Impact of loss of NF-κB1, NF-κB2 or c-REL on SLE-like autoimmune disease and lymphadenopathy in Fas(lpr/lpr) mutant mice.

Defects in apoptosis can cause autoimmune disease. Loss-of-function mutations in the 'death receptor' FAS impair the deletion of autoreactive lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus-like autoimmune disease in mice (Fas(lpr/lpr) (mice homozygous for the lymphoproliferation inducing spontaneous mutation)) and humans. The REL/nuclear factor-κB (NF-κB) transcription factors regulate a broad range of immune effector functions and are also implicated in various autoimmune diseases. We generated compound mutant mice to investigate the individual functions of the NF-κB family members NF-κB1, NF-κB2 and c-REL in the various autoimmune pathologies of Fas(lpr/lpr) mutant mice. We show that loss of each of these transcription factors resulted in amelioration of many classical features of autoimmune disease, including hypergammaglobulinaemia, anti-nuclear autoantibodies and autoantibodies against tissue-specific antigens. Remarkably, only c-REL deficiency substantially reduced immune complex-mediated glomerulonephritis and extended the lifespan of Fas(lpr/lpr) mice. Interestingly, compared with the Fas(lpr/lpr) animals, Fas(lpr/lpr)nfkb2(-/-) mice presented with a dramatic acceleration and augmentation of lymphadenopathy that was accompanied by severe lung pathology due to extensive lymphocytic infiltration. The Fas(lpr/lpr)nfkb1(-/-) mice exhibited the combined pathologies caused by defects in FAS-mediated apoptosis and premature ageing due to loss of NF-κB1. These findings demonstrate that different NF-κB family members exert distinct roles in the development of the diverse autoimmune and lymphoproliferative pathologies that arise in Fas(lpr/lpr) mice, and suggest that pharmacological targeting of c-REL should be considered as a strategy for therapeutic intervention in autoimmune diseases.

Loss of c-REL but not NF-κB2 prevents autoimmune disease driven by FasL mutation.

FASL/FAS signaling imposes a critical barrier against autoimmune disease and lymphadenopathy. Mutant mice unable to produce membrane-bound FASL (FasL(Δm/Δm)), a prerequisite for FAS-induced apoptosis, develop lymphadenopathy and systemic autoimmune disease with immune complex-mediated glomerulonephritis. Prior to disease onset, FasL(Δm/Δm) mice contain abnormally high numbers of leukocytes displaying activated and elevated NF-κB-regulated cytokine levels, indicating that NF-κB-dependent inflammation may be a key pathological driver in this multifaceted autoimmune disease. We tested this hypothesis by genetically impairing canonical or non-canonical NF-κB signaling in FasL(Δm/Δm) mice by deleting the c-Rel or NF-κB2 genes, respectively. Although the loss of NF-κB2 reduced the levels of inflammatory cytokines and autoantibodies, the impact on animal survival was minor due to substantially accelerated and exacerbated lymphoproliferative disease. In contrast, a marked increase in lifespan resulting from the loss of c-REL coincided with a striking reduction in classical parameters of autoimmune pathology, including the levels of cytokines and antinuclear autoantibodies. Notably, the decrease in regulatory T-cell numbers associated with loss of c-REL did not exacerbate autoimmunity in FasL(Δm/Δm)c-rel(-/-) mice. These findings indicate that selective inhibition of c-REL may be an attractive strategy for the treatment of autoimmune pathologies driven by defects in FASL/FAS signaling that would be expected to circumvent many of the complications caused by pan-NF-κB inhibition.

Elevated Mcl-1 inhibits thymocyte apoptosis and alters thymic selection.

T cells developing in the thymus undergo rigorous positive and negative selection to ensure that those exported to peripheral lymphoid organs bear T-cell receptors (TCRs) capable of reacting with foreign antigens but tolerant of self. At each checkpoint, whether a thymocyte survives or dies is determined by antiapoptotic and proapoptotic Bcl-2 family members. We used Mcl-1 transgenic (tg) mice to investigate the impact of elevated expression of antiapoptotic Mcl-1 on thymocyte apoptosis and selection, making a side-by-side comparison with thymocytes from BCL-2tg mice. Mcl-1 was as effective as Bcl-2 at protecting thymocytes against spontaneous cell death, diverse cytotoxic insults and TCR-CD3 stimulation-driven apoptosis. In three different TCR tg models, Mcl-1 markedly enhanced positive selection of thymocytes, as did Bcl-2. In H-Y TCR tg mice, elevated Mcl-1 and Bcl-2 were equally effective at inhibiting deletion of autoreactive thymocytes. However, in the OT-1tg model where deletion is mediated by a peripheral antigen whose expression is regulated by Aire, Mcl-1 was less effective than Bcl-2. Thus, the capacity of Mcl-1 overexpression to inhibit apoptosis triggered by TCR stimulation apparently depends on the thymocyte subset subject to deletion, presumably due to differences in the profiles of proapoptotic Bcl-2 family members mediating the deletion.

