s2fieldsofstudy: Biology Medicine

text:
The Influence of Immunosuppressive Agents on the Risk of De Novo Donor-Specific HLA Antibody Production in Solid Organ Transplant Recipients

Production of de novo donor-specific antibodies (dnDSA) is a major risk factor for acute and chronic antibody-mediated rejection and graft loss after all solid organ transplantation. In this article, we review the data available on the risk of individual immunosuppressive agents and their ability to prevent dnDSA production. Induction therapy with rabbit antithymocyte globulin may achieve a short-term decrease in dnDSA production in moderately sensitized patients. Rituximab induction may be beneficial in sensitized patients, and in abrogating rebound antibody response in patients undergoing desensitization or treatment for antibody-mediated rejection. Use of bortezomib for induction therapy in at-risk patients is of interest, but the benefits are unproven. In maintenance regimens, nonadherent and previously sensitized patients are not suitable for aggressive weaning protocols, particularly early calcineurin inhibitor withdrawal without lymphocyte-depleting induction. Early conversion to mammalian target of rapamycin inhibitor monotherapy has been reported to increase the risk of dnDSA formation, but a combination of mammalian target of rapamycin inhibitor and reduced-exposure calcineurin inhibitor does not appear to alter the risk. Early steroid therapy withdrawal in standard-risk patients after induction has no known dnDSA penalty. The available data do not demonstrate a consistent effect of mycophenolic acid on dnDSA production. Risk minimization for dnDSA requires monitoring of adherence, appropriate risk stratification, risk-based immunosuppression intensity, and prospective DSA surveillance.

D e novo formation of donor-specific antibodies (DSA) directed against HLA has been identified as a major risk factor for antibody-mediated rejection (AMR). 1 Production of de novo DSA (dnDSA) is associated with an increased risk of graft failure in all types of solid organ transplantation: kidney, 2-4 kidney-pancreas, 5 liver, 6 simultaneous liver-kidney, 7 small bowel, 8 heart, 9,10 lung, 11,12 and pancreatic islet 13 transplantation. In the medium-to long-term, although late acute AMR can occur, chronic AMR is more common and repre-sents the most common cause of late allograft dysfunction. 6,14,15 Patients with HLA class II or both class I + II DSA are at the greatest risk for chronic AMR 16 with anti-DQ dnDSA being the predominant specificity in kidney, [17][18][19] liver, 6 heart, 20 and lung 21 transplant patients. This occurs more frequently in nonadherent patients. 22,23 Clinical presentation varies between organs and includes acute and chronic graft dysfunction arising from microvascular injury leading to progressive fibrosis and loss of function. 9,10 Chronic AMR in kidney transplant patients may manifest as subclinical or clinically evident proteinuria with a slow, progressive loss of graft function over several years, 24,25 characterized by histopathologic changes, with or without C4d staining, and the presence of DSA in serum. 26 In kidney transplantation, it is estimated that graft loss may occur in 15% to 20% of cases within 1 year of AMR being diagnosed. 27 Chronic AMR is associated with acute hemodynamic compromise, accelerated transplant coronary artery disease and mortality after heart transplantation, 15,28 and graft injury and fibrosis in liver transplants. 29,30 The dnDSA development in lung transplant recipients is a major risk for progression to bronchiolitis obliterans syndrome and greater severity of and death related to bronchiolitis obliterans syndrome. 14,31,32 Research into the presence and clinical impact of dnDSA received a major impetus after the development of solidphase assays, which improved the sensitivity of detection and characterization of HLA antibodies compared to previous complement-dependent cytotoxicity assays. 33,34 The near-universal adoption of single-antigen beads for specificity testing, moreover, has made it possible to differentiate between dnDSA and non-DSA more accurately. 33 Current techniques also permit investigation of the biological activity and mechanisms of antibody injury. For instance, complement-binding (C1q) dnDSA appears to show a stronger relationship with graft loss than non-C1q-binding antibodies. 1,35,36 Considerable challenges persist, however, including intermanufacturer and lot-to-lot variation, a lack of standardization in cutoff points to define a positive test, and a degree of intralaboratory and interlaboratory variabilities. 34,37 Variability between laboratories using the solid-phase antigen bead assay with Luminex technology can be reduced by standardizing the test protocol and using identical reagents. 34 The DSA measurement using this technique can assess strength, effector function (via analysis of complement fixing properties, although false positive or negative results are possible), and immunoglobulin G subclasses. Furthermore, xenoantibodies, such as rabbit antithymocyte globulin (rATG) and monoclonal antibodies, such as rituximab, may interfere with some antibody detection methods, such as complement-dependent cytotoxicity and flow cytometric crossmatch [37][38][39][40] but not with solid phase antigen bead assays. Thus, comparison of dnDSA results between studies can be confounded by potential differences in the immunosuppression administered or in the timing and type of monitoring techniques used during follow-up.
Because dnDSA development has been convincingly associated with inferior outcomes, 4,41 it is imperative to avoid this undesirable alloimmune response, but simple overimmunosuppression carries significant risks, and may still be insufficient to control a robust antibody response. Therefore, it is essential to understand the risk factors for dnDSA formation and the relative effects that each immunosuppressive agent may have on prevention of dnDSA formation.
Toward the goal of risk-based personalized immunosuppression, this review evaluates the influence of induction and maintenance immunosuppression on the likelihood of dnDSA formation.

IMMUNOSUPPRESSION-INDEPENDENT RISK FACTORS FOR dnDSA PRODUCTION
Donor and recipient characteristics alter the risk of dnDSA formation. In particular, the degree of HLA matching is a major independent predictor of dnDSA formation. 6,41,42 Although only mismatching at HLA A, B, or DR loci has traditionally been evaluated, currently DQ mismatching appears consistently associated with the highest frequency of dnDSA. 6 Across all organs, including pediatric recipients, dnDSA are mostly directed against DQ antigens, in particular DQ β chains. 6,19,28,43 Other HLA loci, however, cannot be neglected. Wiebe and colleagues 41 observed HLA-DRβ1, but not HLA-DQ, to be an independent predictor for dnDSA in a series of 315 kidney transplant patients without preformed DSA, and a significant effect of HLA-DR matching has been described by other authors. 4 Future, more sophisticated, epitope mismatching analyses may prove useful in risk assessments of dnDSA development. [44][45][46] Younger age (typically, <50 years) is consistently associated with increased risk of dnDSA development. 6,42,[47][48][49] This may result from a more robust immune system or may simply be a consequence of greater nonadherence. African American race, 43 male sex, 48 the pre-existence of non-DSA HLA at the time of transplantation, and persistent BK virus infection 50 also appear to influence risk. Other important factors include sensitization events, such as retransplantation, 47,51 pregnancy, 52 and blood transfusions 51,53 or indicators of high immunological risk, such as previous acute rejection. 4,41,47,48,54,55 In the absence of randomized controlled trials evaluating dnDSA development between different immunosuppressive therapies, this array of risk factors complicates comparisons between studies, particularly where multivariate analyses are not performed. They should, however, be taken into account when customizing immunosuppressive strategy. It is critical to also remember that dnDSA develops over time; numerically more patients become dnDSA positive as time posttransplant increases, which must be considered when comparing studies.

EFFECT OF INTENSITY OF IMMUNOSUPPRESSION Nonadherence or Inadequate Immunosuppression
Underimmunosuppression is known to lower kidney allograft survival 56 and, intuitively, would be expected to increase the likelihood of dnDSA production. Two distinct scenarios heighten concern for dnDSA development: (1) nonadherence to the prescribed regimen and (2) underimmunosuppression as a result of weaning strategies. Recent studies of nonadherence to the immunosuppressive regimen 57,58 have shown nonadherence to affect at least a quarter of patients across all organs, and it increases over time after transplantation. Nonadherence is a well-established risk factor for dnDSA 23,41,59 and late acute AMR. 16,22 Data from small single-center series in adult 59,60 and pediatric 61 kidney transplants have described a high rate of nonadherence or prescribed reduction in immunosuppression in the majority of patients with dnDSA 59,61 or AMR. 60 In 1 report of 23 cases of AMR, 4 patients had documented nonadherence, whereas 16 patients had previously received a physician-directed reduction in immunosuppression. 60 Sellarés et al 23 prospectively followed up kidney transplant patients for a median of 31.4 months after indication allograft biopsy. Nonadherent patients at the time of biopsy were more likely to be DSA positive (77% versus 29% in adherent patients, P < 0.001), and more likely to progress to graft failure (32% versus 3%, P = 0.0001) than adherent patients. Similarly, Wiebe et al 41 studied 315 consecutive DSA-negative kidney transplant recipients, 15% of whom developed dnDSA within a median of 4.6 years. Nonadherence was significantly more frequent in those with dnDSA (49% versus 8% in adherent individuals, P < 0.001), a finding confirmed in logistic regression analysis (odds ratio [OR] = 8.75, P < 0.001). In liver transplantation, compliance with the calcineurin inhibitor (CNI) regimen was the most influential factor associated with dnDSA formation. 6 Also, some medications can be associated with dnDSA production, such as interferon in renal allograft recipients. 62 Although the risk of dnDSA has never been tested in liver allograft recipients of interferon therapy, plasma cell hepatitis is a known complication and one can speculate this to be associated with dnDSA formation. 63 Therefore, this may educate us about the role of interferon, either endogenous or exogenous in dnDSA formation.

