought to play a central role in transcriptional control in eukaryotic cells. Other known HDA inhibitors were also evaluated and found to possess antiparasitic activity, suggesting that HDA is an attractive target for the development of novel antiparasitic agents.

In gradients of external chemo-attractant, mammalian neutrophilic leukocytes (neutrophils) 1 and Dictyostelium discoideum amoebae 2 adopt a polarized morphology and selectively accumulate lipid products of phosphatidylinositol-3-OH kinases (PI(3)Ks), including PtdIns(3,4,5)P 3 , at their up-gradient edges; the internal PtdIns(3,4,5)P 3 gradient substantially exceeds that of the external attractant. An accompanying report 3 presents evidence for a positive feedback loop that amplifies the gradient of internal signal: PtdIns(3,4,5)P 3 at the leading edge stimulates its own accumulation by inducing activation of one or more Rho GTPases (Rac, Cdc42, and/or Rho), which in turn increase PtdIns(3,4,5)P 3 accumulation. Here we show that interruption of this feedback by treatment with PI(3)K inhibitors reduces the size and stability of pseudopods and causes cells to migrate in jerky trajectories that deviate more from the up-gradient direction than do those of controls. Moreover, amplification of the internal PtdIns(3,4,5)P 3 gradient is markedly impaired by latrunculin or jasplakinolide, toxins that inhibit polymerization 4,5 or depolymerization 6 of actin, respectively. Thus reciprocal interplay between PtdIns(3,4,5)P 3 and polymerized actin initiates and maintains the asymmetry of intracellular signals responsible for cell polarity and directed motility.

Cyanobacteria are a prolific source of secondary metabolites, including compounds with toxic and enzyme-inhibiting activities. Microcystins and nodularins are the end products of a secondary metabolic pathway comprised of mixed polyketide synthases and nonribosomal peptide synthetases. Both peptides are potent natural toxins produced by distantly related genera of cyanobacteria. Horizontal gene transfer is thought to play a role in the sporadic distribution of microcystin producers among cyanobacteria. Our phylogenetic analyses indicate a coevolution of housekeeping genes and microcystin synthetase genes for the entire evolutionary history of the toxin. Hence they do not corroborate horizontal transfer of genes for microcystin biosynthesis between the genera. The sporadic distribution of microcystin synthetase genes in modern cyanobacteria suggests that the ability to produce the toxin has been lost repeatedly in the more derived lineages of cyanobacteria. The data we present here strongly suggest that the genes encoding nodularin synthetase are recently derived from those encoding microcystin synthetase.

Background-The role of endothelin (ET)-1 in maintenance of basal vascular tone has been demonstrated by local and systemic vasodilatation to endothelin receptor antagonists in humans. Although the constrictor effects mediated by the vascular smooth muscle ET A receptors are clear, the contribution from endothelial and vascular smooth muscle ET B receptors remains to be defined. The present study, in human forearm resistance vessels in vivo, was designed to further investigate the physiological function of ET A and ET B receptor subtypes in human blood vessels and determine the mechanism underlying the vasodilatation to the ET A -selective receptor antagonist BQ-123. Methods and Results-Two studies were performed, each in groups of eight healthy subjects. Brachial artery infusion of BQ-123 caused significant forearm vasodilatation in both studies. This vasodilatation was reduced by 95% (Pϭ.006) with inhibition of the endogenous generation of nitric oxide and by 38% (PϽ.001) with coinfusion of the ET B receptor antagonist BQ-788. In contrast, inhibition of prostanoid generation did not affect the response to BQ-123. Infusion of BQ-788 alone produced a 20% reduction in forearm blood flow (PϽ.001).

Cilengitide, a cyclic RGD pentapeptide, is currently in clinical phase III for treatment of glioblastomas and in phase II for several other tumors. This drug is the first anti-angiogenic small molecule targeting the integrins v 3, v 5 and 5 1. It was developed by us in the early 90s by a novel procedure, the spatial screening. This strategy resulted in c(RGDfV), the first superactive v 3 inhibitor (100 to 1000 times increased activity over the linear reference peptides), which in addition exhibited high selectivity against the platelet receptor IIb 3. This cyclic peptide was later modified by N-methylation of one peptide bond to yield an even greater antagonistic activity in c(RGDf(NMe)V). This peptide was then dubbed Cilengitide and is currently developed as drug by the company Merck-Serono (Germany).