Analysis of thymic stromal cell populations using flow cytometry.

The complexity of the lymphostromal interplay that is essential to alphabetaT-cell development is reflected by the heterogeneity of both lymphocytes and thymic stromal cells. While panels of monoclonal antibodies have described many of the cellular components of these microenvironments, the means to quantify stromal cell subsets using flow cytometry remains poorly defined. This study refines and compares various stromal cell isolation procedures and determines the effects of various digestion enzymes on important surface molecules. Three- and four-color flow cytometry is used to correlate established and novel stromal cell markers to define thymic fibroblasts, epithelium and a unique subset of thymic endothelium that express MHC class II. This work provides a basis for the purification of thymic stromal cells for further phenotypic, functional and genetic analysis.

Review of total hip replacement. The Middlemore Hospital experience, 1980-1991.

AIM: To determine the outcome of 1951 total hip arthroplasties performed between 1980 and 1991 at Middlemore Hospital. METHODS: An independent retrospective review was carried out between 1993 to 1995 giving a minimum follow-up of two years. Useable data were obtained for 96.8% of cases. RESULTS: The overall wound infection rate was 3.4% (n=65), and of these seven (10.8%) required revision. The revision rate for infection for all patients was 1.16% (n=22). The in-hospital dislocation rate was 2.6% (n=49), and of these five (9.8%) required revision. Patient pain, activity and satisfaction were acceptable. Revision was required for 163 patients (8.7%), mainly for loosening. A survival analysis of the commonly used implants at seven years matched results from other studies. (Stem survival: Spectron 99.5%, Charnley 95.6%. Cup survival: Spectron 97.9%, Charnley 98.1%). Analysis of outcome predictors showed that youth and weight both influence the rate of revision. CONCLUSIONS: Results in terms of patient satisfaction and revision rates were comparable with other published series. The wound infection rate was higher than desirable, but not unexpected in view of the number of surgeons involved (73) and the lack of special theatre facilities.

Posterior surgical treatment for the rheumatoid cervical spine.

Twenty-six patients with rheumatoid disease affecting the cervical spine underwent surgical treatment for neck pain, neurological deficit, or both. Atlantoaxial subluxation (n=13), subaxial subluxation (n=7) and vertical migration of the odontoid (n=6) were treated. Arthrodesis with autologous bone was augmented with wire, Ransford loop, Hartshill rectangle or Magerl technique. Pain relief occurred in 92% of patients. Neurological deficit improved in 89% and was unchanged in the remainder. Radiographic stability was achieved in all but one patient. Posterior surgery effectively relieved pain and neurological deficit, and the complications encountered did not jeopardize the outcome.

Reamed versus unreamed femoral nails. A randomised, prospective trial.

We performed a randomised, prospective trial to evaluate the use of unreamed titanium nails for femoral fractures. Of 48 patients with 50 femoral fractures 45 were followed to union; 23 with an unreamed and 22 with a reamed nail. The study was stopped early because of a high rate of implant failure. The fractures in the unreamed group were slower to unite (39.4 weeks) than those in the reamed group (28.5 weeks; p = 0.007). The time to union was over nine months in 57% of the unreamed group and in 18% of the reamed group. In the unreamed group 14 secondary procedures were required in ten patients to enhance healing compared with three in three patients in the reamed group. Six implants (13%) failed, three in each group. Four of these six fractures showed evidence of delayed union. To achieve quicker union and fewer implant failures we recommend the use of reamed nails of at least 12 mm in diameter for female patients and 13 mm in males.

Matchett Brown hemiarthroplasty for displaced subcapital fractures of the hip.

BACKGROUND: Matchett Brown hemiarthroplasty has been routinely performed at Middlemore Hospital in elderly patients following subcapital fracture of the hip. The outcome of patients undergoing Matchett Brown hemiarthroplasty was evaluated. METHODS: Matchett Brown hemiarthroplasties performed at Middlemore Hospital during 1987 were retrospectively reviewed. Medical records were reviewed and where possible patients were interviewed, examined and radiographs of their hip taken. RESULTS: The overall survival at follow up was 34%, with the greatest predictor of survival being whether the patient had been living alone prior to the accident. The majority of patients who survived the 4 year follow up had excellent mobility at the time of fracture. At follow up most patients had little or no pain from their hip, but three complained of constant pain. CONCLUSIONS: Hemiarthroplasty proved to be a satisfactory form of replacement in this group. If one were to select a patient for total hip replacement, rather than hemiarthroplasty, then age alone is not as important as other factors such as degree of mobility and independence of living.

