A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese.

AIMS: In this study, we compare seven different methods which have been designed or modified to extract total DNA from raw milk and raw milk cheese with a view to its subsequent use for the PCR of bacterial DNA. MATERIALS AND RESULTS: Seven extraction methods were employed to extract total DNA from these foods, and their relative success with respect to the yield and purity of the DNA isolated, and its quality as a template for downstream PCR, was compared. Although all of the methods were successful with respect to the extraction of DNA naturally present in cheese, they varied in their relative ability to extract DNA from milk. However, when milk was spiked with a representative Gram-positive (Listeria monocytogenes EGDe) or Gram-negative (Salmonella enterica serovar Typhimurium LT2) bacterium, it was established that all methods successfully extracted DNA which was suitable for subsequent detection by PCR. CONCLUSIONS: Of the seven approaches, the PowerFood™ Microbial DNA Isolation kit (MoBio Laboratories Inc.) was found to most consistently extract highly concentrated and pure DNA with a view to its subsequent use for PCR-based amplification and also facilitated accurate detection by real-time quantitative PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurately assessing the bacterial composition of milk and cheese is of great importance to the dairy industry. Increasingly, DNA-based technologies are being employed to provide an accurate assessment of this microbiota. However, these approaches are dependent on our ability to extract DNA of sufficient yield and purity. This study compares a number of different options and highlights the relative success of these approaches. We also highlight the success of one method to extract DNA from different microbial populations as well as DNA which is suitable for real-time PCR of microbes of interest, a challenge often encountered by the food industry.

Second-generation cementless total hip arthroplasty. Eight to eleven-year results.

BACKGROUND: Second-generation cementless femoral components were designed to provide more reliable ingrowth and to limit distal osteolysis by incorporating circumferential proximal ingrowth surfaces. We examined the eight to eleven-year results of total hip arthroplasty with a cementless, anatomically designed femoral component and a cementless hemispheric acetabular component. METHODS: Ninety-two consecutive primary total hip arthroplasties with implantation of a femoral component with a circumferential proximal porous coating (Anatomic Hip) and a cementless hemispheric porous-coated acetabular component (Harris-Galante II) were performed in eighty-five patients. These patients were prospectively followed clinically and radiographically. Six patients (seven hips) died and five patients (seven hips) were lost to follow-up, leaving seventy-four patients (seventy-eight hips) who had been followed for a mean of ten years (range, eight to eleven years). The mean age at the time of the arthroplasty was fifty-two years. RESULTS: The mean preoperative Harris hip score of 51 points improved to 94 points at the time of final follow-up; 86% of the hips had a good or excellent result. Thigh pain was reported as mild to severe after seven hip arthroplasties. No femoral component was revised for any reason, and none were loose radiographically at the time of the last follow-up. Two hips underwent acetabular revision (one because of dislocation and one because of loosening). Kaplan-Meier survivorship analysis was performed with revision or loosening of any component as the end point. The ten-year survival rate was 96.4% +/- 2.1% for the total hip prosthesis, 100% for the femoral component, and 96.4% +/- 2.1% for the acetabular component. Radiolucencies adjacent to the nonporous portion of the femoral component were seen in sixty-eight (93%) of the -seventy-three hips with complete radiographic follow-up. Femoral osteolysis proximal to the lesser trochanter was noted in four hips (5%). No osteolysis was identified distal to the lesser trochanter. Periacetabular osteolysis was identified in twelve hips (16%). Five patients underwent exchange of the acetabular liner because of polyethylene wear. CONCLUSIONS: This second-generation cementless, anatomically designed femoral component provided excellent clinical and radiographic results with a 100% survival rate at ten years. The circumferential porous coating of this implant improved ingrowth and prevented distal osteolysis at a mean of ten years after the arthroplasty.

Cementless acetabular reconstruction after acetabular fracture.

