Active and latent forms of transforming growth factor beta activity in synovial effusions.

We have evaluated the possible involvement of TGF-beta in rheumatoid arthritis by assay of 16 cell-free synovial fluids for the presence of its active and "latent" forms. Evidence has been obtained for TGF-beta-like activity in synovial effusions by four criteria: (a) TGF-beta receptor competition, (b) soft-agar colony formation of AKR-2B and NRK-49F indicator cells, (c) immunological neutralization of the biological activity, and (d) biochemical activation of a latent form.

Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia.

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.

Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue.

Vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF), is a potent microvascular permeability enhancing cytokine and a selective mitogen for endothelial cells. It has been implicated in tumor angiogenesis and ascites fluid accumulation. Since development of the destructive synovial pannus in rheumatoid arthritis (RA) is associated with changes in vascular permeability (synovial fluid accumulation), synovial cell hyperplasia, and angiogenesis, we examined synovial fluids (SFs) and joint tissue for the expression and local accumulation of VPF/VEGF. VPF/VEGF was detected in all of 21 synovial fluids examined and when measured by an immunofluorimetric assay, ranged from 6.9 to 180.5 pM. These levels are biologically significant, since < 1 pM VPF/VEGF can elicit responses from its target cells, endothelial cells. Levels of VPF/VEGF were highest in rheumatoid arthritis fluids (n = 10), with a mean value (+/- SEM) of 59.1 +/- 18.0 pM, vs. 21.4 +/- 2.3 pM for 11 SFs from patients with other forms of arthritis (p = 0.042). In situ hybridization studies that were performed on joint tissues from patients with active RA revealed that synovial lining macrophages strongly expressed VPF/VEGF mRNA, and that microvascular endothelial cells of nearby blood vessels strongly expressed mRNA for the VPF/VEGF receptors, flt-1 and KDR. Immunohistochemistry performed on inflamed rheumatoid synovial tissue revealed that the VPF/VEGF peptide was localized to macrophages within inflamed synovium, as well as to microvascular endothelium, its putative target in the tissue. Together, these findings indicate that VPF/VEGF may have an important role in the pathogenesis of RA.

Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40.

The ligand for the CD40 antigen is a 39-kilodalton protein, gp39, expressed on the surface of activated CD4+ T cells and is essential for thymus-dependent humoral immunity. The role of gp39-CD40 interactions in autoimmune disease was investigated in vivo with the use of an antibody that blocks their interactions (anti-gp39). Arthritis induced in mice by immunization with type II collagen was inhibited by anti-gp39. Anti-gp39 blocked the development of joint inflammation, serum antibody titers to collagen, the infiltration of inflammatory cells into the subsynovial tissue, and the erosion of cartilage and bone. Thus, interference with gp39-CD40 interactions may have therapeutic potential in the treatment of autoimmune disease.

PEN-2 gene mutation in a familial Alzheimer's disease case.

Genetic evidence indicates a central role of cerebral accumulation of beta-amyloid (Abeta) in the pathogenesis of Alzheimer's disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with gamma-secretase activity, the enzymatic complex generating Abeta. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age-matched controls (n=253), sporadic AD (SAD, n=256) and familial AD (FAD, n=140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele epsilon4 of apolipoprotein E than controls. The pathogenic role of the PEN-2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.

Annexin-1 localization in human skin: possible association with cytoskeletal elements in keratinocytes of the stratum spinosum.