Weaning of Immunosuppression
Calcineurin inhibitor and steroid-sparing strategies have been widely investigated in all types of solid organ transplantation. Graft injury from antibody-mediated damage may increase if CNI therapy is withdrawn or becomes subtherapeutic. 64 A complete understanding of this effect, however, has to date been hampered by the lack of DSA data collection in the majority of earlier randomized CNI-sparing trials, and the paucity of longterm data on dnDSA monitoring after CNI reduction or withdrawal in more recent studies. Steroid withdrawal or avoidance may not increase the risk of dnDSA if adequate immunosuppression is otherwise maintained. 65,66 In a 5-year longitudinal study of 37 kidney transplants randomized to steroid withdrawal at day 7 or to standard steroid therapy, all of whom received rATG induction, tacrolimus and mycophenolate mofetil (MMF), Delgado et al 65 found that only one patient in the standard-steroids group developed dnDSA, and none in the steroid-withdrawal arm.

IMMUNOLOGICAL EFFECTS OF IMMUNOSUPPRESSIVE AGENTS
Although B-cells and plasma cells produce antibodies, T-cell help is essential for the development of dnDSA. Effective T-cell suppression is therefore crucial to prevent dnDSA formation. Figure 1 shows a schematic overview of the key immunosuppressant agents and classes and their targets, including helper T-cells, each of which is discussed in more detail below.

Biological Therapies
Rabbit antithymocyte globulin targets peripheral Tlymphocytes, B-lymphocytes, natural killer cells, and plasma cells, and to a lesser extent monocytes and macrophages. 67 Administration of rATG at a cumulative dose of 6 mg/kg depletes T-cells for up to 12 months 68 and may also reduce B-cells. 69 The monoclonal antibody alemtuzumab depletes both T-and B-cells for up to a year, 69,70 and primate models suggest that depletion is more complete than that with rATG. 71 The IL-2 receptor antagonist (IL-2RA) agents block activated T-cells without affecting T-cell or B-cell numbers.
Rituximab, a chimeric anti-CD20 monoclonal antibody, inhibits development of memory T-cells and modulates the B-cell response by depleting memory B-cells. 72 Recent publications show that rituximab could be of benefit in the induction of sensitized patients, and in abrogating rebound antibody response in patients undergoing desensitization or treatment for AMR. 73,74 The proteasome inhibitor bortezomib profoundly inhibits activated B-cells and induces plasma cell apoptosis. 75 Both agents have been used to treat refractory AMR 27 and for desensitization in the scenario of preformed antibodies. 76,77 Maintenance Therapies The CNI agents cyclosporine (CsA) and tacrolimus suppress the humoral immune response by interfering with T-helper cell signaling 78 and are potent suppressors of antibody-mediated natural killer cell activation in vitro. 64 CNI agents also attenuate T-cell-dependent B-cell immune responses by reducing levels of stimulatory cytokine mRNA in activated T-cells. 78 Mycophenolic acid (MPA) inhibits both T-and B-cell proliferation (by blocking guanosine nucleotide production and preventing DNA synthesis 79 ) and T-cell trafficking through the transcription of GMP-dependent cell adhesion molecules. 79,80 The mechanistic target of rapamycin (mTOR) inhibitors everolimus and sirolimus block growth factor-mediated proliferation of T-cells and interfere with T-helper cell signaling. [81][82][83] They also suppress B-cell proliferation, B-cell immunoglobulin production in the early phase of the B-cell immune reaction, 84 and B-cell activation 85 and differentiation 86,87 and inhibit intracellular signaling implicated in AMR-induced allograft damage. 22,85,88,89 In a study comparing the immunologic effects of sirolimus, CsA and tacrolimus in a porcine model of arterial transplantation, dnDSA formation by day 30 was suppressed only in the sirolimus group. 90 Clinically, memory and regulatory T-cell recovery 91,92 during immune reconstitution after rATG or alemtuzumab induction is greater in kidney transplant patients treated with an mTOR inhibitor compared to CNI therapy. [91][92][93][94][95] Experimental models suggest that mTOR inhibition reduced noncomplement-mediated vascular injury by DSA. 96 Corticosteroids exert a multifaceted immunomodulatory effect, altering T-cell function and redistributing cell subsets. 97 The B-cell antibody production is suppressed indirectly by steroids, through various mechanisms arising from a modified effect of T-cell function on allogeneic B-cell activation. 97

IMMUNOLOGICAL EFFECTS OF IMMUNOSUPPRESSIVE DRUGS: CLINICAL EVIDENCE
The following sections assess the available data for individual immunosuppressive agents on dnDSA formation. Interpretation of these data is hampered by differences in intensity of overall immunosuppression, a paucity of prospective clinical trials and an abundance of multidrug cocktails. Despite these shortcomings, several hypothesis-generating possibilities can be considered. We will discuss drug classes and therapies separately.

Biological Therapies Antithymocyte Preparations and IL-2RA Monoclonal Antibodies
In kidney transplantation, randomized controlled trials of antithymocyte globulins and IL-2RA agents have not reported data on dnDSA rates, although they have demonstrated a significant reduction in acute rejection with T-cell depletion therapy. [98][99][100] Rabbit antithymocyte globulin is an established induction agent in transplantation, and a possible treatment for AMR. 27 The effect of rATG induction therapy on propensity to produce dnDSA has been assessed in some nonrandomized trials (Table 1). In a recent single-center analysis of 114 consecutive DSA-positive kidney transplant patients with a negative crossmatch who received rATG or basiliximab induction therapy, rATG was associated with a lower risk of dnDSA (hazard ratio [HR], 0.16; 95% confidence interval [95% CI], 0.04-0.50; P = 0.003) and AMR (HR, 0.16; 95% CI, 0.05-0.60; P = 0.006) in multivariate analysis. 101 Other retrospective studies observed no significant difference in the risk of dnDSA using IL-2RA versus rATG induction after kidney transplantation. 42,55 However, these studies were not designed to determine the role of induction immunosuppression on the incidence of dnDSA and rATG was used in higher immunological risk patients.
Overall, rATG appears to achieve a short-term decrease in dnDSA production in moderately sensitized patients. Prospective randomized controlled trials are needed to assess the role of IL2-RA and antithymocyte globulins on the incidence of dnDSA.

Alemtuzumab
Randomized controlled trials of IL-2RAversus alemtuzumab have demonstrated a significant reduction in acute rejection with alemtuzumab 98,105 but did not provide information on the rate of dnDSA. When alemtuzumab induction was compared to basiliximab with low-dose rATG in a matchedcohort single-center study of kidney transplant patients, the incidence of dnDSA at 1 year after transplantation was higher in patients receiving alemtuzumab (50% [8/16]) than in the basiliximab/rATG control group (12.5% [4/32]; P = 0.011). 102 Other authors have observed a high rate of AMR with alemtuzumab after kidney-pancreas transplantation in patients receiving CNI maintenance therapy, 106 and an increased risk of AMR and dnDSA formation in alemtuzumab-treated kidney transplant patients receiving CNI-free immunosuppression. 69,107 Together, these data suggest that alemtuzumab as an induction agent may not be effective in preventing the early appearance of dnDSA, although new interventional randomized studies are needed to specifically address this question.