BACKGROUND: Total hip arthroplasty in patients with posttraumatic arthritis has produced results inferior to those in patients with nontraumatic arthritis. The use of cementless acetabular reconstruction, however, has not been extensively studied in this clinical context. Our purpose was to compare the intermediate-term results of total hip arthroplasty with a cementless acetabular component in patients with posttraumatic arthritis with those of the same procedure in patients with nontraumatic arthritis. We also compared the results of arthroplasty in patients who had had prior operative treatment of their acetabular fracture with those in patients who had had prior closed treatment of their acetabular fracture. METHODS: Thirty total hip arthroplasties were performed with use of a cementless hemispheric, fiber-metal-mesh-coated acetabular component for the treatment of posttraumatic osteoarthritis after acetabular fracture. The median interval between the fracture and the arthroplasty was thirty-seven months (range, eight to 444 months). The average age at the time of the arthroplasty was fifty-one years (range, twenty-six to eighty-six years), and the average duration of follow-up was sixty-three months (range, twenty-four to 140 months). Fifteen patients had had prior open reduction and internal fixation of their acetabular fracture (open-reduction group), and fifteen patients had had closed treatment of the acetabular fracture (closed-treatment group). The results of these thirty hip reconstructions were compared with the intermediate-term results of 204 consecutive primary total hip arthroplasties with cementless acetabular reconstruction in patients with nontraumatic arthritis. RESULTS: Operative time (p < 0.001), blood loss (p < 0.001), and perioperative transfusion requirements (p < 0.001) were greater in the patients with posttraumatic arthritis than they were in the patients with nontraumatic arthritis. Of the patients with posttraumatic arthritis, those who had had open reduction and internal fixation of their acetabular fracture had a significantly longer index procedure (p = 0.01), greater blood loss (p = 0.008), and a higher transfusion requirement (p = 0.049) than those in whom the fracture had been treated by closed methods. Eight of the fifteen patients with a previous open reduction and internal fixation required an elevated acetabular liner compared with one of the fifteen patients who had been treated by closed means (p = 0.005). Two of the fifteen patients with a previous open reduction and internal fixation required bone-grafting of acetabular defects compared with seven of the fifteen patients treated by closed means (p = 0.04). The thirty patients treated for posttraumatic arthritis had an average preoperative Harris hip score of 41 points, which increased to 88 points at the time of follow-up; there was no significant difference between the open-reduction and closed-treatment groups (p = 0.39). Twenty-seven patients (90%) had a good or excellent result. There were no dislocations or deep infections. The Kaplan-Meier ten-year survival rate, with revision or radiographic loosening as the end point, was 97%. These results were similar to those of the patients who underwent primary total hip arthroplasty for nontraumatic arthritis. CONCLUSIONS: The intermediate-term clinical results of total hip arthroplasty with cementless acetabular reconstruction for posttraumatic osteoarthritis after acetabular fracture were similar to those after the same procedure for nontraumatic arthritis, regardless of whether the acetabular fracture had been internally fixed initially. However, total hip arthroplasty after acetabular fracture was a longer procedure with greater blood loss, especially in patients with previous open reduction and internal fixation. Previous open reduction and internal fixation predisposed the hip to more intraoperative instability but less bone deficiency.

Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo.

CD26 or dipeptidyl peptidase IV (DP IV) is expressed on various cell types, including T cells. Although T cells can receive activating signals via CD26, the physiological role of CD26/DP IV is largely unknown. We used the reversible DP IV inhibitor Lys[Z(NO(2))]-pyrrolidide (I40) to dissect the role of DP IV in experimental autoimmune encephalomyelitis (EAE) and to explore the therapeutic potential of DP IV inhibition for autoimmunity. I40 administration in vivo decreased and delayed clinical and neuropathological signs of adoptive transfer EAE. I40 blocked DP IV activity in vivo and increased the secretion of the immunosuppressive cytokine TGF-beta1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF-alpha production, I40 consistently up-regulated TGF-beta1 secretion. A neutralizing anti-TGF-beta1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DP IV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-beta1-mediated antiinflammatory effect at the site of pathology.

Hybrid total hip arthroplasty with a precoated offset stem. Four to nine-year results.

