Characterization of process air emissions in automotive production plants.

During manufacturing, particles produced from industrial processes become airborne. These airborne emissions represent a challenge from an industrial hygiene and environmental standpoint. A study was undertaken to characterize the particles associated with a variety of manufacturing processes found in the auto industry. Air particulates were collected in five automotive plants covering ten manufacturing processes in the areas of casting, machining, heat treatment and assembly. Collection procedures provided information on air concentration, size distribution, and chemical composition of the airborne particulate matter for each process and insight into the physical and chemical processes that created those particles.

Characterization of murine pregnancy decidua transforming growth factor beta. I. Transforming growth factor beta 2-like molecules of unusual molecular size released in bioactive form.

A novel type of bioactive transforming growth factor beta 2 (TGF beta 2)-related immunosuppressive activity, lower in molecular mass (20-23 kDa) than a conventional TGF beta 2 standard (25 kDa), has been shown to be released by non-T non-B suppressor cells of murine decidua into fetal bovine serum (FBS)-containing tissue culture medium during a 48-h incubation at 37 degrees C. Substitution of a serum-free medium has allowed direct PAGE-Western blotting and the demonstration in supernatant (prior to incubation at 37 degrees C) of a TGF beta 2-immunoreactive doublet at a higher molecular mass (26 and 27 kDa) than the standard but with apparent immunosuppressive activity. When decidua were incubated in serum-free medium at 37 degrees C, immunosuppressive activity in the supernatant increased to peak at 18 h in association with the appearance of the previously described lower molecular mass species of molecule. The doublet did not appear to be the result of glycosylation of a conventional 25-kDa TGF beta 2 and could be converted into the lower molecular mass form by incubation for 18 h at 37 degrees C, with pH 4.5 but not with pH 7.5 buffer; this incubation in the absence of decidual cells was not accompanied by increased immunosuppressive activity. This transformation could be blocked by a brief heating of the supernatant to 80 degrees C for 10 min to destroy enzymes present in supernatant prior to mixing with the acidic buffer, but biologic activity neutralizable by anti-TGF beta 2 was retained.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycosylated recombinant human tumor necrosis factor binding protein-1 reduces mortality, shock, and production of tumor necrosis factor in rabbit Escherichia coli sepsis.

OBJECTIVE: To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. DESIGN: Prospective, randomized, controlled trial. SETTING: University hospital research laboratory. SUBJECTS: Eighteen female, New Zealand white rabbits. INTERVENTIONS: Anesthetized rabbits, infused with E. coli (10(9) organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion of E. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. MEASUREMENTS AND MAIN RESULTS: Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p < .05). Treatment with r-hTNF binding protein-1 was associated with 100% survival, as compared with 55.6% of the saline-treated rabbits (p < .05). Approximately 85% of r-hTNF binding protein-1 was cleared from the circulation 1 hr after the bolus injection (from 171 +/- 27 micrograms/mL at time = 0, to 27 +/- 4 micrograms/mL at 60 mins, decreasing to 6 +/- 2 micrograms/mL for the next 3 hrs). The r-hTNF binding protein-1-treated rabbits had lower serum TNF bioactivity during the first 2 hrs (p < .01). The decreased bioactivity of TNF was confirmed by a specific radioimmunoassay for rabbit TNF. However, at 4 hrs, the vehicle-treated rabbits had lower serum bioactive TNF concentrations (p < .05). The decrease in TNF concentrations in the r-hTNF binding protein-1-treated rabbits resulted from decreased production and, in part, from carry-over of r-hTNF binding protein-1 into the bioassay. CONCLUSIONS: Treatment with r-hTNF binding protein-1 improved hemodynamic variables and survival of E. coli-challenged rabbits. Administration of r-hTNF binding protein-1 suppressed bioactivity of TNF in the circulation of these rabbits, and the production of TNF as well.

Interleukin-10 stimulates hematopoiesis in murine osteogenic stroma.

