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.

Bacterial cell wall-induced immunosuppression. Role of transforming growth factor beta.

Group A streptococcal cell wall (SCW)-injected rats exhibit a profound immunosuppression that persists for months after the initial intraperitoneal injection of SCW. The goal of this study was to determine the mechanisms for the suppressed T lymphocyte proliferative responses in this experimental model of chronic inflammation. When spleen cell preparations were depleted of adherent cells, restoration of T cell proliferative responses to Con A and PHA occurred, implicating adherent macrophages in the regulation of immunosuppression. Furthermore, macrophages from SCW-treated animals, when cocultured with normal spleen cells in the presence of Con A or PHA, effectively inhibited the proliferative response. Supernatants from suppressed spleen cell cultures were found to inhibit normal T cell mitogenesis. Taken together, these results implicated a soluble macrophage-derived suppressor factor in the down regulation of T cell proliferation after exposure to SCW in vivo. Subsequent in vitro studies to identify this suppressor molecule(s) revealed the activity to be indistinguishable from the polypeptide transforming growth factor beta (TGF-beta). Furthermore, TGF-beta was identified by immunolocalization within the spleens of SCW-injected animals. The cells within the spleen that stained positively for TGF-beta were phagocytic cells that had ingested, and were presumably activated by, the SCW. These studies document that TGF-beta, previously shown to be a potent immunosuppressive agent in vitro, also effectively inhibits immune function in chronic inflammatory lesions in vivo.

Suppression of arthritis by an inhibitor of nitric oxide synthase.

Nitric oxide (NO), a toxic radical gas produced during the metabolism of L-arginine by NO synthase (NOS), has been implicated as a mediator of immune and inflammatory responses. A single injection of streptococcal cell wall fragments (SCW) induces the accumulation of inflammatory cells within the synovial tissue and a cell-mediated immune response that leads destructive lesions. We show here that NO production is elevated in the inflamed joints of SCW-treated rats. Administration of NG-monomethyl-L-arginine, an inhibitor of NOS, profoundly reduced the synovial inflammation and tissue damage as measured by an articular index and reflected in the histopathology. These studies implicate the NO pathway in the pathogenesis of an inflammatory arthritis and demonstrate the ability of a NOS inhibitor to modulate the disease.

Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa.

The mechanism by which Helicobacter pylori, a noninvasive bacterium, initiates chronic antral gastritis in humans is unknown. We now show that H. pylori releases products with chemotactic activity for monocytes and neutrophils. This chemotactic activity was inhibited by antisera to either H. pylori whole bacteria or H. pylori-derived urease. Moreover, surface proteins extracted from H. pylori and purified H. pylori urease (a major component of the surface proteins) exhibited dose-dependent, antibody-inhibitable chemotactic activity. In addition, a synthetic 20-amino acid peptide from the NH2-terminal portion of the 61-kD subunit, but not the 30-kD subunit, of urease exhibited chemotactic activity for monocytes and neutrophils, localizing the chemotactic activity, at least in part, to the NH2 terminus of the 61-kD subunit of urease. The ability of leukocytes to chemotax to H. pylori surface proteins despite formyl-methionyl-leucyl-phenylalanine (FMLP) receptor saturation, selective inhibition of FMLP-mediated chemotaxis, or preincubation of the surface proteins with antiserum to FMLP indicated that the chemotaxis was not FMLP mediated. Finally, we identified H. pylori surface proteins and urease in the lamina propria of gastric antra from patients with H. pylori-associated gastritis but not from uninfected subjects. These findings suggest that H. pylori gastritis is initiated by mucosal absorption of urease, which expresses chemotactic activity for leukocytes by a mechanism not involving N-formylated oligopeptides.

Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta.

After intraarticular injection of TGF-beta 1 or TGF-beta 2, marked swelling and erythema of the injected joints were apparent within 12-24 h. On a scale of 0 to 4, by day 3, the TGF-beta-treated joints had articular indices (AI) of 3.6 +/- 0.5 to 4.0 +/- 0.0 compared with no response for the vehicle-injected contralateral joints. Histopathologic evaluation revealed a predominantly mononuclear phagocyte infiltrate with some neutrophils and T lymphocytes, consistent with active inflammation. The monocytic pattern of leukocyte infiltration at 2-3 d was comparable to that seen in animals with antigen-induced arthritis after 2-3 wk. Extensive synovial fibroblast hyperplasia became apparent within 48 h, likely as a result of TGF-beta induction of growth factor synthesis by the accumulating monocytes. TGF-beta 2, a homologue of TGF-beta 1, was found to induce a similar level of synovitis and synovial hyperplasia consistent with its parallel monocyte and fibroblast chemotactic properties and ability to induce transcription and translation of monocyte/macrophage-derived growth factors. These data suggest that TGF-beta, released by platelets and activated inflammatory cells, may play a direct role in leukocyte recruitment and activation in arthritic and other chronic inflammatory lesions.