BACKGROUND: Use of modern cementing techniques for fixation of femoral components in total hip arthroplasty has had excellent clinical and radiographic results in most patients. However, several authors have described early loosening of femoral components with roughened and precoated finishes. The purpose of this study was to examine the performance of the precoated Iowa stem, which has increased offset, and to compare the results with those of another cemented precoated femoral component with standard offset used at our institution. METHODS: We carried out a prospective analysis of 102 primary hybrid total hip arthroplasties (a cementless acetabular component and a cemented femoral component) performed with use of the Iowa femoral component in ninety-five patients at our institution. The Iowa stem was used in hips that required greater offset than is available with standard stems as determined by preoperative templating. The average age of the patients at the time of the index procedure was sixty-nine years. Sixteen patients (seventeen hips) died before the forty-eight-month minimum follow-up period had elapsed. Two patients were lost to follow-up, and radiographic follow-up was incomplete for one. The mean duration of clinical and radiographic follow-up of the remaining eighty-two hips in the seventy-six surviving patients was sixty-five months (range, forty-eight to 104 months). RESULTS: The average preoperative Harris hip score of 47 points (range, 16 to 69 points) improved to an average of 87 points (range, 24 to 100 points) at the time of the review. Two hips underwent femoral component revision. Four femoral stems were radiographically loose at an average of thirty-four months. Femoral osteolysis was seen in five hips (6 percent) at an average of fifty-four months postoperatively. No acetabular component was revised because of aseptic loosening. According to Kaplan-Meier analysis, the seven-year survival rate, with an end point of femoral revision, osteolysis, or stem debonding, was 90.6 percent (95 percent confidence interval, 0.87 to 0.94). CONCLUSIONS: The prevalence of revision, osteolysis, and loosening after total hip arthroplasty with the Iowa femoral component at our institution was higher than that seen in our series of Harris Precoat stems, which had a survival rate of 98.4 percent (95 percent confidence interval, 0.97 to 1.00) at ten years with the same end points. The design of the Iowa stem may make it difficult to achieve a good cement mantle, and, in combination with the geometry and increased offset of the stem, may compromise the long-term survival of this cemented femoral component.

Dipeptidyl peptidase IV in inflammatory CNS disease.

Current pathogenic concepts of inflammatory demyelinating disorders such as multiple sclerosis (MS) are based on the hypothesis that a T cell-mediated autoimmune response is involved in the disease process. One of the primary goals in the in the development of immunotherapies for autoimmune diseases has been to achieve inactivation of disease-inducing lymphocytes either by direct inhibition or suppression through regulatory cells and/or cytokines. The CD26 antigen is identical with the cell surface ectopeptidase dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) which is involved in regulating T cell activation and growth. Activated T cells, including those specific for myelin antigens, express high levels of CD26/DP IV. In vitro, reversible DP IV inhibitors suppress T cell proliferation and pro-inflammatory cytokine production in response to myelin antigens. Further studies will evaluate the role of DP IV inhibition in T cell-mediated inflammatory disease of the central nervous system.

Effects of substance dependence on outcomes of patients in a randomized trial of two case management models.

OBJECTIVE: The effectiveness of a community-based intensive clinical case management program was compared with that of a hospital-based expanded brokerage case management program for seriously mentally ill adults with and without substance dependence. METHODS: The sample of 268 frequently hospitalized psychiatric patients was recruited during acute psychiatric hospitalization. More than half of the sample (53 percent) was diagnosed as having at least one current DSM-III-R substance dependence disorder co-occurring with their primary major mental disorder. Subjects were stratified by substance dependence status and randomly assigned to one of the case management programs. They were interviewed before hospital discharge and at one, two, and six months after discharge to assess psychosocial and drug use variables. Subjects' service use was examined for the six months before and after hospitalization. RESULTS: The hypothesis that substance-dependent subjects would benefit more from intensive clinical case management was not supported. Substance dependence predicted negative outcomes independent of the case management intervention. The hypothesis that the two case management approaches would be equally effective for subjects not dependent on substances was also not borne out. Intensive clinical case management was the superior treatment for subjects who were not dependent on substances. Fewer of them required psychiatric hospitalization in the six-month postdischarge period than in the six-month period before hospital admission. CONCLUSIONS: The negative outcomes for substance-dependent subjects in both programs suggest that the two case management models were relatively ineffective for these patients. Results suggest that intensive clinical case management can be effective within the first six months for nondependent adults with serious mental illness.

Cementless acetabular revision. Evaluation at an average of 10.5 years.

This study reviews the clinical and radiographic results of 138 consecutive cementless acetabular revisions in 131 patients performed for aseptic loosening at a mean of 10.5 years followup. Kaplan-Meier survivorship of these components was 84% at 11.5 years. Two components (1.8%) in two patients were considered aseptically loose based on radiographic criteria; one patient was symptomatic but the component was not revised because of the patient's poor health, and the other patient was asymptomatic. Pelvic osteolysis was present in 19 hips (17%), appearing at a mean of 103 months. All but two of these were small lesions (< 2 cm) at the periphery of the components. Nevertheless, the incidence of osteolysis has increased with time, and continued followup is warranted. Separation or fragmentation of the fiber-metal porous pads was uncommon (8.3%), but was significantly associated with pelvic osteolysis; this finding has not been reported before with this component. Five patients underwent late revision surgery (after 100 months), one for deep infection, one for periprosthetic femur fracture, and three for late recurrent dislocation. Harris hip scores averaged 81 points (good) at final followup, which is unchanged from the authors' last report on this group. Acetabular revision with a fiber-metal hemispherical component appears durable at a mean followup of more than 10 years.

The fate of stable cemented acetabular components retained during revision of a femoral component of a total hip arthroplasty.

BACKGROUND: The decision as to whether to revise or retain a well fixed cemented acetabular component during revision of a femoral component is especially difficult; the rate of loosening of cemented acetabular components is high, whereas that of porous-coated acetabular components inserted during revision is low. However, removal of a well fixed cemented acetabular component can result in increased operative morbidity and cost and in loss of acetabular bone. Data that can be used to predict the long-term survival of retained well fixed cemented acetabular components are therefore needed. METHODS: We studied the five to thirteen-year clinical and radiographic results in a group of twenty-six consecutive patients in whom a well fixed cemented acetabular component had been retained during revision of a femoral component. Typical demographic data on the patients and information about the components were recorded, and the cemented acetabular components were graded as A through F, according to the system of Ranawat et al., at the time of the femoral revision. The average duration of follow-up was 8.4 years (range, 5.0 to 12.7 years). No patient was lost to follow-up. RESULTS: Four acetabular components (15 percent) had progressive radiolucency (at forty-eight, forty-eight, fifty-nine, and seventy-five months after the femoral revision) and were considered radiographically loose despite not being associated with symptoms. All four components were graded as either E or F at the time that they were retained during the femoral revision; radiographic loosening was significantly related to these two grades (p < 0.01). No acetabular component with a grade of A, B, C, or D loosened. The components that loosened had been in vivo for a relatively shorter, as opposed to longer, duration before the femoral revision compared with the components that did not loosen (p < 0.05). CONCLUSIONS: Retention of the well fixed cemented acetabular components was associated with good clinical results but with a 15 percent rate of loosening. Revision of a cemented acetabular component solely on the basis of the duration that it was in vivo or whether a previous revision had been done does not appear to be warranted. Our findings suggest that acetabular components with a grade of A, B, C, or D at the time of a femoral revision may be retained, as these components continued to function at the time of the five to thirteen-year follow-up in the current study.

Impaction allografting with cement for revision of the femoral component. A minimum four-year follow-up study with use of a precoated femoral stem.

BACKGROUND: Cancellous impaction allografting with cement for revision of the femoral component has conventionally been performed with a polished, tapered implant, which was designed to allow subsidence of the component. However, subsidence has been associated with pain in the thigh, dislocation of the hip, and revision of the component. This prospective study tested the hypothesis that good clinical results can be achieved--without subsidence of the component--with use of impaction allografting and a precoated, collared, straight stem for difficult femoral revisions. METHODS: Twenty-nine patients had revision of the femoral component with use of impaction allografting with cement and a Harris Precoat stem. Impaction allografting was performed when loss of metaphyseal and diaphyseal bone precluded revision with more straightforward techniques or when reconstitution of bone was considered a specific goal of the reconstruction (as was sometimes the case with revision of the component in younger patients). The patients were followed prospectively and were evaluated with use of the Harris hip score and serial radiographs. The patients were followed for a minimum of four years (mean, sixty-three months), except for four who died. RESULTS: Four patients died before the minimum four-year follow-up period had elapsed; all four had the prosthesis in place at the time of death. The Harris hip scores improved from a preoperative mean of 54 points (poor) (range, 21 to 91 points) to a mean of 87 points (good) (range, 41 to 100 points) at the time of the most recent follow-up. Kaplan-Meier survivorship analysis, with aseptic loosening as the end point, was 92 percent (95 percent confidence interval, 82 to 100 percent) at six years; one additional hip failed because of a hematogenous infection at seventy-three months postoperatively, for an overall failure rate of 12 percent (three of twenty-five patients) at the time of the most recent follow-up. Two hips needed a repeat revision; one was revised because of subsidence of the stem with recurrent osteolysis and the other, in a patient who had hemodialysis, because of late sepsis. A third femoral component subsided and failed but was not revised. Radiographic evidence of bone-stock reconstitution was observed in six (29 percent) of the twenty-one patients for whom radiographs were available. As in other series of patients managed with impaction allografting, the complication rate was high; excluding the revisions, three reoperations were performed, and six patients had either intraoperative femoral fracture or perforation necessitating cerclage wiring or cortical strut allografting and cerclage wiring at the time of the procedure. There were six nonunions in eighteen patients who had been operated on with a transtrochanteric approach. CONCLUSIONS: Difficult revisions of the femoral component with use of impaction allografting and a precoated stem provided satisfactory clinical and radiographic results at the time of intermediate-term follow-up. However, the high rate of complications in our series led us to refine our indications for the procedure.

In vivo survival of viral antigen-specific T cells that induce experimental autoimmune encephalomyelitis.

A peptide derived from the human papillomavirus L2 protein is recognized by a myelin basic protein (MBP)-specific T cell clone from a multiple sclerosis patient and by MBP-specific autoantibodies purified from multiple sclerosis brain tissue. We now show in mice that low doses of this papillomavirus peptide were optimal in selecting a subpopulation of papillomavirus peptide-specific T cells that cross-reacted with MBP(87-99) and with an unrelated viral peptide derived from the BSLF1 protein of Epstein-Barr virus (EBV). These low dose viral peptide- specific T cell lines were highly encephalitogenic. Splenocytes from mice transferred with viral peptide-specific T cells showed a vigorous response to both the papillomavirus and MBP peptides, indicating that viral antigen-specific T cells survived for a prolonged time in vivo. The EBV peptide, unable to prime and select an autoreactive T cell population, could still activate the low dose papillomavirus peptide-specific cells and induce central nervous system (CNS) autoimmunity. Cytokine profiles of papillomavirus peptide-specific encephalitogenic T cells and histopathology of CNS lesions resembled those induced by MBP. These results demonstrate conserved aspects in the recognition of the self-antigen and a cross-reactive viral peptide by human and murine MBP-specific T cell receptors. We demonstrate that a viral antigen, depending on its nature, dose, and number of exposures, may select autoantigen-specific T cells that survive in vivo and can trigger autoimmune disease after adoptive transfer.

Screening and brief intervention for high-risk college student drinkers: results from a 2-year follow-up assessment.

This randomized controlled trial evaluated the efficacy of a brief intervention designed to reduce the harmful consequences of heavy drinking among high-risk college students. Students screened for risk while in their senior year of high school (188 women and 160 men) were randomly assigned to receive an individualized motivational brief intervention in their freshman year of college or to a no-treatment control condition. A normative group selected from the entire screening pool provided a natural history comparison. Follow-up assessments over a 2-year period showed significant reductions in both drinking rates and harmful consequences, favoring students receiving the intervention. Although high-risk students continued to experience more alcohol problems than the natural history comparison group over the 2-year period, most showed a decline in problems over time, suggesting a developmental maturational effect.

Study of relapsing remitting experimental allergic encephalomyelitis SJL mouse model using MION-46L enhanced in vivo MRI: early histopathological correlation.

MION-46L, a superparamagnetic iron oxide contrast agent, was investigated for its ability to increase the sensitivity of in vivo 3D MRI in the detection of brain lesions in a chronic experimental allergic encephalomyelitis (crEAE) mouse model. Lesion conspicuity on postcontrast 3D MRI was dramatically enhanced as compared to precontrast images corresponding to areas of inflammatory and demyelinating lesions. MION-46L could be detected on Prussian blue iron stain in the vascular endothelium, the perivascular space, and in macrophages within perivascular cuffs and areas of inflammation and demyelination. By taking advantage of the MION-46L induced macroscopic susceptibility effect, acute early lesions measuring only 100 microm in diameter could be detected. MION-46L enhanced MRI may be used to 1) provide a unique sensitivity in EAE lesion detection and correlate imaging to histopathology; 2) help to understand EAE lesion development and its underlying pathophysiology; and 3) eventually assist in preclinical screening of new experimental therapies directed at patients with multiple sclerosis (MS).

Chronic relapsing experimental autoimmune encephalomyelitis: effects of insulin-like growth factor-I treatment on clinical deficits, lesion severity, glial responses, and blood brain barrier defects.

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE), a model for multiple sclerosis, was used to test 2 regimens of insulin-like growth factor-I (IGF-I) treatment. We induced crEAE by injecting 3x10(7) myelin basic protein-(MBP) sensitized lymph node cells into adult female SJL/J mice. Fifty-one mice, divided randomly into 4 groups, were used in the first trial. Two groups received IGF-I (a gift of Cephalon, Inc.) 0.6 mg/kg/d subcutaneously from day 7 to day 16 and the other two groups received placebo injections. IGF-I treatment reduced clinical deficits during the first attack and during 2 subsequent relapses. Image analysis of immunostained and histological sections showed that IGF-I treatment reduced BBB defects and both the numbers and sizes of inflammatory, demyelinating, and demyelinated lesions. Twelve mice that had recovered from their first attack were used in our second trial to evaluate possible adverse effects of prolonged treatment with a higher dose of IGF-I. Six received 1.2 mg/kg/d for 6 weeks (days 19-63). No adverse effects of IGF-I treatment were identified. The eyes, hearts, livers, and kidneys of IGF-I-treated mice were normal histologically and their spleens also appeared normal except for mild to moderate microscopic increases in lymphopoesis. Our results suggest that prolonged IGF-I treatment is well tolerated and that the anti-inflammatory effects of IGF-I have a major role in reducing clinical deficits and lesion severity in crEAE. These effects, if present in multiple sclerosis, may benefit patients with this disease.

In vivo three-dimensional MR microscopy of mice with chronic relapsing experimental autoimmune encephalomyelitis after treatment with insulin-like growth factor-I.

PURPOSE: The purpose of this study was to determine the ability of three-dimensional in vivo MR microscopy to depict the treatment effects of insulin-like growth factor-I (IGF-I) in SJL mice with chronic relapsing experimental autoimmune encephalomyelitis (crEAE). METHODS: The experiments were performed at 4.7-T on 10 crEAE mice and on one set of control animals. Five crEAE mice were treated with IGF-I and five were treated with a placebo. RESULTS: In the crEAE mice treated with the placebo, in vivo MR microscopy showed areas of abnormal signal throughout the cerebrum, brain stem, and cerebellum. These findings were not present in either the IGF-I-treated mice or the normal control animals. The diffuse alterations in signal intensity in the placebo-treated crEAE mice were not identified on histologic sections of the same areas. CONCLUSION: Differences between the IGF-I- and placebo-treated groups may reflect changes in stabilization or permeability of cell membranes and/or of the blood-brain barrier, although other alternative contrast mechanisms could be playing a role. In vivo MR microscopy depicted changes resulting from treatment of crEAE with IGF-I.

Primary cementless acetabular reconstruction in patients younger than 50 years old. 7- to 11-year results.

The efficacy of primary cementless acetabular reconstruction in patients younger than 50 years of age was analyzed in 79 consecutive cementless, hemispheric, porous coated acetabular reconstructions (Harris-Galante-I). The average age was 37 years at surgery (range, 20-49 years). The average followup was 106 months (range, 78-126 months). No acetabular reconstructions were revised for aseptic loosening. Two stable acetabular reconstructions were revised during femoral revision. Two excessively worn polyethylene liners were exchanged and one acetabular osteolytic area was debrided and grafted; these procedures retained the metal shell. At final followup, all 72 acetabular reconstructions were radiographically stable. Acetabular osteolysis occurred in five cases (7.4%), from 84 to 104 months. Acetabular or femoral osteolysis occurred in patients with increased polyethylene wear. Polyethylene wear was inversely related to the patient's age. Using revision and loosening, the Kaplan-Meier 10 year survival of the acetabular reconstruction was 98.8% (95% confidence interval, 96.6%-100.%). The intermediate results of cementless, hemispheric, porous coated acetabular reconstruction in younger patients was excellent with no radiographic loosening. At 7- to 11-year followup, osteolysis was the most common problem and increased in frequency and extent with continued in vivo duration.

