Systemic gene transfer of binding immunoglobulin protein (BiP) prevents disease progression in murine collagen-induced arthritis.

Summary Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production.

Combination gene therapy targeting on interleukin-1ß and RANKL for wear debris-induced aseptic loosening.

This study investigated the efficacy of a combination gene therapy to repress interleukin-1 (IL-1) and receptor activator of nuclear factor NF-kappa B ligand (RANKL) for the treatment of particulate debris-induced aseptic loosening, and tried to explore the molecular mechanism of the exogenous gene modifications on osteoclastogenesis. RAW cells activated by titanium particles were transduced with DFG-IL-1Ra (retroviral vector encoding IL-1 receptor antagonist) and AAV-OPG (adeno-associated viral vectors-osteoprotegerin) individually or in combination for 4 weeks. Pro-inflammatory cytokines in culture media were determined by enzyme-linked immunosorbent assay, and gene expressions of RANK, IL-1ß, c-Fos, TRAF6, JNK1 and CPK were examined using real-time PCR. An established knee-implant-failure mouse model was employed to evaluate the efficacy of the in vivo double-gene therapy. The surgical implantation of a titanium alloy pin into the proximal tibia was followed by monthly challenge with titanium debris. Peri-implant gene transfers of IL-1Ra and OPG (respectively or in combination) were given 3 weeks after surgery. The combination of OPG and IL-1Ra gene transfer exhibited strong synergetic effects in blockage of inflammation and osteoclastogenesis at 8 weeks after gene modification. The combination therapy reversed peri-implant bone resorption and restored implant stability when compared with either single gene transduction. Real-time PCR data indicated that the action of IL-1Ra gene therapy may be mediated via the JNK1 pathway, while the reduction of osteoclastogenesis by OPG gene modification may be regulated by c-Fos expression. In addition, both gene modifications resulted in significant diminishment of TRAF6 expression.

Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model.

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone-implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1ß, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Tumour necrosis factor receptor gene therapy affects cellular immune responses in collagen induced arthritis in mice.

BACKGROUND: Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) amenable to immunotherapy directed against tumour necrosis factor alpha (TNFalpha). OBJECTIVE: To evaluate whether local TNF receptor (TNF-R) gene therapy in DBA/1 mice exerts an influence beyond anti-inflammatory effects. Two measures of CIA pathogenesis were investigated-namely, immunity to collagen II (CII) 245-270 peptide (the major immunodominant epitope within bovine CII) and the preferential activation of T cell Vbeta8.2 variable region receptors in arthritic DBA/1 mice. METHODS: DBA/1 mice received single periarticular injections of media or retroviral vectors containing LacZ or human TNF-R into affected arthritic paws at disease onset. Disease severity was monitored, immune responses towards the immunodominant bovine CII 245-270 and subdominant CII 334-360 peptide epitopes were assessed by ELISA, and T cell Vbeta usage was analysed by real time polymerase chain reaction for the LacZ transduced, TNF-R, and viral-free media treated control animals. The therapeutic influence of TNF-R gene transduction was compared with other groups at different times after treatment. RESULTS: Reduced disease severity was seen 15-35 days after treatment, with a concomitant increase in immunity towards the subdominant CII 334-360 peptide epitope rather than the immunodominant CII 245-270 peptide in TNF-R treated animals. Early in the disease, TNF-R treated animals demonstrated a reduction of bias towards the otherwise predominant Vbeta8.2 T cell subset. CONCLUSIONS: TNF-R gene therapy influences cellular immunity in CIA, leading to overall disease amelioration, thus suggesting that TNF inhibition may have therapeutic potential beyond the control of inflammation in RA.

A novel murine model of orthopaedic wear-debris associated osteolysis.

OBJECTIVE: To develop a mouse model of bone resorption to quantitatively evaluate wear-debris induced osteolysis. METHODS: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of femur or calvaria from a syngeneic mouse donor. One group of bone-implanted pouches was stimulated with ultra-high molecular weight polyethylene (UHMWPE) debris, and the remaining bone-implanted pouches received saline alone as controls. The tissues were harvested at 2, 7, and 14 days after bone implantation for molecular and histological analyses. RESULTS: Marked inflammatory responses (thicker membrane and increased cellular infiltration) were observed in UHMWPE-stimulated pouches, compared with the saline control. Intensive tartrate-resistant acid phosphatase (TRAP) staining was identified in the UHMWPE-stimulated pouches, especially at the attachment site of inflammatory tissue with implanted bone, where active osteolysis occurred. Image analysis showed that the bone collagen loss was closely related to the amount of UHMWPE within the tissue, and was most prevalent at the contact site of bone with inflammatory tissue. UHMWPE stimulation also significantly increased the release of free calcium into the pouch fluids. CONCLUSION: This model demonstrates a sensitive, rapid, and reproducible method for studying wear-debris induced osteolysis seen in patients with aseptic loosening.

Increased dermal elastic fibers in the tight skin mouse.

OBJECTIVE: The tight skin (Tsk-1) mouse has been proposed as a model for systemic sclerosis on the basis of increased accumulation of collagen and glycosaminoglycans in the skin, and by the presence of serum autoantibodies. The genetic basis of the mutation has been identified as a genomic duplication within the fibrillin-1 (Fbn-1) gene that results in a larger than normal Fbn-1 transcript, but the mechanism that leads to dermal fibrosis is unclear Fibrillin molecules associate into a polymer that is coated with elastin molecules to form elastic fibers. To further evaluate the Tsk-1 mouse model of scleroderma, we have studied elastic fibers in the skin of these mice. METHODS: Skin sections obtained from C57BL/6-TSK+ (Tsk-1) and C57BL6-pa/+ (control) mice were stained with Masson's trichrome for evaluation of collagen and Gomori's aldehyde fuchsin stain for elastic tissue. Computer assisted image analysis was performed to quantify differences in histologic sections. RESULTS: Tsk-1 mice had a highly significant increase in the percentage of elastic fibers (19.6%) in the dermis compared to control mice (7.9%) [p < 0.001]. This correlates with the findings in the skin of systemic sclerosis patients where increased elastic fibers have been observed. In addition, an increased level of dermal collagen staining was also observed in the Tsk-1 dermis (82.9%) compared with the level in normal sections (73.7%) [p < 0.01]. CONCLUSION: These data support the use of the Tsk-1 mouse as a model for the connective tissue abnormalities of human scleroderma.

Aseptic loosening.

Although total joint replacement surgery is one of the most successful clinical procedures performed today, bone loss around knee and hip implants (osteolysis), resulting in aseptic loosening of the prosthesis, remains a major problem for many patients. Over the last decade much has been learned about this process, which is caused by wear debris particles that simulate a local inflammatory response and osteoclastic bone resorption. Aseptic loosening cannot be prevented or treated by existing nonsurgical methods. Gene transfer, however, offers novel possibilities. Here, we review the current state of the field and the experimental gene therapy approaches that have been investigated toward a solution to aseptic loosening of prosthetic implants.

Protective effects of IL-1Ra or vIL-10 gene transfer on a murine model of wear debris-induced osteolysis.

The current study evaluated the protective effects of anti-inflammatory cytokine gene transfer on osteolysis provoked by orthopedic biomaterial particles using a murine model of inflammatory bone loss. A section of bone was surgically implanted into an air pouch established on a syngeneic recipient mouse. Inflammation was provoked by introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles into the pouch, and retroviruses encoding for interleukin-1 receptor antagonist (hIL-1Ra), viral interleukin-10 (vIL-10), or LacZ genes were injected. Pouch fluid and tissue were harvested 7 days later for histological and molecular analyses. The results indicated that IL-1Ra or vIL-10 gene transfer significantly inhibited IL-1beta and tumor necrosis factor (TNF) expression at both mRNA and protein levels. There were significantly lower mRNA expressions of calcitonin receptor and cathepsin K in RNA isolated from hIL-1Ra- or vIL-10-transduced pouches than LacZ-transduced and virus-free controls. Both anti-inflammatory cytokine gene transfers significantly reduced the mRNA expression of M-CSF (70-90%) and RANK (>65%) in comparison with LacZ- and virus-free controls. Histological examination showed that hIL-1Ra or vIL-10 gene transfer dramatically abolished UHMWPE-induced inflammatory cellular infiltration and bone pit erosion compared to LacZ-transduced and virus-free controls. Histochemical staining revealed significantly fewer osteoclast-like cells in samples treated with IL-1Ra or vIL-10 gene transfer. In addition, bone collagen content was markedly preserved in the groups with anti-inflammatory cytokine gene transfers compared with the other two groups. Overall, retrovirus-mediated hIL-1Ra or vIL-10 gene transfer effectively protected against UHMWPE-particle-induced bone resorption, probably due to the inhibition of IL-1/TNF-induced M-CSF production and the consequent osteoclast recruitment and maturation.

IFNgamma deficient C57BL/6 (H-2b) mice develop collagen induced arthritis with predominant usage of T cell receptor Vbeta6 and Vbeta8 in arthritic joints.

BACKGROUND: Transgenic deficiency in interferon gamma (IFNgamma) or IFNgamma receptor makes resistant strains of mice bearing H-2(b) or H-2(d) susceptible to collagen induced arthritis (CIA). OBJECTIVE: To determine whether the escape from regulation of disease susceptibility at the major histocompatibility complex level involves a new use of autoimmune T cells expressing T cell receptor (TCR) Vbeta that vary from the cell populations previously identified within arthritic joints. METHODS: Arthritis was induced by a standard protocol with type II bovine collagen (CII) in complete Freund's adjuvant. Clinical features, histopathology, immunological responses, and TCR profile in arthritic joints in IFNgamma knockout C57BL/6 (B6.IFNgamma KO) mice (H-2(b)) were compared directly with those in DBA/1 mice (H-2(q)). RESULTS: 60-80% of B6.IFNgamma KO mice developed a progressive arthritis with a similar clinical course to classical CIA in DBA/1 mice. The affected joints in B6.IFNgamma KO mice had an erosive form of arthritis with similar features to joint disease in DBA/1 mice. B6.IFNgamma KO mice produced significantly higher levels of IgG2b and IgG1 autoantibodies to murine CII and showed increased proliferative response to CII compared with B6 mice. Comparable levels of interleukin 1beta and tumour necrosis factor alpha expression were detected in arthritic joints from beta6.IFNgamma KO and DBA/1 mice. B6.IFNgammaKO mice used predominantly TCR Vbeta6 and Vbeta8 in arthritic joints. This TCR Vbeta profile is similar to that found in DBA/1 mice with CIA. CONCLUSIONS: C57BL/6 mice deficient in IFNgamma production can develop arthritis that resembles classical CIA. These data suggest that IFNgamma is a key factor mediating susceptibility to CIA.

TNF receptor gene therapy results in suppression of IgG2a anticollagen antibody in collagen induced arthritis.

BACKGROUND: Therapeutic strategies to block tumour necrosis factor alpha (TNFalpha) activity in experimental autoimmune arthritis models and rheumatoid arthritis (RA) have proved highly successful, and provide sustained beneficial effects. OBJECTIVE: To examine whether TNFalpha inhibition has immunological activity beyond the reduction of inflammation in collagen induced arthritis (CIA), an established experimental model of RA. METHODS: Arthritic DBA/1 mice received single periarticular injections of retroviral constructs encoding human TNF receptor (TNF-R) into the affected arthritic paw, at the onset of arthritis. Severity of arthritis, antibodies to collagen type II (CII), and extent of pathological joint damage of arthritic paws were compared between TNF-R and media treated (control) animals 3, 7, 14, 21, and 49 days after disease onset. RESULTS: Severity of CIA was significantly decreased in TNF-R treated animals compared with controls, 14-34 days after disease onset. Joint destruction was reduced in TNF-R injected joints and in the uninjected contralateral and ipsilateral paws of TNF-R treated animals. Seven days after disease onset, TNF-R treated mice had lower levels of inflammatory Th1 driven IgG2a antibodies to CII (p<0.05) than controls. This altered the anticollagen IgG2a:IgG1 ratio towards Th2 driven IgG1. CONCLUSIONS: Local TNF-R gene therapy in CIA appears to have systemic effects on the anti-CII antibodies. The overall influence of TNF-R gene therapy is that it inhibits the progression of CIA mainly by suppressing the inflammatory Th1 response rather than by stimulating a Th2 response. Therefore, periarticular TNF-R gene therapy may have excellent therapeutic potential in RA.

IL-1Ra and vIL-10 gene transfer using retroviral vectors ameliorates particle-associated inflammation in the murine air pouch model.

OBJECTIVE: This study examined anti- inflammatory gene therapy to ameliorate tissue responses to ultra high molecular weight polyethylene (UHMWPE) particles in the murine air pouch. METHODS: Retroviruses encoding human interleukin- 1 receptor antagonist (IL-1Ra), viral interleukin-10 (vIL-10), or LacZ (reporter) genes were injected into murine air pouches stimulated by UHMWPE particles. Pouch membranes and fluids were harvested at 1, 3 and 7 days post gene-transduction, and assayed for markers of inflammation using histological, molecular, and immunological techniques. RESULTS: Real time RT-PCR and ELISA showed a strong production of IL-1beta in pouch tissue and lavage fluid induced by particle stimulation, accompanied by a lower expression of IL-6, TNF-alpha and IL-4. Transduction of IL-1Ra or vIL-10 genes resulted in a significant reduction of IL-1beta both at the mRNA and at the protein level. The gene therapy also resulted in diminution of IL-6 and TNF-alpha expression. In addition, significant elevation of TGF-beta expression was observed in IL-1Ra transduced pouches. Histological analysis revealed that the membranes of pouches transduced with vIL-10 or IL-1Ra were significantly less inflamed than the membranes of non-viral and LacZ-transduced pouches, with less cellular proliferation and lowered monocyte/macrophage influx. CONCLUSIONS: IL-1Ra or vIL-10 gene transduction was effective in ameliorating local inflammation by reducing the IL-1 production and subsequent cellular events elicited in response to UHMWPE particles in this model. These findings suggest that IL-1 directed gene therapy might be excellent therapeutic candidates to prevent or retard the inflammatory response to wear debris that contributes to the pathology of aseptic loosening.

Effects of cytokine gene therapy on particulate-induced inflammation in the murine air pouch.

Retroviral vectors encoding the human IL-1 antagonist (IL-1Ra) gene and the human tumor necrosis factor soluble receptor (sTNF-R) gene were investigated using an in vivo model of the inflammatory response to orthopedic wear debris. Air pouches established in BALB/c mice were injected with polymethylmethacrylate (PMMA) particles to provoke an inflammatory reaction, and infected with retroviral vectors expressing IL-1Ra, sTNF-R or a LacZ marker gene. Pouch membranes and fluids were harvested after 48 or 72 hours for analyses. Positive PCR reactions for Neo genes were observed specifically in DNA extracted from the membrane of retroviral-infected pouches. ELISA assays revealed the presence of human IL-1 Ra in pouch fluid from DFG-IRAP-Neo transduced mice, but not control animals. Histological evaluation indicated that the IL-1Ra gene transfer was associated with markedly decreased inflammation in the model, with resolution of the edematous phase of the reaction, decreased pouch fluid accumulation, and lowered macrophage influx. The data suggest that the air pouch model represents a useful tool to evaluate gene therapy, and demonstrate that IL-1Ra gene therapy may be an appropriate therapeutic approach to inflammation.

Widespread susceptibility among inbred mouse strains to the induction of lupus autoantibodies by pristane.

Unlike other agents associated with drug-induced lupus, the isoprenoid alkane pristane induces autoantibodies pathognomonic of lupus, including anti-Sm, anti-dsDNA, and anti-ribosomal P in BALB/c and SJL/J mice. The susceptibility of other strains of mice to pristane-induced lupus is unknown and is the focus of the present study. Anti-nRNP/Sm, anti-Su, and anti-ribosomal P autoantibodies were produced by most strains of mice surveyed within several months of pristane treatment, although there was marked interstrain variability in their frequencies, levels, and times of onset. In sharp contrast, the production of autoantibodies against the double-stranded RNA binding proteins NF45/NF90/p110 was restricted to B6 and B10.S mice. We conclude that pristane selectively induces lupus-specific autoantibodies in virtually any strain of mouse regardless of its genetic background. However, H-2-linked as well as non-H2 genes influenced the expression of individual autoantibody markers. The widespread susceptibility of pristane-treated mice to lupus autoantibody production and the relatively small effect of MHC are unique features of this chemically induced lupus syndrome, with potential implications for understanding the pathogenesis of autoantibodies in idiopathic human systemic lupus erythematosus.

Polymorphism in the vitamin D receptor gene and bone mass in African-American and white mothers and children: a preliminary report.

OBJECTIVE: To evaluate the contribution of polymorphisms in the vitamin D receptor (VDR) gene to ethnic variations in bone mass in mother and children from different ethnic origins. METHODS: VDR genotypes and bone mass in 43 African-American and white women, mean age 38.2 years, and 41 of their children were studied. All children had a whole body bone mass measurement at age 9, and 39 had follow up measurements at age 11, while all the mothers had a single measurement. DNA was extracted from peripheral blood samples, subjected to polymerase chain reactions using primers specific for the VDR gene, and the Bsm1 restriction fragment length polymorphism defined. RESULTS: There was a significant ethnic difference in the VDR genotype frequencies among the adults and the children. No African-American subjects had the genotype "BB". In contrast, there was a 25% frequency of the "BB" genotype in the white adults and 24% in the white children. After pooling the ethnic groups, the mean bone mass in the "bb" genotype was significantly higher than in the "BB" genotype among the mothers, but this was not found in the children at baseline. However, by age 11, those with the "Bb" or "bb" genotypes had a larger gain in bone mass than those with "BB". CONCLUSION: These data support the suggestion that the ethnic difference in VDR genotype frequencies, together with the association between the genotypes and bone mass, may help to explain the well known ethnic differences in bone mass. Further, our observations suggest that VDR polymorphism may have an effect on bone mass during puberty as peak bone mass is accumulated.

The influence of silicone implantation on murine lupus in MRL lpr/lpr mice.

OBJECTIVE: The use of silicone breast implants has been implicated in the development of autoimmune connective tissue diseases including systemic lupus erythematosus (SLE). We examined the influence of implanted silicones in MRL lpr/lpr and MRL +/+ mice, to determine whether silicone increases autoimmunity and exacerbates experimental lupus. METHODS: Mice were implanted with either silicone gel or silicone oil (polydimethylsiloxane; PDMS), while saline injected mice were used as controls. Proteinuria levels, palpation of lymphadenopathy, serum autoantibodies, circulating cytokines, and weight change were monitored for 18 weeks, when terminal glomerulonephritis was evaluated by histopathological techniques. Proteins were extracted from the surface of recovered implants, and the composition and immune reactive status of the silicone-binding proteins (SBP) were investigated. RESULTS: No adverse influence of silicone gel or silicone oil on the clinical aspects of lupus was observed. However, anti-DNA antibodies were significantly increased in MRL mice implanted with silicone gel compared to the control animals, and rheumatoid factor titers were modestly increased in implanted MRL lpr/lpr mice. Serum cytokine levels were influenced by silicone implantation in MRL lpr/lpr mice (but not MRL +/+ mice), with interleukin 1 (IL-1) levels increased in gel implanted animals and IL-2 levels elevated in PDMS (silicone oil) implanted mice. Different SBP were detected on implants recovered from MRL lpr/lpr mice compared with MRL +/+ mice, and Western blotting revealed the presence of strong autoantibodies to SBP in sera from MRL lpr/lpr mice, but not MRL +/+ mice. CONCLUSION: These findings suggest that silicone implantation may influence immunological responses during murine lupus, including the provocation or exacerbation of autoantibodies. However, these immune modifications did not appear to influence the clinical variables of this experimental lupus model.

Influence of long term silicone implantation on type II collagen induced arthritis in mice.

OBJECTIVES: The use of silicone implants in cosmetic and reconstructive surgery has been implicated in the development of autoimmune connective tissue diseases. Previous investigation of the influence of short-term silicone implantation using an experimental model of rheumatoid arthritis revealed no adverse influence upon disease despite the generation of autoantibodies against silicone bound proteins. This study was designed to examine the influence of long term implantation of different forms of silicone in collagen induced arthritis. METHODS: DBA/1 mice were surgically implanted with silicone elastomers, gel or oil nine months before immunisation with type II collagen emulsified in Freund's incomplete adjuvant. The incidence and severity of arthritis, antibodies to type II collagen, and serum cytokines were assessed and compared with sham implanted mice. Silicone implants were recovered, and autoantibodies to silicone bound proteins evaluated in arthritic and non-arthritic mice. RESULTS: Immunisation with CII/FIA resulted in a 30% arthritis incidence in sham implanted DBA/1 mice. Long term silicone implantation resulted in an increased incidence of arthritis, with a significant increase of 90% arthritis in animals implanted with silicone elastomers. Animals implanted with silicone elastomer also developed foreign body sarcomas during the study. Serum concentrations of interleukin 10 were increased in mice implanted with elastomers and immunised with CII/FIA, while interleukin 5 concentrations were significantly diminished in these mice. The production of autoantibodies to autologous silicone bound proteins, including anti-type I collagen antibody, was also attributed to the implantation of either silicone gel or silicone elastomer in type II collagen immunised animals. CONCLUSIONS: These data suggest that long term silicone implantation results in both the production of autoantibodies to connective tissue antigens and increased susceptibility to an experimental model of autoimmune disease.

Experimental acute hematogenous osteomyelitis in mice. I. Histopathological and immunological findings.

This study investigated immunological responses to Staphylococcus aureus bone infection. Because considerable immunological information is available on the mouse, a murine model of acute hematogenous osteomyelitis was established. Osteomyelitis was created in the proximal tibia of C3H/HeJ mice by a tibial epiphyseal fracture followed by intravenous bacterial inoculation with Staphylococcus aureus (strain LS-1). Swelling and warmth of the limb was found, and following limb exposure, abscess formation was evident in the proximal tibia. Histological examination revealed distortion primarily at the hypertrophic zone of the physis and polymorphonuclear leukocyte infiltration throughout the damaged area of the proximal tibia. Local infection was demonstrated at the fracture site, evidenced by the recovery of Staphylococcus aureus following microbiological analysis of tissue specimens. Polymerase chain reaction was utilized to detect 16S ribosomal prokaryotic nucleic acid to demonstrate that the diagnosis of osteomyelitis could be established in the absence of conventional microbiological techniques. The infected mice had an increase of circulating large leukocytes (granulocytes) and an elevation of total serum immunoglobulin. Flow cytometry revealed significant increases in splenic B lymphocytes and in lymph-node CD4+ T lymphocytes. These results indicate that an experimental model of acute hematogenous osteomyelitis that closely resembles the pathology of the disease in humans may be consistently induced in mice. Furthermore, marked immunological changes may be observed in response to the Staphylococcus aureus bone infection.

Experimental acute hematogenous osteomyelitis in mice. II. Influence of Staphylococcus aureus infection on T-cell immunity.

A murine model of acute hematogenous osteomyelitis was used to study the immune response following Staphylococcus aureus infection and to examine the hypothesis that the bacteria may modify T-cell responses due to the production of bacterial enterotoxins with mitogenic or superantigenic activity. Lymph-node T cell-receptor expression was assessed with use of flow cytometry and reverse transcription-polymerase chain reaction techniques, and increased apoptosis (programmed cell death) in T-cell subsets was monitored. The expression and levels of circulating cytokines and T-cell cytokines within tissues surrounding the damaged area of the proximal tibia were also investigated. Analysis of T-cell receptors in experimental osteomyelitis revealed two distinct patterns of T-cell evolution during the disease. Certain T-cell subsets (Vbeta2, Vbeta3, Vbeta9, and Vbeta10) were activated and expanded during the first 24 hours after infection; they reached maximum levels 6 days after infection, followed by a return to pre-infection levels. In contrast, other T-cell subsets (Vbeta11, Vbeta12, Vbeta13, Vbeta14, and Vbeta16) contracted during the first 24 hours after infection, followed by expansion to a maximum level 9 days after infection. Activation and proliferation of T-cell subsets (notably Vbeta14 T cells) was followed by apoptosis, suggesting that staphylococcal bone infection caused superantigenic-like effects on the mouse immune system. Analysis of cytokine responses in local tissue revealed that the T-cell cytokines interleukin-2 and interferon-gamma showed a late and relatively short activation pattern compared with the inflammatory cytokines interleukin-1, interleukin-6, and tumor necrosis factor-alpha. The results suggest that Staphylococcus aureus bone infection may undermine the antibacterial immune response through downregulation of T-cell immunity and immune-cytokine production, which could increase the severity of the systemic infection and local osseous destruction that occur with acute hematogenous osteomyelitis.

Proteins bound to polyethylene components in patients who have aseptic loosening after total joint arthroplasty. A preliminary report.

BACKGROUND: Immunological responses to proteins that adhere to ultra-high molecular weight polyethylene have not, to our knowledge, been examined previously in patients who have aseptic loosening. In the current study, polyethylene components from forty-nine failed prostheses recovered during revision procedures were examined for the presence of antibodies that were bound to the polyethylene surface or that were reactive with other proteins that were bound to the polyethylene surface. METHODS: The polyethylene components consisted of thirty acetabular cups recovered during revision total hip arthroplasties and nineteen tibial components recovered during revision total knee arthroplasties. After extensive washing, bound proteins were extracted from the polyethylene components with use of 0.1-molar glycine-hydrogen chloride solution followed by four-molar guanidine hydrochloride solution. RESULTS: Sufficient protein for analysis was recovered from forty-two polyethylene components. Polyacrylamide gel electrophoresis demonstrated a minimum of one and a maximum of twelve protein bands, with molecular weights ranging from thirteen to 231 kilodaltons. Immunoblotting revealed the presence of type-I collagen in most (thirty-four) of the forty-two explants, whereas aggrecan proteoglycans were detected in eight samples. Immunoglobulin also was detected in most (thirty-three) extracts, whereas type-II collagen was consistently absent. The presence of autologous antibodies directed against polyethylene-bound proteins in sera drawn at the time of the revision was investigated. Antibodies that were reactive against the ultra-high molecular weight polyethylene-bound proteins were detected in twenty-six of the forty-two patients with use of the Western blot technique. The number of reactive bands ranged from one to six, and the strongest binding was directed against a 103-kilodalton protein. Assays for specificity revealed that these sera autologous antibodies were reactive against the type-I collagen that was present in the explant solutions. CONCLUSIONS: We hypothesize that immunoglobulin complexed with polyethylene may fix complement and that the complement cascade may in turn attract inflammatory cells to the polyethylene surface. Our data support the hypothesis that an immunological response to antigens bound to the polyethylene surface may contribute to aseptic loosening. CLINICAL RELEVANCE: Despite improvements in materials and designs of prostheses, aseptic loosening is the most common complication of total joint replacement, frequently leading to revision operations. We examined the immunological response to proteins that bind to ultra-high molecular weight polyethylene in patients who had aseptic loosening and discovered a high prevalence of antibodies to polyethylene-bound proteins. This immunological response may contribute to an inflammatory reaction in the periprosthetic tissue, ultimately leading to increased bone resorption around the prosthesis.

Pristane-induced arthritis in mice. V. Susceptibility to pristane-induced arthritis is determined by the genetic regulation of the T cell repertoire.

OBJECTIVE: Pristane-induced arthritis (PIA) is an experimental seropositive arthritis that is characterized by serologic and cellular immune abnormalities and is dependent on the presence of a competent CD4+ T cell population. We examined the regulation of PIA by genes of the major histocompatibility complex (MHC) and the Mls-1 loci to determine whether the selection of the T cells that infiltrate arthritic joints is a critical factor in disease susceptibility. METHODS: Genetic regulation of PIA was investigated using F1 hybrid and congenic strain analysis to determine the influence of MHC and Mls-1 genes. The T cell receptor Vbeta phenotypes of lymph node cells and T cells infiltrating arthritic joints were examined with 2-color flow cytometry and reverse transcription-polymerase chain reaction techniques. RESULTS: F1 hybrid offspring from 2 major PIA-susceptible strains (DBA/1 x BALB/c) were resistant to the induction of arthritis because of the interaction between genes of the MHC and the Mls-1 loci, which modified the T cell repertoire. This conclusion was supported by the observed resistance to PIA in BALB/ c-Mls-1a mice, where T cells expressing the Vbeta8.1 and Vbeta6 phenotypes were absent. The receptor phenotype of T cells infiltrating arthritic joints in DBA/1 mice was markedly skewed toward Vbeta8.1 and Vbeta6 compared with the population observed in lymph nodes from either PIA or normal control DBA/1 mice. CONCLUSION: The data support the hypothesis that PIA is a T cell-mediated disease. While pristane causes a polyclonal T cell expansion that gives rise to lymphadenopathy, the development of arthritis in susceptible strains of mice occurs due to the preservation of specific T cell subsets with the capacity to infiltrate synovial joints.