Bacterial cell wall-induced hepatic granulomas. An in vivo model of T cell-dependent fibrosis.

In vitro studies implicate a molecular link between inflammatory mononuclear cells and alterations in fibroblast growth and function. We have extended these observations in an experimental animal model in which we document the T cell-dependence of fibrosis that occurs after activation of the cell-mediated immune system by specific antigen. Chronic granulomatous lesions were induced in the livers of susceptible rats by the intraperitoneal injection of group A streptococcal cell walls (SCW). The development of granulomas that are composed primarily of lymphocytes and macrophages was associated with the recruitment and proliferation of connective tissue cells. Furthermore, this expanded population of fibroblasts generated a collagenous structure consisting primarily of types I and III collagen around the granuloma. The progression of these chronic inflammatory lesions leads to the formation of fibrotic nodules throughout the livers of the treated animals. Intact granulomas, as well as mononuclear cells derived from the granulomas, spontaneously elaborated a soluble factor(s) that stimulates fibroblast proliferation. Physicochemical analysis revealed that the primary granuloma-derived peak of fibroblast growth activity corresponded to an apparent Mr of 40,000, which is consistent with a previously described T lymphocyte--derived fibroblast-activating factor (FAF) in guinea pig and human. Furthermore, the fibrosis that occurs in the granuloma is apparently T cell--dependent, since no fibrotic lesions developed in SCW-injected athymic nude rats nor in SCW-injected animals treated with the T cell inhibitor, cyclosporin A (CsA). Mononuclear cells from neither of these functionally T cell--deficient animals could generate FAF activity. These data show a role for T lymphocyte--derived cytokines in the development of hepatic fibrosis in SCW-injected rats.

Macrophage- and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome.

The multifunctional cytokine, transforming growth factor beta (TGF-beta), was identified by immunocytochemistry in the brain tissues of four patients with acquired immune deficiency syndrome (AIDS), but not in control brain tissue. The TGF-beta staining was localized to cells of monocytic lineage as well as astrocytes, especially in areas of brain pathology. In addition, the brain tissues from the AIDS patients contained transcripts for human immunodeficiency virus 1 (HIV-1) by in situ hybridization, suggesting a correlation between the presence of HIV-1 in the brain and the expression of TGF-beta. However, the expression of TGF-beta was not limited to HIV-1-positive cells, raising the possibility of alternative mechanisms for the induction of TGF-beta in these AIDS patients' brains. To investigate these mechanisms, purified human monocytes were infected in vitro with HIV-1 and were shown to secrete increased levels of TGF-beta. In addition, HIV-1-infected monocytes released a factor(s) capable of triggering cultured astrocytes that are not infected with HIV-1 to secrete TGF-beta. The release of TGF-beta, which is an extremely potent chemotactic factor, may contribute to the recruitment of HIV-1-infected monocytic cells, enabling viral spread to and within the central nervous system (CNS). Moreover, TGF-beta augments cytokine production, including cytokines known to be neurotoxic. The identification of TGF-beta within the CNS implicates this cytokine in the immunopathologic processes responsible for AIDS-related CNS dysfunction.

Selective elimination of HIV-1-infected cells with an interleukin-2 receptor-specific cytotoxin.

Infection by human immunodeficiency virus type 1 (HIV-1) is associated with cellular activation and expression of the interleukin-2 (IL-2) receptor. A genetically engineered fusion toxin, DAB486 IL-2, that contains the enzymatic site and translocation domain of diphtheria toxin and the receptor binding domain of IL-2 specifically kills cells that express high-affinity IL-2 receptors. This toxin selectively eliminated the HIV-1-infected cells from mixed cultures of infected and uninfected cells and inhibited production of viral proteins and infectious virus. Thus, cellular activation antigens present a target for early antiviral intervention.

