A preliminary in vitro and in vivo study of the effects of new anthraquinones on neutrophils and bone remodeling.

Osteolysis, that is, progressive periprosthetic bone loss, is responsible for approximately 70% of aseptic loosening and implant failure. Usually, it is due to a granulomatous reaction wear-induced, leading to macrophage and osteoclast-mediated bone resorption. At present, there is no established prophylaxis or treatment for this process. For this purpose, as a preliminary investigation, we aimed to study the effects in two directions, inhibition of proinflammatory signals, and bone remodeling activity, of two newly synthesized anthraquinone molecules [N,N'-Diethylamino-2,6-anthraquinone-disulfonamide (GR375) and N,N'-(p-ethoxyphenyl)-2,6-anthraquinone-disulfon amide (GR377)]. Among the pro-inflammatory signals, the ability of the two anthraquinones to interfere with the production of superoxide anion (O(2) (-)), which was assumed as a marker of reactive oxygen species (ROS), was evaluated in an in vitro cell model by testing phagocytes, such as human neutrophils, challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP). Both compounds inhibited O(2) (-) production, in a dose-dependent way, without exerting scavenger effects. An in vivo model was applied to investigate their effect on bone remodeling. Fifty-four female Wistar rats were divided into eight groups of six animals each, and a 4-week treatment was applied in two phases. A 25 mg/kg/os dose in the first phase and 12.5-6.25 mg/kg/os doses in the second one were employed. The tibia trabecular bone at the secondary spongiosa level was analyzed, and trabecular bone volume (%TBV), trabecular thickness (TbTh), and apatite lattice parameters were measured. At the highest doses of GR375 and GR377 the %TBV and the TbTh increased by 33.2, 34.6%, and 3.6 and 9.1%, respectively, whereas crystallographic parameters were not significantly different from the untreated group. Our results suggest a simultaneous antiinflammatory and antiosteoclastic activity of both drugs that encourages to perform further research. If it will be confirmed, they could be proposed in a variety of bone diseases, in particular, when acute inflammation is associated to osteolytic processes and, eventually, in the prevention and treatment of periprosthetic osteolysis.

A dual pathway model of daily stressor effects on rheumatoid arthritis.

This study evaluated the initial promise of a dual-pathway conceptual model linking daily event stressors to rheumatoid arthritis (RA) disease activity through changes in immune system activation and mood. Fifty individuals, who were studied on five occasions two weeks apart, reported daily event stressors on the Daily Life Experience Checklist, daily mood on an abbreviated version of the Profile of Mood States-B, and daily joint pain on the Rapid Assessment of Disease Activity in Rheumatology. Serial clinical examinations comprised ratings of joint tenderness and swelling, and blood drawn during exams was analyzed for sedimentation rate (an indicator of systemic inflammation) and soluble interleukin-2 receptors (a marker of immune system activation known to correlate with RA disease activity). Across-person analyses failed to establish links from daily event stressors to either disease activity or composites of joint pain and joint inflammation when associations were adjusted for the effect of neuroticism on self-report measures. Pooled within-person analyses, however, were generally consistent with the relations predicted by the dual-pathway model. Increases in daily event stressors during the week preceding each clinical exam were associated with increased joint pain (regardless of changes in mood). At the same time, increased daily stressors were indirectly associated with decreased joint inflammation through reduction in levels of soluble interleukin-2 receptors. The dual-pathway model, which may be limited to short-term psychological and psychoimmunologic processes, underscores the importance of distinguishing potentially opposing effects of stress on pain versus inflammation in individuals with rheumatoid arthritis.

Effects of orally administered bisphosphonates on bone loss in a disuse osteopenia model involving the rat.

OBJECTIVE: This study was carried out to evaluate the effectiveness of two newly synthesized bisphosphonates (BPs) [Alendronate (4-amino-1-hydroxybutylidene-1, 1-bisphosphonic acid (AHBuBP)) and Neridronate (6-amino-1-hydroxyhexylidene-1,1-bisphosphonic acid (AHHexBP)], administered orally, in reducing experimentally induced bone loss. METHODS: Unilateral sciatic nerve section was performed on the Sprague-Dawley rat to induce osteopenia in one of the hind limbs. Histomorphometric measurements of the tibial trabecular bone and femur ash content determinations were effected to assess the degree of osteopenia. For comparison Chlodronate (dichloromethylene-1-bisphosphonic acid (Cl2MBP) was employed as the reference drug. CONCLUSIONS: The results of this investigation show that both BPs were significantly active in reducing the osteopenic process in the involved limb and were more active than Chlodronate.

Effects of bisphosphonate derivatives on macrophage function.

