Regulation of proliferation of peritoneal tissue repair cells by peritoneal macrophages.

Macrophages produce soluble mediators which modulate fibroblast growth during tissue repair. Interaction between tissue repair fibroblasts (TRC) and regulatory proteins from surgically elicited macrophages is important for peritoneal reepithelialization. In this study, we compared the effects of an extract from postsurgical macrophage spent medium with those of known growth factors on TRC collected from injured peritoneum to evaluate certain characteristics of macrophage secretory products on peritoneal healing. Rabbits underwent a midline laparotomy followed by resection and reanastomosis of the ileum or abrasion of the abdominal wall. TRC were then collected at various times after surgery. Peritoneal macrophages recovered from nonsurgical or postsurgical rabbits were cultured for 2 days in vitro. The peak of thymidine incorporation by TRC occurred on day 5 after surgery; this gradually decreased with extended postsurgical times. Fibroblast growth factor and epidermal growth factor stimulated, whereas TGF-beta inhibited, [3H]thymidine incorporation into TRC. Maximal thymidine incorporation occurred when TRC from Postsurgical Day 5 were cultured with an extract from postsurgical macrophage spent medium. However, when TRC recovered from Postsurgical Days 2 and 10 were cultured with an extract of postsurgical macrophage spent medium, they showed greater stimulation than Day 5 TRC. These data suggest that postsurgical macrophages may produce an array of factors that stimulate fibroblast growth and differentiation and may in turn affect tissue repair throughout the wound healing process.

Follicle regulatory protein: a novel marker for granulosa cell cancer patients.

Follicle regulatory protein (FRP) is secreted by the granulosa cell of the ovary and plays a role in modulating follicle development. A dual epitope immunoassay using two murine monoclonal antibodies, isotype IgG1 (raised against porcine FRP), in tandem was developed to measure FRP in serum. The levels of FRP in the serum of women with granulosa cell tumors, normal, menstruating women, and postmenopausal women were determined. The levels of FRP were elevated in the serum of 79% of the women with granulosa cell tumors compared to the normal controls. FRP levels in serial samples from women with granulosa cell tumors generally correlated with the clinical course of the disease. Thus, FRP may provide a useful marker for granulosa cell tumors.

The mitogenic activity of peritoneal tissue repair cells: control by growth factors.

The purpose of this study was to determine the proliferative activity of tissue repair fibroblasts recovered directly from injured peritoneum at various times after surgery and to test the mitogenic response of tissue repair cells (TRC) to growth factors. Rabbits underwent bilateral peritoneal abrasion (5 X 5 cm) with sterile gauze until punctate bleeding developed. Postsurgical (Days 2, 5, 7, and 10) tissue repair cells were recovered from the injured peritoneum by scraping with a scalpel blade. Although tissue repair cells consisted of a mixed cell type after 4 days in culture, recovered cells were essentially fibroblasts. These TRC were then pulsed with [3H]thymidine after 4 days in culture. The incorporation of thymidine into Postsurgical Day 5 TRC increased significantly compared to that of Day 2 TRC (P less than 0.05). Incorporation then decreased with time following surgery. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) stimulated the incorporation of thymidine into TRC. However, the response of Postsurgical Day 7 and 10 TRCs to 1 microgram/ml EGF was significantly greater than those of Postsurgical Day 2 and 5 TRCs (Day 2 TRC, 166 +/- 7.4; Day 10 TRC, 420 +/- 96% of control cells without EGF, P less than 0.05). Platelet-derived growth factor (PDGF, 10 ng/ml) also stimulated the incorporation of thymidine into Day 10 TRCs, but this stimulatory activity (129.9 +/- 8.5% of control) was less than EGF or FGF. IL-1 alpha and IL-2 did not stimulate the incorporation of thymidine into TRC at a concentration of 100 pg/ml, but these cytokines did stimulate protein synthesis by TRC.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitogenic and protein synthetic activity of tissue repair cells: control by the postsurgical macrophage.

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophages secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [3H]thymidine, [3H]proline, and [35S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and reanastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [3H]thymidine and [3H]proline into untreated TRC gradually decreased with culture duration. Conversely, [35S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [3H]thymidine incorporation by the TRC. [3H]Proline and [35S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.

Secretion of follicle-regulatory protein by porcine granulosa cells.

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of follicle regulatory protein in the porcine ovary.

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.

Angiogenic, mitogenic, and chemotactic activity of human follicular fluid.

Human follicular fluid was found to induce the formation of new blood vessels on the chick chorioallantoic membrane 3 to 5 days after implantation. To characterize this response more fully, the effects of human follicular fluid on a continuous line of bovine aortic arch endothelial cells were studied. Human follicular fluid showed strong mitogenic activity toward endothelial cells, ranging from a threefold increase in thymidine incorporation at a dilution of 1:10 to a 1.4-fold increase at a dilution of 1:3200. Endothelial cells also exhibited a directional migration (chemotaxis) toward human follicular fluid. A 1:10 dilution of human follicular fluid induced a 6.8-fold increase in directional cellular migration through membranes with 8 microns pores, and a 1:800 dilution induced a 1.8-fold increase in migration. Endothelial cells responding to gradients of human follicular fluid also demonstrated a marked change in morphologic structure when compared with control cells. Human plasma and fibrinogen were angiogenic and chemotactic (but not mitogenic) toward endothelial cells, which suggested that fibrinogen may account for at least part of the angiogenic activity of human follicular fluid. These results indicate that human follicular fluid is strongly angiogenic and that this biologic activity can be associated with effects directly on endothelial cells in vitro.

Trehalose-containing lipooligosaccharides from mycobacteria: structures of the oligosaccharide segments and recognition of a unique N-acylkanosamine-containing epitope.

The structures of the oligosaccharide segments of nine trehalose-containing lipooligosaccharides (LOS) of Mycobacterium kansasii have been established by positive and negative fast-atom bombardment mass spectrometry, acetolysis, partial acid hydrolysis, methylation analyses, and nuclear magnetic resonance. Upon acetolysis, all produce the alpha,alpha-trehalose-containing tetraglucose (Glc4) "core" -beta-D-Glcp(1----3)-beta-D-Glcp(1----4)-alpha-D-Glcp(1----1)-alpha-D-Gl cp. The simplest (LOS I') contains an additional alpha 1----3-linked 3-O-methyl-L-rhamnopyranose (3-O-Me-L-Rhap) unit; those of intermediate complexity (LOS I-III) contain an additional D-xylopyranose (D-Xylp) residue or xylobiose in beta 1----4 linkage; and those of ultimate complexity (LOS IV-VIII) contain further D-Xylp residues and the distal N-acylkanosamine- (KanNAcyl) and fucopyranosyl- (Fucp) containing disaccharide KanNAcyl(1----3)Fucp. Thus, the structure of the oligosaccharide from LOS VII is KanNAcyl(1----3)Fucp(1----4) [-beta-D-Xylp(1----4)]6-alpha-L-3-O-Me-Rhap(1----3)Glc4++ +. Polyclonal rabbit and murine monoclonal antibodies react only with the more complex KanNAcyl-Fucp-containing lipooligosaccharides, indicating that the KanNAcyl distal end, not the trehalose end, contains the antibody binding site unique to M. kansasii and is responsible for the serological distinctiveness of M. kansasii among mycobacterial species.

Enzyme-linked immunosorbent assay of glycolipid antigens for identification of mycobacteria.

Enzyme-linked immunosorbent assays which are based on species- or type-specific glycolipids antigens and in which rabbit antisera are prepared with homologous strains are capable of distinguishing among serological variants of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex, Mycobacterium chelonei subspecies chelonei and abscessus, Mycobacterium simiae I and II, Mycobacterium kansasii, Mycobacterium szulgai, Mycobacterium xenopi, and Mycobacterium fortuitum biovariant peregrinum. The immunoreactive glycolipids can be divided into two classes. Those resistant to alkali, the C-mycoside glycopeptidolipids, are present in the M. avium-M. intracellulare-M. scrofulaceum, the M. chelonei subspecies chelonei and abscessus, and the M. simiae I and II complexes and in M. fortuitum biovariant peregrinum. The alkali-labile glycolipid antigens, the lipooligosaccharides, are present in M. kansasii, M. szulgai, and M. xenopi. In one study, the combination of enzyme-linked immunosorbent assay and alkaline susceptibility was compared with seroagglutination in the identification of 60 clinical isolates of nontuberculous mycobacteria: 45 showed perfect concordance, 9 could be narrowed to one, two, or three possibilities, and the rest did not correspond. In a second study involving 43 clinical isolates that were untypable by seroagglutination or were autoagglutinable, the results of enzyme-linked immunosorbent assay and thin-layer chromatography of glycolipid antigens were compared: 21 showed clear concordance. The results demonstrate that enzyme-linked immunosorbent assay is particularly useful in assessing the antigenicity of lipids, and sensitivity, ease, and rapidity recommend it as an adjunct to seroagglutination and thin-layer chromatography for the identification of nontuberculous mycobacteria.

Serological specificity of phenolic glycolipid I from Mycobacterium leprae and use in serodiagnosis of leprosy.

The serological activities of the specific phenolic glycolipid I from Mycobacterium leprae, its dissected parts, and related glycolipids from other mycobacteria were examined by enzyme-linked immunosorbent assay against hyperimmune anti-M. leprae rabbit antiserum and sera from patients with leprosy and other mycobacterial diseases. High anti-phenolic glycolipid I immunoglobulin M antibodies were found in 23 of 24 (96%) of lepromatous leprosy patients on short term chemotherapy and in 8 of 13 tuberculoid leprosy patients (62%). Sera from patients with tuberculosis or atypical mycobacterial infections were devoid of anti-phenolic glycolipid I activity. The structurally related phenolic glycolipids from Mycobacterium kansasii and Mycobacterium bovis and the aglycone segments of the M. leprae product showed no significant activity. Thus, the trisaccharide determinant of phenolic glycolipid I is specific in its structure, serological activity, and, to a lesser extent, the antibody class it evokes.