Annexin-1 (also called lipocortin-1 or p35), a putative substrate of the epidermal growth factor/receptor kinase, protein kinase C, and transglutaminase, was immunolocalized in embryonic, neonatal, adult, and diseased human epidermis. In embryonic skin intense annexin-1 immunoreactivity was found in the periderm at 54 d estimated gestational age (EGA). Later (EGA = 91-143 d), annexin-1 immunoreactivity was restricted to basal keratinocytes. In neonatal skin, basal cells were often more heavily stained than were suprabasal keratinocytes, which were also stained. Only basal keratinocytes stained in adult plantar skin, but in thin skin annexin-1 was present in the basal, suprabasal, and sometimes even in the granular layers of the epidermis. Often, annexin-1 appeared concentrated around the perimeter of cells, especially tonofilament/desmosome-rich keratinocytes of the spinous-cell layer. At high magnification, annexin-1 appeared associated with distinct structures and was very granular in appearance in the intensely stained ductal keratinocytes of eccrine sweat glands, cells that are very highly enriched in keratin tonofilaments. This striking distribution in certain keratinocytes enriched in tonofilaments suggests a role for annexin-1 in cytoskeletal functions.

Histological distribution of the 35-KD protein substrate of the epidermal growth factor receptor/kinase in thymus.

Rat thymus has been identified as a tissue comparatively enriched in a 35-KD substrate of the epidermal growth factor receptor/kinase (lipocortin-1) (J Biol Chem 261:13784, 1986). A polyclonal antiserum prepared against the 35-KD protein was used to determine histological distribution of the protein in thymus. Frozen sections of rat thymus were examined after indirect labeling of the 35-KD protein with a rhodamine conjugate of secondary antibody. The antigen was localized primarily in the reticular network of the thymic epithelium, with no detectable labeling of resident thymocytes. Immunoblotting (Western blots) of cytosol extracts also demonstrated that thymocytes did not contain detectable amounts of the antigen. Cultured thymic epithelial cells (TEC), however, contained an abundance of two immunologically related protein bands with molecular weights similar but not identical to the antigen from the parental cell line (human A-431 carcinoma). Paraffin sections of rat and human thymus were subjected to an immunoperoxidase staining procedure, and it was observed that Hassall's corpuscles (keratinized epithelial cells) and other cortical and medullary TECs were intensely stained. The demonstration that the antigen is primarily associated with TEC in thymus, in conjunction with its distribution in other tissues, will aid in deducing its physiological role.

Primidone in the long-term treatment of essential tremor: a prospective study with computerized quantitative analysis.

The long-term efficacy of primidone (375-750 mg/day) in essential tremor was evaluated prospectively in 11 patients who had shown a favorable response to 4-week treatment with the drug under placebo-controlled conditions. On accelerometric evaluation, the magnitude of tremor after 3, 6, and 12 months on primidone was still significantly reduced compared with the initial placebo period. After discontinuation of primidone, tremor amplitude reverted to the placebo levels. Some loss of efficacy during long-term administration, however, was suggested by the results of self-assessment, physician's assessment, and performance tests. Three patients discontinued prematurely the drug because the sedative effects outweighed the potential therapeutic benefit. Side effects (especially drowsiness and sedation) were common at 4 weeks and 3 months but tended to subside thereafter. It is concluded that primidone retains at least part of its tremorolytic effect for up to 1 year, although the overall clinical benefit is limited in most patients.

Effect of propranolol in head tremor: quantitative study following single-dose and sustained drug administration.

The effect of the beta-adrenoceptor antagonist propranolol has been investigated in nine patients suffering from isolated (six patients) or prominent (three patients) essential tremor of the head. In a double-blind, placebo-controlled study the tremorolytic efficacy of propranolol has been assessed by a quantitative accelerometric method after a single oral dose (120 mg) and following 2 weeks of sustained treatment with two different dosage regimens of the drug (120 and 240 mg daily). As compared with placebo, a significant reduction in tremor magnitude was found following a single oral dose but not on sustained administration of the beta-blocker at either dosage. The results suggest that the efficacy of sustained propranolol on isolated or prominent essential head tremor is less predictable and satisfactory than expected on the basis of the single-dose response, as compared with hand tremor.

Specific membrane receptors for diferric-transferrin in cultured rat skeletal myocytes and chick-embryo cardiac myocytes.

