Preliminary biomechanical results of a novel pin configuration for external fixation of vertical shear pelvic fractures.

BACKGROUND: Vertical shear fractures are unstable and potentially life-threatening injuries that require urgent reduction and stabilization. The aim of this study was to compare the biomechanical efficacy of three different external fixation pin configurations for vertical shear pelvic fractures in a cadaveric model. We hypothesized that a modified external fixation pin configuration with a crestal (CR) pin in the stable hemipelvis and bilateral supra-acetabular (SA) pins provides the greatest overall stability to axial loading. METHODS: The force to failure within a standard standing axial load (maximum 650 N) was tested on 10 human cadaveric pelvises with vertical shear fractures. Three pin configurations were compared including iliac crest (IC), SA and a modified SA frame with a third CR pin on the stable hemipelvis. Both displacement at the posterior pelvis at 650 N and force to failure of >25 mm displacement was recorded. RESULTS: The mean force to failure was highest with CR (499 N), then IC (350 N) and then SA (265 N) pin configurations, being statistically non-significant (P = 0.165). The minimum force to failure followed a similar trend with 296, 68 and 43 N for CR, IC and SA, respectively. About 1/4 CR, 1/4 IC and 2/9 SA pins sustained 650 N or more without failure. CONCLUSION: It was shown that this new design may reliably withstand a seated physiological load of 250 N. However, none of the three pin configurations tested can reliably withstand a standing load of 650 N. Further experiments are needed to quantify these findings under physiological loading.

Minor Antigen Vaccine-Sensitized DLI: In Vitro Responses Do Not Predict In Vivo Effects.

UNLABELLED: We reported on a pilot study of minor histocompatibility antigen vaccination using constructs expressing male-specific gene disparities of selected mouse CDNA on Y and sex determining region Y in the canine model. We performed reduced-intensity hematopoietic cell transplantation with female donors and male recipients, producing stable mixed donor-recipient hematopoietic chimeras. We then performed a vaccine series in three female transplant donors followed by donor lymphocyte infusion (DLI) into their respective mixed chimeras. One mixed chimera experienced a significant shift in the percentage of donor chimerism, but no response occurred in the other 2 recipients. We then hypothesized that inadequate donor sensitization was responsible for these results. METHODS: To test this hypothesis, we added 4 monthly booster vaccinations to 2 of the original hematopoietic cell transplantation donors, including the donor that drove the partial response, followed by a second DLI. RESULTS: Strong T cell responses were shown by ELISpot and confirmed by intracellular cytokine staining in both donors. A second DLI resulted in a further increase in donor chimerism in the same mixed chimera that experienced the previous increase, but no change in donor chimerism was again seen in the other recipient. Evaluation of RNA expression of the target antigens demonstrated that conversion occurred in the recipient that expressed both selected mouse CDNA on Y and sex determining region Y. CONCLUSIONS: T cell responses against Y chromosome-encoded disparities were not necessarily sufficient to drive in vivo female antimale responses. Other factors including the presence of specific haplotypes or the heterogeneous expression of the target antigen may affect T cell responses against minor histocompatibility antigens. These results warrant future vaccine studies in a larger transplant cohort using epigenetic modulation of the recipient to promote target gene expression.

Pediatric Tibial Osteomyelitis.

BACKGROUND: Osteomyelitis shows a strong predilection for the tibia in the pediatric population and is a significant source of complications. The purpose of this article is to retrospectively review a large series of pediatric patients with tibial osteomyelitis. We compare our experience with that in the literature to determine any factors that may aid diagnosis and/or improve treatment outcomes. METHODS: A 10-year retrospective review was performed of clinical records of all cases of pediatric tibial osteomyelitis managed at the 2 children's orthopaedic departments in the Auckland region. The Osteomyelitis Database was used to identify all cases between 1997 and 2007, at Starship Children's Hospital, and 1998 and 2008 at Middlemore's Kids First Hospital. RESULTS: One hundred ninety-one patients fulfilled the inclusion criteria, and had a review of clinical notes and relevant investigations. The average duration of symptoms before presentation to hospital was 5.7 days. Less than 40% of patients had a recent episode of trauma. Almost 60% of patients could not bear weight on admission. Over 40% of patients had a temperature above 38°C. Erythrocyte sedimentation rate was elevated in 78% and the C-reactive protein was elevated in 90% of patients. In total, 42% of blood cultures and almost 75% of tissue cultures were positive, with Staphylococcus aureus being the most commonly cultured organism. X-rays, bone scans, and magnetic resonance imaging were all used to aid the diagnosis. About 43% of patients had surgery. Treatment length was an average of 2 weeks 6 days of intravenous antibiotics followed by 3 weeks 2 days of oral treatment. Six postsurgical complications and 46 readmissions were noted: 25 for relapse, with the remainder due to social and antibiotic-associated complications. CONCLUSIONS: Although generally diagnosed on presentation, pediatric tibial osteomyelitis can require more sophisticated investigations and prolonged management. Treatment with intravenous and oral antibiotics and surgical debridement where indicated can lead to a good clinical outcome, although complications are often noted. LEVEL OF EVIDENCE: Level IV-Prognostic study.

