Unique CD8+ T Cell-Mediated Immune Responses Primed in the Liver.

BACKGROUND: The liver immune environment is tightly regulated to balance immune activation with immune tolerance. Understanding the dominant immune pathways initiated in the liver is important because the liver is a site for cell transplantation, such as for islet and hepatocyte transplantation. The purpose of this study is to examine the consequences of alloimmune stimulation when allogeneic cells are transplanted to the liver in comparison to a different immune locale, such as the kidney. METHODS: We investigated cellular and humoral immune responses when allogeneic hepatocytes are transplanted directly to the recipient liver by intraportal injection. A heterotopic kidney engraftment site was used for comparison to immune activation in the liver microenvironment. RESULTS: Transplantation of allogeneic hepatocytes delivered directly to the liver, via recipient portal circulation, stimulated long-term, high magnitude CD8 T cell-mediated allocytotoxicity. CD8 T cells initiated significant in vivo allocytotoxicity as well as rapid rejection of hepatocytes transplanted to the liver even in the absence of secondary lymph nodes or CD4 T cells. In contrast, in the absence of recipient peripheral lymphoid tissue and CD4 T cells, CD8-mediated in vivo allocytotoxicity was abrogated, and rejection was delayed when hepatocellular allografts were transplanted to the kidney subcapsular site. CONCLUSIONS: These results highlight the CD8-dominant proinflammatory immune responses unique to the liver microenvironment. Allogeneic cells transplanted directly to the liver do not enjoy immune privilege but rather require immunosuppression to prevent rejection by a robust and persistent CD8-dependent allocytotoxicity primed in the liver.

A Novel Radiographic Indicator of Developmental Cervical Stenosis.

BACKGROUND: Developmental cervical stenosis of the spinal canal predisposes patients to neural compression and loss of function. The Torg-Pavlov ratio has been shown to provide high sensitivity but low specificity for identifying developmental cervical stenosis. A more sensitive and specific radiographic index has not been reported to our knowledge. The objective of this study was to develop and provide an objective, sensitive, and specific radiographic index to assess for developmental cervical stenosis. METHODS: The C3 through C6 levels of the cervical spine were analyzed on lateral radiographs of 150 adult patients to determine the spinolaminar line-to-lateral mass distance (SL), lateral mass-to-posterior vertebral body distance (LM), spinolaminar line-to-vertebral body (canal) diameter (CD), and vertebral body diameter (VB). Ratios of these measurements were calculated to eliminate magnification effects. The corresponding true spinal canal diameter was measured using computed tomography (CT) midsagittal sections. Receiver operating characteristic (ROC) curve analysis was performed to identify a radiographic measurement ratio with optimal sensitivity and specificity, using a true canal diameter of <12 mm to define developmental cervical stenosis. RESULTS: Several of the measured ratios demonstrated a strong correlation with the true canal diameter at all cervical levels. However, ROC curve analysis showed that only an LM/CD ratio of ≥0.735 indicated a canal diameter of <12 mm (developmental cervical stenosis). The sensitivity of this ratio at C5 was 83% and its specificity at C5 was 74%. An LM/CD ratio of ≥0.735 measured only at the C5 level also indicated developmental cervical stenosis at any cervical level from C3 through C6 with 76% sensitivity and 80% sensitivity. Other ratios, including the Torg-Pavlov ratio, did not demonstrate an adequate statistical profile to indicate developmental cervical stenosis. The accuracy of the LM/CD ratio was not adversely affected by the patient's sex. CONCLUSIONS: This analysis provided a novel index for identifying developmental cervical stenosis: the C5 lateral mass/canal diameter (LM/CD) ratio. We believe that this ratio is the best radiographic measurement available to screen for developmental cervical stenosis in the adult spine patient population. It provides an objective radiographic screening tool for physicians to detect developmental cervical stenosis and decide whether additional imaging or surgical referral is appropriate. LEVEL OF EVIDENCE: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Low incidence of symptomatic thromboembolic events after total ankle arthroplasty without routine use of chemoprophylaxis.

