Microstructure of the Advanced Titanium Alloy VT8M-1 Subjected to Rotary Swaging.

In this study, the microstructural behavior of the advanced Ti-5.7Al-3.8Mo-1.2Zr-1.3Sn-0.15Si (VT8M-1) alloy during rotary swaging (RS) was investigated. VT8M-1 has increased heat resistance and is considered a replacement for the Ti-6Al-4V alloy. It was shown that, during RS, the evolution of the primary a phase is characterized by the formation of predominantly low-angle boundaries according to the mechanism of continuous dynamic recrystallization. The density of low-angle boundaries increases three times: from 0.38 µm-1 to 1.21 µm-1 after RS. The process of spheroidization of the lamellar (a + b) component is incomplete. The average size of globular a and b particles was 0.3 µm (TEM). It is shown that the microstructures after RS (ε = 1.56) and equal-channel angular pressing (ECAP) (ε = 1.4) are significantly different. The temperature-velocity regime and the predominance of shear deformations during ECAP contributed to a noticeable refinement of the primary a-phase and a more complete development of globularization of the lamellar (a+b) component. EBSD studies have shown that RS leads to the formation of a structure with a higher density of low- and high-angle boundaries compared to the structure after ECAP. The results are useful for predicting alloy microstructure in the production of long rods that are further used in forging operations.

Evaluation of LFA-1 Peptide-Methotrexate Conjugates in Modulating Endothelial Cell Inflammation and Cytokine Regulation.

Interactions between vascular endothelial cells and inflammatory leukocytes are intermediated via cell adhesion molecules and they become one of the key events for vascular cell injury and development of atherosclerosis. This study evaluated the effects of MTX-peptide conjugates as anti-inflammatory agents on human coronary artery endothelial cells (HCAEC) and Molt-3 T cells. Cyclic peptides, cLABL and cLBEL, were derived from the α- and ß-subunits of leukocyte function-associated antigen-1 (LFA-1), respectively. They interact with intercellular adhesion molecule-1 (ICAM-1) to inhibit LFA-1/ICAM-1-mediated homotypic or heterotypic T-cell adhesion. cLABL and cLBEL were linked to the anti-inflammatory drug, methotrexate (MTX), to produce MTX-cLABL and MTX-cLBEL conjugates. This study showed that peptides and MTX-peptide conjugates inhibited T cell adhesion to HCAEC monolayers while MTX alone did not. The conjugates, but not MTX, inhibited binding of anti-ICAM-1 monoclonal antibody (mAb) to ICAM-1 on the HCAEC. This indicates that conjugation of MTX to cLABL and cLBEL peptides did not dramatically change their binding properties to ICAM-1. The conjugates had relatively lower toxicity to cells compared to MTX alone, while they were more toxic than the parent peptides. At low concentrations, MTX, MTX-cLABL and MTX-cLBEL decreased production of IL-6 and IL-8 as inflammatory cytokines. In contrast, higher concentrations of the parent peptides compared to the conjugates were required to inhibit IL-6 and IL-8 productions. Overall, both MTX-cLABL and MTX-cLBEL were more active than both free-peptides. In addition, the conjugates were less toxic than MTX alone. In conclusion, the conjugate can selectively target MTX to ICAM-1-expressing cells to increase cell targeting and to lower MTX toxicity.

Effect of the Texture of the Ultrafine-Grained Ti-6Al-4V Titanium Alloy on Impact Toughness.

In this work, the strength properties and impact toughness of the ultrafine-grained (UFG) Ti-6Al-4V titanium alloy produced by severe plastic deformation (SPD) in combination with upsetting were studied, depending on the direction of crack propagation. In the billets processed by equal-channel angular pressing (ECAP), the presence of anisotropy of ultimate tensile strength (UTS) and ductility was observed, conditioned by the formation of a metallographic and crystallographic texture. At the same time, the ECAP-processed UFG alloy exhibited satisfactory values of impact toughness, ~0.42 MJ/m2. An additional upsetting of the ECAP-processed billet simulated the processes of shape forming/die forging and was accompanied by the development of recovery and recrystallization. This provided the "blurring" of texture and a reduction in the anisotropy of UTS and ductility, but a difference in impact toughness in several directions of fracture was still observed. It is shown that texture evolution during upsetting provided a significant increase in the crack propagation energy. The relationship between microstructure, texture and mechanical properties in different sections of the material under study is discussed.