Rituximab
The chimeric monoclonal anti-CD20 antibody rituximab is an established option for treatment of refractory AMR, 27 although its effect on dnDSA prevention is less certain. In a nonrandomized trial of 320 unsensitized kidney transplant recipients, pretransplant rituximab did not influence dnDSA production. 108 In a prospective, double-blind trial of patients randomized to a single dose of rituximab or placebo induction, the incidences of AMR, biopsy-proven acute rejection, and dnDSA formation (rituximab, 3% [1/33]; placebo, 16% [6/38]) were similar at 3 years. 103 Several other centers have described their use of induction therapy with low-dose or single-dose rituximab in kidney, 109 liver, 110,111 and intestinal or multivisceral 112 transplantation, usually in combination with rATG, and have reported low rates of AMR (<4%) in patients with a positive crossmatch, 109,110 but data on dnDSA formation have not been reported. However, a randomized trial of rituximab versus IL-2RA induction in nonsensitized kidney transplant patients was terminated because of an excess of acute cellular rejection in the rituximab group (83% vs 14%, P = 0.01). 113 In contrast, in a recent retrospective analysis of 281 kidney transplant patients divided into four groups according to whether they had preexisting DSA and whether rituximab was administered, the rate of appearance of de novo HLA antibodies-and the incidence of chronic AMR-was lower in the groups that received rituximab treatment versus their untreated counterparts. 114 A Japanese report found that ABO-incompatible recipients either splenectomized or induced with low-dose rituximab developed dnDSA less frequently at 2 years than ABOcompatible recipients without either treatment (2.2%, 1.7%, and 18.1%, respectively). 109 Finally, preemptive treatment with rituximab and plasmapheresis was shown to increase the chance of clearing early dnDSA in a retrospective study of lung transplant recipients, although this did not appear to be associated with a clinical benefit. 115 Although there is adequate clinical evidence to support rituximab therapy for the treatment of AMR and possibly prevention of dnDSA, more randomized studies are needed to determine how and in whom it should be used.

Bortezomib
Off-label use of bortezomib, a first-in-class proteasome inhibitor, has yielded AMR treatment results. [116][117][118][119][120][121] This has prompted its use as part of induction regimens or after desensitization to target B-cells and plasma cells in combination with rATG. 104,122 In a randomized pilot study, Ejaz and colleagues 104 treated 40 kidney transplant patients at high immunological risk with rATG alone, rATG/rituximab, rATG/ bortezomib, or rATG/rituximab/bortezomib. In total, 10 of 40 patients (25%) developed dnDSA within 1 year. Circulating dnDSA had cleared by 1 year in the rATG and rATG/ bortezomib group, but not in the rituximab-treated cohorts. The incidence of AMR, however, was not significantly different between groups. In a study of 18 patients undergoing clonal depletion with donor-specific transfusion followed by treatment with bortezomib, rATG, rituximab, and steroids, 4 patients developed dnDSA, whereas 4 could be weaned off immunosuppression. 123 Similar results were reported in a small series of pediatric heart transplant cases, where bortezomib has been associated with a marked reduction in dnDSA and resolution of AMR. 124 In summary, the role of proteasome inhibitors in the prevention of dnDSA, while promising, remains to be determined in larger prospective studies.

Belatacept
The costimulation blocker belatacept prevents T-cell activation. 125 In the phase 3 registration trial (BENEFIT), AMR was avoided in all treatment arms, but acute cellular rejection was more frequent and more severe in the intensive belatacept treatment group compared to the CsA treatment group. 126 Similarly, no AMR occurred in the BENEFIT-EXT study of belatacept therapy in recipients of expanded criteria donors. 127 Numerically lower DSA rates at 1 year, 126 2 years, 128 and 3 years 129 were seen in the BENEFIT patients treated with belatacept compared to those treated with CsA. However, no statistical comparison was performed, and preformed DSA were not distinguished from dnDSA. Therefore, belatacept may exert a protective effect on dnDSA formation, but prospective granular data are still needed.

Calcineurin Inhibitors
Calcineurin inhibitor administration, especially early after transplant, is likely protective against dnDSA formation ( Table 2). In 244 consecutive kidney and kidney-pancreas  . This is a potential cause for concern because these levels have been reported during the maintenance phase of some relatively aggressive CNI reduction studies in kidney transplantation. 132,133 Discontinuation of CNI therapy early after kidney transplantation in the absence of suitable induction therapy may result in inadequate immunosuppression to prevent dnDSA formation.
Several studies in kidney and liver transplantation have reported that tacrolimus-based immunosuppression is associated with a lower risk of dnDSA formation than CsA-based regimens. 4,6,47,55,130 In 1 large single-center liver transplant experience, use of CsA versus tacrolimus was significantly associated with an increased risk of dnDSA (OR, 2.5; P = 0.004). 6 Generally, the available data demonstrate a clear signal that compliance with CNI, and adequate CNI trough levels, play a more important role in dnDSA formation than the choice of CNI; however, in compliant patients tacrolimus may provide more protection from dnDSA formation than CsA.

Purine Synthesis Inhibitors and Antimetabolites
Maintenance immunosuppressive therapy with MPA profoundly depresses the primary and secondary humoral response in kidney transplant patients, 134,135 but data on the effect of purine synthesis inhibitors modulation of dnDSA formation are still inconclusive. Studies in kidney transplantation have shown inconsistent results regarding an effect of MMF versus azathioprine in terms of dnDSA onset 130,136 (Table 3). No conclusive results could be reached in a large prospective study by Hourmant and colleagues, 4 although azathioprine was more frequently used than MMF among patients with dnDSA whereas the converse was true in DSA-negative patients. Retrospective analyses in liver transplant populations have also shown mixed findings (Table 3). Until more robust data are available, one cannot definitively conclude an independent beneficial effect of MPA on dnDSA formation. Although the bulk of data may suggest a modest benefit, this may simply result from increased intensity of immunosuppressive regimens that include MPA therapy, rather than a specific mechanism to reduce the risk of dnDSA.

mTOR Inhibitors
Well-designed prospective studies to assess the relationship between mTOR inhibitors and risk of dnDSA are lacking (Table 4). A recent post hoc analysis was performed on 127 kidney transplant patients at a single center randomized to convert from CsA to a CNI-free everolimus-based regimen at 3 to 4.5 months after transplantation or to remain on CsA (ZEUS). 54 All patients received basiliximab induction, MPA and were started on oral steroids; by the end of the observation period, 59% of everolimus-treated patients and 62% of CsA-treated patients were steroid-free. During a median follow-up of 1273 days, dnDSA was detected in 23.0% of patients (14/61) who stopped CsA and switched to everolimus, compared to 10.8% of patients (7/65) who continued CsA (HR = 2.43; P = 0.048). The time to first detection of dnDSA was shorter in the everolimus cohort (median, 551 days vs 1173 days in the CsA group), and AMR occurred in 8 everolimus-treated patients compared to 2 CsA-treated patients (P = 0.036). 54 Five of the 8 AMR patients in the everolimus group received reduced-dose MPA and 2 were steroidfree; the 2 patients in the CsA group with AMR had no MPA or steroids. Although underpowered, everolimus monotherapy was associated with dnDSA and AMR (HR = 5.35; P = 0.036) but since immunosuppression appears to have been inadequate in many cases, interpretation is difficult.
Conflicting results have been reported (so far in abstract form only) by Sommerer et al 139 in patients who received basiliximab induction with CsA to month 3, and were randomized to continue standard CsA, switch to low-dose CsA with everolimus or convert to a CNI-free everolimus regimen, all with MPA and steroids (HERAKLES). At four years after kidney transplantation, the incidence of dnDSA was similar in the standard CsA group (16.7%), the CNI-free group receiving everolimus (17.9%), and the CsA-everolimus cohort (29.6%; P = n.s.).
Kamar et al 49 found no effect on dnDSA formation after converting patients from CNI to everolimus when the switch took place at a later time after transplantation (median, 22 months). In their single center case-control retrospective study, the incidence of dnDSA was compared over a mean of 35 months in a cohort of 61 patients. 49 Controls were matched for age, sex, induction therapy, and date of transplantation. All patients were DSA-free at the initial comparison, and at last follow-up, the proportion of patients with dnDSAwas not significantly different between the everolimus group (9.8% [6/61]) and the CNI-treated controls (5% [3/61]; P = n.s.), with a similar median time to dnDSA detection (9.5 months and 13 months, respectively) and everolimus trough concentrations. 49 Another retrospective study 18 found a significantly higher rate of dnDSA in multivariable analysis of 56 kidney transplant patients converted from tacrolimus to mTOR monotherapy (sirolimus in 84%) at a mean of 1.3 years after transplantation compared to 214 who continued tacrolimus therapy. However, patients converted to sirolimus more than a year after transplantation had a similar rate of dnDSA emergence to the tacrolimus-treated cohort, once again suggesting that early mTOR inhibitor monotherapy conversion (<1 year) provides inadequate immunosuppression. 18 Prevalence data from a series of 267 maintenance kidney transplant patients observed no significant difference in mTOR inhibitor therapy between recipients with or without class I or class II dnDSA. 25 Perbos and colleagues 138 reported no increase in dnDSA onset in a series of kidney, liver, heart, and lung transplant recipients receiving everolimus with lowdose CNI about 6 years after transplantation, consistent with  limited data in kidney transplant patients treated with mTOR inhibitors and low-dose extended-release tacrolimus. 140 In liver transplantation, Del Bello and colleagues 47 found no significant association between mTOR inhibitor therapy and dnDSA formation. Taken together, existing data do not provide conclusive evidence that immunosuppressive regimens based on an mTOR inhibitor and reduced-exposure CNI therapy are invariably associated with increased risk of dnDSA formation. However, an early switch from CNI therapy to mTOR inhibitor monotherapy may increase the risk of dnDSA production, whereas late (>1 year) posttransplant conversion to mTOR inhibitor monotherapy may not increase the risk of dnDSA formation.