Bone marrow from 5-fluorouracil-treated mice support osteogenesis when cultured in the presence of beta-glycerophosphate and vitamin C. These cultures are unable to support the growth of granulocyte/macrophage colony-forming units for longer than 2 weeks. In contrast, granulocyte/macrophage colony-forming units were detected for more than 6 weeks in interleukin-10 (IL-10)-treated cultures. In addition, IL-10-treated cultures contain long-term culture initiating cells, suggesting the presence of pluripotent hematopoietic cells. Apparently, IL-10 does not directly stimulate the proliferation of granulocyte/macrophage colony-forming units. Interleukin-10 is unable to stimulate [3H]-thymidine incorporation or to increase the number of granulocyte/macrophage colony-forming units in cell suspensions harvested from untreated or interleukin-10-treated bone marrow cultures. Interleukin-10 acts via an indirect pathway. Because exogenous transforming growth factor-beta (TGF-beta) reverses IL-10's stimulatory activity on myeloid progenitors, IL-10 most likely works by blocking TGF-beta synthesis, which acts as an endogenous suppressor of hematopoiesis in osteogenic marrow cultures. This is shown further by the increased numbers of granulocyte/macrophage colony-forming units in cultures treated with neutralizing anti TGF-beta antibodies (1D11.16). Interleukin-10 and 1D11.16 change the cultured bone marrow stroma from an osteogenic into a hematopoietic morphology. It may be that by blocking endogenous TGF-beta production, IL-10 drives marrow mesenchymal cells away from osteogenic differentiation toward hematopoietic support.

Growth acceleration and stem cell expansion in Dexter-type cultures by neutralization of TGF-beta.

Hematopoietic lineage-restricted stem cell growth has been shown to be significantly inhibited by the addition of exogenous transforming growth factor-beta (TGF-beta) to Dexter-type long-term murine bone-marrow cultures. In order to examine whether TGF-beta produced by these cells has a role in hematopoietic growth regulation, Dexter cultures have been treated with either 1D11.16, a monoclonal antibody that neutralizes the biological activity of TGF-beta types 1, 2, and 3, or with a control antibody. The composition and cellularity of the nonadherent cell populations in these cultures were assessed weekly. Treatment with anti-TGF-beta antibody resulted in a five- to 20-fold increase in nonadherent cells in the cultures when compared to either the control or untreated cultures by week 4. The majority of these cells were granulocyte/macrophage-lineage cells as assessed by histologic and flow-cytometric analysis. There was also a significant increase of megakaryocytes in cultures treated with anti-TGF-beta antibody. Stem-cell analysis, using a colony-forming unit-spleen (CFU-S) assay that combined both the adherent and nonadherent populations from either 4- or 6-week cultures, showed that there are an equivalent number of hematopoietic stem cells per 10(6) cells regardless of antibody treatment. Therefore, cultures treated with anti-TGF-beta antibody contained at least three times as many stem cells as the control cultures. Finally, kinetics studies show that the presence of anti-TGF-beta antibody is required from the onset of culture to produce these effects. These results suggest that TGF-beta is involved in normal growth regulation of bone-marrow hematopoietic cells. By addition of a neutralizing antibody, the normal TGF-beta negative growth signal is disrupted, allowing for expanded growth of several cell populations.

Interleukin 10 inhibits transforming growth factor-beta (TGF-beta) synthesis required for osteogenic commitment of mouse bone marrow cells.

Interleukin 10 (IL-10) suppressed TGF-beta synthesis in mouse bone marrow cultures. Coincidingly, IL-10 down-regulated the production of bone proteins including alkaline phosphatase (ALP), collagen and osteocalcin, and the formation of mineralized extracellular matrix. The mAb 1D11.16 which neutralizes TGF-beta 1 and TGF-beta 2, induced suppressive effects comparable to IL-10 when administered before the increase of cell proliferation in the culture. It appears that mainly TGF-beta 1 plays a role in this system since (a) TGF-beta 2 levels were undetectable in supernatants from osteogenic cultures, (b) no effect was observed when the anti-TGF-beta 2 neutralizing mAb 4C7.11 was added and (c) the suppressive effect of IL-10 could be reversed by adding exogenous TGF-beta 1. It is unlikely that TGF-beta 1 modulates osteogenic differentiation by changing the proliferative potential of marrow cells since 1D11.16 did not affect [3H]thymidine ([3H]TdR) incorporation or the number of fibroblast colony forming cells (CFU-F) which harbor the osteoprogenitor cell population. Furthermore, 1D11.16 did not alter [3H]TdR uptake by the cloned osteoprogenitor cell lines MN7 and MC3T3. Light and scanning electron microscopy showed that IL-10 and 1D11.16 induced comparable morphological changes in the marrow cultures. Control cultures contained flat adherent cells embedded in a mineralized matrix. In contrast, IL-10 and 1D11.16 treated cultures were characterized by round non-adherent cells and the absence of a mineralized matrix. In this study, the mechanism by which IL-10 suppresses the osteogenic differentiation of mouse bone marrow was identified as inhibition of TGF-beta 1 production which is essential for osteogenic commitment of bone marrow cells.

