Reversal of the detrimental effects of chronic protein malnutrition on long bone fracture healing.

OBJECTIVE: To determine whether dietary intervention in the immediate postfracture period will reverse the detrimental influence of protein deprivation on fracture healing in the rat. DESIGN: Adult Sprague-Dawley rats were maintained on a diet containing either a normal or reduced protein concentration. After five weeks, both femora of each rat were pinned with an intramedullary 0.625-millimeter K-wire. A closed fracture of the right femur was created one week later, by use of a handheld device. Groups of rats were killed and the femora harvested at 14 days for histologic study and at twenty-eight and fifty-six days for mechanical testing. INTERVENTION: Control rats (Group I) were maintained on a 20 percent protein diet. Malnourished (Group II) animals were maintained on a 6 percent protein diet during the six-week prefracture period and throughout the fifty-six-day postfracture period. Malnutrition was confirmed by measurement of serum concentrations of transferrin, immunoglobulin, and albumin. Renourished (Group III) animals were started on the 6 percent protein diet but were fed a 20 percent protein diet in the fifty-six-day postfracture period. RESULTS: When compared with control, well-nourished rats, malnourished animals had callus composed primarily of fibrous-type tissue and had decreased periosteal and external callus as well as callus strength. The callus from renourished animals histologically resembled that from well-nourished animals with large amounts of periosteal and external callus. Based on mechanical testing results, callus from malnourished animals showed reduced strength and stiffness as compared with control renourished animals. In renourished animals, the cross-sectional area of the fracture callus, as well as callus stiffness and strength, were greater than those in malnourished and well-nourished animals. CONCLUSION: Protein deprivation has a profound detrimental effect on fracture healing. The identification of a protein-reduced state and its reversal could result in improved fracture healing and presumably a better clinical outcome in malnourished patients.

In situ complement activation by polyethylene wear debris.

A frequent long-term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris accumulation, synovitis, and osteolysis about the implant-bone or cement-bone interface. Complement, an effector system in plasma, synovial fluid, and tissue, has powerful chemotactic, inflammatory, and osteoclast-activating potentials. This study explored the complement-activating ability of polyethylene, a material used in joint implants. In vitro hemolytic assays using sheep red blood cells (E(sh)), human serum, and particulate polyethylene suggested alternative pathway complement activation, as well as polyethylene adsorption of activated complement components. These results were confirmed by enzyme-linked immunosorbent assay (ELISA) quantification of activated complement factors Bb and C3b. In situ double antibody immunoperoxidase staining for factors Bb, C3a, iC3b, and SC5-9 in synovial tissue from revision hip specimens showed localized alternative pathway activation and component adsorption. These results introduce a likely role for complement activation in particle-mediated recruitment, proliferation, and activation of macrophages during early events in osteolysis and implant loosening.

Carpal bone titanium implant arthroplasty. 10 years' experience.

In 1984, in an effort to address the silicone wear particle problem, titanium implants were developed for the scaphoid, lunate, and trapeziometacarpal joint. The design of these implants closely resembled their silicone counterparts, though some modifications were made to accommodate the properties of unalloyed titanium and enhance their stability. Carpal bone implants act as articulating spacers to help maintain the relationship of adjacent carpal bones after local resection procedures. Their use allows carpal stabilization procedures and provides functional mobility with good strength and pain relief. Their surgical application began in 1985. The 10-year clinical experience seems very promising to date.

An osteolytic lesion associated with polyethylene wear debris adjacent to a stable total knee prosthesis.

Seven years after total knee arthroplasty, a patient with a well-fixed, uncemented femoral component (cobalt-chromium-molybdenum alloy) developed a large cystic lesion in the distal femur adjacent to the femoral component. This lesion contained fibrotic soft tissue, evidence of a foreign-body giant cell reaction, and a large number of polyethylene particles, but no metal wear debris, infection, or malignancy.

Adsorption of collagenase to particulate titanium: a possible mechanism for collagenase localization in periprosthetic tissue.

