In vivo degradation of 14C-labeled small intestinal submucosa (SIS) when used for urinary bladder repair.

The rate of in vivo degradation was determined for a naturally occurring biomaterial derived from the extracellular matrix of the small intestinal submucosa (SIS). The SIS was labeled by giving weekly intravenous injections of 10 microCi of 14C-proline to piglets from 3 weeks of age until the time of sacrifice at 26 weeks. The resultant SIS prepared from these pigs contained approximately 10(3) fold more 14C than unlabeled tissues. The labeled SIS was used to repair experimental defects in the urinary bladder of 10 dogs. The animals were sacrificed at post-operative times ranging from 3 days to 1 year and the remodeled urinary bladder tissue was harvested for evaluation of 14C by a combination of liquid scintillation counting and accelerator mass spectrometry. The remodeled tissue contained less than 10% of the 14C (disintegrations per minute/gram tissue wet weight) at 3 months post-surgery compared to the SIS biomaterial that was originally implanted. The SIS scaffold was replaced by host tissue that resembled normal bladder both in structure and function. After implantation, 14C was detected in highest concentrations in the blood and the urine. The SIS bioscaffold provides a temporary scaffold for tissue remodeling with rapid host tissue remodeling, degradation, and elimination via the urine when used as a urinary bladder repair device.

Retrospective study of the association of stress and smoking during pregnancy in rural women.

Women who continue to smoke during pregnancy put themselves and their fetuses at serious risk for complications. Various smoking cessation programs have been designed that specifically target pregnant smokers. Longitudinal studies, however, have shown that there is a group of women who are unable to quit smoking while pregnant. Women from a rural area of the Mid-West (N=299) were interviewed postpartum to determine the stresses these women experienced prenatally and the association of the stress with continuing to smoke during pregnancy. Subjects were divided into three groups: Nonsmokers, Quitters, Smokers. This study not only confirms other reports that these women are more stressed but also documents some of the major stressors. Statistically significant differences were found between groups for financial worries (P=.0002), problems with the family (P<.001), and domestic violence (P<.001). Assessing pregnant women for stress and, especially, domestic violence should be part of the implementation of the Clinical Practice Guidelines for Smoking Cessation.

Vascular endothelial growth factor in porcine-derived extracellular matrix.

An extracellular matrix (ECM) derived from the submucosa of the porcine small intestine (SIS) has been shown to induce angiogenesis and host tissue remodeling when used as a xenogeneic bioscaffold in animal models of wound repair. In the present study, we compared the in vitro effects of SIS ECM extracts to several purified angiogenic growth factors on human dermal microvascular endothelial cell (HMEC) growth patterns. The SIS ECM was shown to induce tube formation from HMEC in a three-dimensional fibrin-based angiogenesis assay in a manner similar to that caused by the addition of vascular endothelial growth factor (VEGF). This tube formation was blocked in the presence of anti-VEGF neutralizing antibody. Western blots and ELISA procedures showed that the SIS ECM contains as much as 0.77 ng VEGF/g SIS. The closely related endothelial cell mitogen, platelet-derived growth factor (PDGF), was not detectable in the SIS extracts. We conclude that VEGF is present in the SIS extracellular matrix. The role of VEGF in SIS-induced wound repair remains unknown, but its presence in the ECM makes it a possible contributor to the angiogenic effect of SIS when this ECM is used as a tissue repair scaffold in animal models of wound repair.

Galalpha(1,3)Gal epitope in porcine small intestinal submucosa.

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial derived from porcine jejunum, which promotes constructive tissue remodeling when applied as a xenogeneic graft material. Galactosyl-alpha(1,3)galactose (Gal) is a cell-associated epitope responsible for hyperacute rejection of porcine whole-organ xenografts in primates. Because SIS is harvested from porcine tissue, it may contain the Gal epitope. The goals of this study were to determine if Gal is present in SIS and, if it is present, to determine if human serum complement can be activated in vitro following exposure to porcine-derived SIS. SIS was probed for Gal by immunohistochemical methods and by lectin-peroxidase staining. SIS stained strongly positive with human serum, which contains naturally occurring antibodies to Gal, followed by anti-immunoglobulin G (IgG) or anti-IgM peroxidase conjugate. Blocking with the lectin I-B(4), which is specific for the Gal epitope, decreased the intensity of staining. Exposure of SIS to alpha-galactosidase reduced staining to negligible amounts. The Gal epitope is distributed transmurally throughout the SIS material. Subtyping of the immunoglobulins that bind to SIS showed that IgG(2) is the major immunoglobulin of human plasma that binds to SIS. SIS did not activate complement in vitro as measured by radioimmunoassay for C3a.