Transforming growth-factor-alpha and factor-beta(1) levels in plasma and effusions from patients with cancer or hematologic malignancies.

Transforming growth factors-alpha and -beta1 are thought to play a role in carcinogenesis. Using a sandwich linked immunosorbent assay, we have measured TGF-alpha and -beta1 levels in malignant human plasma and effusions. TGF-alpha and -beta1 plasma levels were not significantly different among normal volunteers, patients with solid tumors, and patients with hematologic malignancies (TGF-alpha, p=0.225; TGF-beta1, p=0.354). Statistically significant differences were also not found in levels between malignant and non-malignant effusions (TGF-alpha, p=0.327; TGF-beta1, p=0.095). However, a trend for the majority of elevated TGF-alpha or TGF-beta1 levels to be in malignant effusions warrants further studies with larger numbers of samples.

Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro.

Addition of transforming growth factor-beta 1 (TGF-beta 1) to in vitro cultures of murine B cells activated with bacterial LPS selectively stimulates IgG2b and IgA class switching and decreases cellular proliferation. To assess a possible role for endogenous TGF-beta in modulating the Ig isotypes produced by LPS-activated cells, we utilized a neutralizing anti-TGF-beta mAb to abrogate endogenous TGF-beta activity. Anti-TGF-beta antibody, over a range of relatively low cell densities, strikingly inhibited both IgG3 and IgG2b production in response to LPS, with little or no change in the concentrations of secreted IgM. This effect of anti-TGF-beta antibody was specific, since it did not occur with an isotype-matched control mAb and was completely reversed with exogenous TGF-beta 1. Optimal IgG3 secretion occurred at concentrations of TGF-beta that were approximately eightfold lower than that necessary for maximal synthesis of IgG2b. Neutralization of endogenous TGF-beta in LPS-activated cultures was associated with an approximately twofold increase in proliferation and viable cell yields, a modest decrease in the percentage of membrane (m)IgG2b+ cells, and a modest increase in the percentage of mIgG3+ cells. This latter finding indicated that TGF-beta was not required for IgG3 class switching, but for maturation of mIgG3+ cells into Ig secretors. Highly purified B cells, obtained by electronic cell sorting, released active TGF-beta in response to LPS and showed a similar marked reduction in LPS-mediated IgG3 and IgG2b secretion in the presence of anti-TGF-beta antibody. Abrogation of endogenous TGF-beta activity in LPS-activated cultures also resulted in a striking reduction in IFN-gamma-mediated IgG2a production, and a more modest decrease in the synthesis of IgG1 and IgE in the presence of IL-4. These data indicate that relatively low concentrations of TGF-beta are essential for stimulating optimal IgG secretion by LPS-activated B cells, in an Ig isotype-nonspecific manner, and may regulate these responses in an autocrine fashion.

Immunohistochemical localization of intracellular and extracellular associated TGF beta in the skin of patients with systemic sclerosis (scleroderma) and primary Raynaud's phenomenon.

We have investigated the distribution of TGF beta using antibodies specific for its intracellular and extracellular forms in full-thickness biopsies of patients with SSc, primary Raynaud's phenomenon (PRP), systemic lupus erythematosus (SLE), and from normal subjects. Nine of 11 SSc biopsies demonstrated intracellular TGF beta in endothelial cells while only 6 exhibited extracellular TGF beta. Endothelial cells in skin biopsies of all PRP patients displayed both intracellular and extracellular TGF beta. All other control biopsies were negative. In patients with PRP, some positively staining fibroblasts were found scattered throughout the dermis. Lastly, extracellular TGF beta was localized in the papillary dermis of PRP and SSc biopsies and in all the dermal layers of SLE patients. No significant staining of TGF beta was observed in the endothelial cells, fibroblasts, or in the extracellular matrix of the majority of biopsies from normal subjects. These data suggest that TGF beta may be one of the cytokines involved in the early stages of pathogenesis of SSc, and that endothelial cells in SSc and PRP may be a source and/or a target of TGF beta.