BACKGROUND: There is little evidence regarding the incidence of symptomatic venous thromboembolism (VTE) following total ankle arthroplasty (TAA) to allow formulation of treatment recommendations. The purpose of this study was to determine the incidence of symptomatic VTE events after TAA without use of chemoprophylaxis and to identify risk factors contributing to the occurrence of VTEs. METHODS: We conducted a retrospective chart review of 637 patients (664 ankles) who received a TAA between May 2007 and January 2014 and had a minimum follow-up of 3 months. Chemoprophylaxis was prescribed only in the setting of a history of VTE or active coagulopathy. Patients were continued on chemoprophylactic agents if they were taking these medications preoperatively. A VTE event was defined when clinical signs and symptoms of deep venous thrombosis (DVT) were confirmed with use of Doppler ultrasonography or pulmonary embolism was confirmed with the use of a computed tomography scan. Routine screening for VTE was not performed. RESULTS: The overall incidence of clinically detected VTE events was 0.60% (4/664), with 0.45% (3 patients) developing a DVT and 0.15% (1 patient) developing a nonfatal pulmonary embolism. Moreover, we identified a subset of 434 patients without identifiable preoperative risk factors who were not taking chemoprophylaxis preoperatively and were not prescribed chemoprophylaxis postoperatively. Two of these patients developed a DVT postoperatively (0.46%). Given the low incidence of clinically detected VTE, no significant correlation could be identified between the occurrence of VTE events and risk factors. CONCLUSIONS: Our results suggest that clinically detectable VTE after TAA is uncommon. Patients without identifiable risk factors do not appear to require chemoprophylaxis following TAA. We recommend continuation of antiplatelet or anticoagulation therapy in patients who are taking these medications preoperatively and the initiation of chemoprophylaxis postoperatively in patients with known risk factors for VTE. LEVEL OF EVIDENCE: Level IV, case series.

Targeting pro-inflammatory cytokines following joint injury: acute intra-articular inhibition of interleukin-1 following knee injury prevents post-traumatic arthritis.

INTRODUCTION: Post-traumatic arthritis (PTA) is a progressive, degenerative response to joint injury, such as articular fracture. The pro-inflammatory cytokines, interleukin 1(IL-1) and tumor necrosis factor alpha (TNF-α), are acutely elevated following joint injury and remain elevated for prolonged periods post-injury. To investigate the role of local and systemic inflammation in the development of post-traumatic arthritis, we targeted both the initial acute local inflammatory response and a prolonged 4 week systemic inflammatory response by inhibiting IL-1 or TNF-α following articular fracture in the mouse knee. METHODS: Anti-cytokine agents, IL-1 receptor antagonist (IL-1Ra) or soluble TNF receptor II (sTNFRII), were administered either locally via an acute intra-articular injection or systemically for a prolonged 4 week period following articular fracture of the knee in C57BL/6 mice. The severity of arthritis was then assessed at 8 weeks post-injury in joint tissues via histology and micro computed tomography, and systemic and local biomarkers were assessed in serum and synovial fluid. RESULTS: Intra-articular inhibition of IL-1 significantly reduced cartilage degeneration, synovial inflammation, and did not alter bone morphology following articular fracture. However, systemic inhibition of IL-1, and local or systemic inhibition of TNF provided no benefit or conversely led to increased arthritic changes in the joint tissues. CONCLUSION: These results show that intra-articular IL-1, rather than TNF-α, plays a critical role in the acute inflammatory phase of joint injury and can be inhibited locally to reduce post-traumatic arthritis following a closed articular fracture. Targeted local inhibition of IL-1 following joint injury may represent a novel treatment option for PTA.

Inhibition of recall responses through complementary therapies targeting CD8+ T-cell- and alloantibody-dependent allocytotoxicity in sensitized transplant recipients.

Allospecific T memory cell responses in transplant recipients arise from environmental exposure to previous transplantation or cross-reactive heterologous immunity. Unfortunately, these memory responses pose a significant barrier to the survival of transplanted tissue. We have previously reported that concurrent inhibition of CD154 and LFA-1 suppresses primary CD8-dependent rejection responses that are not controlled by conventional immunosuppressive strategies. We hypothesized that CD154- and LFA-1-mediated inhibition, by targeting activation as well as effector functions, may also be efficacious for the control of alloreactive CD8+ T-cell responses in sensitized hosts. We found that treatment with anti-LFA-1 mAb alone enhanced transplant survival and reduced CD8-mediated cytotoxicity in sensitized CD4 KO recipients. However, treatment with anti-CD154 mAb alone did not have an effect. Notably, when both CD4- and CD8-dependent rejection pathways are operative (wild-type sensitized recipients), LFA-1 significantly inhibited CD8-mediated in vivo allocytotoxicity but did not correspond with enhanced hepatocyte survival. We hypothesized that this was due to alloantibody-mediated rejection. When anti-LFA-1 mAb treatment was combined with macrophage depletion, which we have previously reported impairs alloantibody-mediated parenchymal cell damage, in vivo cytotoxic effector function was significantly decreased and was accompanied by significant enhancement of hepatocyte survival in sensitized wild-type recipients. Therefore, LFA-1 is a potent therapeutic target for reduction of CD8-mediated cytotoxicity in sensitized transplant recipients and can be combined with other treatments that target non-CD8-mediated recall alloimmunity.

Cytotoxic effector function of CD4-independent, CD8(+) T cells is mediated by TNF-α/TNFR.

BACKGROUND: Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms. METHODS: Mice were transplanted with allogeneic hepatocytes. In vivo cytotoxicity assays and various gene-deficient recipient mice and target cells were used to determine the development of Fas-, TNF-α-, and perforin-dependent cytotoxic effector mechanisms after transplantation. RESULTS: CD8(+) T cells maturing in CD4-sufficient hepatocyte recipients develop multiple (Fas-, TNF-α-, and perforin-mediated) cytotoxic mechanisms. However, CD8(+) T cells, maturing in the absence of CD4(+) T cells, mediate cytotoxicity and transplant rejection that is exclusively TNF-α/TNFR-dependent. To determine the kinetics of CD4-mediated help, CD4(+) T cells were adoptively transferred into CD4-deficient mice at various times posttransplant. The maximal influence of CD4(+) T cells on the magnitude of CD8-mediated in vivo allocytotoxicityf occurs within 48 hours. CONCLUSION: The implication of these studies is that interference of CD4(+) T cell function by disease or immunotherapy will have downstream consequences on both the magnitude of allocytotoxicity as well as the cytotoxic effector mechanisms used by allospecific CD8(+) cytolytic T cells.

Direct anterior approach for total hip arthroplasty using the fracture table.

Total hip arthroplasty is a successful procedure for treatment of painful hip arthritides. A large volume of literature is devoted to the patient outcomes and complication profiles of the commonly used surgical approaches to help refine the technique, enhance patient function, and limit cost and patient morbidity. The direct anterior approach has been reported using a fracture table to promote surgical exposure to the proximal femur. This technique is described herein with attention paid to the technical points which facilitate surgical exposure, patient safety, and functional outcome. Following a literature review of recent reports using this procedure are reviewed in context of the reported complications. The results show the direct anterior approach using a fracture table performed by experienced surgeons is an effective technique which provides early postoperative function and reduces the risk of dislocation.

Recipient immune repertoire and engraftment site influence the immune pathway effecting acute hepatocellular allograft rejection.

As novel acute allograft rejection mechanisms are being discovered, determining the conditions that promote or subvert these distinct rejection pathways is important to interpret the clinical relevance of these pathways for specific recipient groups as well as specific tissue and organ transplants. We have employed a versatile hepatocellular allograft model to analyze how the host immune repertoire and immune locale influences the phenotype of the rejection pathway. In addition, we investigated how peripheral monitoring of cellular and humoral immune parameters correlates with the activity of a specific rejection pathway. Complete MHC mismatched hepatocellular allografts were transplanted into immune competent CD4-deficient, CD8-deficient, or C57BL/6 hosts to focus on CD8-dependent, CD4-dependent, or combined CD4 and CD8-dependent alloimmunity, respectively. Hepatocellular allografts were transplanted to the liver or kidney subcapsular space to investigate the influence of the immune locale on each rejection pathway. The generation of donor-reactive DTH, alloantibody, and allospecific cytotoxicity was measured to assess both cellular and humoral immunity. Graft-infiltrating lymphocytes were phenotyped and enumerated in each recipient group. In the presence of CD8+ T cells, cytolytic cellular activity is the dominant mechanism of graft destruction and is amplified in the presence of CD4+ T cells. The absence of CD8+ T cells (CD8 KO) results in potent humoral immunity as reflected by high levels of cytotoxic alloantibody and graft rejection with similar kinetics. Transplant to the liver compared to the kidney site is distinguished by more rapid kinetics of rejection and alloimmunity, which is predominately cell mediated rather than a mix of both humoral and cell-mediated immunity. These studies define several rejection mechanisms occurring in distinct immune conditions, highlighting the plasticity of acute allograft rejection responses and the need to design specific monitoring strategies for these pathways to allow dynamic immune assessment of clinical transplant recipients and targeted immunotherapies.

