Cultures Of Fetal Fibroblasts Research Articles

Substance P, a Neuropeptide, Promotes Wound Healing via Neurokinin-1 Receptor.

Substance P (SP), an endogenous neuropeptide, mediates intracellular signaling, mainly through a tachykinin receptor. The tachykinin receptors family consists of neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 receptors. Our previous studies on SP have shown its wound healing potentials. But the exact mechanism of wound healing by SP is not exactly known. In view of this, the present study was aimed at evaluating the in vitro wound healing effect of SP alone and in the presence of NK-1, NK-2, and both receptor antagonists. Scratch assay, transwell assay, and tumor growth factor-beta 1 (TGF-β1) assay were performed on buffalo fetal fibroblast culture. The cotreatment of fibroblast cultures with SP alone during the 24 h caused the significant proliferation and migrations of cells in both horizontal and vertical directions. The SP in the presence of spantide II (NK-1 antagonist) failed to stimulate this migration. The treatment of cells with SP in the presence of NK-2 antagonist treatment also showed a significant reduction of migration of cells with respect to SP treatment alone. The SP in the presence of both NK-1 and NK-2 antagonists failed to stimulate the horizontal migration of cells and most of the ineffectiveness of SP was observed in this combination. The TGF-β1 levels were significantly higher in the supernatants of cells that were exposed to SP alone. All other treatments have significantly lower TGF-β1 levels than SP alone treatment. It is concluded that different actions on fibroblast cells by SP were mainly mediated through the NK-1 receptor.

About the effectiveness of cell technologies in extensive soft tissue defects plasty

Abstract. Fetal fibroblast culture transplantation results were evaluated in the treatment of 18 burn victims. Comparison group consisted of 18 burn patients received medical care without cellular technologies utilization. The main comparison parameters in the study groups: the timing of the first stage of autodermoplasty; the number of autodermoplasties during the treatment; hospitalization duration; the newly formed epidermis area estimation. Fetal fibroblast culture transplantation in burn patients with extensive skin defects was performed on average 14,883,56 days after the injury. The timing of the first stage of autodermoplasty did not differ in the main and control groups, not exceeding an average of 19,122,01 days (p=0,48). An average of 2,710,67 surgeries using cell technologies performed in patients of the study group. The use of fetal fibroblasts culture in patients with extensive skin defects reduces the need for autodermoplasty by 1,6 times due to the granulation tissue formation and the epidermal growth beginning 7 days after and complete epidermal formation 14 days after transplantation. Regenerative medicine technologies utilization in patients with extensive skin lesions is possible and appropriate. Due to the fetal fibroblasts culture transplantation a kind of temporary biological coating is formed in the wound. It accelerates the wound healing process phase change from exudation to proliferation and the preparation of skin defects for autodermoplasty, expanding the possibilities of effective patients treatment.

Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering.

As a biological tissue material, amniotic membrane (AM) has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional (3D) spongy scaffold made of the extracellular matrix (ECM) of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. In this experimental study, cellular components of human amniotic membrane (HAM) were removed with 0.03% (w/v) sodium dodecyl sulphate (SDS). Quantitative analysis was performed to determine levels of Glycosaminoglycans (GAGs), collagen, and deoxyribonucleic acid (DNA). To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid (HCl 0.1 M) with pepsin (1 mg/ml). In the present experiment 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (H&E) staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scaffolds were cytocompatible. Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment (39 ± 4.06 μg/ml vs. 341 ± 29.60 μg/ml). Differences were observed between cellular and denude AM in matrix collagen (478 ± 18.06 μg/mg vs. 361 ± 27.47 μg/mg).With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% ± 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 μg/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 µm measured from scanning electron microscope (SEM) images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells. The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation.

Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture.

Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

A New Mechanism for Aging: Chemical “Age Spots” in Immortal DNA Strands in Distributed Stem Cells

The existence of immortal DNA strands (IDSs) in distributed stem cells (DSCs) of adult human tissues was first inferred by Cairns. Cairns deduced the existence of IDSs by connecting two seemingly disparate observations - one his own and the other belonging to Lark. Cairns noted a mathematical discrepancy between predicted human tissue cell mutation rates and human cancer incidence. He integrated this insight with Lark's earlier discovery of non-random mitotic chromosome segregation in both plant root tip cells and mouse fetal fibroblast cultures to predict the existence of IDSs as the essential elements of a mutation-defense mechanism in DSCs. Since Cairns' seminal prediction, several laboratories have identified IDSs in diverse mammalian cells with DSC properties. Past studies focused on the potential roles of IDSs as originally envisioned in DSC genetic fidelity or in the maintenance of the DSC phenotype. Another possible consequence of IDSs, aging, has received little attention. Herein, the potential for cumulative chemical modifications and decompositions (i.e., "age spots") of IDSs in DSCs to act as a major determinant of human aging is considered. If accrued chemical alterations of IDSs prove to be essential determinants of aging, then a means to restore IDSs may yield new strategies for tissue rejuvenation.

Slc39a14 Gene Encodes ZIP14, A Metal/Bicarbonate Symporter: Similarities to the ZIP8 Transporter

The mouse and human genomes contain 14 highly conserved SLC39 genes. Viewed from an evolutionary perspective, SLC39A14 and SLC39A8 are the most closely related, each having three noncoding exons 1. However, SLC39A14 has two exons 4, giving rise to Zrt- and Irt-related protein (ZIP)ZIP14A and ZIP14B alternatively spliced products. C57BL/6J mouse ZIP14A expression is highest in liver, duodenum, kidney, and testis; ZIP14B expression is highest in liver, duodenum, brain, and testis; and ZIP8 is highest in lung, testis, and kidney. We studied ZIP14 stably retroviral-infected mouse fetal fibroblast cultures and transiently transfected Madin-Darby canine kidney (MDCK) polarized epithelial cells. Our findings include: 1) ZIP14-mediated cadmium uptake is proportional to cell toxicity, but manganese is not; 2) ZIP14B has a higher affinity than ZIP14A toward Cd(2+) (K(m) = 0.14 versus 1.1 microM) and Mn(2+) uptake (K(m) = 4.4 versus 18.2 microM); 3) ZIP14A- and ZIP14B-mediated Cd(2+) uptake is most inhibited by Zn(2+), and next by Mn(2+) and Cu(2+); 4) like ZIP8, ZIP14A- and ZIP14B-mediated Cd(2+) uptake is dependent on extracellular HCO(3)(-); 5) like ZIP8, ZIP14 transporters are localized on the apical surface of MDCK-ZIP cells; and 6) like ZIP8, ZIP14 proteins are glycosylated. Tissues such as intestine and liver, located between the environment and the animal, show high levels of ZIP14; given the high affinity for ZIP14, Cd(2+) is likely to act as a rogue hitchhiker-displacing Zn(2+) or Mn(2+) and entering the body to cause unwanted cell damage and disease.