Methotrexate (MTX)-cIBR conjugate for targeting MTX to leukocytes: conjugate stability and in vivo efficacy in suppressing rheumatoid arthritis.

Methotrexate (MTX) has been used to treat rheumatoid arthritis at low doses and leukemia at high doses; however, this drug can produce severe side effects. Our hypothesis is that MTX side effects can be attenuated by directing the drug to the target cells (i.e., leukocytes) using (cyclo(1,12)PenPRGGSVLVTGC) peptide (cIBR). To test this hypothesis, MTX was conjugated to the N-terminus of cIBR peptide to give MTX-cIBR conjugate. MTX-cIBR (5.0 mg/kg) suppressed joint arthritis in adjuvant arthritis rats and prevented periarticular inflammation and bone resorption of the limb joints. In vitro, the toxicity of MTX-cIBR peptide against Molt-3 T cells was inhibited by anti-lymphocyte function-associated antigen-1 (LFA-1) antibody and cIBR peptide in a concentration-dependent manner, suggesting that the uptake of MTX-cIBR was partially mediated by LFA-1. Chemical stability studies indicated that MTX-cIBR was most stable at pH 6.0. The MTX portion of MTX-cIBR was unstable under acidic conditions, whereas the cIBR portion was unstable under basic conditions. In biological media, MTX-cIBR had short half lives in rat plasma (44 min) and homogenized rat heart tissue (38 min). This low plasma stability may contribute to the low in vivo efficacy of MTX-cIBR; therefore, there is a need to design a more stable conjugate to improve the in vivo efficacy.

Sequence recognition of alpha-LFA-1-derived peptides by ICAM-1 cell receptors: inhibitors of T-cell adhesion.

Blocking the T-cell adhesion signal from intercellular adhesion molecule-1/leukocyte function-associated antigen-1 interactions (Signal-2) can suppress the progression of autoimmune diseases (i.e. type-1 diabetes, psoriasis) and prevent allograph rejection. In this study, we determined the active region(s) of cLAB.L peptide [cyclo(1,12)Pen-ITDGEATDSGC] by synthesizing and evaluating the biologic activity of hexapeptides in inhibiting T-cell adhesion. A new heterotypic T-cell adhesion assay was also developed to provide a model for the T-cell adhesion process during lung inflammation. Two hexapeptides, ITDGEA and DGEATD, were found to be more active than the other linear hexapeptides. The cyclic derivative of ITDGEA [i.e. cyclo(1,6)ITDGEA] has similar activity than the parent linear peptide and has lower activity than cLAB.L peptide. Computational-binding experiments were carried out to explain the possible mechanism of binding of these peptides to intercellular adhesion molecule-1. Both ITDGEA and DGEATD bind the same site on intercellular adhesion molecule-1 and they interact with the Gln34 and Gln73 residues on D1 of intercellular adhesion molecule-1. In the future, more potent derivatives of cyclo(1,6)ITDGEA will be designed by utilizing structural and binding studies of the peptide to intercellular adhesion molecule-1. The heterotypic T-cell adhesion to Calu-3 will also be used as another assay to evaluate the selectivity of the designed peptides.

Ectopic Agouti protein overexpression increases stimulated corticosterone production without effect on adenylate cyclase activity in mouse adrenal cells.

OBJECTIVE: The antagonism of Agouti protein (AP) and Agouti-related protein on melanocortin receptors suggests an inhibitory role in the regulation of steroidogenesis. However, we have previously demonstrated that ectopic AP overexpression increased restraint-induced corticosterone release and adrenal reactivity to ACTH in mice. A high steroidogenic response to ACTH may be a consequence of a stimulatory AP action on the adenylate cyclase (AC) and/or intracellular steroidogenic enzymes. The aim of the present study was to estimate the effect of ectopic AP overexpression on the activity of AC and steroidogenic intracellular enzymes. METHODS: ACTH and forskolin were used for AC stimulation, and dibutyryl cAMP and progesterone were used for stimulation of intracellular steroidogenic enzymes in isolated adrenal cells in male C57Bl/6J mice of two Agouti genotypes: A(y)/a (ectopic AP overexpression) and a/a (absence of AP in all tissues). RESULTS: ACTH and forskolin increased cAMP accumulation to the same extent in both A(y)/a and a/a mouse adrenal cells (P<0.001; ANOVA), but resulted in higher corticosterone production in A(y)/a mice (P<0.001 for ACTH and P<0.01 for forskolin; ANOVA). Dibutyryl cAMP- and progesterone-induced corticosterone production was higher in A(y)/a mice than in a/a mice (P<0.001 for dibutyryl cAMP and P<0.01 for progesterone; ANOVA). CONCLUSIONS: Ectopic AP overexpression increased stimulated corticosterone production and intracellular steroidogenic enzyme reactivity to cAMP without an effect on AC activity.