Antibodies directed to the Sa antigen are highly specific for rheumatoid arthritis (RA) and can be detected in approximately 40% of RA sera. The antigen, a doublet of protein bands of about 50 kDa, is present in placenta and in RA synovial tissue. Although it has been stated that the Sa antigen is citrullinated vimentin, experimental proof for this claim has never been published. In this study, we investigated the precise nature of the antigen. Peptide sequences that were obtained from highly purified Sa antigen were unique to vimentin. Recombinant vimentin, however, was not recognized by anti-Sa reference sera. In vivo, vimentin is subjected to various post-translational modifications, including citrullination. Since antibodies to citrullinated proteins are known to be highly specific for RA, we investigated whether Sa is citrullinated and found that Sa indeed is citrullinated vimentin. Anti-Sa antibodies thus belong to the family of anticitrullinated protein/peptide antibodies. Th...

Silk fibers have potential biomedical applications beyond their traditional use as sutures. The physical properties of silk fibers and films make it a promising candidate for tissue engineering scaffold applications, particularly where high mechanical loads or tensile forces are applied or in cases where low rates of degradation are desirable. A critical issue for biomaterial scaffolds is biocompatibility. The direct inflammatory potential of intact silk fibers as well as extracts was studied in an in vitro system. The results indicate that silk fibers are largely immunologically inert in short-and long-term culture with RAW 264.7 murine macrophage cells while insoluble fibroin particles induced significant TNF release. Soluble sericin proteins extracted from native silk fibers did not induce significant macrophage activation. While sericin did not activate macrophages by itself, it demonstrated a synergistic effect with bacterial lipopolysaccharide. The low level of inflammatory potential of silk fibers makes them promising candidates in future biomedical applications. r

Blood vessels of tumors carry specific markers that are usually angiogenesis-related 1,2 . We previously used phage-displayed peptide libraries in vivo to identify peptides that home to tumors through the circulation and that specifically bind to the endothelia of tumor blood vessels 3,4 . Here we devised a phage screening procedure that would favor tumor-homing to targets that are accessible to circulating phage, but are not blood vessels. Screening on MDA-MB-435 breast carcinoma xenografts yielded multiple copies of a phage that displays a cyclic 9amino-acid peptide, LyP-1. Homing and binding to tumor-derived cell suspensions indicated that LyP-1 also recognizes an osteosarcoma xenograft, and spontaneous prostate and breast cancers in transgenic mice, but not two other tumor xenografts. Fluorescein-labeled LyP-1 peptide was detected in tumor structures that were positive for three lymphatic endothelial markers and negative for three blood vessel markers. LyP-1 accumulated in the nuclei of the putative lymphatic cells, and in the nuclei of tumor cells. These results suggest that tumor lymphatics carry specific markers and that it may be possible to specifically target therapies into tumor lymphatics.

The cyanobacteria or "blue-green algae", as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins). However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites.

for the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) Study Group Gene-gene and gene-environment interactions are key features in the development of rheumatoid arthritis (RA) and other complex diseases. The aim of this study was to use and compare three different definitions of interaction between the two major genetic risk factors of RA-the HLA-DRB1 shared epitope (SE) alleles and the PTPN22 R620W allele-in three large case-control studies: the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) study, the North American RA Consortium (NARAC) study, and the Dutch Leiden Early Arthritis Clinic study (in total, 1,977 cases and 2,405 controls). The EIRA study was also used to analyze interactions between smoking and the two genes. "Interaction" was defined either as a departure from additivity, as interaction in a multiplicative model, or in terms of linkage disequilibrium-for example, deviation from independence of penetrance of two unlinked loci. Consistent interaction, defined as departure from additivity, between HLA-DRB1 SE alleles and the A allele of PTPN22 R620W was seen in all three studies regarding anti-CCP-positive RA. Testing for multiplicative interactions demonstrated an interaction between the two genes only when the three studies were pooled. The linkage disequilibrium approach indicated a gene-gene interaction in EIRA and NARAC, as well as in the pooled analysis. No interaction was seen between smoking and PTPN22 R620W. A new pattern of interactions is described between the two major known genetic risk factors and the major environmental risk factor concerning the risk of developing anti-CCP-positive RA. The data extend the basis for a pathogenetic hypothesis for RA involving genetic and environmental factors. The study also raises and illustrates principal questions concerning ways to define interactions in complex diseases.

Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14-180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation.

Background: Rheumatoid factor (RF) and autoantibodies against cyclic citrullinated peptide (CCP) are markers that might help physicians diagnose rheumatoid arthritis. Purpose: To determine whether anti-CCP antibody more accurately identifies patients with rheumatoid arthritis and better predicts radiographic progression than does RF. Data Sources: MEDLINE through September 2006 and reference lists of retrieved studies and review articles. Study Selection: Studies in any language that enrolled at least 10 participants and that examined the role of anti-CCP antibody and RF in the diagnosis or prognosis of known or suspected rheumatoid arthritis. Data Extraction: Two authors independently evaluated studies for inclusion, rated methodological quality, and abstracted relevant data. Data Synthesis: The DerSimonian-Laird random-effects method was used to summarize sensitivities, specificities, and positive and negative likelihood ratios from 37 studies of anti-CCP antibody and 50 studies of RF. The pooled sensitivity, specificity, and positive and negative likelihood ratios for anti-CCP antibody were 67% (95% CI, 62% to 72%), 95% (CI, 94% to 97%), 12.46 (CI, 9.72 to 15.98), and 0.36 (CI, 0.31 to 0.42), respectively. For IgM RF, the values were 69% (CI, 65% to 73%), 85% (CI, 82% to 88%), 4.86 (CI, 3.95 to 5.97), and 0.38 (CI, 0.33 to 0.44). Likelihood ratios among IgM RF, IgG RF, and IgA RF seemed to be similar. Results from studies of patients with early rheumatoid arthritis were similar to those from all studies. Three of 4 studies found that risk for radiographic progression was greater with anti-CCP antibody positivity than with IgM RF positivity. Limitations: Many studies had methodological limitations. Studies of RF were heterogeneous and had wide ranges of sensitivity and specificity. Conclusions: Anti-CCP antibodies are more specific than RF for diagnosing rheumatoid arthritis and may better predict erosive disease.

In the context of the renewed interest of peptides as therapeutics, it is important to have an on-line resource for 3D structure prediction of peptides with well-defined structures in aqueous solution. We present an updated version of PEP-FOLD allowing the treatment of both linear and disulphide bonded cyclic peptides with 9-36 amino acids. The server makes possible to define disulphide bonds and any residue-residue proximity under the guidance of the biologists. Using a benchmark of 34 cyclic peptides with one, two and three disulphide bonds, the best PEP-FOLD models deviate by an average RMS of 2.75 Å from the full NMR structures. Using a benchmark of 37 linear peptides, PEP-FOLD locates lowest-energy conformations deviating by 3 Å RMS from the NMR rigid cores. The evolution of PEP-FOLD comes as a new on-line service to supersede the previous server. The server is available at: http://bioserv .rpbs.univ-paris-diderot.fr/PEP-FOLD.

During the past decade, RGD-peptides have become a popular tool for the targeting of drugs and imaging agents to ␣ v ␤ 3 -integrin expressing tumour vasculature. RGD-peptides have been introduced by recombinant means into therapeutic proteins and viruses. Chemical means have been applied to couple RGD-peptides and RGD-mimetics to liposomes, polymers, peptides, small molecule drugs and radiotracers. Some of these products show impressive results in preclinical animal models and a RGD targeted radiotracer has already successfully been tested in humans for the visualization of ␣ v ␤ 3 -integrin, which demonstrates the feasibility of this approach. This review will summarize the structural requirements for RGD-peptides and RGD-mimetics as ligands for ␣ v ␤ 3 . We will show how they have been introduced in the various types of constructs by chemical and recombinant techniques. The importance of multivalent RGDconstructs for high affinity binding and internalization will be highlighted. Furthermore the in vitro and in vivo efficacy of RGD-targeted therapeutics and diagnostics reported in recent years will be reviewed.