The effects of three bisphosphonates (BPs), designated 4-amino-1-hydroxybutylidene-1, 1-bisphosphonate (AHBuBP), 6-amino-1-hydroxylidene-1, 1-bisphosphonate (AHHexBP) and chloromethylenebisphosphonate (Cl2MBP), were evaluated on the basis of their effect on the phagocytic activity, lysosomal enzyme release and superoxide anion production in the rat peritoneal macrophage (M phi). AHBuBP was found to inhibit in a concentration-dependent manner the phagocytosis of sheep red blood cells (SRBC). The same activity was seen with the phagocytosis of latex beads, although this effect was independent of calcium concentration. Conversely, AHHexBP and Cl2MBP were weak inhibitors of phagocytosis of both SRBC and latex beads. Inhibition studies on the phorbol myristate acetate-stimulated production of superoxide anion have shown all three BPs to be active. When compared with Cl2MBP, AHBuBP and AHHexBP were shown to be substantially active in inhibiting the release of beta-glucuronidase from ionophore A23187-stimulated rat peritoneal M phi.

Potential role of heterologous collagen in promoting cutaneous wound repair in rats.

The effect of native bovine tendon type-I collagen sponges (CONDRESS) on wound repair was evaluated by employing an experimental animal (rat) model which utilized subcutaneously implanted collagen and polyurethane sponges. Lesions were also created in the control groups with the exception that implants were omitted. The fusion of the hypodermal layer was selected as the index of wound repair expressed as % healing. In order to assess the extent of the healing process, parameters of clinical evaluation such as exudate volume, number of polymorphonuclears (PMNs) and macrophages (MOs) were also determined. All studies were effected at time intervals of 24, 48 and 72 h post wounding and implantation. The collagen-treated groups showed a greater healing capacity as compared to the polyurethane sponge-treated and control groups. Likewise, the exudate volume, number of leukocytes and mononuclear-type cells were all significantly higher for the collagen-treated animals than those of the polyurethane sponge-treated and control ones. Furthermore, the healthier appearance of the artificially produced wounds in the collagen sponge-treated groups (when compared to the others after 24 h) further confirmed collagen's validity in the treatment of wounds.

Cyclophosphamide and 15(S)-15 methyl PGE1 correct the T/B lymphocyte ratios of NZB/NZW mice.

The lupus of NZB/NZW F1 female mice is associated with immune complex glomerulonephritis and premature death. Cyclophosphamide and 15(S)-15 methyl PGE1 therapy halt disease progression. Fluorescein conjugated antibodies were utilized to label specific leukocytes and the subsets were quantitated using a Fluorescence Activated Cell Sorter. Normal outbred CD-1 female mice showed a decrease in absolute T and B cell numbers with age, but the ratio of T and B cells remained essentially constant through 9 months of age. By contrast the NZB/W female mice showed decreased numbers of total lymphocytes relative to CD-1 controls at all ages. Moreover relative to CD-1s, there was a far greater decrease in T cell numbers (7 x for NZB/W versus 2 x for CD-1) and B cell numbers failed to decrease with age. The characteristic decline in T lymphocyte numbers and relative increase in B cell numbers in NZB/W mice were corrected with cyclophosphamide and PGE1 therapy. However, there was no selective modification of T cell subsets (L3T4+ or Ly2+) with therapy. Our investigation suggests correction of the abnormal T/B cell ratio may be a useful marker of therapeutic activity in NZB/W mice.

Kell blood group activity of gram-negative bacteria.

To understand better the relationships between blood-group antigens and bacterial constituents, examples of 23 gram-negative bacteria (representing the 10 genera Citrobacter, Edwardsiella, Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Serratia, and Shigella) were tested for the presence of Kl-like antigens by hemagglutination-inhibition (HAI) assays against both IgG and IgM anti-Kl. Saline-suspended whole organisms, cell-free culture media, and disrupted organisms were used to test for such antigens in, on, and secreted by the microorganisms examined. Disrupted organisms of an isolate of Shigella sonnei nonspecifically inhibited IgG anti-Kl as well as IgG antibodies of the specificities Kpb, Fya, S, and c. However, only Escherichia coli 0125:B15, subtype 12808, had specific K1-like activity (no activity with other IgG [(k, Kpb, Jka, Fya, S, c] and IgM [A, B, M, P1] antibodies). Disrupted organisms inhibited IgM but not IgG anti-K1 in the HAI assay. A second subtype, E. coli 0125:B15, subtype 12809, exhibited no K1-like activity. These findings support the report of K1 activity in cell-free broth cultures of E. coli 0125:B15 (subtype unspecified). Thus, although not all E. coli 0125:B15 possesses K1-like activity, the finding of such activity in at least one E. coli subtype confirms the idea that bacterial components may play a role in the production of naturally occurring antibodies directed against non-ABO red cell antigens.

An ELISA method for detecting unexpected antibodies.

This paper reports a new horseradish peroxidase enzyme linked immunosorbant assay (ELISA) to detect patient serum alloantibodies. The new methodology utilizes a water-soluble chromogen, O-dianisidine and a temporary solid antibody support, osmotically lysed reagent erythrocytes. In a double blind study the horseradish peroxidase ELISA was found to be as sensitive as the conventional antiglobulin test. In parallel dilution studies on serum samples containing anti-Kell antibodies, the horseradish peroxidase-ELISA was found to be substantially more sensitive than the conventional antiglobulin test. While the procedure is at present impractical for routine use, these results indicate a potential for improved sensitivity which warrants further research. Possible applications and prospective areas of research are discussed.