Screening of homing and tissue-penetrating peptides by microdialysis and in vivo phage display.

In vivo phage display is a method used for identification of organ- or disease-specific vascular homing peptides for targeted delivery of pharmaceutics. It is agnostic as to the nature and identity of the target molecules. The current in vivo biopanning lacks inbuilt mechanisms to select for peptides capable of vascular homing that would also be capable of tissue penetration to reach therapeutically relevant cells in the tissue parenchyma.

Epitope-specific antibody fragments block aggregation of AGelD187N, an aberrant peptide in gelsolin amyloidosis.

Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist.

Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

Background: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries.

Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro.

Dimeric IgA specifically disables intracellular mutated oncodrivers.

A prevailing belief in the immunotherapy field has been that antibody therapy can effectively target only extracellular antigens. In this issue of Immunity, Biswas et al. demonstrate therapeutically effective targeting, neutralization, and removal of mutated oncodriver proteins from within epithelial cancer cells by treatment with pIgR-dependent, transcytosing dimeric-IgA antibodies.

Selection of biophysically favorable antibody variants using a modified Flp-In CHO mammalian display platform.

Mammalian display enables the selection of biophysically favorable antibodies from a large IgG antibody library displayed on the plasma membrane of mammalian cells. We constructed and validated a novel mammalian display platform utilizing the commercially available Flp-In CHO cell line as a starting point. We introduced a single copy of a landing pad for Bxb1 integrase-driven recombinase-mediated cassette exchange into the FRT site of the Flp-In CHO line to facilitate the efficient single-copy integration of an antibody display cassette into the genome of the cell line.

Dual drug delivery collagen vehicles for modulation of skin fibrosis.

Single molecule drug delivery systems have failed to yield functional therapeutic outcomes, triggering investigations into multi-molecular drug delivery vehicles. In the context of skin fibrosis, although multi-drug systems have been assessed, no system has assessed molecular combinations that directly and specifically reduce cell proliferation, collagen synthesis and transforming growth factor1 (TGF1) expression. Herein, a core-shell collagen type I hydrogel system was developed for the dual delivery of a TGFtrap, a soluble recombinant protein that inhibits TGFsignalling, and Trichostatin A (TSA), a small molecule inhibitor of histone deacetylases.

Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies.

Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available fibrosis models. Indeed, customarily utilised scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology.

Systemically Administered Homing Peptide Targets Dystrophic Lesions and Delivers Transforming Growth Factor-β (TGFβ) Inhibitor to Attenuate Murine Muscular Dystrophy Pathology.

Muscular dystrophy is a progressively worsening and lethal disease, where accumulation of functionality-impairing fibrosis plays a key pathogenic role. Transforming growth factor-β1 (TGFβ1) is a central signaling molecule in the development of fibrosis in muscular dystrophic humans and mice. Inhibition of TGFβ1 has proven beneficial in mouse models of muscular dystrophy, but the global strategies of TGFβ1 inhibition produce significant detrimental side effects.

Exposed CendR Domain in Homing Peptide Yields Skin-Targeted Therapeutic in Epidermolysis Bullosa.

Systemic skin-selective therapeutics would be a major advancement in the treatment of diseases affecting the entire skin, such as recessive dystrophic epidermolysis bullosa (RDEB), which is caused by mutations in the COL7A1 gene and manifests in transforming growth factor-β (TGF-β)-driven fibrosis and malignant transformation. Homing peptides containing a C-terminal R/KXXR/K motif (C-end rule [CendR] sequence) activate an extravasation and tissue penetration pathway for tumor-specific drug delivery. We have previously described a homing peptide CRKDKC (CRK) that contains a cryptic CendR motif and homes to angiogenic blood vessels in wounds and tumors, but it cannot penetrate cells or tissues.

The practicalities of establishing a porcine isolated heart model.

Background: The isolated heart apparatus is over 100 years old, but remains a useful research tool today. While designs of many large animal systems have been described in the literature, trouble-shooting and refining such a model to yield a stable, workable system has not been previously described. This paper outlines the issues, in tabular form, that our group encountered in developing our own porcine isolated heart rig with the aim of assisting other workers in the field planning similar work.

Role of carbonic anhydrases in skin wound healing.