Macrophages are typically stimulated by components of microbial cell walls. Surprisingly, cell wall-less mycoplasmas can also very efficiently stimulate macrophages. We showed recently that mycoplasma-derived lipopeptides constitute the active principle. We have now isolated a clone of Mycoplasma fermentans expressing mainly one macrophage-stimulating lipopeptide. This lipopeptide was detergent-extracted and isolated by reversed-phase high-performance liquid chromotography, using nitric oxide release from C3H/HeJ mouse macrophages as bioassay for detection. In contrast to "conventional" bacterial lipoproteins, this lipopeptide had a free NH 2 terminus. Amino acid composition, sequence, and the molecular weight of 2,163.3 are consistent with the following structure: S-(2,3-bisacyloxypropyl)cysteine-GNNDESNISFKEK with one mole C16:0, and a further mole of a mixture of C18:0 and C18:1 fatty acid per lipopeptide molecule. The sequence could not be found in either the protein identification resource nor the Swiss Prot data bank. We named this 2-kD lipopeptide, macrophage-activating lipopeptide-2 (MALP-2). Synthetic dipalmitoyl MALP-2 and mycoplasma-derived MALP-2 were compared with the bioassay. Both lipopeptides showed an identical dose dependency with a half-maximal response at 10 Ϫ 11 M concentration. MALP-2 may be one of the most potent natural macrophage stimulators besides endotoxin.

Retinal neovascularization is the major cause of untreatable blindness. The role of growth hormone (GH) in ischemia-associated retinal neovascularization was studied in transgenic mice expressing a GH antagonist gene and in normal mice given an inhibitor of GH secretion (MK678). Retinal neovascularization was inhibited in these mice in inverse proportion to serum levels of GH and a downstream effector, insulin-like growth factor-l (IGF-I). Inhibition was reversed with exogenous IGF-I administration. GH inhibition did not diminish hypoxia-stimulated retinal vascular endothelial growth factor (VEGF) or VEGF receptor expression. These data suggest that systemic inhibition of GH or IGF-I, or both, may have therapeutic potential in preventing some forms of retinopathy. membrane (arrows). (B) Cross section Of an eye from a GH antagonist GI 19K transgenic mouse. No R. G. Smith and J. M. Schaeffer, Merck Research Labo-vascular cell nuclei are apparent internal to the inner limiting membrane. (C) Nontransgenicflat-mounted ratories, Rahway, NJ 07065, USA.

Insect antimicrobial peptides (AMPs) are an important group of insect innate immunity effectors. Insect AMPs are cationic and contain less than 100 amino acid residues. According to structure, insect AMPs can be divided into a limited number of families. The diverse antimicrobial spectrum of insect AMPs may indicate different modes of action. Research on the model organism Drosophila indicate that insect AMPs gene regulation involves multiple signaling pathways and a large number of signaling molecules.

It is generally assumed that type A lantibiotics primarily kill bacteria by permeabilization of the cytoplasmic membrane. As previous studies had demonstrated that nisin interacts with the membrane-bound peptidoglycan precursors lipid I and lipid II, we presumed that this interaction could play a role in the pore formation process of lantibiotics. Using a thin-layer chromatography system, we found that only nisin and epidermin, but not Pep5, can form a complex with [ 14 C]-lipid II. Lipid II was then purified from Micrococcus luteus and incorporated into carboxyfluoresceinloaded liposomes made of phosphatidylcholine and cholesterol (1:1). Liposomes supplemented with 0.05 or 0.1 mol% of lipid II did not release any marker when treated with Pep5 or epilancin K7 (peptide concentrations of up to 5 mol% were tested). In contrast, as little as 0.01 mol% of epidermin and 0.1 mol% of nisin were sufficient to induce rapid marker release; phosphatidylglycerol-containing liposomes were even more susceptible. Controls with moenomycin-, undecaprenol-or dodecaprenolphosphate-doped liposomes demonstrated the specificity of the lantibiotics for lipid II. These results were correlated with intact cells in an in vivo model. M. luteus and Staphylococcus simulans were depleted of lipid II by preincubation with the lipopeptide ramoplanin and then tested for pore formation. When applied in concentrations below the minimal inhibitory concentration (MIC) and up to 5-10 times the MIC, the pore formation by nisin and epidermin was blocked; at higher concentrations of the lantibiotics the protective effect of ramoplanin disappeared. These results demonstrate that, in vitro and in vivo, lipid II serves as a docking molecule for nisin and epidermin, but not for Pep5 and epilancin K7, and thereby facilitates the formation of pores in the cytoplasmic membrane.