Corticosteroids
In an attempt to prevent or minimize steroid-associated side effects, some immunosuppressive strategies avoid the use of steroids or enable their early discontinuation (Table 5). In a prospective, single-center analysis by Lachmann and colleagues, 130 no significant difference in the development of dnDSA could be seen in kidney transplant patients with or without steroid therapy at the time of antibody testing. In a retrospective analysis of 749 liver transplant patients by Kaneku et al, 6 the risk of dnDSA was reduced in the presence of steroid therapy in univariate but not multivariate analysis. Similarly, in a retrospective analysis of 232 liver transplant patients tested annually for dnDSA, multivariate analysis did not show steroid therapy to be independently associated with dnDSA. 46 Despite consistent data, these findings are potentially confounded; steroids may have been preferentially discontinued in patients with well-functioning, rejection-free grafts.
Planned early steroid discontinuation may not influence the risk of dnDSA, depending on the immunosuppressive regimen used. Delgado and colleagues treated 37 kidney transplant patients with rATG induction, tacrolimus and MMF who were randomized to steroid withdrawal at week 1 posttransplant versus standard steroid therapy in a double-blind trial with annual follow-up of up to 5 years with no difference in dnDSA formation. 65 Consistent with these results, Li et al 66 reported no dnDSA with excellent graft function in 13 pediatric transplant patients at high immunological risk who received entirely steroid-free immunosuppression with a similar regimen (rATG induction with tacrolimus/MMF maintenance therapy).
Intriguing data regarding the influence of steroids comes from a series of 72 living-donor kidney transplant patients given a tolerance protocol comprising clonal depletion by total lymphoid irradiation or bortezomib followed by low-dose maintenance immunosuppression (steroids with or without one other agent). 141 Donor-specific antibodies were assessed every 1 to 2 months. At 3 months, patients tapered to less than 10 mg/day had a high rate of dnDSA (53%), compared to patients maintained on 10 to less than 20 mg/day (22%), and the lowest risk (0%) was seen in patients continued on 20 mg/day or greater or steroids plus another immunosuppressant agent. In multivariate analysis, steroid dose was inversely associated with dnDSA production, with an adjusted risk ratio of 0.92 (95%, 0.85-0.99; P = 0.03) for every 2.5 mg/day increase.
Thus, existing data present a complex picture regarding an association between steroid therapy and risk of dnDSA production. Current findings do not suggest an adverse effect of early steroid withdrawal or steroid-free immunosuppression in selected patients, but firm conclusions cannot be drawn.

FUTURE DIRECTIONS
Understanding how to appropriately regulate the B-cell compartment is critical to prevent dnDSA and achieve decadeslong survival for solid organ transplants. Careful evaluation of the known impact of immunosuppressive agents on dnDSA has highlighted the major shortcomings of existing reports. As a consequence, we are currently unable to "personalize" immunosuppressive treatment to prevent dnDSA.
New studies should consider the addition of novel anti-B-cell agents that have recently emerged in other relevant fields, such as hematopoietic stem cell transplantation, 142 autoimmunity, 143 and multiple myeloma. 144 However, careful target selection may be critical to success because simple B-cell annihilation is likely not the answer given the difficulty of reaching niche resident plasma cells and the critical importance of regulatory B-cells. 145 Recently, several new interesting B-cell-directed strategies have attracted attention, which can be divided into direct and indirect B-cell agents. 143 Novel direct B-cell agents include variants of anti-CD20 monoclonal antibodies (mAbs), such as ocrelizumab, which more thoroughly deplete B-cells, 146 and anti-CD19 mAbs that also deplete memory B-cells and short-lived plasma cells while CD19 − plasma cells remain unaffected. 147 Furthermore, the anti-CD22 mAb epratuzumab is able to modulate B-cell function by altering adhesion molecule expression, interfering with migration, 148 and inhibiting BCR-dependent B-cell activation. 149 Indirect B-cell targeting can be accomplished through modulation of the BAFF/APRIL pathway, primarily produced by macrophages, neutrophils, and dendritic cells and also B-cells and activated T-cells, comprising 3 cellular receptors (BAFF-R, BCMA, and TACI) differentially expressed by different subpopulations of B-cells. 150 In particular, BAFF-R is critical for immature B-cell survival/maturation, BCMA is required for plasma cell survival, and TACI is necessary for T-cell-independent B-cell responses, regulation of B-cells and Ig class-switching. Therefore, the BAFF/APRIL pathway appears to lie at a critical immune intersection that may regulate the B-cell compartment. Accordingly, targeting such a complex system with novel and specific interventions has gained considerable interest. Kwun and colleagues 151 recently documented simultaneous neutralization of BAFF and APRIL using a fusion protein composed of the TACI receptor and Ig Fc (TACI-Ig, atacicept), which prevented early DSA formation and AMR in a depletion-induced preclinical AMR model.
Plasma cell targeting represents another area of active research. Interference with BAFF/APRIL using the anti-BAFF mAb tabalumab is being explored. 152 In addition, secondgeneration proteasome inhibitors are being evaluated 153 and other plasma cell depleting agents that target cell surface molecules (CD38 and CD138) are currently under investigation. 144 In addition, some bortezomib-recalcitrant multiple myeloma patients have responded to novel proteasome inhibitors targeting different sites. 154 Additionally, research in the complex ubiquitin proteasome system is generating novel molecules that target protein degradation upstream of the proteasome and drug combinations with enhanced therapeutic capacity. Finally, orally available proteasome inhibitors may soon become available. 155 An effective B-cell response is also considerably influenced by the critical contribution of helper T-cells. As a consequence, novel costimulatory blockers, such as CTLA4-Ig Included both preformed and de novo DSA. ARR, adjusted risk ratio.
(belatacept), that prevent T-cell help to B-cells may represent an important adjunct to prevent dnDSA. It should be noted, however, that an inhibitory role of belatacept on regulatory T-cells has been reported, 156 and in 1 study resulted in an increased risk of cell-mediated rejection. 157 Because regulatory T-cell generation is independent of the CD40-CD154 pathway, an anti-CD40 mAb may represent an alternative. 158 Although cellular destruction has remained the dominant mechanism to treat dnDSA formation, prevention remains a superior approach. Interference with cellular trafficking to essential locations for costimulation has the potential to prevent dnDSAwhile allowing the regulatory cell populations to remain intact. Despite a lack of data and the withdrawal of efalizumab from the market, this type of approach remains attractive. 159 Taken together, these data point to a large number of novel immunosuppressive agents that, in theory, hold the potential to prevent dnDSA. If combined with a better understanding of how to appropriately risk-stratify patients to receive these potent immunosuppressive agents, this critical combination of knowledge may be able to substantially decrease the risk of dnDSA, resulting in improved allograft survival in the not-too-distant future. However, to achieve this, we need well-designed prospective, multicenter, randomized clinical trials across organ transplant types that collect detailed donor and recipient genetic information, immunosuppression serum levels, and compliance data in addition to frequent HLA and non-HLA DSA testing 160,161 with different riskbased immunosuppressive strategies.