Development of a Minor Histocompatibility Antigen Vaccine Regimen in the Canine Model of Hematopoietic Cell Transplantation.

BACKGROUND: Minor histocompatibility antigen (miHA) vaccines have the potential to augment graft-versus-tumor effects without graft-versus-host disease (GVHD). We used mixed hematopoietic chimerism in the canine model of major histocompatibility complex-matched allogeneic hematopoietic cell transplantation as a platform to develop a miHA vaccination regimen. METHODS: We engineered DNA plasmids and replication-deficient human adenovirus type 5 constructs encoding large sections of canine SMCY and the entire canine SRY gene. RESULTS: Priming with replication-deficient human adenovirus type 5 constructs and boosting with ex vivo plasmid-transfected dendritic cells and cutaneous delivery of plasmids with a particle-mediated epidermal delivery device (PMED) in 2 female dogs induced antigen-specific T-cell responses. Similar responses were observed after a prime-boost vaccine regimen in three female hematopoietic cell transplantation donors. Subsequent donor lymphocyte infusion resulted in a significant change of chimerism in 1 of 3 male recipients without any signs of graft-versus-host disease. The change in chimerism in the recipient occurred in association with the development of CD4+ and CD8+ T-cell responses to the same peptide pools detected in the donor. CONCLUSIONS: These studies describe the first in vivo response to miHA vaccination in a large, outbred animal model without using recipient cells to sensitize the donor. This model provides a platform for ongoing experiments designed to define optimal miHA targets and develop protocols to directly vaccinate the recipient.

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations.

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins.

Identification of novel HLA class II target epitopes for generation of donor-specific T regulatory cells.

Therapies capable of generating host T regulatory cells (T(R)) responsive to donor-specific HLA-class II minor histocompatibility antigens have the potential to promote tolerance of a transplanted organ. Our group has developed a novel approach for the identification of potentially therapeutic T(R) target antigens. We perform parallel non-synonymous SNP genotyping of HLA-identical subject pairs to identify peptide variations expressed by only one of the two subjects. Variant peptide pairs are then evaluated for binding a shared HLA-class II allele. Minor peptides predicted to bind HLA-class II with greater affinity than the common variant peptide are tested for HLA class II binding and in vitro induction of suppressive CD4+ T cells. Using this approach we have identified multiple pairs of variant peptides capable of differential binding and induction of suppressive CD4+ T cells. These data demonstrate the feasibility of identifying potentially therapeutic HLA class II minor antigens for generation of donor-specific T(R).

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

Absence of SPARC leads to impaired lens circulation.

SPARC is a matricellular glycoprotein involved in regulation of extracellular matrix, growth factors, adhesion, and migration. SPARC-null mice have altered basement membranes and develop posterior sub-capsular cataracts with cell swelling and equatorial vacuoles. Exchange of fluid, nutrients, and waste products in the avascular lens is driven by a unique circulating ion current. In the absence of SPARC, increased circulation of fluid, ions, and small molecules led to increased fluorescein distribution in vivo, loss of resting membrane polarization, and altered distribution of small molecules. Microarray analysis of SPARC-null lenses showed changes in gene expression of ion channels and receptors, matrix and adhesion genes, cytoskeleton, immune response genes, and cell signaling molecules. Our results confirm the hypothesis that the regulation of SPARC on cell-capsular matrix interactions can increase the circulation of fluid and ions in the lens, and the phenotype in the SPARC-null mouse lens is the result of multiple intersecting functional pathways.

Adoptive immunotherapy against allogeneic kidney grafts in dogs with stable hematopoietic trichimerism.

Dogs given nonmyeloablative conditioning and marrow grafts from 2 dog leukocyte antigen (DLA)-identical littermate donors developed stable trichimerism and stably accepted a subsequent kidney graft from one of the marrow donors without the need for immunosuppression. In this study, we used trichimeras to evaluate strategies for adoptive immunotherapy to solid tumors, using the kidney as a tumor surrogate. Three DLA-identical trichimeric recipients were established by simultaneously infusing marrow from 2 DLA-identical donor dogs into a DLA-identical recipient conditioned with 2 Gy of total body irradiation (TBI) and given a short course of postgraft immunosuppression. After stable hematopoietic engraftment was confirmed, a kidney was transplanted from 1 of the 2 marrow donors into each respective trichimeric recipient. Peripheral blood lymphocytes from each kidney donor were then used to sensitize the alternate marrow donor. The trichimeric recipients were given donor lymphocyte infusions (DLIs) from the sensitized dogs and monitored for chimerism, graft-versus-host disease (GVHD), and kidney rejection. After DLI, we observed both prompt rejection of the transplanted marrow and donor kidney and disappearance of corresponding hematopoietic chimerism. Presumably due to shared minor histocompatibility antigens, host chimerism also disappeared, and GVHD in skin, gut, and liver developed. The native kidneys, although exhibiting lymphocytic infiltration, remained functionally normal. This study demonstrates that under certain experimental conditions, the kidney--an organ ordinarily not involved in graft-versus-host reactions--can be targeted by sensitized donor lymphocytes.

Standard treatments induce antigen-specific immune responses in prostate cancer.

PURPOSE: Prostate tumors express antigens that are recognized by the immune system in a significant proportion of patients; however, little is known about the effect of standard treatments on tumor-specific immunity. Radiation therapy induces expression of inflammatory and immune-stimulatory molecules, and neoadjuvant hormone therapy causes prominent T-cell infiltration of prostate tumors. We therefore hypothesized that radiation therapy and hormone therapy may initiate tumor-specific immune responses. EXPERIMENTAL DESIGN: Pretreatment and posttreatment serum samples from 73 men with nonmetastatic prostate cancer and 50 cancer-free controls were evaluated by Western blotting and SEREX (serological identification of antigens by recombinant cDNA expression cloning) antigen arrays to examine whether autoantibody responses to tumor proteins arose during the course of standard treatment. RESULTS: Western blotting revealed the development of treatment-associated autoantibody responses in patients undergoing neoadjuvant hormone therapy (7 of 24, 29.2%), external beam radiation therapy (4 of 29, 13.8%), and brachytherapy (5 of 20, 25%), compared with 0 of 14 patients undergoing radical prostatectomy and 2 of 36 (5.6%) controls. Responses were seen within 4 to 9 months of initiation of treatment and were equally prevalent across different disease risk groups. Similarly, in the murine Shionogi tumor model, hormone therapy induced tumor-associated autoantibody responses in 5 of 10 animals. In four patients, SEREX immunoscreening of a prostate cancer cDNA expression library identified several antigens recognized by treatment-associated autoantibodies, including PARP1, ZNF707 + PTMA, CEP78, SDCCAG1, and ODF2. CONCLUSION: We show for the first time that standard treatments induce antigen-specific immune responses in prostate cancer patients. Thus, immunologic mechanisms may contribute to clinical outcomes after hormone and radiation therapy, an effect that could potentially be exploited as a practical, personalized form of immunotherapy.

Stable trichimerism after marrow grafting from 2 DLA-identical canine donors and nonmyeloablative conditioning.

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial "trichimerism," which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.

Immunoediting of cancers may lead to epithelial to mesenchymal transition.

Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.

Identification of autoantibodies elicited in a patient with prostate cancer presenting as dermatomyositis.

OBJECTIVES: Dermatomyositis is an uncommon autoimmune disease distinguished by proximal muscle weakness and a characteristic skin rash. Dermatomyositis has also frequently been associated with malignancy, typically heralding the diagnosis of ovarian, lung, gastric, or colorectal cancer. We report an unusual case of prostate adenocarcinoma preceded by a diagnosis of dermatomyositis. We hypothesized that in this particular patient, proteins produced by the neoplastic prostatic tissue, which might be normally expressed in muscle tissue, were immunologically recognized as autoantigens. METHODS: Serum from this patient was used to screen a cDNA lambda phage expression library from normal prostate tissue for prostate protein-specific IgG. RESULTS: We identified several immunoreactive plaques encoding known autoantigens, and several encoding known muscle-related proteins, including aldolase C, eukaryotic translation elongation factor 1 alpha 1, transgelin, and acetyl-coenzyme A acyltransferase 1. IgG specific for these proteins were not specifically recognized in sera from other patients with prostate cancer compared with male control blood donors, and were not specifically recognized in a small panel of sera from patients with breast or ovarian cancer and dermatomyositis. CONCLUSIONS: Our results demonstrate that this patient with prostate cancer presenting as dermatomyositis had autoantibodies to specific proteins, possibly associated with his autoimmune myopathy. Moreover, given this patient's history and the multiple treatment options for prostate cancer, the identification of dermatomyositis in men should prompt an evaluation to exclude a concurrent diagnosis of prostate cancer.

Application of Bayesian modeling of autologous antibody responses against ovarian tumor-associated antigens to cancer detection.

Biomarkers for early detection of epithelial ovarian cancer (EOC) are urgently needed. Patients can generate antibodies to tumor-associated antigens (TAAs). We tested multiplex detection of antibodies to candidate ovarian TAAs and statistical modeling for discrimination of sera of EOC patients and controls. Binding of serum antibody of women with EOC or healthy controls to candidate TAA-coated microspheres was assayed in parallel. A Bayesian model/variable selection approach using Markov Chain Monte Carlo computations was applied to these data, and serum CA125 values, to determine the best predictive model. The selected model was subjected to area under the receiver-operator curve (AUC) analysis. The best model generated an AUC of 0.86 [95% confidence interval (95% CI), 0.78-0.90] for discrimination between sera of EOC patients and healthy patients using antibody specific to p53, NY-CO-8, and HOXB7. Inclusion of CA125 in the model provided an AUC of 0.89 (95% CI, 0.84-0.92) compared with an AUC of 0.83 (95% CI, 0.81-0.85) using CA125 alone. However, using TAA responses alone, the model discriminated between independent sera of women with nonmalignant gynecologic conditions and those with advanced-stage or early-stage EOC with AUCs of 0.71 (95% CI, 0.67-0.76) and 0.70 (95% CI, 0.48-0.75), respectively. Serum antibody to p53 and HOXB7 is positively associated with EOC, whereas NY-CO-8-specific antibody shows negative association. Bayesian modeling of these TAA-specific serum antibody responses exhibits similar discrimination of patients with early-stage and advanced-stage EOC from women with nonmalignant gynecologic conditions and may be complementary to CA125.

Serologic analysis of ovarian tumor antigens reveals a bias toward antigens encoded on 17q.

We utilized SEREX immunoscreening to identify a set of novel tumor antigens that are associated with human serous ovarian cancer and may prove useful for the early detection and treatment of this disease. Extensive screening with a panel of sera from 25 late-stage ovarian cancer patients against 3 independent cDNA libraries identified a set of 9 antigens that were immunogenic in more than 1 patient and not in a panel of 20-45 normal female serum donors. These antigens include p53, NY-ESO-1, UBQLN1, HOXB6, TOP2A, putative helicase-RUVBL (RUVBL), HMBA-inducible (HEXIM1), DDX5 and HDCMA. Ten of 25 ovarian cancer patients (40%) expressed serum IgG to at least 1 of these antigens, while 14% (4/25) had antibodies to 2 or more antigens. Unexpectedly, 4 antigens identified in this screen, DDX5, HEXIM1, TOP2A and HOXB6, are encoded within a region of 17q that also includes the genes for HER2/neu, Homeobox-B7 and BRCA1. Real-time RT-PCR analysis showed that mRNA for HER2/neu and 3 SEREX-defined antigens, TOP2A, HOXB6 and DDX5, was more abundant in ovarian tumors than most normal tissues, including normal and benign ovarian tissues, suggesting that elevated expression of genes encoded within this region of chromosome 17 is a common event in ovarian tumors. Thus, these abnormal expression patterns combined with the endogenous immune response suggests that these antigens represent potential targets for immunotherapy.