A classical approach was used to determine whether myocyte cultures exhibited diferric-transferrin binding phenomena consistent with the presence of specific, high-affinity membrane receptors. Experiments were performed using a continuous cell line, designated L-6, derived from rat skeletal muscle, as well as primary cultures of chick-embryo cardiac myocytes. Rat transferrin isolated from pooled serum was used in experiments involving L-6 cells, and ovotransferrin isolated from hen's egg white was used with the chick-embryo cardiac myocytes. The data from equilibrium binding experiments, corrected for nonspecific binding, and analyzed by Scatchard analysis indicated that there were approximately 2 x 10(5) transferrin receptors per L-6 myocyte and 2 x 10(4) ovotransferrin receptors per cardiac myocyte present, under the conditions used for the equilibrium binding experiments. Whereas the L-6 myocytes grew exponentially under the assay conditions, the cardiac-myocyte cultures were in a non-dividing state. It is thought that the differences in receptor number per cell reflect changes arising form the differing ion demand made by the cells, under these two growth conditions. It is clear that myocytes acquire iron from diferric (ovo)transferrin in a process that involves high-affinity, specific binding to membrane receptors.

[Our experience with a new material for skin graft areas]. / La nostra esperienza su un nuovo materiale da medicazione per le aree di prelievo cutaneo.

The authors describe a new method of medicating disepidermised areas from which a dermo-epidermic graft has been removed to cover burn areas or following post-traumatic loss of substance. This method consists of the use of a thin microfibrillar film of a polysaccharidic type, which serves as a temporary substitute for the skin, offering selective permeability, the possibility of transpiration and gaseous exchange, but at the same time being impermeable to liquids and microorganisms. A study was performed to evaluate the efficacy and tolerability of this new dressing, involving 30 patients of both sexes aged between 9 and 87 years old. The site, dimensions, and type of graft were assessed in each patient together with the duration of pain and the time taken for the lesion to heal. This study has highlighted the positive function performed by medication with microfibrillar film in facilitating the cicatricial process, achieving complete re-epithelisation within an average of 8-9 days, with a considerable reduction in pain and satisfactory esthetic and functional result. No collateral effects or complications relating to the use of this material are reported in any of the cases studied.

Fibronectin-associated transforming growth factor.

We have studied the ability of fibronectins to induce anchorage-independent growth of NRK-49F cells in serum-free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage-independent growth at concentrations in 20-100 micrograms/ml range. The ability of exogenously supplied fibronectins to promote anchorage-independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin-agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum-free assay system. The TGF-like activity appears to either co-purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity "dissociates" from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF-like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical-chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type-beta transforming growth factor/growth inhibitor (beta-TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at micrograms/ml concentrations.

Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cells.

We have isolated a soluble 35-kDa protein from A-431 cells that in the presence of calcium can serve as a substrate for the epidermal growth factor (EGF)-receptor/kinase. The purification procedure exploits the reversible, Ca2+-dependent binding of the 35-kDa protein to the A-431 total particulate fraction. The 35-kDa protein was purified by 1) Ca2+-dependent adsorption to A-431 particulate fractions, 2) release by chelation of Ca2+ with ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 3) chromatography on Sephadex G-100, DEAE-cellulose, and CM-cellulose columns. When a plasma membrane preparation from A-431 cells is used as a source of the EGF-receptor/kinase, the phosphorylation of the 35-kDa protein occurs on tyrosine, is greatly enhanced in the presence of EGF, and occurs only when Ca2+ is added to the standard reaction mixture for phosphorylation. Autophosphorylation of the receptor does not require Ca2+. We have postulated that one of the roles of Ca2+ is to facilitate the interaction of the 35-kDa protein with cellular membranes. Ca2+ enhances, but apparently is not essential for, the direct phosphorylation of the 35-kDa protein by the Triton X-100-solubilized, purified EGF-receptor/kinase. Incubation of intact A-431 cells with EGF at 37 degrees C (but not 0 degrees C) enhances the ability of the particulate fraction prepared from these cells to bind and/or phosphorylate the 35-kDa protein. We suggest that this enhancement in the phosphorylation of the 35-kDa protein, a presumed physiological substrate, is associated with the clustering and internalization of the EGF receptor/kinase complex.

Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells.

We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex.

Lipocortin I (p35) is abundant in a restricted number of differentiated cell types in adult organs.

Lipocortin-I (p35) is a unique calcium- and phospholipid-binding protein of the lipocortin/calpactin family. Although several possibilities have been suggested, functions for the individual proteins of this family are not yet known with certainty. As an initial step in the identification of the biological function(s) of p35, we have used immunohistochemical methods to define precisely many of the cellular phenotypes that contain p35 in vivo. In all organs where p35 is found, we have observed a striking distribution of p35-positive cells. Typically it is highly enriched in a limited range of differentiated cell types while apparently totally absent from most others. Our identification of specific p35-positive cell types in vivo will now set limitations on likely possibilities for functions of this protein and thereby permit a more logical approach to the determination of its true function.

Lipocortin-independent effect of dexamethasone on phospholipase activity in a thymic epithelial cell line.

In the cloned rat thymic endocrine epithelial cell line TEA3A1, treatment with dexamethasone leads to decreased levels of prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2. Dexamethasone treatment also leads to a decrease of both calcium-dependent and calcium-independent phospholipase A2 activity measured in a cell-free assay. Dexamethasone-treated cells also have increased levels of lipocortin-I, a putative modulator of phospholipase A2 activity. The property of calcium-dependent binding of lipocortin to the particulate fraction was used to prepare cytosolic and particulate subcellular fractions which contained phospholiphase A2 activity but no lipocortin-I. Dexamethasone decreased phospholipase A2 activity in both cytosolic and particulate fractions even in the absence of lipocortin, suggesting the presence of a lipocortin-independent mechanism.

Critical role of peripheral blood phagocytes and the involvement of complement in tumour necrosis factor enhancement of passive collagen-arthritis.

Studies have implicated tumour necrosis factor-alpha (TNF-alpha) in type-II collagen (CII)-induced arthritis (CIA), a well established animal model of human rheumatoid arthritis. Precisely how TNF is involved in CIA is not yet clear. In this study the effects of TNF on CIA were examined, independent of its potential effects on the immune response, by performing peri-articular injection of TNF in combination with passive immunization of rats. A sub-arthritic dose (5 mg) of affinity-purified anti-CII IgG, which alone was insufficient to induce spontaneous clinical arthritis, was used throughout the study. Obvious clinical arthritis that persisted for several days was rapidly induced by injections of 100 ng TNF into hindpaws of rats that were passively immunized shortly before the TNF injection. Injections of TNF in non-immunized control rats did not induce clinical arthritis, nor did buffer-only injections in passively immunized controls. The clinical arthritic response was a local phenomenon, limited only to the TNF-injected hindpaws. No swelling was observed in the opposite, buffer-injected hindpaws, indicating the effects of TNF were not systemic. Depletion of peripheral blood phagocytes with anti-rat neutrophil antiserum before passive immunization completely abolished the ability of TNF to induce clinical arthritis, identifying phagocytic cells as the essential target cells in evoking this arthritic response. A role for complement activation was also demonstrated in this model through the use of a soluble recombinant version of CD35, the cell surface complement receptor type-1 (sCR1, BRL55730), which significantly reduced TNF-induced arthritis in phagocyte-replete rats.

Clinical and computer-based assessment of long-term therapeutic efficacy of propranolol in essential tremor.

Despite a large number of studies demonstrating the effectiveness of propranolol in relieving essential tremor (ET) the long-term therapeutic outcome of these patients remains poorly defined. The results of a one-year follow-up study in 18 patients with mild to severe ET performed by using clinical and computer-based methods of assessment indicate that the initial therapeutic benefit of propranolol is apparently retained. However, following 3-6 months of sustained treatment a proportion of patients required an increase in daily dosage of propranolol in order to maintain adequate symptomatic control of tremor, indicating a relative decrease in tremorolytic efficacy of the drug possibly due to long-term tolerance.