Ribonucleotide reductase: regulation, regulation, regulation.

Ribonucleotide reductase (RNR) catalyses the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. It is composed of two dissimilar subunits, R1, the large subunit containing the allosteric regulatory sites, and R2, the small subunit containing a binuclear iron center and a tyrosyl free radical. Recent isolation of the mammalian and yeast RNR genes has shown that, in addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. The mRNAs for both subunits are cell-cycle regulated and, in yeast, inducible by DNA damage. Yeast encode a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. This regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes.

Finding prospective partners in the library: the two-hybrid system and phage display find a match.

The two-hybrid system uses the efficacy of yeast genetic assays to identify protein-protein interactions. It permits the rapid cloning of genes encoding products that interact with a given protein of interest. Also being developed are phage display methods that allow direct physical selection of binding proteins. These methods have significantly altered strategies for analysing signaling and regulatory pathways.

Thymus-dependent and -independent regulation of Ia antigen expression in situ by cells in the synovium of rats with streptococcal cell wall-induced arthritis. Differences in site and intensity of expression in euthymic, athymic, and cyclosporin A-treated LEW and F344 rats.

Euthymic LEW rats, when injected with streptococcal cell walls, exhibited rapid onset development of acute exudative arthritis coincident with enhanced synovial expression of Ia antigen. By 21 d after injection, the expression of Ia was markedly increased compared with basal conditions and paralleled the severity of the later developing proliferative and erosive disease. Immunodeficient athymic and cyclosporin A-treated LEW rats developed only the early phase arthritis, which was again paralleled by synovial Ia expression. Chronic expression of high levels of Ia antigen was not observed. Histocompatible F344 rats, both athymic and euthymic, developed minimal, if any, clinically significant arthritis and did not exhibit the enhanced Ia expression demonstrated in the LEW rats. Our results indicate that enhanced synovial Ia expression parallels clinical disease severity and varies by rat strain, and that the rapid onset enhanced synovial Ia expression is thymus independent, whereas the markedly enhanced chronic phase Ia expression is thymus dependent.

Transforming growth factor beta 1 suppresses acute and chronic arthritis in experimental animals.

Systemic administration of the cytokine, TGF beta 1, profoundly antagonized the development of polyarthritis in susceptible rats. TGF beta 1 administration (1 or 5 micrograms/animal), initiated one day before an arthritogenic dose of streptococcal cell wall (SCW) fragments, virtually eliminated the joint swelling and distortion typically observed during both the acute phase (articular index, AI = 2.5 vs. 11; P less than 0.025) and the chronic phase (AI = 0 vs. 12.5) of the disease. Moreover, TGF beta 1 suppressed the evolution of arthritis even when administration was begun after the acute phase of the disease. Histopathological examination of the joint revealed the systemic TGF beta 1 treatment greatly reduced inflammatory cell infiltration, pannus formation, and joint erosion. Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF beta 1 reversed the leukocytosis associated with the chronic phase of the arthritis. Control animals subjected to the same TGF beta 1 dosing regimen displayed no discernable immunosuppressive or toxic effects even after 4 wk of treatment. These observations not only provide insight into the immunoregulatory effects of TGF beta, but also implicate this cytokine as a potentially important therapeutic agent.

Role of the thymus in streptococcal cell wall-induced arthritis and hepatic granuloma formation. Comparative studies of pathology and cell wall distribution in athymic and euthymic rats.

Systemic administration of an aqueous suspension of group A streptococcal cell wall fragments to susceptible rats induces acute and chronic polyarthritis, as well as noncaseating hepatic granulomas. To gain insight into the role of the thymus in the pathogenesis of this experimental model, pathologic responses and cell wall tissue distribution were compared in congenitally athymic rats (rnu/rnu) and their euthymic littermates (NIH/rnu). Within 24 h, both rat strains developed acute arthritis, characterized by polymorphonuclear leukocytic exudate in the synovium and joint spaces. This acute process was maximal at day 3 and gradually subsided. Beginning 2-3 wk after injection, the euthymic, but not the athymic, rats developed the typical exacerbation of arthritis, characterized by synovial cell hyperplasia with villus formation and T helper/inducer lymphocyte-rich mononuclear cell infiltration. This process eventually resulted in marginal erosions and destruction of periarticular bone and cartilage. Parallel development of acute and chronic hepatic lesions was observed. Bacterial cell wall antigen distribution and persistence were similar in the athymic and euthymic rats. Cell wall antigens were demonstrated in the cytoplasm of cells within subchondral bone marrow, synovium, liver, and spleen, coincident with the development of the acute lesions, and persisted in these sites, although in decreasing amounts, for the duration of the experiment. Our findings provide evidence that the acute and chronic phases of the experimental model are mechanistically distinct. The thymus and functional thymus derived-lymphocytes appear not to be required for the development of the acute exudative disease but are essential for the development of chronic proliferative and erosive disease. Induction of disease is dependent upon cell wall dissemination to and persistence in the affected tissues.