CONCLUSIONS
De novo DSA risk reduction is essential to prevent chronic AMR and improve long-term graft survival in all solid organ transplant patients. Utilization of solid-phase single-antigen assays as part of risk-based monitoring for circulating DSA has increased. However, personalizing immunosuppression precisely based on an individual's risk for dnDSA production remains in development.
Patients with a history of nonadherence, greater HLA mismatching (particularly for class II DQ or DR) or an increased propensity to sensitization due to previous transplant, transfusion, pregnancy, or previous acute rejection, are more likely to develop dnDSA. These individuals may need more intensive immunosuppression than those without risk factors. Especially in the first year after transplantation, rATG induction appears to attenuate dnDSA production in moderately sensitized patients. Administration of newer agents, such as rituximab or bortezomib, as induction therapy in atrisk patients may be of interest, but the benefits are unproven in adequately powered studies. Early CNI withdrawal, especially in the absence of depleting induction, is not advisable in patients with risk factors for dnDSA. Although late CNI withdrawal in lower-risk patients can be undertaken, close DSA surveillance is recommended. Mammalian target of rapamycin inhibitor monotherapy early after transplant is not recommended, but the combination of an mTOR inhibitor with reduced-exposure CNI has not been associated with increased risk of dnDSA. The available data do not indicate a consistent effect of MPA on dnDSA production. Early withdrawal of steroid therapy appears feasible with no increased risk of dnDSA when combined with induction and nonsteroid-based maintenance immunosuppression. Overall, the priority in at-risk individuals is to maintain adequate immunosuppression, establish an appropriate DSA monitoring protocol, and enforce adherence. The future demands DSA monitoring to become a routine component of randomized immunosuppression trials in all organs to improve our understanding of the relative risk of dnDSA production between regimens.


extfieldsofstudy:
Medicine
Materials Science

s2fieldsofstudy: Biology Medicine Engineering

text:
Preparation and Characterization of a Novel Decellularized Fibrocartilage “Book” Scaffold for Use in Tissue Engineering

At the tendon-to-bone insertion, there is a unique transitional structure: tendon, non-calcified fibrocartilage, calcified fibrocartilage, and bone. The reconstruction of this special graded structure after defects or damage is an important but challenging task in orthopedics. In particular, reconstruction of the fibrocartilage zone has yet to be successfully achieved. In this study, the development of a novel book-shape scaffold derived from the extracellular matrix of fibrocartilage was reported. Specifically, fibrocartilage from the pubic symphysis was obtained from rabbits and sliced into the shape of a book (dimensions: 10 mm × 3 mm × 1 mm) with 10 layers, each layer (akin to a page of a book) with a thickness of 100-μm. These fibrocartilage “book” scaffolds were decellularized using sequentially 3 freeze-thaw cycles, 0.1% Triton X-100 with 1.5 M KCl, 0.25% trypsin, and a nuclease. Histology and DNA quantification analysis confirmed substantial removal of cells from the fibrocartilage scaffolds. Furthermore, the quantities of DNA, collagen, and glycosaminoglycan in the fibrocartilage were markedly reduced following decellularization. Scanning electron microscopy confirmed that the intrinsic ultrastructure of the fibrocartilage tissue was well preserved. Therefore, the results of this study suggest that the novel “book” fibrocartilage scaffold could have potential applications in tissue engineering.

Introduction
At the tendon-to-bone insertion, there is a physiologically unique transitional fibrocartilage zone, with calcified collagen fibers connecting to the bone and non-calcified collagen fibers connecting to the tendon. This graded transitional construction is formed under physiological loading conditions and is closely related to its mechanical properties [1][2][3]. Reconstruction of the tendon-to-bone insertion following defects or damage is a challenge for orthopedic surgeons. Previously, treatment involved using a suspending device to fix the tendon or ligament to the bone through surgical operations or using biodegradable interference fit fixation. Despite appropriate surgical management during such treatment, the original tendon-bone architecture may not be regenerated where fibrovascular scars have been formed. Because of incomplete regeneration of the original characteristic fibrocartilage zone between the tendon and bone, it is possible that the resultant tendon-bone junction may not be able to transfer load efficiently between the soft and hard tissues, which can lead to re-tearing or failure to heal [4][5][6]. Recently, growth factors and cytokines, physical stimulation, and stem cell transplantation have been used at the repair site with the aim of improving tendon-to-bone healing. However, to date, the graded transitional structure has yet to be successfully reconstructed [6,7].
The development of tissue engineering technology, which often involves transplanting cells into synthetic or natural polymer scaffolds, has enhanced the study of tendon-to-bone interface regeneration. For example, Lu et al. [8] developed a three-phase poly(lactic-co-glycolic acid)/ extracellular matrix (ECM) scaffold, with implanted fibroblasts, chondrocytes, and osteoblasts, which mimicked the transitional structure of the tendon-to-bone insertion. Distinct mineral and fibrocartilage-like tissue regions were formed on the scaffold, and collagen type II and aggrecan were detected. Chen et al. [9] developed a silk-RADA peptide scaffold, with implanted bone marrow stromal cells used to regenerate the cartilage-subchondral bone interface. They found that hypertrophic chondrocytes were embedded in the calcified ECM and that glycosaminoglycans (GAGs) and collagen were expressed. Despite some success, the synthetic biomaterials that were used in these studies could not fully simulate the original structure and matrix components of tendon-to-bone insertion. The anisotropy properties of the fibrocartilage zone at the tendon-to-bone insertion are closely related to its mechanical bearing capabilities and hence the successful reconstruction of the original structure of the fibrocartilage zone is particularly important.
Several studies have reported the use of decellularized cartilage in tissue engineering for articular cartilage reconstruction [10][11][12]. It is believed that a decellularized cartilage may be a viable option as a replacement, as the antigenic cellular material will be removed while preserving the relatively non-immunogenic extracellular matrix [13]. However, it is difficult to achieve cell migration into acellular cartilage scaffolds because they are highly dense with limited space [14]. Cartilage sheets and cartilage particles are often used in cartilage tissue engineering. For example, Gong et al. developed a "sandwich model" with 10-and 30-μm acellular cartilage sheets [11], whereas Yang et al. developed a 3D porous acellular scaffold by using acellular cartilage particles with diameters ranging from 500 nm to 5 μm [15]. Although these scaffolds were multi-aperture and cells could migrate into the acellular cartilage scaffold by a series of treatments, the original structure of the cartilage matrix was destroyed. Furthermore, a thin single-layer tissue chip is difficult to be operated in polyphase integration.
In this study, a decellularized fibrocartilage with a "book" design was developed for use in tissue engineering. The characteristics of the "book" scaffold were investigated by using various techniques: histology, to detect cellular components and structures; scanning electron microscopy, to determine the ultrastructure; and quantification assays, to measure levels of DNA, collagen, and GAG.

Experimental animals
Samples of fibrocartilage of the pubic symphysis were collected from 20 New Zealand white rabbits (18 weeks old, 2-3 kg), supplied by the Experimental Animal Center of Central South University. All rabbits were maintained under specific pathogen free conditions and fed standard rodent chow ad libitum. All experimental procedures conformed to the Chinese National Health and were approved by the Ethics Committee of the Center for Scientific Research with Animal Models of Central South University (2013-3-13). The pubic symphysis fibrocartilage samples were harvested from 20 New Zealand white rabbits which were euthanized (overdose of sodium pentobarbital, 100 mg/kg). None surgery was performed before euthanization. All efforts were made to minimize suffering.