282 CHARACTERIZATION OF THE DEVELOPMENTAL POTENTIAL OF FETAL SOMATIC STEM CELLS ISOLATED FROM DIFFERENT TISSUES

Recently we discovered a novel type of stem cells which can be derived from primary cultures of fetal connective tissue using a high-density culture (HD) system (Kues et al. 2005 Biol. Reprod. 70, 1020–1028). This cell type shows most of the characteristics of true embryonic stem cells and is a promising alternative for developing therapeutically useful cells. The goal of the present study was to analyze and characterize stem cell populations obtained from five different tissues, i.e., brain, heart, lung, liver, and adrenal gland, using this HD culture system. Explant cultures of the double transgenic strain OG2/Rosa26 were established from mouse fetuses at Day 13.5 post-conception. In this strain, cells with pluripotent properties are characterized by the green fluorescence of the green fluorescent protein (GFP) which is under the transcriptional control of the Oct-4 promoter. In a total of five replicates, cells were subcultivated and reseeded at a concentration of 25 000 cells/cm–2. Cell culture conditions were as recently reported for fetal somatic stem cell derivation (Kues et al. 2005 Biol. Reprod. 72, 1020–1028). After colony formation and/or observation of green fluorescence under UV illumination, mRNA was isolated for RT-PCR analysis of the expression of genes known to be involved in pluripotency, i.e., Oct-4, Nanog, Stat3, TNAP, Rex1, and Sox2. Under these culture conditions, 12.5% of the liver and 80% of the brain explants showed senescence and could not be seeded at the required concentration. No colony formation was observed in brain and heart cultures. Cells from heart explants showed a three-dimensional growth over the whole surface of the attached cells and were difficult to separate. On average, colony formation was observed after 11 days � 8 (mean � SD) (fetal fibroblasts), 10 days � 7 (lung), 18 days � 11 (liver), and 4 days � 1 (adrenal gland). Green fluorescence was detected after 17 days � 9 (fetal fibroblasts), 17 days � 9 (lung), 35 days � 13 (heart, randomly distributed), 26 days � 10 (brain), 26 days � 8 (liver), and 8 days � 5 (adrenal gland). A single large clump of stem-like cells formed in 43% of the fetal fibroblast, 35% of the lung, and 41% of the liver derivatives. Oct-4 expression was not observed in any of the cultures. Nanog and Stat3 were expressed in all cell cultures, and TNAP was expressed in all cultures from heart, brain, lung, and adrenal gland. Rex1 was expressed in all cultures from brain and adrenal gland, and in 25% of the heart cultures and in 16.6% of the fetal fibroblasts. Sox2 was expressed in all cultures from brain and in 16.6% of fetal fibroblast cultures. These preliminary results show that cells with some of the typical features of stem cells can be derived from various tissues of the body, but that the efficiency is different among tissues. Only cells derived from the adrenal gland exhibited better colony formation than the original fibroblasts. This work was funded by DFG grant Ni 256/28-1.

ZIP8, Member of the Solute-Carrier-39 (SLC39) Metal-Transporter Family: Characterization of Transporter Properties

Cadmium is a dangerous metal distributed widely in the environment. Members of our laboratory recently identified the ZIP8 transporter protein, encoded by the mouse Slc39a8 gene, to be responsible for genetic differences in response to cadmium damage of the testis. Stable retroviral infection of the ZIP8 cDNA in mouse fetal fibroblast cultures (rvZIP8 cells) leads to as much as a 10-fold increase in the rate of intracellular cadmium influx and accumulation. In the present study, we showed that cadmium uptake operated maximally at pH 7.5 and a temperature of 37 degrees C and was inhibited by cyanide. Of more than a dozen cations tested, manganese(II) was the best competitive cation for cadmium uptake. The Km for Cd2+ uptake was 0.62 microM, and the Km for Mn2+ uptake was 2.2 microM; thus, manganese is probably the physiological substrate for ZIP8. Cadmium uptake was independent of sodium, potassium or chloride ions, but strongly dependent on the presence of bicarbonate. By Western blot analysis of rvZIP8 cells, we showed that ZIP8 protein was glycosylated. Using Z-stack confocal microscopy in Madin-Darby canine kidney polarized epithelial cells, we found that ZIP8 was localized on the apical side-implying an important role for manganese or cadmium uptake and disposition. It is likely that ZIP8 is a Mn2+/HCO3- symporter, that a HCO3- gradient across the plasma membrane acts as the driving force for manganese uptake, and that cadmium is a rogue hitchhiker displacing manganese to cause cadmium-associated disease.

Hemimethylation and Non-CpG Methylation Levels in a Promoter Region of Human LINE-1 (L1) Repeated Elements

DNA methylation within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition. Determining methylation patterns on the complementary strands of repeated sequences is difficult using standard bisulfite methylation analysis. Evolutionary changes in each repeat and the variations between cells or alleles of the same repeat lead to a heterogeneous population of sequences. Potential sequence biases can arise during analyses that are different for the converted sense and antisense strands. These problems can be avoided with hairpin-bisulfite PCR, a double-stranded PCR method in which complementary strands of individual molecules are attached by a hairpin linker ligated to genomic DNA. Using human L1 elements to study methylation of repeated sequences, (i) we distinguish valid L1 sequences from redundant and contaminant sequences by applying the powerful new method of molecular barcodes, (ii) we resolve a controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts in favor of relatively little hemimethylation, (iii) we report that human L1 sequences in different cell types also have primarily concordant CpG methylation patterns on complementary strands, and (iv) we provide evidence that non-CpG cytosines within the regions analyzed are rarely methylated.