Characterization of the ion transport responses to ADH in the MDCK-C7 cell line.

The Madin-Darby canine kidney (MDCK) cell line expresses many characteristics of the renal collecting duct. The MDCK-C7 subclone forms a high-resistance, hormone-responsive model of the principal cells, which are found in distal sections of the renal tubule. The electrophysiological technique of short-circuit current measurement was used to examine the response to antidiuretic hormone (ADH) in the MDCK-C7 clone. Three discrete electrogenic ion transport phenomena can be distinguished temporally and by the use of inhibitors and effectors. Initially the cells exhibit anion secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). The presence of CFTR was confirmed by immunoprecipitation followed by Western blotting. The CFTR-mediated anion secretion is transient and is followed, in time, by a verapamil- and Ba(+)-sensitive anion secretion or cation absorption and, finally, by Na+ reabsorption via epithelial Na+ channels (ENaC). In contrast to other studies of MDCK cells, we see no indication that the presence of CFTR functionally inhibits ENaC. The characterization of the various ion transport phenomena substantiates this cell line as a model renal epithelium that can be used to study the hormonal and metabolic regulation of ion transport.

Endothelial cell adherence to small intestinal submucosa: an acellular bioscaffold.

Degradable biomaterials to be used as scaffolds for tissue repair will ideally be able to support new blood vessel growth. The present study evaluated the adherence of human dermal microvascular endothelial cells (HMECs) to an acellular resorbable scaffold material derived from the small intestinal submucosa (SIS). HMECs were exposed to hydrated and dehydrated forms of SIS and to plastic surfaces coated with one of four different known components of the SIS extracellular matrix: collagen Type I, collagen Type IV, fibronectin, and laminin. Results showed that adherence of HMECs to hydrated SIS was greater than to any of the other tested surfaces (P < 0.05). Exposure of HMECs to either soluble collagen Type IV or soluble fibronectin prior to exposure of these cells to hydrated SIS showed only partial inhibition of HMEC attachment. We conclude that HMECs find hydrated SIS to be a suitable substrate for adherence and that dehydration of SIS adversely affects the ability of HMECs to adhere in vitro. The cause of HMEC adherence to SIS appears to be a combination of both its composition and architecture.

Small intestinal submucosa: a substrate for in vitro cell growth.

The extracellular matrix (ECM) of the small intestinal submucosa (SIS) was harvested by removing the superficial layers of the mucosa and the external muscular layers. The remaining 80 microns thick sheet was disinfected and sterilized by methods which removed all cellular components. The SIS-ECM, retaining its native 3-dimensional microarchitecture and composition, was evaluated for its ability to support in vitro cell growth. Six separate cell types were seeded either alone or in coculture with other cells upon this matrix, grown in selected media, a examined daily for time periods ranging from 48 h to 2 weeks. The six cell types tested were NIH Swiss mouse 3T3 fibroblast, NIH 3T3/j2 fibroblasts, primary human fibroblasts, primary human keratinocytes, human microvascular endothelial cells (HMECs), and an established rat osteosarcoma (ROS) cell line. All cell types showed the ability to attach a proliferate. All fibroblast cell line and the keratinocytes proliferated and/or migrated into the 3-dimensional scaffold of the SIS matrix. The ROS cells and the HMECs were confined in their growth pattern to the surface of the matrix. Coculturing of NIH 3T3/J2 fibroblasts and primary human keratinocytes resulted in a distinctive spatial orientation of the two cell types. The fibroblast populated the mid-substance of the 3-dimensional matrix and the keratinocytes formed an epidermal structure with rete ridge-like formation and stratification when the composite was lifted to an air liquid interface in culture. In summary, SIS provides a substratum with a 3-dimensional scaffold that allows for cell migration and spatial organization. The substratum is suitable for in vitro studies of the interaction between epithelial or mesenchymal cells and a naturally occurring extracellular matrix.

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells.

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 approximately 6 microM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

The future on ice.

Reproductive Tissue Banking: Scientific Principles Edited by Armand M. Karow and John D. Critser. San Diego, Academic Press, 1997, $99.00 (xviii+472 pages), ISBN 0-12-399770-4.

Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms.

The individual effects of aldosterone and insulin on amiloride-sensitive Na+ transport in model renal epithelia have been well characterized. However, in the physiological state, many hormones are present concurrently and their interactions need to be addressed. We have found that, over 5 h, the effects of insulin and aldosterone are additive. This indicates that the biochemical pathways are largely independent. To delineate the signaling pathways, we examined the requirement for tyrosine kinases by using genistein, a tyrosine kinase inhibitor. Genistein blocks basal (constitutive) Na+ transport and inhibits insulin- and aldosterone-stimulated Na+ transport. From these results, we conclude that a tyrosine phosphorylation is an important component of amiloride-sensitive Na+ transport.

Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line.

The Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na+ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na+ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na+ transport.

The heterotrimeric G-protein Gi is localized to the insulin secretory granules of beta-cells and is involved in insulin exocytosis.

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells. Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on beta-TC3 homogenate and ISG demonstrated that G alpha i was dramatically enriched in ISG. Levels of G alpha o and G alpha q were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha i to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas. In contrast to G alpha o and G alpha q, G alpha was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha i located in the ISG of beta-cells to stimulate insulin secretion.

Insulin induces an unmasking of the carboxyl terminus of G(i) proteins in rat adipocytes.

Several groups have shown a relationship between the insulin receptor and inhibitory G proteins, G(i). An antisera, 8729, to a peptide sequence (KNNLKDCGLF) corresponding to the carboxyl termini of G(i)alpha subunits was used to investigate this relationship by immunoelectron microscopy. Rat adipocytes were incubated in the absence or presence of 100 ng/ml insulin for 1 h and fixed for immunoelectron microscopy. Insulin-treated adipocytes stained with 8729 were labeled at the cell surface at a much higher density than control adipocytes. Subcellular fractionation of insulin-treated and control cells was followed by PAGE and Western blots of the plasma membrane and low-density microsomes with 8729. The density of the bands did not change in response to insulin treatment. Antibodies to noncarboxyl terminus sequences of the alpha subunit were used for immunoelectron microscopy and no difference was noted between insulin-treated and control adipocytes. These results indicated that 8729 was detecting a conformational change in the structure of G(i)alpha subunit in the plasma membrane in response to insulin. This unmasking of the carboxyl terminus was also seen in response to treatment with phenylisopropyladenosine and prostaglandin E2. Pertussis toxin-catalyzed ADP ribosylation also unmasked the carboxyl terminus. In contrast, isoproterenol, an agonist of stimulatory G proteins (Gs), did not induce an unmasking of the carboxyl terminus. These results support the hypothesis that some of insulin's effects are mediated through G(i) proteins in adipocytes.

Convergence of the endocytic and lysosomal pathways in soybean protoplasts.

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.

A study of the fertility of mothers of twins.

Data on all births to Birmingham residents during a ten-year period were used to compare the reproductive history of mothers immediately before and after a twin maternity with that of mothers of similar age and parity before and after a single birth. It was found that mothers of twins were less likely than mothers of singletons to have a subsequent birth if both twins survived, but if only one survived there was little difference between the two groups. If both twins died, however, the likelihood of a later birth was increased. The interval before the next birth was longer than expected when both twins survived and much shorter when both died. The sex of the twins appeared to have little influence on subsequent reproduction, in spite of the fact that for singleton-containing fraternities a further pregnancy was more likely if the first two children were of the same sex than if they were of opposite sexes. The mean interval preceding a twin maternity was almost the same as that preceding a single birth when both twins survived, but it was shorter when one twin died (being similar to that preceding the birth of a singleton who died) and it was much shorter when both twins died.

Relationship between childhood infections and measured intelligence.

The possibility that some of the common childhood infections lead to unrecognized impairments of neurological function was examined in 43 820 Birmingham children whose intelligence was assessed in the 11-plus examination. Mean verbal reasoning scores were lower for children who had had measles or pertussis than for those who had had neither of these diseases. However, since attack rates and measured intelligence are related inversely to social class, the lower scores of children with measles and pertussis may be due to class differences which are not eliminated completely by standardization for maternal age and birth order. Mean scores were a little higher for children who had had rubella than for those who had not, and it is suggested that this difference may be due to more frequent reporting of the disease by the more intelligent mothers.

The influence of the birth of a malformed child on the mother's further reproduction.

Births which occurred in Birmingham in 1964-70 were assembled into fraternities by computer linkage. By calculating the frequency with which one birth was followed by another and the interval between births the reproductive behavior of mothers after the birth of a malformed child was compared with that of all mothers, taking account of differences in maternal age, parity, and period of observation. It was found that malformations which resulted in stillbirth or early death were more frequently followed by another birth and that the interval to the following birth was shorter than usual. In this respect malformations did not differ in their effect from other causes of stillbirth and infant death. The birth of children were severe malformations who survived, however, acted as a slight deterrent to further reproduction. The malformation rate among children born after a malformation was double the usual rate; the recurrence rate was particularly high for neural tube defects, 5% of subsequent children being affected. In spite of this parents of children with central nervous system malformations were not deterred from further reproduction unless the affected child survived.