Preparation of "book" cartilage
After removing the soft tissue and bone, the fibrocartilage was trimmed into segments measuring approximately 10 mm × 3 mm × 2 mm, including part of the subchondral bone, which would be advantageous for the successful completion of the next freeze-sectioning step. The trimmed cartilage samples were fixed to the cutting base plate of a cryostat (Leica CM1950; Nussloch, Germany) with optimum cutting temperature (OCT) compound (polyvinyl alcohol and polyethylene glycol, Tissue-Tek1; Sakura Finetek USA, Inc., Torrance, CA, USA) and placed for 20 min at a presetting temperature of −22°C. The cartilage segments were then sliced to a thickness of 100 μm but left uncut at one end, which was achieved by using the near spin button with a single click representing a precession of 20 μm. Thus, a book-type fibrocartilage tissue scaffold was achieved by 5 clicks for each layer, and the thickness of each layer of the "book" was 100 μm. Ten layers were added to each "book" scaffold, and its final size was approximately 10 mm × 3 mm × 1 mm (Fig 1).

Preparation of the acellular fibrocartilage scaffold
An improved decellularization method was developed according to published methods, which had previously been used to decellularize articular cartilage [16][17][18]. The sliced fibrocartilage samples were washed twice with phosphate-buffered saline (PBS) (3 × 30 min), followed by 3 freeze-thaw cycles (one cycle: freezing for 10 min in liquid nitrogen was followed by thawing for 10 min in a water bath at 37°C). Subsequently, the samples were immersed in 0.1% Triton X-100 with 1.5 M KCl for 12 h at 4°C with gentle agitation. They were then washed with 10 mM Tris buffer (pH 8.0) for 3 h, followed by another wash with PBS for 3 h. After these washes, the samples were digested in 0.25% trypsin (Genview, USA) for 24 h at 37°C with gentle agitation. Following a further 24-h wash in PBS, with rinsing repeated every 4 h during this period, the samples were incubated in nuclease solution (including 500 U/mL deoxyribonuclease I and 1 mg/mL ribonuclease A; Genview) for 12 h at 37°C with agitation to remove nuclear materials. Finally, the samples were repeatedly rinsed in PBS with agitation for 24 h, with rinsing repeated as described above.

Histological analyses
The decellularized fibrocartilage scaffolds (n = 2 for each group) were placed flat onto filter papers to remove superfluous water from the samples' surfaces. They were then fixed to the cutting base plate of the Cryostat Microtome System with OCT compound, where they were frozen at −22°C for 20 min. Subsequently, the samples were cut into 8-μm-thick sections in a direction perpendicular to the "book" and then placed onto glass slides. Before microscopic examination, sections were stained using hematoxylin and eosin (H&E) stain, Masson's trichrome stain (KeyGEN Biotech, China), and 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) to detect the cellular components and collagen fibrous structures.

Scanning electron microscopy
Fibrocartilage "book" sheets with or without cells (n = 2 for each group) were rinsed with PBS and fixed overnight in 0.05% glutaraldehyde at 4°C. Following dehydration via a graded series of acetone, and replacement via a graded series of isoamyl acetate, samples were critical-point dried. Scaffolds were then coated with gold/palladium by using a sputter coater (EiKO-IB-5, Japan), and their surface morphology was visualized by using a scanning electron microscope (SEM) (Hitachi S-3400N, Japan).

Quantification of residual DNA, collagen, and GAG in the ECM scaffolds
After the decellularization treatment, the samples (n = 8 for each group) were frozen overnight at −80°C. Subsequently, the samples were freeze-dried using a lyophilizer (SIM International Group, USA) at −70°C for 24 h. The freeze-dried samples were weighed and minced. According to the DNeasy Blood & Tissue protocol (Qiagen, Germany), 10 mg of lyophilized sample was digested with proteinase K (supplied with the kit) at 56°C for 3 h. The concentration of the purified DNA was measured by a spectrophotometer (MaestroNano, USA), and the quantity of DNA was calculated according to the DNA concentration. Standard DNA (Sigma-Aldrich) was used to make a standard curve. For the analysis of collagen, the Blyscan collagen assay kit (Biocolor, UK) was used. The lyophilized samples were digested in pepsin (Sigma-Aldrich) (1.0 mg/mL 0.5 M acetic acid) at 4°C for 48 h. The amount of collagen was measured using a Multiskan MK3 microplate reader (Thermo Scientific, USA). Fluorescence intensities were used to calculate the amount collagen in the scaffolds based on a standard curve produced by using standard collagen supplied with the kit. A Sircol GAG assay (Biocolor) was used to quantify the GAG that remained in the ECM scaffolds. The lyophilized samples were digested in 0.2 mg/mL papain (Sigma-Aldrich) in PBS (pH 6.4) at 65°C for 18 h. The amount of GAG was measured using the Multiskan MK3 microplate reader. Fluorescence intensities were also used to calculate the amount of GAG based on a standard curve produced using standard GAG supplied with the kit.

Statistical analysis
The data are given as mean±standard deviation. One-way analysis of variance was used to determine the statistical significance between the different groups. Data were analyzed using SPSS17.0, and P < 0.05 was considered statistically significant.

Characterization of the book-shape cartilage scaffold
The "book" fibrocartilage scaffolds were produced using freeze-sectioning. The size of the scaffold was 10 mm × 3 mm × 1 mm (Fig 2A-2C). The thickness of a layer was 100 μm. After decellularization, the scaffolds retained their original shape and size but were more transparent and white in color (Fig 2D-2F).

Histological analyses
H&E and Masson's trichrome staining demonstrated that the cellular component was removed from the scaffolds following decellularization, while the native collagen structure was preserved. Furthermore, DAPI-positive cell nuclei were rarely observed after treatment (Fig 3A-3P).

SEM analyses
Using SEM, empty lacunar structures were observed in all decellularized scaffolds. High-magnification micrographs revealed that the characteristic cartilage lacuna and native collagen structure were well preserved after decellularization (Fig 4A-4D).

Quantification of DNA, collagen, and GAG
The quantity of DNA in the fibrocartilage samples was 0.97±0.29 μg/mg (fibrocartilage dry weight) before decellularization and 0.08±0.02 μg/mg after decellularization. The amount of DNA in the decellularized fibrocartilage ECM scaffolds was significantly lower than in the fresh fibrocartilage (P < 0.05). The quantity of collagen in the fibrocartilage samples was 228.68±72.32 μg/mg (dry weight) before decellularization and 158.70±50.17 μg/mg after decellularization (P < 0.05). This represented a 30.3% loss of collagen in the decellularized fibrocartilage ECM scaffolds compared with the fresh fibrocartilage. The quantity of GAG in the fibrocartilage samples was 55.43±6.11 μg/mg (dry weight) before decellularization and 28.41±4.83 μg/mg after decellularization (P < 0.05). Therefore, there was a 48.7% loss of GAG in decellularized ECM scaffolds compared with fresh samples (Figs 5 and 6).