Association of circulating receptor Fc gamma RIII-positive monocytes in AIDS patients with elevated levels of transforming growth factor-beta.

Monocytes in the circulation of normal individuals express two receptors for the constant region of immunoglobulin, Fc gamma RI and Fc gamma RII. In contrast, we have observed that AIDS monocytes express significant levels of a third Fc gamma R, Fc gamma RIII (CD16), which is normally associated with activation or maturation of the monocyte population. By dual-fluorescence analysis using a monoclonal antibody specific for Fc gamma RIII (MAb 3G8), 38.5 +/- 3.2% of the LeuM3 (CD14)-positive monocytes in AIDS patients were CD16 positive as compared to 10.4 +/- 1.0% for healthy individuals (n = 29; P less than 0.005). Furthermore, AIDS monocytes expressed Fc gamma RIII-specific mRNA which is expressed minimally or not at all in control monocytes. As a recently identified inducer of Fc gamma RIII expression on blood monocytes, transforming growth factor-beta (TGF-beta) was found to be elevated in the serum and/or plasma of AIDS patients. Moreover, incubation of normal monocytes with AIDS serum or plasma induced CD16 expression which correlated with serum TGF-beta levels (r = 0.74, P less than 0.001) and was inhibited with a neutralizing antibody to TGF-beta. Thus, the increased CD16 expression on peripheral blood monocytes in AIDS patients may be the consequence of elevated circulating levels of the polypeptide hormone TGF-beta.

Expression of interleukin 2 receptors by monocytes from patients with acquired immunodeficiency syndrome and induction of monocyte interleukin 2 receptors by human immunodeficiency virus in vitro.

A population of circulating mononuclear cells from patients with AIDS was identified which expressed interleukin 2 receptors (IL-2R). By dual-fluorescence flow microfluorometry, the patients' IL-2R+ cells were further identified as Leu M3+ monocytes (29.4 +/- 5.2% of the Leu M3+ cells were IL-2R+, n = 15), whereas Leu M3+ monocytes from normal subjects were IL-2R negative (2.0 +/- 0.42%; P less than 0.001). By Northern analysis, monocytes from AIDS patients, but not control subjects, constitutively expressed steady-state levels of IL-2R mRNA. Functionally, the IL-2R+ monocytes were capable of depleting IL-2 from culture supernatants, suggesting a mechanism for the reduced IL-2 levels commonly seen in AIDS patients. IL-2R+ monocytes also expressed increased levels of surface HLA-DR which may favor monocyte T-cell interactions and the transmission of human immunodeficiency virus (HIV). In additional studies, normal monocytes were infected with a macrophage-tropic HIV isolate in vitro and monitored for IL-2R and HLA-DR expression. Within 24-48 h after exposure to HIV in vitro, but before evidence of productive infection, greater than 25% of the monocytes became IL-2R+ with increasing numbers of IL-2R+ cells and HLA-DR levels through day 6. These early signaling effects of HIV could be mimicked by adding purified HIV envelope glycoprotein gp120 to the monocytes. This stimulation of monocytes before or independent of productive infection of the cells by HIV is consistent with in vivo observations of activated and/or abnormal functions by monocytes that do not appear to be infected with HIV in AIDS patients.

Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.