Agouti yellow mutation increases adrenal response to ACTH in mice.

OBJECTIVE: Agouti protein (AP) and agouti-related protein with a similar sequence and action are endogenous antagonists of melanocortin receptors, implicated in the control of the hypothalamo-pituitary-adrenal (HPA) axis. Dominant mutation of the agouti gene (agouti yellow (A(y))) in heterozygous A(y)/a mice leads to ectopic overexpression of AP and produces an obese phenotype. The existing data on the HPA function in A(y)/a-mice are equivocal; therefore, the present study aimed to assess HPA function in 3-month-old male C57Bl/6J mice of two agouti genotypes: A(y)/a (ectopic AP overexpression) and a/a (absence of AP). DESIGN AND METHODS: In order to evaluate the HPA function, activating (15-min restriction, ACTH-induced corticosterone production in vitro) and inhibiting (i.p. injection of dexamethasone, 0.02 microg/g body weight) stimuli were employed. To estimate the effect of obesity on some HPA functions, A(y)/a males were subdivided into obese and non-obese groups. RESULTS: Basal plasma concentrations of ACTH and corticosterone; basal corticosterone production in vitro; and feedback inhibition of resting corticosterone levels by dexamethasone were similar in A(y)/a- and a/a-mice. Restraint-induced plasma corticosterone was greater in obese and non-obese A(y)/a-mice than in a/a-mice, whereas restraint-induced plasma ACTH levels were similar. Adrenal cell responses to ACTH (10(-13)-10(-10) M) were higher in obese and non-obese A(y)/a-mice than in a/a-mice. Dexamethasone, injected 3 h prior to stress, inhibited stress-induced corticosterone levels by a significantly greater amount in A(y)/a-mice than in a/a-mice. CONCLUSIONS: AP may have both stimulating and inhibiting influences on the HPA axis. AP overproduction increased the response of the HPA to short-restraint stress due to increased adrenal responsiveness to ACTH; this result was not effected by obesity development.

Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases.

This review focuses on providing insights into the structural basis and clinical relevance of LFA-1 and VLA-4 inhibition by peptides and small molecules as adhesion-based therapeutic strategies for inflammation and autoimmune diseases. Interactions of cell adhesion molecules (CAM) play central roles in mediating immune and inflammatory responses. Leukocyte function-associated antigen (LFA-1, alpha(L)beta(2), and CD11a/CD18) and very late antigen (VLA-4, alpha(4)beta(1), and CD49d/CD29) are members of integrin-type CAM that are predominantly involved in leukocyte trafficking and extravasation. LFA-1 is exclusively expressed on leukocytes and interacts with its ligands ICAM-1, -2, and -3 to promote a variety of homotypic and heterotypic cell adhesion events required for normal and pathologic functions of the immune systems. VLA-4 is expressed mainly on lymphocyte, monocytes, and eosinophils, but is not found on neutrophils. VLA-4 interacts with its ligands VCAM-1 and fibronectin (FN) CS1 during chronic inflammatory diseases, such as rheumatoid arthritis, asthma, psoriasis, transplant-rejection, and allergy. Blockade of LFA-1 and VLA-4 interactions with their ligands is a potential target for immunosuppression. LFA-1 and VLA-4 antagonists (antibodies, peptides, and small molecules) are being developed for controlling inflammation and autoimmune diseases. The therapeutic intervention of mostly mAb-based has been extensively studied. However, due to the challenging relative efficacy/safety ratio of mAb-based therapy application, especially in terms of systemic administration and immunogenic potential, strategic alternatives in the forms of peptide, peptide mimetic inhibitors, and small molecule non-peptide antagonists are being sought. Linear and cyclic peptides derived from the sequences of LFA-1, ICAM-1, ICAM-2, VCAM-1, and FN C1 have been shown to have inhibitory effects in vitro and in vivo. Finally, understanding the mechanism of LFA-1 and VLA-4 binding to their ligands has become a fundamental basis in developing therapeutic agents for inflammation and autoimmune diseases.