Discussion
Tendons and ligaments attach to bone through a transitional fibrocartilage zone that has complex biomechanical properties. This unique tissue structure is often not regenerated during the healing of an injury and/or surgical reattachment [19]. A natural, porous, easy-to-use, multilayered, decellularized "book" fibrocartilage scaffold was developed. This was achieved by developing fibrocartilage into a book-type decellularized bio-scaffold, a structure that was completely natural and had good biocompatibility. By engineering tissues, donor site morbidity can be avoided and large quantities of tissue with good histocompatibility can be produced  [14]. Scaffolds play an important role in tissue engineering because they provide tissue-specific compositions that influence the attachment and growth of cells. Common musculoskeletal tissue engineering scaffolds are primarily divided into synthetic and natural biomaterials. The application of scaffolds made from synthetic biomaterials is somewhat limited by their inherent lack of biological activity [20,21]. However, scaffolds made from natural biomaterials simulate the natural structure of biological tissue. Therefore, they may exhibit superior histocompatibility to synthetic scaffolds. In recent years, ECM-based scaffolds have shown great potential in tissue engineering [20]. Decellularized ECM from donor tissues has been used for constructing many complex tissues and organs [22][23][24][25]. Decellularization is an important process in the preparation of ECM scaffolds. By using chemical and physical processing, cell membranes and nuclei are disrupted, and the majority of cells and residual cellular components are removed from the tissues. This ensures that inflammatory and immune responses directed toward the scaffold are minimized, while the naturally occurring structure, organization, and biomechanical properties  of the ECM are maintained [14]. Therefore, decellularized ECM scaffolds closely mimic the natural tissue environment and have better porosity and biocompatibility, as well as adequate mechanical strength, which promote cellular attachment, proliferation, and differentiation [14,26]. The decellularized "book" bio-scaffold developed in this study, which was also found to mimic the natural tissue well, would be novel supplementary to the decellularized ECM scaffolds currently available.
It is difficult to remove all native cells due the dense nature of cartilage. Many methods have been developed to decellularize cartilage, and physical, chemical, and/or enzymatic techniques are usually used in combination. However, currently, there is no standard method for cartilage decellularization [14]. Consequently, in this study, a combination of physical, chemical, and enzymatic methods was used to decellularize the fibrocartilage scaffolds. Physical methods, which include thermal shock (freeze-thaw cycles), agitation in solution, physical factors, and manual disruption (pulverization or cutting to thin slices), can rupture cell membranes and facilitate the transport of decellularization solution to the cells and cellular material from the tissue [13,14,27]. Furthermore, by cutting tissues into particles or thin pieces, cells can easily be removed, while the natural composition of the ECM remains largely preserved [11,15,18]. Chemical treatments include detergents, solvents, acidic and alkaline solutions, and ionic solution. Sodium dodecyl sulfate, an ionic detergent, and Triton X-100, a nonionic detergent, are effective for the decellularization of cartilage tissue, but exposing tissues to these agents for too long can alter the mechanical properties of the cartilage ECM. It is often advantageous to use several chemicals in a series of short wash cycles to increase efficiency and reduce the time that tissue is exposed to any individual chemical [27]. DNases and RNases are also commonly used to remove nucleic acids from the material [14,28,29]. On the basis of the known efficacy of these existing techniques, the fibrocartilage in this study was first sliced into a "book" shape, a thin sheet structure with 100-μm-thick layers, and then decellularized by a combination of freeze-thaw cycles and treatments with Triton X-100, trypsin, and a nuclease, with continuous gentle agitation. Following this procedure, DNA quantification and histology analyses confirmed that cells were substantially removed from the scaffolds. Moreover, SEM analysis confirmed that the intrinsic ultrastructure of fibrocartilage tissue was well preserved. However, minimal nuclear material remained in the fibrocartilage at the end of the decellularization process. It is possible that this remaining material could result in mild host immune reaction, and this feature will be investigated in further in vivo study. A significant reduction in GAG and collagen content was also observed in fibrocartilage after decellularization, and this finding is consistent with the results of previous decellularization studies [24,28].
Fibrocartilage is mainly distributed in intervertebral and articular discs and the pubic symphysis. The pubic symphysis is a unique joint consisting of a fibrocartilaginous disc sandwiched between the articular surfaces of the pubic bones. Histologically, the fibrocartilage at the tendon-bone interface is similar to the fibrocartilage-bone interface at the symphysis pubis [30]. Therefore, in this research, fibrocartilage of the pubic symphysis was used as the donor for tissue engineering. The results of the current study provide a strong foundation for the production of a new three-phased multilayered decellularized "book" scaffold, which will include bone, cartilage, and tendon, and into which mesenchymal stem cells can be implanted and cultured. In future research, the related molecular biological mechanisms of the scaffolds will be explored using in vitro and animal experiments.
This study had some limitations, which are as follows. The investigation was performed in vitro; therefore, the biocompatibility of the natural scaffold in tendon-to-bone healing in vivo remains unknown. Secondly, the mechanical properties of the scaffold were not tested, nor were the experiments conducted using human subjects. Finally, GAGs is one of the main components of cartilage and play an important role in maintaining the structure of cartilage. The content of GAG was reduced in the current study. It was unknown how the biological activity were affected by the reduced GAG content.

Conclusions
In summary, a natural, porous, easy-to-use, multilayered, decellularized "book" fibrocartilage scaffold was developed in this ex vivo study. The acellular "book" scaffold represents an optimal carrier and effectively mimics the native environment and the structure of fibrocartilage. We expect its successful application in future tissue engineering, particularly for the reconstruction of the tendon-to-bone insertion.


extfieldsofstudy: Biology Medicine

text:
Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

Background The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. Methods and Results To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. Conclusions and Significance We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

DHAV-1 disease is a fatal, rapidly spreading viral infection of young ducklings that is characterized primarily by hepatitis [7]. DHAV-1 disease has spread worldwide and continues to be a threat to duck farms because of the high mortality associated with the disease. Mortality in the field often exceeds 50% and can reach 95% [8]. The hepatic clinical signs caused by DHAV-1, DHAV-2, and DHAV-3 are difficult to differentiate [2,3,5].
In this study, we used a mAb against VP1 protein (2D10) to screen a phage-display random 12-mer peptide library for the linear B-cell epitope of VP1. This report thus documents the first epitope analysis of the VP1 protein of DHAV-1. By furthering our understanding of the antigenic structure of VP1, our findings will facilitate the specific serologic diagnosis of DHAV-1 infection and will contribute to the rational design of effective vaccines.

Ethics Statement
Care of laboratory animals and animal experimentation were performed in accordance with animal ethics guidelines and approved protocols. All animal studies were approved by the Animal Ethics Committee of the Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences.

Indirect Immunofluorescence Assay
Detection of the VP1 protein of DHAV-1 or DHAV-3 by means of an immunofluorescence assay was performed according as previously described with modifications [19]. Briefly, confluent DEF cells were infected with DHAV-1 HP-1 or DHAV-3 JT virus (10 M.O.I.) and then incubated at 37°C for 72 h. The cells were fixed with cold methanol for 10 min and then probed with mAb 2D10 or uninfected mouse serum (as a control) for 1 h at 37°C. Then, 50 μL/well of FITC-conjugated goat anti-mouse IgG (KPL, MD, USA) at a 1:400 dilution was added and the cells were incubated for 1 h at 37°C. To confirm the location of the VP1 protein, the cells were then stained with DAPI as described previously [20]. The stained cells were viewed by using a Zeiss Axioplan-2 or a confocal LSM 700 (Carl Zeiss) fluorescence microscope.

In Vitro Neutralization Assay
Neutralizing antibody titers of mAb 2D10 were detected by using a virus-based neutralization assay as previously described [18]. Briefly, 100 μL of serial diluted mAb (initial dilution, 1:10; 2-fold dilutions to 1:320) was incubated with 100 μL 1×10 2.5 TCID 50 of DHAV-1 HP-1 for 2 h at 37°C. The virus-mAb mixture (200 μL) was then transferred onto a monolayer of DEF cells in a 96-well plate (triplicate wells). Sera from uninfected healthy mice (diluted in phosphatebuffered saline, PBS) and uninfected DEF cells served as controls. Cells were observed daily for cytopathic effects (CPE) for 7 days. Neutralization titers were read as the highest mAb dilution that protected >95% of the cells from CPE.

Epitope Mapping
The Ph.D-12 Phage Display Peptide Library Kit (New England BioLabs Inc) was used in this study. The mAb 2D10 was purified from mice ascites fluid by using Protein G Agorose (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Three successive rounds of biopanning were carried out in accordance with the manufacturer's instructions. Briefly, each well of a 96-well plate was coated with 10 μg/mL of mAb 2D10 in coating buffer overnight at 4°C, and then blocked with blocking buffer for 2 h at 4°C. The phage library was added to the plate and incubated for 1 h at room temperature. The unbound phages were removed by successive washings with TBS buffer containing gradually increased concentrations (0.1%, 0.3%, and 0.5%) of Tween-20, and the bound phages were eluted by 0.2 M glycine-HCl containing 1 mg/mL BSA and immediately neutralized with 1 M Tris-HCl. The eluted phages were amplified and titered on LB/IPTG/Xgal plates for the subsequent rounds of selection. The ratio of output to input was calculated as follows: titer of the amplified output phages/titer of the input phages (1.5×10 11 )×100%.

Phage ELISA and Sequencing of DNA Inserts Displayed by Phage Clones
After three rounds of biopanning, 15 individual phage clones were selected for target binding in the ELISA as described in manufacturer's instructions. Briefly, 96-well plates were coated with 100 ng of purified mAb 2D10, or anti-porcine IFN-c mAb (Sigma, St Louis, MO, USA) as negative a control overnight at 4°C. The coated wells were blocked for 2 h at room temperature and then the phages (10 10 pfu/100 μL/well) diluted in blocking solution were added. The plates were incubated for 1 h at room temperature and were then washed ten times with TBST. Bound phages were subjected to reaction with horseradish peroxidase (HRP)-conjugated sheep anti-M13 antibody (Pharmacia, Piscataway, NY, USA), followed by color development with the substrate solution containing o-phenylenediamine (OPD). The positive phage clones were sequenced with the sequencing primer as described in the manufacturer's instructions.

