High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors.

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.

Relative distribution and crosslinking of collagen distinguish fetal from adult sheep wound repair.

BACKGROUND/PURPOSE: Collagen deposition in midgestation fetal skin wounds occurs rapidly and in a normal reticular pattern unlike adult scar. Although collagen types I, III, and V are present in both fetal and adult skin wounds, their relative distribution and pattern of crosslinking are unknown. We compared the quantity, distribution, and crosslinking of specific collagen types in fetal and adult sheep wounds. METHODS: Nine fetal lambs at 75, 100, and 120 days' gestation (term, 145 days) and their ewes received subcutaneous polyvinylalcohol (PVA) sponge implants. PVA sponges were harvested at 3, 7, or 14 days after implantation, were processed, and then analyzed for collagen content, distribution, and crosslinking by two-dimensional cyanogenbromide (2-D CNBr) peptide mapping. Collagen types were further analyzed in normal skin of fetal sheep at 75, 90, 125, and 140 days' gestation and in their ewes. RESULTS: Between days 3 and 14 after implantation, total collagen deposition within PVA sponges increased 25-fold in fetal lambs but only 10-fold in adult sheep. The type I to III ratios inside 14-day sponges of 75-day gestation fetuses and adult ewes were 6.4 and 1.3, respectively. Thus, by day 14 in both fetal and adult sponges, type I collagen emerged as the major constituent. Although type V comprised less than 2% of normal skin collagen, alpha1(V) chains constituted the greatest collagen fraction in 3-day fetal implants, whereas within 3-day adult sponges only alpha2(V) collagen was detected. The total collagen content of unwounded fetal sheep skin increased twofold from 75 to 90 days' gestation. However, noncrosslinked forms of collagen type I diminished rapidly after 90 days' gestation, corresponding with the transition to scarring of fetal sheep wounds. CONCLUSIONS: Collagen types I, III, and V are deposited rapidly in fetal wounds and display an ontogenic transition in their metabolism from a fetal to an adult phenotype. Crosslinking of type I collagen increases during development and corresponds with the transition to scarring of fetal wounds after midgestation. These observations may help design strategies that induce a more fetallike repair of adult wounds.

Acylated ascorbate stimulates collagen synthesis in cultured human foreskin fibroblasts at lower doses than does ascorbic acid.

Acylated derivatives of ascorbic acid were found to be active in a number of biochemical and physiological processes. In the present study we investigated the effects of 6-O-palmitoyl ascorbate on collagen synthesis by cultured foreskin human fibroblasts. Our observations indicate a marked stimulatory effect on collagen synthesis by 6-O-palmitoyl ascorbate in the concentration range of 5-20 microM, while the synthesis stimulated by ascorbic acid was maximal at concentrations of 20-100 microM. Cells treated with 10 microM palmitoyl ascorbate for 36 h exhibited a production of collagen threefold greater than those in the presence of 10 microM ascorbic acid, and it was about the same as in cells treated with 100 microM ascorbic acid. By 48 h differences were not significant. Acylated ascorbate impaired vitality of the treated fibroblasts at concentrations exceeding 20 microM in media supplemented with 0.5% FCS. However, most of the cytotoxic effect was neutralized by FCS at a concentration of 10%. The resistance of acylated ascorbate against oxidative degradation as well as the role of free radicals in the modulation of collagen synthesis by ascorbic acid and by its derivatives is discussed.

Biochemical composition of collagen in continent and stress urinary incontinent women.

This study was designed to assess the relationship between the amount of collagen type III in the pelvic supportive tissues and stress urinary incontinence (SUI) with or without pelvic relaxation. Fourteen women agreed to participate in the study: 6 had stress urinary incontinence and pelvic relaxation (group 1); 4 had no pelvic relaxation and no sign or symptoms of SUI (group 2); 4 had pelvic relaxation without SUI (group 3). All patients underwent gynecologic surgical procedures for benign pathology and at that time biopsies were taken from perineal skin, uterosacral ligaments and round ligaments of the uterus. Collagen type III content was measured in the specimens and compared between the groups. Each subject preoperatively underwent complete urodynamic workup. A t test was used for the statistical analysis. Collagen type III content was significantly reduced (p < 0.05) in the specimens from patients with SUI (group 1) as compared independently with each of the other two groups (groups 2 and 3). Tissues from women without SUI (groups 2 and 3) had a similar content of collagen type III. These findings suggest that women with SUI show an altered collagen profile in the skin, the uterosacral, and the round ligaments. This seems unrelated to secondary damage of the supportive tissues and degree of pelvic relaxation.

Whole organ evaluation of collagen in the developing human larynx and adjoining anatomic structures (hyoid and trachea).

The collagen composition (types I, II, and III) of the normal developing human larynx and trachea was examined by biochemical methods. Autopsy specimens of larynges with attached upper tracheal rings were obtained from 28 humans ranging in age from birth to 44 years. The specimens were randomly collected, but excluded if laryngeal disease existed. The age, sex, and cause of death were documented. Collagen is important in the growth, development, repair, regeneration, and structural and functional integrity of the laryngeal framework. A preliminary report of selected cartilaginous components of the larynx was previously published by the authors, which studied the changes in the phenotypic expression of the collagen genes in children from the newborn period to 5 years 10 months of age. The current study included all of the functioning components of the skeletal larynx and trachea. The results of biochemical examination of these tissues are reported, and the potential clinical significance of the results of the study is discussed.

Collagen in the developing larynx. Preliminary study.