Adjuvant chemotherapy for non-metastatic osteosarcoma of the extremities in two New Zealand cancer centres.

BACKGROUND: Adjuvant chemotherapy significantly improves survival of patients with non-metastatic osteosarcoma but most of the data come from trials conducted in major international cancer centres. AIM: To review the efficacy and toxicity of an adjuvant chemotherapy regimen used in two regional cancer centres in New Zealand. METHODS: Retrospective review of patients treated for non-metastatic high-grade osteosarcoma of the extremities. The regimen (POMA) consists of high-dose-methotrexate 8 g/m2 and vincristine 1.5 mg/m2 (maximum 2 mg) on days 1 and 8 followed by folinic acid then doxorubicin 50 mg/m2 and cisplatin 100 mg/m2 on day 15. This cycle was repeated every 35 days. Following amputation patients received six cycles while in selected patients two cycles were planned prior to limb salvage surgery followed by a further four cycles. Actuarial survival was calculated using the Kaplan-Meier method. RESULTS: Twenty patients were treated with POMA between 1986 and 1993. Amputation was performed in 16 patients and limb-salvage surgery in four. Sixteen patients (80%) remain alive with no evidence of disease at a median follow-up of 40 months. Thirteen patients (65%) have been continuously disease-free. Actuarial survival at five years is 70%. Seven patients relapsed, six in lungs, of whom four underwent pulmonary metastasectomy; three of these remain free of disease 31, 35 and 40 months later. There was no local relapse. The toxicity of POMA is significant but tolerable. CONCLUSION: The results obtained at two regional cancer centres in New Zealand using POMA compare favourably to those achieved in clinical trials performed at major international cancer centres.

Serum blocks the osteolytic effect of cortisol in neonatal mouse calvaria.

Chronic glucocorticoid excess is associated with the development of osteoporosis and, in human subjects, there is histomorphometric evidence of increased bone resorption. Paradoxically, most in vitro studies have suggested that glucocorticoids inhibit bone resorption but recently two groups have demonstrated increased osteolysis in glucocorticoid-treated bone organ cultures. The present study reexamines the effect of cortisol on basal bone resorption in neonatal mouse calvaria with particular emphasis on the effect of serum supplementation of the media. In the absence of serum, 45Ca release was significantly stimulated by 10(-7) M cortisol (treatment/control 1.37 +/- 0.06, P less than 0.005) and by 10(-6) M cortisol (treatment/control 1.27 +/- 0.08, P less than 0.005). The stimulation of resorption by 10(-7) M hydrocortisone was progressive from 24 to 96 hours of incubation. In contrast, when calvaria were incubated in the presence of 5% serum, bone resorption was not increased by cortisol (10(-8) M-10(-6) M). In the presence of 5% charcoal-stripped, heat-inactivated serum, there was a small stimulation of 45Ca release at 10(-6) M hydrocortisone only (treatment/control 1.19 +/- 0.06, P less than 0.01). Incubation of bones with indomethacin did not modify the effect of cortisol in either the presence or absence of serum. In serum-free conditions, cortisol 10(-8) M significantly inhibited the rate of thymidine incorporation, though at higher concentrations this effect was not seen. Cortisol produced a dose-related inhibition of serum-stimulated thymidine incorporation. It is concluded that the presence of serum substantially modifies the effect of cortisol on basal bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Orthopaedic training in private practice.

The background philosophy, structure and training experience offered by a new free-standing clinic set up purely to facilitate orthopaedic post-graduate training is described. Emphasis is placed on the ethical issues involved. The educational programme, which includes involvement in all local and national activities, is summarized. The registrars' operative experience is recorded using log-sheet criteria. This venture has predated the major restructuring of the health care system in Auckland, which is anticipated in the next few years, and will therefore allow the training programme to evolve appropriately, employing the educational opportunities of all sectors of health care.

Adenylate cyclase blockers dissociate PTH-stimulated bone resorption from cAMP production.