327 CHARACTERIZATION OF FETAL AND ADULT FIBROBLASTS FROM NELORE BOVINE FOR NUCLEAR TRANSFER

Cloning by nuclear transfer is a technology that has provided major advances in reproductive sciences. It is an efficient method: (1) to produce transgenic embryos, fetuses, and animals; (2) to multiply genetically superior adult animals; and (3) to generate proteins, tissues, and organs for xenotransplants of human interest. Basic studies on nuclear transfer have contributed to understanding how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies. The aim of this study was to establish a stable lineage of Nelore adult and fetal fibroblasts for embryo reconstruction. Cultures were characterized by morphological aspects, and by immunocytochemical and ultrastructural analysis. Cultures of fetal and adult fibroblasts at passage 2 stained positively for cytokeratin, a typical protein from the intermediate filament of epithelial cells, in some cells but staining was not found in subsequent passages. A dense and organized network occurred in adult and fetal fibroblast cultures presenting positive immunostainig for vimentin in pass 5, 10, and 15 subcultures, but did not stain for cytokeratin (negative control). The results demonstrated that fibroblasts continued expressing vimentin and maintained their morphological aspects in culture. The ultrastructural analysis showed the presence of organelles involved in protein synthesis including dilated rough endoplasmatic reticulum, Golgi cisternae, and polyribosomes. Elongate mitochondria were distributed around the Golgi complex, suggesting high metabolic activity. These results demonstrated that adult and fetal fibroblasts continued to develop biological activities and maintained specific characteristics under these culture conditions. In conclusion, adults and fetal fibroblasts can be successfully used as donor cells for nuclear transfer. This work was supported by FAPESP 01/11931-8 and 01/13944-0.

196PREVENTION AND TREATMENT OF BOVINE VIRAL DIARRHEA VIRUS INFECTIONS IN FETAL FIBROBLAST CELLS

Unnoticed infections with bovine viral diarrhea virus (BVDV) can occur in cultured cells used for somatic cell nuclear transfer. Aromatic cationic molecules have exhibited inhibitory activity against in vitro replication of BVDV. The purpose of this research was to evaluate the ability of aromatic cationic compounds to prevent or treat noncytopathic BVDV infections of fetal fibroblast cells. Aromatic compounds tested were 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan (DB606); 2-(2-benzimidazolyl)-5-[4-(2-imidazolino) phenyl]furan dihydrochloride (DB772); and 2-(1-methyl-2-benzimidazolyl)-5-[4’-(2-imidazolino)-2’-methylphenyl]furan dihydrochloride (DB824). To evaluate prevention of BVDV infections, 10 cell lines in the absence or presence of 7 dilutions of each of the 3 compounds were inoculated with BVDV. The concentrations of BVDV in medium and cell lysates were determined by serial dilution and virus isolation. Samples were obtained 72 hours post-inoculation. Bovine viral diarrhea virus in cell culture medium and cell lysate samples was evaluated by comparison to equivalent samples from control cultures in which no compound was added (percent of control=cell culture infective doses (50%; CCID50) of BVDV in compound sample/CCID50 of BVDV in control sample lacking compound). The viral inhibitory concentrations (99%) of compounds were calculated with JMP software by least-squares regression techniques. Cumulatively, the 99% endpoints for inhibition of viral replication in fetal fibroblast cell lines for the 3 compounds were 0.1μM, 0.007μM and 0.028μM, respectively. To evaluate therapeutic treatment of established BVDV infections, the concentration of BVDV in medium and cell lysates of 2 fetal fibroblast cell lines were evaluated. The cell lines were previously determined to be infected with a genotype 1a strain of BVDV. Samples were obtained during 4 sequential passages in the absence or presence of 0.04μM and 4μM concentrations of DB772 or DB824. Presence of BVDV was determined by reverse transcription nested polymerase chain reaction and virus isolation. While BVDV persisted in cultures supplemented with no aromatic compound or 0.04μM, both DB772 and DB824 effectively cured BVDV infections after 1 passage in 4μM, and cells remained viable. Results indicate that BVDV infections can be effectively prevented or treated in fetal fibroblast cultures. Further research is needed to determine if exposed cells are competent for production of normal embryos via nuclear transfer.