Transforming growth factor beta 1 selectivity stimulates immunoglobulin G2b secretion by lipopolysaccharide-activated murine B cells.

Bacterial lipopolysaccharide (LPS) has been reported to induce immunoglobulin (Ig)G2b class switching, yet we observed strain differences in IgG2b secretion in response to this mitogen. Specifically, BALB/c B cells, unlike those from DBA/2, synthesized relatively low amounts of IgG2b relative to IgG3, IgG1, or IgM. This report demonstrates that transforming growth factor (TGF) beta 1, previously shown to induce IgA class switching, selectively stimulates IgG2b secretion by BALB/c resting B cells activated with LPS. This activity was specifically reversed with a neutralizing anti-TGF-beta 1 antibody. The ability of TGF-beta 1 to act directly on highly purified membrane (m)IgM+ mIgG2b- cells to stimulate IgG2b production, stimulate an increase in IgG2b-secreting cells, and selectively increase the steady-state levels of germline gamma 2b RNA, suggests that it promotes IgG2b class switching. In this regard, addition of anti-TGF-beta antibody to cultures of DBA/2-derived resting B cells activated by LPS, alone, led to selective reduction in IgG2b secretion, indicating that endogenous TGF-beta 1 accounts for the high IgG2b secretory response observed in that strain. Finally, TGF-beta 1 failed to stimulate IgG2b secretion by B cells activated with dextran-conjugated anti-IgD antibody. We propose that TGF-beta 1 is a switch factor for the murine IgG2b subclass for appropriately activated B cells. In combination with other data, this would show that all six non-IgM, non-IgD isotypes in the mouse can be selectively induced by specific cytokines.

Reversal of acute and chronic synovial inflammation by anti-transforming growth factor beta.

Transforming growth factor beta (TGF-beta) induces leukocyte recruitment and activation, events central to an inflammatory response. In this study, we demonstrate that antagonism of TGF-beta with a neutralizing antibody not only blocks inflammatory cell accumulation, but also tissue pathology in an experimental model of chronic erosive polyarthritis. Intraarticular injection of monoclonal antibody 1D11.16, which inhibits both TGF-beta 1 and TGF-beta 2 bioactivity, into animals receiving an arthropathic dose of bacterial cell walls significantly inhibits arthritis. Inhibition was observed with a single injection of 50 micrograms antibody, and a 1-mg injection blocked acute inflammation > 75% compared with the contralateral joints injected with an irrelevant isotype control antibody (MOPC21) as quantitated by an articular index (AI = 0.93 +/- 0.23 for 1D11.16, and AI = 4.0 +/- 0 on day 4; p < 0.001). Moreover, suppression of the acute arthritis achieved with a single injection of antibody was sustained into the chronic, destructive phase of the disease (on day 18, AI = 0.93 +/- 0.07 vs. AI = 2.6 +/- 0.5; p < 0.01). The decreased inflammatory index associated with anti-TGF-beta treatment was consistent with histopathologic and radiologic evidence of a therapeutic response. These data implicate TGF-beta as a profound agonist not only in the early events responsible for synovial inflammation, but also in the chronicity of streptococcal cell wall fragment-induced inflammation culminating in destructive pathology. Interrupting the cycle of leukocyte recruitment and activation with TGF-beta antagonists may provide a mechanism for resolution of chronic destructive lesions.

Characterization of monoclonal antibodies recognizing bovine bone osteoglycin.

Six monoclonal antibodies (mAb) are described that bound to the bovine bone glycoprotein, osteoglycin. The protein osteoglycin was originally called Osteoinductive Factor (OIF). The antibodies were characterized with respect to their reaction patterns in Western blots, indirect immunoprecipitation, and binding epitope. The antibodies bound to one of two sequential determinants, either residues 62-76 or 95-105, in the C-terminal region of mature osteoglycin. One mAb, 2C11, was found to be useful for affinity purification of osteoglycin. Another mAb, 3B2, was able to immunohistochemically stain osteoblasts, osteocytes, chondrocytes, occasional osteoclasts and nail bed epithelial cells in rat neonatal forelimb. The mAbs will provide an essential tool for the further characterization of this unique glycoprotein.

Regulation of synovial cell growth. Coexpression of transforming growth factor beta and basic fibroblast growth factor by cultured synovial cells.