The primary purpose of this study was to determine the types of collagen in the developing human larynx that contribute to the structural framework and function of various components of this organ. The infant larynx is much more than a mere miniature of the adult "voice box." There are many age-related differences that occur in the larynx from the newborn period to the adult period of life. While collagen has been studied in numerous tissues, both normal and diseased, there have been no studies of the whole organ content, types, and/or changes of collagen in the developing human larynx that may account for many of the clinical findings. This study may at least in part explain whether collagen differences may account for the structural changes and responses that are seen in clinical practice.

A highly specific and quantitative method for determining type III/I collagen ratios in tissues.

The distribution of type I and type III collagens in rat, bovine and human skin were examined by a quantitative 2-D CNBr peptide mapping method. The procedure involved the solubilization of tissues by digestion with CNBr, radioactive labeling in vitro by [3H]-NaBH4 in dimethylformamide, reduction by mercaptoethanol, a second CNBr digestion and 2-D (isoelectric focusing and NaDodSO4 electrophoresis) mapping. The amounts of type I and type III collagen peptide spots in the fluorographs of 2-D maps were analyzed by 2-D scanning densitometer/analyzer. Mixtures containing various ratios of purified type I and type III collagen were used to obtain a standard curve. Using this procedure we were able to determine that in adult human skin (age range 35-65 years) 22% (+1.3%) of the labelled collagen is type III. This value is significantly higher than that was previously estimated by less accurate methods.

Alteration of collagen composition and cross-linking in keloid tissues.

Collagen composition and cross-linking in human keloid and normal skin tissues were analyzed biochemically. CNBr peptides were separated by 2-dimensional (2-D) mapping and high performance liquid chromatography (HPLC). The amounts of type I and type III collagen was quantified by 2-D scanning densitometry of fluorographs of 2-D maps derived from samples radioactively labelled in vitro by [3H]-NaBH4 in dimethylformamide. Keloid tissues contained 31.6 +/- 2.2 percent type III collagen as compared to 21.4 +/- 2.7 percent type III present in normal human skin dermis. HPLC profiles of CNBr peptides showed that approximately 5 percent of the high molecular weight material in keloids is mercaptoethanol reducible, compared to insignificant amounts in normal skin. 2-D maps derived from CNBr peptides of keloid collagen demonstrated thiol reduction sensitive alpha 1(III)-CB9 dimer as well as 24,000- and 32,000-dalton CNBr peptides, which were not mercaptoethanol reduction sensitive in normal skin due to cross-linking via the lysyl oxidase pathway. Also, a group of 20,000- to 25,000-dalton CNBr peptides, in the alpha 1(I)-CB6 cross-linking region were prominent in keloid tissues.

The effect of gamma-irradiation on collagen molecules, isolated alpha-chains, and crosslinked native fibers.

This study shows how collagen molecules are readily damaged by gamma-radiation at dosages commonly used for sterilizing biomedical products. At 1 Mrad, while the reported effectiveness of the radiation at such a low dosage to completely sterilize a material is questionable, less damage was caused to the collagen peptide backbone. Above such dosage, however, significant damage was clearly demonstrated with collagen alone and collagen in a chemically crosslinked tissue matrix. The enzyme digestion study showed that the material exposed to a very high dosage of radiation resisted degradation by pronase. However, molecular weight analysis showed a significant number of peptide bonds being cleaved by the radiation which could cause considerable changes in the long-term characteristics of the material. Therefore, tissues exposed to high dosages of gamma-radiation should be tested for long term functional changes. We want to caution against the usage of the enzyme degradation assay as a universal test for all bioprosthetic derived from biological tissues.

Mechanism of crosslinking of proteins by glutaraldehyde. IV: In vitro and in vivo stability of a crosslinked collagen matrix.

The use of native or reconstituted collagen as a bioprothesis for tissue augmentation requires the introduction of exogenous synthetic crosslinks. The degree of crosslinking determines the rate of resorption or replacement of the implanted materials by the host. Since biophysical and chemical methods to quantify these crosslinks have in general been difficult to evaluate, we have developed in vitro enzymatic approaches which enable us to correlate the degree of crosslinking with the rates of enzymatic degradation. When the number of stable crosslinks formed is large it is essential to partially unfold the collagen fibrils by heating or by exposure to denaturing agents to enhance their susceptibility to hydrolysis. In the present study we demonstrate that increasing the number of reactive amino groups on collagen by coupling 1,6-diaminohexane to carboxyl groups using a water soluble carbodiimide can significantly enhance the number of crosslinks introduced by glutaraldehyde. We also show that the enzymatic method developed correlates well with the biodegradation of radiolabeled crosslinked collagenous tissues implanted subcutaneously in rats.

A model of the mechanism of enzyme action in terms of protein conformational relaxation.

A theoretical model of enzymatic reaction is formulated in which the modulation of the reaction coordinates by low-frequency conformational motions of the enzyme molecule causes the lowering of the activation energy barriers until they completely disappear. If the rates of electron transitions in the enzyme-substrate complex exceed the characteristic frequencies of conformational motions then the rate of the elementary enzymatic reaction shows hysteresis dependence on temperature and substrate concentration.

Mechanism of crosslinking of proteins by glutaraldehyde III. Reaction with collagen in tissues.

Bovine pericardium, a dense collagenous connective tissue, was crosslinked with glutaraldehyde using different modalities of fixation. The degree of crosslinking was evaluated as a function of the ability of CNBr and pronase to solubilize collagen. Our results suggest that glutaraldehyde fixes primarily the surface of the fibers and creates a polymeric network which hinders the further crosslinking of the interstitium of the fiber. When a low concentration of glutaraldehyde was used, a slow time-dependent crosslinking process was observed. This slow process is maintained over a long period of time, greatly beyond that required for the actual penetration of glutaraldehyde to occur.