Critical role of effector macrophages in mediating CD4-dependent alloimmune injury of transplanted liver parenchymal cells.

Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.

Disparate primary and secondary allospecific CD8+ T cell cytolytic effector function in the presence or absence of host CD4+ T cells.

The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.

Targeting LFA-1 and cd154 suppresses the in vivo activation and development of cytolytic (cd4-Independent) CD8+ T cells.

Short-term immunotherapy targeting both LFA-1 and CD40/CD154 costimulation produces synergistic effects such that long-term allograft survival is achieved in the majority of recipients. This immunotherapeutic strategy has been reported to induce the development of CD4+ regulatory T cells. In the current study, the mechanisms by which this immunotherapeutic strategy prevents CD8+ T cell-dependent hepatocyte rejection in CD4 knockout mice were examined. Combined blockade of LFA-1 and CD40/CD154 costimulation did not influence the overall number or composition of inflammatory cells infiltrating the liver where transplanted hepatocytes engraft. Expression of T cell activation markers CD43, CD69, and adhesion molecule CD103 by liver-infiltrating cells was suppressed in treated mice with long-term hepatocellular allograft survival compared to liver-infiltrating cells of untreated rejector mice. Short-term immunotherapy with anti-LFA-1 and anti-CD154 mAb also abrogated the in vivo development of alloreactive CD8+ cytotoxic T cell effectors. Treated mice with long-term hepatocyte allograft survival did not reject hepatocellular allografts despite adoptive transfer of naive CD8+ T cells. Unexpectedly, treated mice with long-term hepatocellular allograft survival demonstrated prominent donor-reactive delayed-type hypersensitivity responses, which were increased in comparison to untreated hepatocyte rejectors. Collectively, these findings support the conclusion that short-term immunotherapy with anti-LFA-1 and anti-CD154 mAbs induces long-term survival of hepatocellular allografts by interfering with CD8+ T cell activation and development of CTL effector function. In addition, these recipients with long-term hepatocellular allograft acceptance show evidence of immunoregulation which is not due to immune deletion or ignorance and is associated with early development of a novel CD8+CD25high cell population in the liver.

CD4+ T-cell-dependent immune damage of liver parenchymal cells is mediated by alloantibody.

BACKGROUND: Allogeneic hepatocytes initiate both CD4- and CD8-dependent rejection responses. The current studies address the hypothesis that acute damage of allogeneic liver parenchymal cells by the CD4-dependent pathway is alloantibody-mediated and examines immune conditions which promote activation of this pathway. METHODS: The role of alloantibody in CD4-dependent hepatocyte rejection was evaluated by assessing hepatocyte (FVB/N, H-2q) survival in CD8-depleted B-cell knockout (KO) (H-2b) recipients and by monitoring hepatocyte survival in C57BL/6.SCID (H-2b) recipients transfused with donor-reactive alloantibody. The development of donor-reactive alloantibody in C57BL/6 (H-2b), CD8-depleted C57BL/6, CD8 KO (H-2b), IFN-gamma KO (H-2b), perforin KO (H-2b), and FasL mutant gld/gld (H-2b) hepatocyte recipients was assessed. RESULTS: Hepatocyte rejection in B-cell KO mice was significantly delayed by CD8+ T-cell depletion (median survival time [MST], 35 days) when compared to untreated (MST, 8 days) and CD4-depleted (MST, 10 days) recipient mice. Transfusion of donor-reactive alloantibody into SCID recipients with functional hepatocellular allografts was sufficient to precipitate rejection in a dose-dependent fashion. Donor-reactive alloantibody was minimal in the serum of C57BL/6 hepatocyte recipients, but was produced in significant quantities in hepatocyte recipients genetically deficient in or depleted of CD8+ T cells and in recipients with impaired cytotoxic effector mechanisms. In addition, recipients with defects in Th1 immunity, such as IFN-gamma KO recipients, also produced readily detectable alloantibody. CONCLUSIONS: Collectively, these data support the hypothesis that acute immune damage of allogeneic hepatocytes by the CD4-dependent pathway is mediated by alloantibody and that this pathway is favored when Th1- or cell-mediated cytotoxic effector immune mechanisms are impaired.