In an experimental model of arthritis, increased leukocyte adhesion is associated with the evolution of acute and chronic synovial inflammation. Whereas peripheral blood mononuclear cells (PBMC) from control animals bind minimally to fibronectin matrices, PBMC from animals receiving arthropathic doses of bacterial cell walls demonstrate increased integrin mRNA expression and enhanced adhesion. To determine whether this augmented adhesion was causal in the development of synovial pathology, peptides synthesized from several fibronectin domains which inhibited leukocyte adhesion in vitro were administered to arthritic animals either as free peptides or coupled to a carrier molecule. Not only were peptides containing either the RGD or CS-1 cell-binding domains inhibitory to chronic synovial pathology (articular index = 10.5 +/- 0.3 for untreated animals compared to 1.25 +/- 0.25 for RGD and 2.5 +/- 0.7 for CS-1), but three peptides synthesized from the carboxy-terminal 33-kD heparin-binding domain of fibronectin were also found to significantly inhibit leukocyte recruitment and the evolution of arthritis. Based on these data, which are the first to explore the therapeutic potential of heparin-binding fibronectin peptides in chronic inflammation, it appears that antagonism of cellular adhesion and recruitment by fibronectin peptides may provide an important mechanism for modulating the multi-step adhesion process and attenuating aberrant inflammatory responses.

Sailing and sports medicine: a literature review.

Sailing medicine has been mainly addressed by healthcare professionals who happen to sail. Although there has been an increase in the number of studies of various aspects of sailing over the last 15 years, efforts to advance evidence based knowledge of sailing and sports medicine face unique obstacles. Recent interest in research by groups such as Olympic and America's Cup teams has produced beneficial changes.

The effects of glucose on nonmemory cognitive functioning in the elderly.

The present study evaluated the role of glucose in the performance of a series of memory and nonmemory neuropsychological tasks. Each of the 28 healthy (no evidence of diabetic or dementing disease), elderly (mean age = 73), subjects participating in the study were tested under each of two conditions (50 g glucose or 27.3 mg saccharin) in a counterbalanced fashion. While actively manipulating glucose levels in participants, blood glucose (BG) levels were monitored to evaluate the effectiveness of individual subject's regulation of ingested glucose. It was hypothesized that glucose would enhance performance on a memory measure (Rey/Taylor Figure) as well as other nonmemory measures (e.g. design fluency, trail making test, etc.). A repeated measures, counterbalanced design was used to evaluate the direct manipulation of glycemic condition. Results reveal an enhancement effect on both the recall of the Rey/Taylor Figure as well as verbal fluency and figural fluency. A significant correlation between glycemic regulation and performance on a test of divided attention (dichotic listening) was also evident (r = -0.47). These findings suggest that a specific facilitation of retrieval mechanisms may account for enhancement of both memory and fluency performance.

Pneumococcal sepsis in childhood leukemia and lymphoma.

Forty episodes of sepsis in 28 patients with leukemia or lymphoma were reviewed. Streptococcus pneumoniae accounted for 14 episodes and was the most frequent etiologic agent isolated. Escherichia coli and Staphylococcus aureus accounted for an additional 16 episodes. No patients with pneumococcal sepsis died whereas the overall mortality was 25%. Five of the patients with pneumococcal sepsis had polymorphonuclear leukocyte counts less than 500/cu mm. All patients in remission who had pneumococcal sepsis had a leukocytosis in response to their infection. Foci in patients with pneumococcal sepsis were identified in 50% of the episodes and all were confined to the respiratory tract.

Arachidonic acid metabolism in the neonatal platelet.

An assessment of arachidonic acid metabolism in the platelet of the neonate was performed. The uptake of [14C]arachidonic acid into platelets of both the neonate and the adult were similar. Neonatal platelets, however, released a significantly greater amount (P less than .001) of prelabeled arachidonic acid (24.7% +/- 2.8%) in response to the physiologic agent thrombin when compared with platelets from adult control subjects (14.6% +/- 0.8%). When the activities of the lipoxygenase (12-L-hydroxy-5,8,10,14-eicosatetraenoic acid) and cyclooxygenase pathways (12-L-hydroxy-5,8,10-heptadecatrienoic acid and thromboxane B2) were evaluated following incubation of platelets with [14C]arachidonic acid, significant differences were observed between adult and neonatal platelets. Platelets from the neonate produced less (P less than .01) thromboxane B2 (11.1% +/- 1.7%) when compared with platelets from adult control subjects (19% +/- 1.7%). In contrast, the lipoxygenase product 12-L-hydroxy-5,8,10,14-eicostatetraenoic acid was increased (P less than .005) in the platelet from the neonate (41.5% +/- 2%), when compared with the adult (31.2% +/- 2.1%). The observation that the availability of substrate arachidonic acid is increased in the platelet of the neonate may have general implications in neonatal pathophysiologic processes.