OBJECTIVE: To demonstrate expression of transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) by cultured rheumatoid arthritis (RA) synovial cells and to investigate their role as synovial cell mitogens. METHODS: Polypeptide growth factors were detected and identified by immunocytochemical staining and Western blot analysis. Messenger RNA (mRNA) transcripts encoding TGF beta and bFGF were identified by polymerase chain reaction analysis. The influence of neutralizing growth factor monoclonal antibodies (MAb) on RA synovial cell growth was investigated. TGF beta bioactivity was determined by Mv1Lu assay. RESULTS: Lysates of RA, as compared with normal, synovial cells contained greater amounts of TGF beta and bFGF. Western blot analysis identified a single TGF beta band (MW approximately 25 kd) in each of the cell lysates examined. Western blot analysis using MAb DE6 identified a doublet of bFGF bands (MW approximately 18.0 kd) in normal synovial cell lysates and 4 bFGF bands (MW approximately 18.0, 22.0, 22.6, and 25.2 kd) in RA synovial cell lysates. RA and normal synovial cells expressed mRNA transcripts encoding TGF beta 1 but not TGF beta 2, and FGF-2 (basic FGF). Additional mRNA transcripts encoding FGF-5 and FGF-7 were expressed by RA, but not normal, synovial cells in culture. In contrast to MAb 1D11.16, which caused a dose-dependent decrease in RA synovial cell growth, MAb DG2 (up to 100 micrograms/ml) had no effect on cell growth. CONCLUSION: RA and normal synovial cells cultured in serum-free medium express TGF beta 1 and native bFGF. However, only RA synovial cells in culture express higher molecular weight isoforms of bFGF. TGF beta 1 appears to regulate synovial cell growth in vitro through an external autocrine loop. Despite expression of high-affinity bFGF receptors on cultured synovial cells, the mechanisms by which bFGF modulates synovial cell growth are unknown.

Skin distribution and differential expression of transforming growth factor beta 1 and beta 2.

Transforming growth factor beta (TGF-beta) 1 and 2 have both become increasingly important in cutaneous biology, but their expression and distribution in human skin are not entirely clear. In this report, normal forearm skin from four volunteers was investigated for TGF-beta 1 and beta 2 immunostaining with antibodies that detect preferentially either cell- or matrix-associated forms of these peptides. Marked cell-associated TGF-beta 1 was found in the dermis, particularly around blood vessels and ducts; cellular TGF-beta 2 immunostaining was less prominent, and was predominantly around blood vessels. Neither TGF-beta 1 nor -beta 2 could be detected in the epidermis or epithelial structures, and the dermal matrix contained minimally detectable amounts of the two isoforms. In all cases, dermal matrix and cells contained greater amounts of TGF-beta 1 than TGF-beta 2. Previous studies have shown that both TGF-beta 1 and -beta 2 can induce dramatic increases in extracellular matrix, and both peptides have been implicated in the pathogenesis of fibrosis. We therefore investigated TGF-beta 1 and -beta 2 immunostaining in involved forearm skin of four patients with systemic sclerosis. Compared to normal skin, fibrotic specimens showed increased amounts of matrix and epidermal TGF-beta 1, but not TGF-beta 2. We conclude that TGF-beta 1 and -beta 2 expression in human skin is differentially regulated, and that their distribution is varied and complex.

Purification and characterization of transforming growth factor-beta 2.3 and -beta 1.2 heterodimers from bovine bone.

A unique form of transforming growth factor-beta (TGF-beta), TGF-beta 2.3 heterodimer, has been purified from bovine bone extract. TGF-beta 2.3 migrated as a single 25-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas under reducing conditions it migrated as a 12.5 kDa band. The TGF-beta 2.3 reacted positively with anti-TGF-beta 2 and anti-TGF-beta 3 antibodies on immunoblots. Equal levels of TGF-beta 2 and TGF-beta 3 sequences were detected by N-terminal sequencing. TGF-beta 2.3 eluted as a single sharp peak by reverse-phase high performance liquid chromatography. However, prior reduction of the protein with dithiothreitol resulted in the protein eluting in two peaks, one containing predominantly TGF-beta 3 and the other containing predominantly TGF-beta 2. TGF-beta 2.3 inhibited proliferation of mink lung epithelial cells and promoted the formation of colonies of normal rat kidney fibroblasts in culture with specific biological activity similar to those of TGF-beta 1 and TGF-beta 2. These results demonstrate that the protein is TGF-beta 2.3 heterodimer, consisting of one polypeptide chain each of TGF-beta 2 and TGF-beta 3 linked by one or more disulfide bonds. In addition, TGF-beta 1.2 heterodimer, previously found only in porcine platelets, has also been purified from bovine bone extract.