Therapeutic potential of phosphodiesterase type 4 inhibition in chronic autoimmune demyelinating disease.

It was recently demonstrated that selective phosphodiesterase type 4 (PDE4) inhibition suppresses the clinical manifestations of acute experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and inhibits the production of tumor necrosis factor-alpha (TNF-alpha), a pathogenetically central cytokine. Since the most common presentation of MS in humans is a relapsing-remitting course, we investigated the therapeutic potential of PDE4 inhibition in the relapsing-remitting EAE model of the SJL mouse. Administration of rolipram, the prototypic PDE4 inhibitor, reduced the clinical signs of EAE during both the initial episode of disease and subsequent relapses. In parallel, there was marked reduction of demyelination and also less inflammation throughout the central nervous system (CNS) of rolipram-treated animals. Gene expression of proinflammatory cytokines in the CNS was reduced in most of the rolipram-treated animals. Additional experiments demonstrated that PDE4 inhibition acted principally by inhibiting the secretion of Th1 cytokines, however, the encephalitogenic potential of myelin basic protein-specific T cells was not impaired. Our findings suggest that PDE4 inhibitors are a promising cytokine-directed therapy in chronic demyelinating disease.

Isolation and Characterization of Autoreactive T Cells in Experimental Autoimmune Encephalomyelitis of the Mouse

Experimental autoimmune encephalomyelitis (EAE) can be adoptively transferred by T cells that are specific for autoantigens of the central nervous system. A variety of autoantigens and their derived peptides have been shown to be excellent stimulators for encephalitogenic T-cell lines and clones. This article describes protocols for the establishment and characterization of autoreactive T-cell lines and clones and for the study of EAE induced by these cells. The rationale for using transferred EAE models is discussed, together with advantages and disadvantages of using in vitro cultured T cells compared with Freund's adjuvant-based immunization for the induction of EAE.

Intravenous antigen administration as a therapy for autoimmune demyelinating disease.

Experimental allergic encephalomyelitis is a prototypic autoimmune disease characterized by central nervous system inflammation and demyelination. Previously, we demonstrated that intravenous administration of high doses of myelin basic protein abrogated the clinical and pathological signs of experimental allergic encephalomyelitis by causing the deletion of encephalitogenic, CD4+, myelin basic protein-specific T cells through antigen-induced programmed cell death. In the present study, we further characterized the ability of intravenous antigen administration to attenuate an immune response by myelin basic protein-reactive encephalitogenic T cells. We demonstrated that multiple injections of myelin basic protein are required to achieve a therapeutic response, and that this form of therapy is effective even after prolonged chronic disease. These studies showed that although interleukin-2-stimulated cell cycling is an important factor leading to T-cell death, the administration of exogenous interleukin-2 with antigen can result in the aggravation of clinical disease compared to administration of antigen alone. More importantly, administration of myelin basic protein alone without interleukin-2 was sufficient to reduce autoreactive T cells and clinical disease in experimental autoimmune encephalomyelitis. Our experiments support the rationale for antigen-specific therapy aimed at inducing the programmed death of autoreactive T cells in autoimmune diseases, potentially including the human demyelinating disease multiple sclerosis.

Distinct roles for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis.

The activation and differentiation of T cells require both antigen/MHC recognition and costimulatory signals. The present studies examined the role of B7-1 (CD80) and B7-2 (CD86) costimulation in the prototypic autoimmune disorder, experimental allergic encephalomyelitis (EAE). In adoptively transferred EAE, in vitro activation of myelin basic protein (MBP)-specific lymph node cells was inhibited by the combination of anti-CD80 plus anti-CD86, but not individually. However, in actively induced disease, one injection of anti-CD80 significantly reduced disease, while anti-CD86 exacerbated disease. Interestingly, one injection of CTLA-4Ig suppressed disease, while multiple injections resulted in enhanced disease. Thus, the costimulation provided by B7-1 molecules appears to be important for the development of encephalitogenic T cells. The enhanced disease caused by multiple injections of CTLA-4Ig or a single injection of anti-CD86 suggests an inhibitory function for CD86 interaction with its counterreceptors CD28 and CTLA-4 in EAE. Alternatively, these results are consistent with an essential timing requirement for the coordinated interaction of B7 and CD28 family receptors, and that disruption of this critical timing can have opposing results on the outcome of an immune response.