Osteolysis is a central feature of aseptic loosening of orthopaedic joint prostheses. This destructive process is believed to result from phagocytosis of implant wear debris by periprosthetic and synovial macrophages and the subsequent release of proinflammatory mediators, including collagenase. Isolated murine macrophages were cultured in vitro with particulate titanium in order to explore the mechanism of macrophage activation by particulate wear debris. The results, in which the amount of secreted, soluble collagenase in culture supernatants was inversely proportional to titanium concentration, suggested that titanium strongly adsorbed secreted collagenase. This inference was confirmed by direct binding assays in which particulate titanium coated with adsorbed collagenase bound an alkaline phosphatase conjugated anti-collagenase antibody, but not a conjugated anti-IgG antibody. Adsorption of collagenase was not influenced by preincubation of titanium particles with albumin. The adsorbed collagenase remained enzymatically active as indicated by its ability to hydrolyze a synthetic peptide substrate. These results demonstrate that particulate titanium stimulates collagenase production by macrophages and then strongly adsorbs the secreted proinflammatory enzyme. The process of macrophage stimulation, collagenase secretion, and adsorption may represent an important mechanism for localization and concentration of collagenase in periprosthetic and synovial tissue, a mechanism that ultimately triggers bone resorption through osteoclast activation.

Metabolism of C4 and factor B in rheumatoid arthritis. Relation to rheumatoid factor.

Metabolic turnover determined by radioiodide labeled C4 and Factor B was studied in 18 patients with rheumatoid arthritis (RA) and 19 normal control subjects as a means of estimating the relative ratio of consumption of components in the classical and alternative pathways of complement activation. Predominance of fractional catabolic rate (FCR) of C4 over Factor B was demonstrated with differentially labeled C4 and Factor B. The hypercatabolism occurred in the extravascular space. C4 FCR correlated significantly with rheumatoid factor (RF) determined in a hemolytic assay (rs = 0.72), measured as IgG RF (rs = 0.57), and as IgM RF (rs = 0.45). There were no significant correlations with several other antibodies measured. These results are consistent with the hypothesis that RA is a systemic, extravascular immune complex disease, in which RF immune complexes play a significant pathogenetic role principally via activation of the classical pathway of complement.

Delineation of spontaneous erythrocyte autoantibody responses of NZB and other strains of mice.

Four anti-erythrocyte autoantibody responses (anti-X, anti-HB, anti-HOL, and anti-I) that occur spontaneously in mice have been characterized with regard to antigenic specificities, predominant immunoglobulin class, and pathogenetic importance. Each autoantibody response exhibits specificity for an independent erythrocyte membrane autoantigen (X, HB, HOL, or I) or a soluble analogue (SEA-X or SEA-HB) present in the plasma. The anti-X response, unique to NZB mice, is directed to a normally exposed murine erythrocyte autoantigen, whereas the anti-HB response is directed to a cryptic erythrocyte autoantigen exposed by limited enzymatic cleavage of the membrane. The anti-I response also is directed to a cryptic but distinct autoantigen, and anti-HOL autoantibodies react with an erythrocyte autoantigen located at the cytoplasmic surface of the membrane. Analysis of the predominant immunoglobulin class of each of the autoantibodies has demonstrated that anti-HB and anti-I antibodies are predominantly of IgM class, whereas anti-X and anti-HOL antibodies are IgG immunoblobulins. Only anti-X and anti-HB autoantibodies are recovered from Coombs' positive erythrocytes from NZB mice and erythrocytes with surface C3 are detected only in NZB mice greater than 9 months of age. These data suggest that only the anti-X and anti-HB responses are pathogenetically implicated in the autoimmune hemolytic anemia of NZB mice.

Evidence for a B lymphocyte defect underlying the anti-X anti-erythrocyte autoantibody response of NZB mice.