Skin wound closure occurs when keratinocytes migrate from the edge of the wound and re-epithelialize the epidermis. Their migration takes place primarily before any vascularization is established, that is, under hypoxia, but relatively little is known regarding the factors that stimulate this migration. Hypoxia and an acidic environment are well-established stimuli for cancer cell migration.

R-Ras deficiency does not affect papain-induced IgE production in mice.

Introduction: R-Ras GTPase has recently been implicated in the regulation of immune functions, particularly in dendritic cell (DC) maturation, immune synapse formation, and subsequent T cell responses.

Methods: Here, we investigated the role of R-Ras in allergen-induced immune response (type 2 immune response) in Rras deficient (R-Ras KO) and wild type (WT) mice.

Results: Initially, we found that the number of conventional DC's in the lymph nodes (LNs) was reduced in R-Ras KO mice.

T-cell-expressed proprotein convertase FURIN inhibits DMBA/TPA-induced skin cancer development.

Proprotein convertases (PCSK) have a critical role in the body homeostasis as enzymes responsible for processing precursor proteins into their mature forms. FURIN, the first characterized member of the mammalian PCSK family, is overexpressed in multiple malignancies and the inhibition of its activity has been considered potential cancer treatment. FURIN has also an important function in the adaptive immunity, since its deficiency in T cells causes an impaired peripheral immune tolerance and accelerates immune responses.

Lack of R-Ras Leads to Increased Vascular Permeability in Ischemic Retinopathy.

Purpose: The role of R-Ras in retinal angiogenesis and vascular permeability was evaluated in an oxygen-induced retinopathy (OIR) model using R-Ras knockout (KO) mice and in human diabetic neovascular membranes.

Methods: Mice deficient for R-Ras and their wild-type (WT) littermates were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. At P17 retinal vascularization was examined from whole mounts, and retinal vascular permeability was studied using Miles assay.

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist.

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences.

Decorin: A Growth Factor Antagonist for Tumor Growth Inhibition.

Decorin (DCN) is the best characterized member of the extracellular small leucine-rich proteoglycan family present in connective tissues, typically in association with or "decorating" collagen fibrils. It has substantial interest to clinical medicine owing to its antifibrotic, anti-inflammatory, and anticancer effects. Studies on DCN knockout mice have established that a lack of DCN is permissive for tumor development and it is regarded as a tumor suppressor gene.

Systemically Administered, Target Organ-Specific Therapies for Regenerative Medicine.

Growth factors and other agents that could potentially enhance tissue regeneration have been identified, but their therapeutic value in clinical medicine has been limited for reasons such as difficulty to maintain bioactivity of locally applied therapeutics in the protease-rich environment of regenerating tissues. Although human diseases are treated with systemically administered drugs in general, all current efforts aimed at enhancing tissue repair with biological drugs have been based on their local application. The systemic administration of growth factors has been ruled out due to concerns about their safety.

Resistance of R-Ras knockout mice to skin tumour induction.

The R-ras gene encodes a small GTPase that is a member of the Ras family. Despite close sequence similarities, R-Ras is functionally distinct from the prototypic Ras proteins; no transformative activity and no activating mutations of R-Ras in human malignancies have been reported for it. R-Ras activity appears inhibitory towards tumour proliferation and invasion, and to promote cellular quiescence.

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2.

Structure and function of the human Gly1619Arg polymorphism of M6P/IGF2R domain 11 implicated in IGF2 dependent growth.

The mannose 6-phosphate/IGF 2 receptor (IGF2R) is comprised of 15 extra-cellular domains that bind IGF2 and mannose 6-phosphate ligands. IGF2R transports ligands from the Golgi to the pre-lysosomal compartment and thereafter to and from the cell surface. IGF2R regulates growth, placental development, tumour suppression and signalling.

Structural insights into the interaction of insulin-like growth factor 2 with IGF2R domain 11.

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R.

Functional evaluation of novel soluble insulin-like growth factor (IGF)-II-specific ligand traps based on modified domain 11 of the human IGF2 receptor.

Ligands transported by the mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor (IGF2R) include IGF-II- and mannose 6-phosphate-modified proteins. Increased extracellular supply of IGF-II, either secondary to loss of the clearance function of IGF2R, loss of IGF binding protein function, or increased IGF2 gene expression, can lead to embryonic overgrowth and cancer promotion. Reduced supply of IGF-II is detrimental to tumor growth, and this suggests that gain of function of IGF-II is a molecular target for human cancer therapy.

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues.

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity.