Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by Inducing Interleukin-10-Producing Natural Killer Cells.

Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8+ T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8+ T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.

Discrete class I molecules on brain endothelium differentially regulate neuropathology in experimental cerebral malaria.

Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Chemogenetic manipulation of CX3CR1+ cells transiently induces hypolocomotion independent of microglia.

Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1CreER/+:R26hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1+ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1+ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119CreER/+:R26hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4+ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1CreER/+ mouse line to manipulate microglia.

The Impact of Preoperative Weight Loss Timing on Surgical Outcomes in Total Hip Arthroplasty.

BACKGROUND: Elevated body mass index (BMI) increases surgical complications post-total hip arthroplasty (THA). However, the effects of rapid weight loss pre-THA remain unclear. This study evaluated patients who had initial BMIs between 40 and 50, and then achieved a BMI under 35 at various intervals before their THA. Comparisons were made with consistent obese and nonobese groups to understand potential complications. METHODS: Using a national database, we categorized THA patients based on initial BMI and weight loss timing before the surgery. These were contrasted with those maintaining a steady BMI of 20 to 30 or 40 to 50. We monitored outcomes like periprosthetic joint infections (PJI), surgical site infections (SSI), and noninfectious revisions for 2 years postsurgery, incorporating demographic considerations. Statistical analyses utilized Chi-square tests for categorical outcomes and Student's t-tests for continuous variables. RESULTS: Among patients who had a BMI of 45 to 50, weight loss 3 to 9 months presurgery increased PJI risks at 90 days (Odds Ratios [OR]: 2.15 to 5.22, P < .001). However, weight loss a year before the surgery lowered the PJI risk (OR: 0.14 to 0.27, P < .005). Constantly obese patients faced heightened PJI risks 1 to 2 years postsurgery (OR: 1.64 to 1.95, P < .015). Regarding SSI, risks increased with weight loss 3 to 9 months before surgery, but decreased when weight loss occurred a year earlier. In the BMI 40 to 45 group, weight loss 3 to 6 months presurgery showed higher PJI and SSI at 90 days (P < .001), with obese participants consistently at greater risk. CONCLUSIONS: While high BMI poses THA risks, weight loss timing plays a crucial role in postoperative complications. Weight loss closer to the surgery (0 to 9 months) can heighten risks, but shedding weight a year in advance seems beneficial. Conversely, initiating weight loss approximately a year before surgery offers potential protective effects against postoperative issues. This highlights the importance of strategic weight management guidance for patients considering THA, ensuring optimal surgical results and reducing potential adverse outcomes.

Experience Using Adjuvant Bacteriophage Therapy for the Treatment of 10 Recalcitrant Periprosthetic Joint Infections: A Case Series.

Periprosthetic joint infections are a devastating complication of joint replacement surgery. One novel therapeutic that has potential to change the current treatment paradigm is bacteriophage therapy. Herein, we discuss our experiences with bacteriophage therapy for 10 recalcitrant periprosthetic joint infections and review the treatment protocols utilized to achieve successful outcomes.

Brain resident memory T cells rapidly expand and initiate neuroinflammatory responses following CNS viral infection.

The contribution of circulating verses tissue resident memory T cells (TRMs) to clinical neuropathology is an enduring question due to a lack of mechanistic insights. The prevailing view is TRMs are protective against pathogens in the brain. However, the extent to which antigen-specific TRMs induce neuropathology upon reactivation is understudied. Using the described phenotype of TRMs, we found that brains of naïve mice harbor populations of CD69+ CD103- T cells. Notably, numbers of CD69+ CD103- TRMs rapidly increase following neurological insults of various origins. This TRM expansion precedes infiltration of virus antigen-specific CD8 T cells and is due to proliferation of T cells within the brain. We next evaluated the capacity of antigen-specific TRMs in the brain to induce significant neuroinflammation post virus clearance, including infiltration of inflammatory myeloid cells, activation of T cells in the brain, microglial activation, and significant blood brain barrier disruption. These neuroinflammatory events were induced by TRMs, as depletion of peripheral T cells or blocking T cell trafficking using FTY720 did not change the neuroinflammatory course. Depletion of all CD8 T cells, however, completely abrogated the neuroinflammatory response. Reactivation of antigen-specific TRMs in the brain also induced profound lymphopenia within the blood compartment. We have therefore determined that antigen-specific TRMs can induce significant neuroinflammation, neuropathology, and peripheral immunosuppression. The use of cognate antigen to reactivate CD8 TRMs enables us to isolate the neuropathologic effects induced by this cell type independently of other branches of immunological memory, differentiating this work from studies employing whole pathogen re-challenge. This study also demonstrates the capacity for CD8 TRMs to contribute to pathology associated with neurodegenerative disorders and long-term complications associated with viral infections. Understanding functions of brain TRMs is crucial in investigating their role in neurodegenerative disorders including MS, CNS cancers, and long-term complications associated with viral infections including COVID-19.

Tobacco and Cannabis Use Have a Synergistic Association on Infection Risk Following Total Knee Arthroplasty.

BACKGROUND: Studies suggest an increase in the number of combined users of tobacco and cannabis. Therefore, we specifically assessed tobacco, cannabis, and combined users who underwent primary total knee arthroplasty (TKA) to determine 90-day to 2-year: (1) odds of periprosthetic joint infection; (2) odds of revision; and (3) medical complications. METHODS: We queried a national, all payer database of patients undergoing primary TKA between 2010 and 2020. Patients were stratified according to current use of tobacco products (n = 30,000), cannabis (n = 400), or a combination (n = 3,526). These were defined according to International Classification of Disease codes, Ninth and Tenth Editions. Patients were tracked from the 2 years before TKA through 2 years afterwards. A fourth group of TKA recipients who did not have tobacco nor cannabis use was used as a matching cohort. Periprosthetic joint infections (PJIs), revisions, and other medical/surgical complications from 90 days through 2 years were evaluated between these cohorts using bivariate analyses. Multivariate analyses assessed independent risk factors for PJI at 90 days through 2 years, adjusted for patient demographics and health metrics. RESULTS: Combined tobacco and cannabis use were associated with the highest rates of PJI following TKA. The odds of 90-day PJI risk among cannabis, tobacco, and combined users was 1.60, 2.14, and 3.39, respectively, as compared to the matched cohort (P < .001). Co-users had the highest and significantly increased revision odds at 2 years following TKA (odds ratio = 1.52, 95% confidence interval, 1.15 to 2.00). At 1 and 2 years following TKA, cannabis, tobacco, and co-users had higher rates of myocardial infarctions, respiratory failures, surgical site infections, and manipulations under anesthesia when compared to the matched cohort (all P < .001). CONCLUSION: Tobacco and cannabis use before primary TKA demonstrated a synergistic association on PJI risk from 90 days through 2 years. Although the harms of tobacco use are well-known, this additional knowledge about cannabis should be incorporated in the shared decision-making discussions in the pre-operative setting to best prepare for expected risks following primary TKA.

Conditional Silencing of H-2Db Class I Molecule Expression Modulates the Protective and Pathogenic Kinetics of Virus-Antigen-Specific CD8 T Cell Responses during Theiler's Virus Infection.

Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2Db The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2Db LoxP-transgenic mouse system using otherwise MHC class I-deficient C57BL/6 mice, thereby conditionally ablating MHC class I-restricted Ag presentation in targeted APC subpopulations. We observed that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130 Loss of H-2Db on CD11c+ APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2Db-deficient LysM+ APCs retained early priming of Db:VP2121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2Db-restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c+ cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell-mediated viral control and immunopathology.

GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts.

Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell-associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF-deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Brain cancer induces systemic immunosuppression through release of non-steroid soluble mediators.

Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

How Does Conversion Total Hip Arthroplasty Compare to Primary?

BACKGROUND: Recent institutional evidence suggests that conversion total hip arthroplasty (THA) incurs higher complication rates and costs when compared to primary THA. These findings contrast with the current reimbursement system as conversion and primary THAs are classified under the same diagnosis-related group. Thus, a national all-payer database was utilized to compare complication rates up to 2 years, 30-day readmission rates, and 90-day costs between conversion THA and matched primary THA patients. METHODS: A retrospective review of the PearlDiver database between 2010 and second quarter of 2018 was performed using Current Procedural Terminology (CPT) codes to compare conversion THA (CPT 27132) to primary THA (CPT 27130). Patients were matched at a 1:3 ratio based on age, gender, Charlson Comorbidity Index, body mass index, tobacco use, and diabetes (conversion = 8369; primary = 25,081 patients). RESULTS: Conversion THA had higher rates of periprosthetic joint infections (conversion: 7.7% vs primary: 1.4%), hip dislocations (4.5% vs 2.0%), blood transfusions (2.0% vs 1.0%), mechanical complications (5.5% vs 1.0%), and revision surgeries (4.0% vs 1.5%) (P < .001 for all) by 90 days. The 30-day readmission rate for conversion THA was significantly higher compared to the primary group (7.3% vs 3.3%) (P < .001). Median cost at 90 days for conversion THA was significantly higher compared to primary THA ($18,800 vs $13,611, P < .001). CONCLUSION: This study revealed increased complication rates, revisions, readmissions, and costs among conversion THA patients compared to matched primary THA patients. These results support the reclassification of conversion into a diagnosis-related group separate from primary THA.

A Biomechanical Comparison of the Effect of Baseplate Design and Bone Marrow Fat Infiltration on Tibial Baseplate Pullout Strength.

BACKGROUND: Early clinical results of a new total knee arthroplasty (TKA) implant design show promise for improved outcomes and patellofemoral function scores. However, reports of early tibial component-cement interface debonding requiring revision have been published. This study investigated the biomechanical properties of three different tibial baseplates to understand potential causes of failure. METHODS: PFC Sigma (control), Attune (1st generation) and Attune S+ (2nd generation) tibial baseplates were implanted into 4th generation sawbone tibia models using a standardized technique. Three of each baseplate were cemented with and without additional bovine bone marrow fat. All models were tested to failure with measured axial distraction force. Implant type, presence or absence of bovine marrow and load to failure were all recorded and compared. Two-way ANOVA followed by post-hoc pairwise comparisons were used to determine statistical significance, which was set to P < .05. RESULTS: The 2nd generation tibial baseplates required significantly more force to failure. The presence of bovine marrow significantly reduced the pullout force of the implant designs overall. No significant difference was detected between the 1st generation and control baseplates. Failure mode for each model was also noted to be different irrespective of the presence or absence of bone marrow fat. CONCLUSION: The 2nd generation baseplates required significantly more force to failure compared with older designs. The presence of bone marrow during cementation of a tibial base plate significantly decreased axial pullout strength of a tibial baseplate in this laboratory model. All 1st generation baseplates exhibited debonding at the cement-implant interface.

Assessment of Splints Applied for Pediatric Fractures in an Emergency Department/Urgent Care Environment.

BACKGROUND: Fractures are common in the pediatric population. The initial evaluation is rarely by an orthopaedic surgeon, but commonly an emergency room or urgent care center physician/extender. This typically involves splint application by a nonorthopaedist to immobilize the extremity and provide stabilization. Iatrogenic injuries from inappropriate splint placement are a potential public health and legal concern that can lead to complications. The primary purpose of this study was to prospectively evaluate the adequacy of all splints placed on patients who presented to a pediatric orthopaedic office; secondary outcomes included assessing prevalence and types of complications that were associated with inadequate splints. METHODS: Patients aged 0 to 18 years who presented with a splint were prospectively enrolled. Information was obtained regarding demographics of the patient and splint placement. Splints were evaluated for functional position, appropriate length, and presence of elastic bandage on the skin. Photographs were taken of each splint, and the extremity was examined for any soft tissue complications. Splints were not removed in 31 patients who had undergone fracture reduction. RESULTS: In total, 275 patients were prospectively enrolled. Splints were improperly placed in 93%, with application of elastic bandage directly to the skin accounting for 77%. Improper positioning was observed in 59%, and inappropriate splint length was present in 52%. Skin and soft tissue complications were observed in 40%. The most common iatrogenic splint-related complication was excessive edema, seen in 28%. Direct injury to the skin and soft tissue was seen in 6%. CONCLUSIONS: Many practitioners incorrectly apply splints, potentially leading to suboptimal results or causing injury. Complications of poor splint placement include excessive swelling, skin breakdown, and poor immobilization. Health care workers who treat pediatric fractures may benefit from more extensive education regarding proper splinting techniques. LEVEL OF EVIDENCE: Level 2-therapeutic study.

Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the Db class I molecule. FVB/Db and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/Db mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/Db mice elicited a strong CD8 T-cell response toward the immunodominant Db-restricted TMEV-derived peptide, VP2121-130, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

Perforin Expression by CD8 T Cells Is Sufficient To Cause Fatal Brain Edema during Experimental Cerebral Malaria.

Human cerebral malaria (HCM) is a serious complication of Plasmodium falciparum infection. The most severe outcomes for patients include coma, permanent neurological deficits, and death. Recently, a large-scale magnetic resonance imaging (MRI) study in humans identified brain swelling as the most prominent predictor of fatal HCM. Therefore, in this study, we sought to define the mechanism controlling brain edema through the use of the murine experimental cerebral malaria (ECM) model. Specifically, we investigated the ability of CD8 T cells to initiate brain edema during ECM. We determined that areas of blood-brain barrier (BBB) permeability colocalized with a reduction of the cerebral endothelial cell tight-junction proteins claudin-5 and occludin. Furthermore, through small-animal MRI, we analyzed edema and vascular leakage. Using gadolinium-enhanced T1-weighted MRI, we determined that vascular permeability is not homogeneous but rather confined to specific regions of the brain. Our findings show that BBB permeability was localized within the brainstem, olfactory bulb, and lateral ventricle. Concurrently with the initiation of vascular permeability, T2-weighted MRI revealed edema and brain swelling. Importantly, ablation of the cytolytic effector molecule perforin fully protected against vascular permeability and edema. Furthermore, perforin production specifically by CD8 T cells was required to cause fatal edema during ECM. We propose that CD8 T cells initiate BBB breakdown through perforin-mediated disruption of tight junctions. In turn, leakage from the vasculature into the parenchyma causes brain swelling and edema. This results in a breakdown of homeostatic maintenance that likely contributes to ECM pathology.

Finding a Balance between Protection and Pathology: The Dual Role of Perforin in Human Disease.

Perforin is critical for controlling viral infection and tumor surveillance. Clinically, mutations in perforin are viewed as unfavorable, as lack of this pore-forming protein results in lethal, childhood disease, familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). However, many mutations in the coding region of PRF1 are not yet associated with disease. Animal models of viral-associated blood-brain barrier (BBB) disruption and experimental cerebral malaria (ECM) have identified perforin as critical for inducing pathologic central nervous system CNS vascular permeability. This review focuses on the role of perforin in both protecting and promoting human disease. It concludes with a novel hypothesis that diversity observed in the PRF1 gene may be an example of selective advantage that protects an individual from perforin-mediated pathology, such as BBB disruption.

Modulatory effects of perforin gene dosage on pathogen-associated blood-brain barrier (BBB) disruption.

BACKGROUND: CD8 T cell-mediated blood-brain barrier (BBB) disruption is dependent on the effector molecule perforin. Human perforin has extensive single nucleotide variants (SNVs), the significance of which is not fully understood. These SNVs can result in reduced, but not ablated, perforin activity or expression. However, complete loss of perforin expression or activity results in the lethal disease familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). In this study, we address the hypothesis that a single perforin allele can alter the severity of BBB disruption in vivo using a well-established model of CNS vascular permeability in C57Bl/6 mice. The results of this study provide insight into the significance of perforin SNVs in the human population. METHODS: We isolated the effect a single perforin allele has on CNS vascular permeability through the use of perforin-heterozygous (perforin+/-) C57BL/6 mice in the peptide-induced fatal syndrome (PIFS) model of immune-mediated BBB disruption. Seven days following Theiler's murine encephalomyelitis virus (TMEV) CNS infection, neuroinflammation and TMEV viral control were assessed through flow cytometric analysis and quantitative real-time PCR of the viral genome, respectively. Following immune-mediated BBB disruption, gadolinium-enhanced T1-weighted MRI, with 3D volumetric analysis, and confocal microscopy were used to define CNS vascular permeability. Finally, the open field behavior test was used to assess locomotor activity of mice following immune-mediated BBB disruption. RESULTS: Perforin-null mice had negligible CNS vascular permeability. Perforin-WT mice have extensive CNS vascular permeability. Interestingly, perforin-heterozygous mice had an intermediate level of CNS vascular permeability as measured by both gadolinium-enhanced T1-weighted MRI and fibrinogen leakage in the brain parenchyma. Differences in BBB disruption were not a result of increased CNS immune infiltrate. Additionally, TMEV was controlled in a perforin dose-dependent manner. Furthermore, a single perforin allele is sufficient to induce locomotor deficit during immune-mediated BBB disruption. CONCLUSIONS: Perforin modulates BBB disruption in a dose-dependent manner. This study demonstrates a potentially advantageous role for decreased perforin expression in reducing BBB disruption. This study also provides insight into the effect SNVs in a single perforin allele could have on functional deficit in neurological disease.