Age-Related Molecular Polymorphism of the Heterodimeric Proteoglycan Bisdermican

Bisdermican (PG760) is a large, heterodimeric, dermatan sulfate proteoglycan found in selected basement membranes, smooth muscle cell layers, and different extracellular matrices. Age-dependent and developmentally regulated alterations in glycosaminoglycan structure and quantity have been shown to be functionally relevant for a number of physiological and pathological processes. Bisdermican was purified from human skin fibroblast cultures of different age and confluency. Following β-elimination, glycosaminoglycan chains were analyzed by Sephacryl-S-300 chromatography. Glycosaminoglycan chains of Bisdermican from infantile fibroblasts had a molecular weight of 19 kDa, whereas the glycosaminoglycan chain of the large Bisdermican subunit purified from confluent fetal fibroblast secretions was slightly larger (Mr = 24 kDa). Bisdermican derived from subconfluent cultures of fetal fibroblasts displayed the largest glycosaminoglycan chains with a molecular weight of 31.5 kDa for the large subunit, and a molecular weight of 22 kDa for the small subunit. Thus, Bisdermican displays a molecular polymorphism that is related to its chronological age and proliferative state.

Production of a cloned calf from a fetal fibroblast cell line.

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Epithalon peptide induces telomerase activity and telomere elongation in human somatic cells.

Addition of Epithalon peptide in telomerase-negative human fetal fibroblast culture induced expression of the catalytical subunit, enzymatic activity of telomerase, and telomere elongation, which can be due to reactivation of telomerase gene in somatic cells and indicates the possibility of prolonging life span of a cell population and of the whole organism.

Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts.

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost during the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived including one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.

A novel large dermatan sulfate proteoglycan from human fibroblasts

Human skin fibroblasts express, in addition to versican, a second large chondroitin sulfate/dermatan sulfate proteoglycan, which has been investigated with the aid of a specific antiserum in cultures of fetal fibroblasts. Its core protein, obtained after chondroitin ABC lyase treatment, exhibits an apparent molecular mass of about 740 kDa in the absence of a reducing agent whereas reduction produces two core proteins of 460 and 300 kDa, respectively. Both subunits carry one or very few dermatan sulfate chains of about 20 kDa which are of similar chemical composition irrespective of the type of subunits to which they are attached. Tryptic peptide maps of [35S]methionine-labeled core proteins indicated that both subunits are related neither to each other nor to versican, suggesting that the proteoglycan exists predominantly as a heterodimeric molecule. It is insensitive to collagenase and does not interact with hyaluronan. Pulse-chase experiments suggested that the core proteins are different gene products. Dimerization begins soon after core protein synthesis but requires more than 2 h for completion. Glycosaminoglycan synthesis occurs immediately prior to secretion. A small proportion of both subunits may be secreted in form of a monomeric proteoglycan. The heterodimeric proteoglycan is a major proteoglycan species of fetal fibroblasts. The secreted product represents 10-20% of [35S]methionine and about 5-10% of [35S]sulfate incorporated into secreted proteoglycans.

Antenatal Diagnosis of Recessive Dystrophic Epidermolysis Bullosa: Collagenase Expression in Cultured Fibroblasts as a Biochemical Marker

We performed fetoscopy and skin biopsy on a 19-week fetus at risk for recessive dystrophic epidermolysis bullosa (RDEB). Ultrastructural analysis of the tissue revealed dermolytic blister formation in the skin characteristic of the disease. To develop a biochemical test for use in antenatal diagnosis of RDEB, we established skin fibroblast cultures from the 20-week aborted fetus. The collagenase production by fetal RDEB fibroblast cultures was greater than seen in normal fetal fibroblast cultures. The concentration in culture medium from fetal RDEB cultures was 5.42 +/- 0.74 micrograms/ml (mean +/- SE) compared with 2.24 +/- 1.11 micrograms/ml in normal adult control cultures and 2.05 +/- 0.61 micrograms/ml in cultures from patients with other genetic forms of epidermolysis bullosa (p less than 0.025). In contrast, the concentration of collagenase in the fetal RDEB culture medium was not different from that seen in cell cultures from known patients with RDEB (5.34 +/- 1.12 micrograms/ml). Collagenase activity of the fetal RDEB medium was also increased approximately 3.5-fold. These data indicate that enhanced expression of collagenase by fetal RDEB skin fibroblasts can serve as a biochemical adjunct, and possibly an alternative, to morphologic examination of tissue for antenatal diagnosis.