It is uncertain whether adenosine 3',5'-cyclic monophosphate (cAMP) or the inositol-calcium pathway mediates the stimulation of bone resorption by parathyroid hormone (PTH). Incubation of bone organ cultures with cAMP analogues and forskolin has not resolved this question because of the cellular inhomogeneity of bone and the consequent presence of adenylate cyclase-linked receptors for both PTH and calcitonin, hormones with opposite effects on bone resorption. We have used two new inhibitors of adenylate cyclase, 9-(tetrahydro-2-furyl)adenine (SQ 22536) and 2',5'-dideoxyadenosine (DDA), to directly reassess the role of cAMP in PTH-stimulated osteolysis. SQ 22536 (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blocked PTH stimulation of cAMP production measured in the absence of a phosphodiesterase blocker. In the presence of 1 mM 3-isobutyl-1-methylxanthine, half-maximal inhibition of PTH-induced cAMP production occurred with 0.2 mM SQ and 0.1 mM DDA, respectively. These concentrations of SQ and DDA had no effect on PTH-stimulated 45Ca release from calvaria, although both agents inhibited bone resorption when present at concentrations of 1-2 mM. At these levels, SQ and DDA caused equivalent inhibition of 45Ca release stimulated by 1,25-dihydroxyvitamin D3 but did not affect basal 45Ca release or [3H]-phenylalanine incorporation. It is concluded that substantial blockade of PTH-induced cAMP production does not affect this hormone's stimulation of bone resorption, which is therefore likely to be mediated by another intracellular messenger system, possibly calcium. In millimolar concentrations, SQ and DDA appear to be nonspecific blockers of osteoclastic bone resorption.

Stimulation of the synthesis of collagenase activator protein in cartilage by a factor present in synovial-conditioned medium.

We have purified a low molecular weight protein from medium conditioned by calf synovium with physical and biological properties similar to the leukocyte cytokine interleukin 1 (IL-1). The factor is active in stimulating the synthesis (three- to fivefold) of collagenase activator protein (CAP) by the surface (1-2 mm) of articular cartilage while CAP synthesis in the deeper zones of articular cartilage is not affected. Recombinant mouse IL-1 and commercially available purified human IL-1 are also capable of stimulating cartilage to synthesize and secrete CAP. The synthesis of other proteins, including collagenase, appeared to be unaffected by either the synovial factors or the human and mouse IL-1.

The in vitro effect of gold complexes on bone resorption.

Mouse calvaria were maintained in organ culture without serum additives. The effects of three gold complexes--aurothioglucose, aurothiomalate, and auranofin--on active bone resorption (45Ca release) and hydroxyproline synthesis were determined. The influence of these compounds on DNA and protein synthesis and lysosomal enzyme release from calvaria was also assessed. All gold complexes reduced bone resorption to some extent, with auranofin being the most potent within a narrow concentration range (10(-6) M). This concentration of auranofin also significantly inhibited collagen synthesis, although DNA and protein synthesis were unaffected. None of the compounds tested appeared to mediate their action via significant inhibition of lysosomal enzyme release.

The effects of hydrocortisone, parathyroid hormone and the bisphosphonate, APD, on bone resorption in neonatal mouse calvaria.

The effects of hydrocortisone and parathyroid hormone (PTH) upon bone resorption rates in neonatal mouse calvaria have been studied. Bone resorption (measured as 45Ca release) was significantly increased by hydrocortisone (10(-7) M and 10(-6) M) and there was a dose-dependent rise with PTH (0.3-0.9 micrograms/liter). When both PTH 0.3 micrograms/liter and hydrocortisone 10(-8) M were present in the incubating medium, bone resorption did not differ from control, but increasing the hydrocortisone concentration to 10(-7) M augmented 45Ca release by 25% (P less than 0.02) and doubling of the PTH level was associated with a 10% increase (nonsignificant). When both PTH and hydrocortisone were present in the higher concentrations (0.6 micrograms/liter and 10(-7) M, respectively) 45Ca release increased by 39% (P less than 0.005) above that resulting from the lower levels of both hormones (0.3 micrograms/l and 10(-8) M, respectively). (3-Amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) in concentrations of 3 X 10(-5) M and 10(-4) M, produced inhibition of basal and hydrocortisone/PTH-stimulated bone resorption without evidence of toxicity. These results indicate that hydrocortisone stimulates bone resorption in neonatal mouse calvaria in vitro, in contrast to the results found in fetal rat bone culture systems. PTH has a similar effect, which is additive to that of hydrocortisone and the combined stimulation can be overcome by APD. The possible relevance of these results to the development and prevention of glucocorticoid-induced osteoporosis is discussed.

Inhibition of prostaglandin action and bone resorption by copper.