Krüppel-like factor KLF10 regulates transforming growth factor receptor II expression and TGF-ß signaling in CD8+ T lymphocytes.

KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-ß1 (TGF-ß1) signaling in CD4(+) T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-ß receptor II (TGF-ßRII) expression with functional relevance in antiviral immune response. Specifically, we show that KLF10-deficient mice have an increased number of effector/memory CD8(+) T cells, display higher levels of the T helper type 1 cell-associated transcription factor T-bet, and produce more IFN-γ following in vitro stimulation. In addition, KLF10(-/-) CD8(+) T cells show enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly isolated CD8(+) T cells from the spleen of adult mice express lower levels of surface TGF-ßRII (TßRII). Congruently, in vitro activation of KLF10-deficient CD8(+) T cells upregulate TGF-ßRII to a lesser extent compared with wild-type (WT) CD8(+) T cells, which results in attenuated Smad2 phosphorylation following TGF-ß1 stimulation compared with WT CD8(+) T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-ßRII promoter in T cells, leading to enhanced gene expression. In vivo viral infection with Daniel's strain Theiler's murine encephalomyelitis virus (TMEV) also led to lower expression of TGF-ßRII among viral-specific KLF10(-/-) CD8(+) T cells and a higher percentage of IFN-γ-producing CD8(+) T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the transcriptional activation of TGF-ßRII in CD8(+) T cells. Thus, KLF10 regulation of TGF-ßRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-ß1/TGF-ßRII signaling pathway is crucial.

Nonequivalence of classical MHC class I loci in ability to direct effective antiviral immunity.

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b) gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b) transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b) but encoding the peptide binding domain of D(b), develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(b)α1α2D(b) gene (low) and D(b) (high) in the CNS of infected mice mirror expression levels of their endogenous H-2(q) counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

Theiler's murine encephalomyelitis virus as an experimental model system to study the mechanism of blood-brain barrier disruption.

Theiler's murine encephalomyelitis virus is a widely used model to study the initiation and progression of multiple sclerosis. Many researchers have used this model to investigate how the immune system and genetic factors contribute to the disease process. Current research has highlighted the importance of cytotoxic CD8 T cells and specific major histocompatibility complex (MHC) class I alleles. Our lab has adopted this concept to create a novel mouse model to study the mechanism of blood-brain barrier (BBB) disruption, an integral feature of numerous neurological disorders. We have demonstrated that epitope-specific CD8 T cells cause disruption of the tight junction architecture and ensuing CNS vascular permeability in the absence of neutrophil support. This CD8 T cell-initiated BBB disruption is dependent on perforin expression. We have also elucidated a potential role for hematopoietic factors in this process. Despite having identical MHC class I molecules, similar inflammation in the CNS, and equivalent ability to utilize perforin, C57BL/6 mice are highly susceptible to this condition, while 129 SvIm mice are resistant. This susceptibility is transferable with the bone marrow compartment. These findings led us to conduct a comprehensive genetic analysis which has revealed a list of candidate genes implicated in regulating traits associated with BBB disruption. Future studies will continue to define the underlying molecular mechanism of CD8 T cell-initiated BBB disruption and may assist in the development of potential therapeutic approaches to ameliorate pathology associated with BBB disruption in neurological disorders.