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 x 10(6) BMC containing 6 to 12 x 10(3) X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15-30 x 10(6) BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks, or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-theta antiserum had no effect on the development of X-PFC; Tlymphocyte ablation was evidenced by the absence of theta+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.

In vivo suppression of the primary immune response by a species of low density serum lipoprotein.

An apparent subspecies of normal human serum low density lipoprotein (LDL-In) has been identified with suppressive activity for early or facilitating events of human lymphocyte mitogen and allogenic cells stimulation in vitro. This report describes the effects of in vivo administration of LDL-In on the mouse anti-SRBC immune response. Human LDL-In is not species specific and was capable of suppressing the in vivo mouse anti-sheep erythrocyte (SRBC) hemagglutination response by 88% after the administration of 500 to 600 mug LDL-In IV, whereas human serum high density lipoproteins and fibrinogen had no effect. Maximal suppression occurred only when LDL-In was injected 24 to 48 hr before antigen administration. Simultaneous or subsequent injection of LDL-In had no effect. The activity of LDL-In was influenced by antigen dose and maximal at low antigen doses. The number of splenic plaque-forming cells was also reduced indicating a suppression of the clonal expansion of primary B cells to antibody-secreting cells rather than only suppression of antibody synthesis by differentiated B cells and their progeny. These observations suggest the hypothesis that endogenous LDL-In could play an important immunoregulatory role in the maintenance of immune homeostasis and the "natural" suppression of non-productive lymphocyte proliferation.

Specific antigen-binding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice.

The receptor characteristics as well as incidence of antigen-binding lymphocytes (ABL) or B and T cell classes with membrane receptors specific for the exposed (X) and cryptic (HB) murine erythrocyte autoantigens were examined in NZB and nine control strains of mice. Whereas only NZB and NZB hybrid mice synthesize anti-X autoantibody pathogenetically implicated in the genetically determined autoimmune hemolytic anemia, the NZB as well as control strains synthesize the ubiquitous anti-HB anti-erythrocyte autoantibody. By utilizing immunocytoadherence assays, maximum numbers of specific ABL of both B and T lymphocyte classes were optimally demonstrated at erythrocyte:lymphocyte ratios of 20:1 and after lymphocyte fixation at 56 degrees C for 20 min. Surface membrane receptor specificity was established by inhibition with semi-purified soluble X or HB autoantigen. Inhibition of immunocyto-adherence with class specific antisera to mouse immuno-globulins demonstrated that the receptors on both B and T cells were of IgM class. Specific receptors regenerated in vitro after trypsinization which excluded the role of cytophilic antibody in the immunocytoadherence reactions. B lymphocyte ABL reactive with the X autoantigen were demonstrable in NZB, NZB hybrid, and control mice. Only in NZB and NZB hybrid mice, strains that uniformly synthesize anti-X autoantibody, were X ABL of T lymphocyte class demonstrated. The presence and incidence of T lymphocyte X ABL is compatible with the expression of a single dominant gene carried by the NAB strain. The incidence of B lymphocyte X ABL increased with age, suggesting proliferation of this cell population. HB ABL of both B and T lymphocyte classes were observed in all strains, concordant with the ubiquitous presence of humoral anti-HB autoantibodies. Differentiation of precursor B cells are evaluated by PFC assay of cells secreting specific autoantibodies. Anti-X PFC were observed only in NZB and NZB hybrid mice; and the observed frequency suggested that less than 3.5% of the specific ABL were differentiated for the secretion of anti-X autoantibody. Anti-HB PFC were observed in all strains and represented as high as 11.8% of specific ABL. Genetic determination of the anti-X anti-erythrocyte autoantibody response does not prescribe the presence of precursors of the antibody-forming cell, but rather appears to influence regulation of the differentiation of these cells. These data suggest that circumvention of immunologic tolerance to this specific erythrocyte autoantigen may occur at the level of the T lymphocyte; or alternatively, that T lymphocytes as well as B lymphocytes, are induced to proliferate and differentiate in the NZB strain.