Mouse calvaria were maintained in organ culture without serum additives. The effects of Cu2+ on bone resorption and on the synthesis and action of prostaglandins were studied. Non-toxic concentrations of copper sulphate (5 microM) were found to decrease active resorption, measured by 45Ca release, to 54% control values (p less than 0.001), while prostaglandin F (PGF), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha, (6-keto-PGF1 alpha), determined by radioimmunoassay, were increased above controls (p less than 0.05). These effects of Cu2+ on prostaglandin synthesis were confirmed by the isolation and quantitation of [3H]-labelled metabolites released from calvaria which had been pre-labelled with [3H]-arachidonic acid. PGE2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2 (TxB2) were all higher in copper-exposed calvaria, but their relative amounts remained unchanged. There was no evidence that Cu2+ influenced the mobilisation of [3H]-arachidonic acid from prelabelled calvaria. The stimulation of bone resorption by exogenous prostaglandins was decreased in the presence of Cu2+ (p less than 0.005), while parathormone-mediated bone resorption was virtually unaffected. Cu2+ also increased the inhibition of bone resorption seen with indomethacin (p less than 0.05). In addition to the effects of the metal on prostaglandin action Cu2+ also decreased beta-glucuronidase activity in the media to 86% of the control values (p less than 0.001). The action of Cu2+ in inhibiting bone resorption in vitro appears complex but does not involve inhibition of prostaglandin synthesis. It is likely that Cu2+ has more than one inhibitory locus.

The effect of blood transfusion on the incidence of deep vein thrombosis.

The incidence of postoperative deep vein thrombosis in 120 patients undergoing elective total hip replacement was determined venographically. Significantly more blood was administered to those developing thrombosis, particularly in the subgroups given general anaesthesia (P less than 0.05). There were no differences in the postoperative haemoglobin values in any of these groups. The distribution of other risk factors identified, namely previous thrombo-embolism, malignancy and previous vein surgery or injections did not influence this finding. The use of TED stockings (Kendall) was effective. It is suggested that greater emphasis should be placed on techniques that reduce blood loss such as regional anaesthesia and the posterior approach to the hip. Further research into the fluids used for blood volume maintenance will be beneficial.

The in vitro effect of indomethacin on basal bone resorption, on prostaglandin production and on the response to added prostaglandins.

Mouse calvaria were maintained in organ culture without serum additives. Basal active resorption, as measured by 45Ca and hydroxy-proline release, was significantly inhibited to 74% control levels by indomethacin (1.4 X 10(-7) M). Prostaglandin F and prostaglandin E2 production, determined by radioimmunoassay, were both significantly lowered by this concentration of indomethacin. DNA, protein and hydroxyproline synthesis, as indices of cell toxicity, were unaffected by low concentrations of indomethacin, while concentrations of 1.4 X 10(-6) M inhibited protein synthesis (p less than 0.005). In the presence of indomethacin (1.4 X 10(-7) M) both PGE2 and PGF2 alpha stimulated resorption in a dose-dependent manner, with PGE2 being the more potent. Neither prostaglandin affected hydroxyproline synthesis at low concentrations, but PGE2 had a marked inhibitory action at a higher concentration (10(-6) M). In combination, the effects of PGE2 and PGF2 alpha showed no evidence of synergism or any antagonistic action. The study shows that in vitro calcium and hydroxyproline resorption in the unstimulated mouse calvaria are inhibited by indomethacin at concentrations measured in serum during human therapy. The decreased PGF and PGE2 production associated with this decreased bone resorption in the presence of non-toxic concentrations of indomethacin would suggest a role for these prostaglandins in maintaining the basal resorption of cultured bone.

The detection of deep venous thrombosis by impedance plethysmography in hip surgery patients.

Cuff impedance plethysmography has been performed on 120 patients undergoing total hip replacement surgery, all of whom have had venography. The low incidence of thrombosis (17.5%) precluded any accurate data on sensitivity, but specificity was 85%, positive accuracy 48%, negative accuracy 92% and validity 82%. There was a high false positive rate in the first postoperative week (40% of 25 negative venograms). The technique is of value for the exclusion of thrombi in such patients after the eighth postoperative day. Venography will still be required for the positive identification of thrombi in any research protocol.

Orthopaedic training in New Zealand.

This paper illustrates some of the problems associated with a nationally organised training programme. The limit on entry numbers is on the 'best guess' for manpower requirements and not on the number of training posts available and hence a challenge can always be made on the grounds that a 'closed shop' is being operated. Experience has shown that to date this is not a valid criticism. However, there is little room for expanding the number of training posts available so that the annual intake cannot be expanded significantly beyond present levels. The selection criteria and methodology are discussed in detail, representing our current practice without any claim to have established an ideal technique. Finally, when a trainee is seconded from an overseas programme and the question of approval of his time in this country is raised, it is most important that the host institution have data relevant to both the local and the national scene.