Two novel patterns of transforming growth factor beta (TGF-beta) binding to cell surface proteins are dependent upon the binding of TGF-beta 1 and indicate a mechanism of positive cooperativity.

Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF-beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex.

Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.

IGG-stimulated and LPS-stimulated monocytes elaborate transforming growth factor type beta (TGF-beta) in active form.

Mononuclear cells (MNC) stimulated either with lipopolysaccharide (LPS) or with surface-adsorbed IgG elaborated significant amounts of tumor necrosis factor (TNF) bioactivity, as well as immunoenzymatically detectable TNF-alpha and interleukin-1 beta. (IL1-beta). In contrast, IgG-stimulated cells released little IL1 bioactivity, but released an IL1 inhibitor, as determined by the thymocyte costimulatory assay (LAF assay). This inhibition was not due to an inhibitory effect of cyclooxygenase products, e.g. prostaglandin-E2 in the LAF assay. In contrast, antibodies against transforming growth factor type beta (TGF-beta), which is an important inhibitor of the LAF assay, augmented the LAF activity of supernatants from LPS-stimulated and IgG-stimulated MNC. Anti-TGF-beta-modulated LAF inhibition was enhanced by acid treatment of supernatants from mononuclear cells, but not of those from purified monocytes. Antibody blocking experiments point for the first time to a TGF-beta species other than type 1 as a monocyte-derived TGF-beta activity. Thus, TGF-beta released in active form from monocytes may be the more important antagonist of IL1 than cyclooxygenase-derived mediators. It implies that the LAF assay, in the absence of anti-TGF-beta antibodies, is an inadequate indicator of IL1 activity.

A novel polyclonal antibody (CL-B1/29) for immunolocalization of transforming growth factor-beta 2 (TGF-beta 2) in adult mouse.

A polyclonal antibody (CL-B1/29) raised against a synthetic peptide with an amino acid sequence identical to the first 29 N-terminal residues of bovine bone-derived transforming growth factor-beta 2 (TGF-beta 2) was characterized and used for immunolocalization of TGF-beta 2 in adult mice. Reduced staining of immunoblots and tissue after absorption of the antiserum with the immunizing peptide or with TGF-beta 2 but not with purified TGF-beta 1 demonstrated that the reagent is specific for TGF-beta 2, with little or no crossreactivity with TGF-beta 1. The immunolocalization of TGF-beta 2 was investigated in formalin-fixed, paraffin-embedded cultured cells and murine tissue. Specimens pre-digested with testicular hyaluronidase demonstrated immunostaining predominantly of extracellular connective tissue matrix, whereas specimens pre-digested with pronase E demonstrated primarily cytoplasmic staining. Immunoreactivity was widely distributed in connective tissue, muscle, adsorptive and secretory epithelia, especially of endocrine tissue, and neural tissue of adult mice.

Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives.

Whereas xenogeneic collagen has provided a safe and effective biomaterial for numerous medical applications, there are few instances in which data permit the correlation of the immunologic profile of well-defined devices with their clinical sequelae. A major exception is the use of injectable bovine dermal collagen for soft-tissue contour correction. The low incidence of hypersensitivity has been studied in the context of clinical efficacy and safety with several devices. The findings indicate that such immunity usually results in the manifestation of local symptoms of dermal inflammation at sites of treatment that resolve as the implant is resorbed by the host. In contrast, more immunogenic hemostatic agents may elicit a more frequent or vigorous immune response that is not clinically visible or relevant in that application. Recent experiences with collagen-based devices for the repair and regeneration of bone have also demonstrated that the presence of immunity to their collagenous or non-collagenous components does not necessarily predict adverse clinical sequelae. Indeed, numerous specific data indicate that this immunity can exist as an epiphenomenon with no effect on osteogenesis. To get a true composite picture of biocompatibility, significant steps must be taken to characterize biomaterials properly and to ensure that immunologic, clinical, histologic, and other pertinent laboratory data are viewed in relation to one another and not in isolation.