Immunological Analysis of Synthetic Peptides
Dot blotting was performed by spotting a synthesized peptide solution onto a nitrocellulose membrane. Approximately 1 μg of each synthesized peptide diluted with TNE buffer was spotted onto the nitrocellulose membrane. The membrane was then incubated with mAb 2D10 (diluted 1:2,000 in PBS) or anti-porcine IFN-c mAb (negative control) and duck-positive anti-DHAV-1 sera or healthy uninfected duck sera (negative control) (diluted 1:100 in PBS) at 37°C for 1 h. After being washed three times with PBST, the membranes were probed with a 1:500 dilution of HRP-conjugated goat anti-mouse IgG (KPL, MD, USA) or goat anti-duck IgG (KPL, MD, USA) at 37°C for 1 h, respectively.

Competitive Inhibition Binding Assay of mAb 2D10 to Synthetic Peptide
To test for synthetic peptide inhibition of mAb 2D10 binding to VP1, 100 μL of VP1 antigen (10μg/mL) was coated onto 96-well plates (at 4°C overnight). Plates were then blocked as described previously. The synthetic peptide LPAPTS (final peptide concentrations 0, 5, 10, 20, 40, 80, and 160 μg/ml) or an unrelated control peptide ( 19 YIRTPACWD 27 , from duck reovirus σB protein. [21]) was mixed with blocking mAb 2D10 (0.2 μg/ml diluted in PBST) and incubated at room temperature for 45 min; these peptide/antibody mixtures were then added to the VP1 antigen-coated 96-well plates and incubated at room temperature for 1 h. After the plate was washed with PBST, HRP-conjugated goat anti-mouse IgG was added and binding was assessed.

Sequence Analysis
To assess the conservation of the epitope among the DHAV viruses, we performed sequence alignment of the corresponding epitope regions on the VP1 proteins of seven DHAV-1 strains (Table 1), two DHAV-3 strains, and one DHAV-2 strain by using the DNASTAR Lasergene program (DNASTAR Inc., Madison, WI, USA).

Characterization and Neutralization Analysis of mAb 2D10
The immunofluorescence assay revealed that mAb 2D10 reacted with DHAV-1 HP-1-infected DEF cells (Fig. 1A), but not with DHAV-3 JT-infected or uninfected DEF cells (Fig. 1B). Green fluorescence was visualized in DHAV-1 HP-1-infected cells, indicating that mAb 2D10 detected the native-form of the VP1 protein in the DHAV-1 HP-1 infected cells. DAPI staining indicated that VP1 was mainly located in the cytoplasm of the infected cells (Fig. 1A), although some VP1 were detected in the nucleus, which is consistent with a previous report on the VP1 protein of FMDV [22]. The neutralizing activity of mAb 2D10 was then determined by use of an in vitro neutralization assay on DEF cells; mAb 2D10 neutralized the DHAV-1 HP1 virus with a neutralization titer (NT 50 ) of 40.

Phage Enrichment by Biopanning
To determine the epitope recognized by mAb 2D10, bio-panning of a phage display 12-mer random peptide library was performed with the purified mAb. After three rounds of biopanning, enrichment of phages bound to the mAb was obtained. The output to input ratios of the three rounds of bio-panning were 0.00009%, 0.037% and 0.78%.

Epitope Prediction
Fifteen phage clones were selected for their reactivity with mAb 2D10 after three rounds of bio-panning. These selected clones were further evaluated in a Phage ELISA. As shown in Fig. 2, eight phage clones (p1-p4 and p9-p12) showed specific reactivity with 2D10 (OD450 nm. 1.20) but did not react with anti-porcine IFN-c mAb (OD450 nm, <0.33). The other  7 phage (p5-p8 and p13-p15) showed less reactivity with 2D10 (OD450 nm, OD<0.55). The eight phage clones with high OD values were sequenced and found to contain the consensus sequence LPAPTS, which is identical to the sequence 173 LPAPTS 178 of the VP1 protein of DHAV-1 HP-1 ( Table 2).

Competitive Inhibition of Synthetic Peptide LPAPTS Binding to mAb 2D10
To confirm that peptide LPAPTS of VP1 is the epitope of 2D10, competitive binding assays were performed. These assays showed that the reactivity of mAb 2D10 with VP1 protein was inhibited   markedly by the synthetic antigen peptide LPAPTS in a dose-dependent manner (p < 0.05, Fig. 3). The control peptide ( 19 YIRTPACWD 27 , from duck reovirus σB protein) showed no inhibition.

Precise Epitope Defining
To verify that the identified motif represented the epitope recognized by mAb 2D10 and duck anti-DHAV-1 sera, peptides representing the motif LPAPTS were synthesized. Dot blot analysis showed that the peptide LPAPTS was recognized by mAb 2D10 and duck anti-DHAV-1 sera (Fig. 4), but did not reacted with mAb anti-porcine IFN-c or healthy duck sera, indicating that the motif represented a linear B-cell epitope of the VP1 protein of DHAV-1. To define the epitope precisely, C-and N-terminal deletion mutants of the LPAPTS motif were synthesized. We found that only the full-length LPAPTS peptide was recognized by mAb 2D10 or duck anti-DHAV-1 sera. Removal of one or two amino acids at either the N-or C-terminus of the peptides abolished antibody binding (Fig. 4), indicating that the peptide LPAPTS represented the minimal requirement for the reactivity of the epitope with mAb 2D10.

Epitope LPAPTS is Highly Conserved among DHAV-1 Strains
To investigate the conservation of the LPAPTS epitope, we aligned the VP1 partial sequence, including the epitope region identified in this study, with other DHAV-1 sequences available in GenBank ( Table 2). The alignment results showed that all of the amino acids in the motif region were identical among DHAV-1 strains (Fig. 6), but differed from the amino acids in the corresponding regions of DHAV-2 and DHAV-3, indicating that the motif represented a conserved epitope on the VP1 protein of DHAV-1.

Discussion
The VP1 protein of DHAV-1 is associated with virus neutralization and is, therefore, a major candidate antigen for vaccine development and disease serological diagnosis [18,23]. Epitope mapping using monoclonal antibodies has become a powerful means to study protein structure and has proved to be an effective strategy for vaccine development and disease diagnosis [24][25][26]. To our knowledge, there have been no previous reports of the linear epitope mapping of the VP1 of DHAV.
In this investigation, we first characterized mAb 2D10 against VP1 of DHAV-1. IFA showed that mAb 2D10 recognized DHAV-1 virus-infected DEF cells but not DHAV-3 virus-infected cells, confirming previous reports that DHAV-1 shows no cross reactivity with DHAV-3 [4,5,7,18]. IFA also showed that the VP1 protein was located mainly in the cytoplasm of DHAV-1-infected DEF cells, but DAPI staining revealed that some VP1 protein was also distributed in the nucleus of infected cells, which is consistent with the location of VP1 in FMDVinfected cells [22]. Most proteins enter the nucleus via nuclear localization signals (NLSs). NLSs have been shown to contain a continuous motif of basic amino acid residues [20,27,28]; When we scanned the VP1 protein sequence, we noticed a cluster of basic amino acids ( 189 KMKKRWKPR 197 ) (basic amino acids underlined) (S2 Table) at the C-terminal of the VP1 protein, which may represent an NLS. Whether this basic motif serves some nuclear import function should be evaluated. The linear epitope LPAPTS recognized by mAb 2D10 was identified by using a random phage display peptide library. Sequence analysis confirms that the epitope sequence was identical to 173 LPAPTS 178 of the VP1 protein of DHAV-1. Alignment of DHAV-1, DHAV-2, and DHAV-3 virus strain sequences demonstrated that this epitope sequence was highly conserved among DHAV-1 viruses, but not among DHAV-2 and DHAV-3 viruses, indicating that it is a DHAV-1 type-specific epitope. This finding supports previous reports that there is no cross reactivity between DHAV-1 and DHAV-2 or DHAV-1 and DHAV-3 [2,4,5,7,18]. Dot blotting assays with mAb 2D10 and N-or C-terminal deletion mutants of the epitope demonstrated that the peptide LPAPTS was the minimal unit required for 2D10 recognition (i.e. maximal binding to mAb 2D10). The peptide LPAPTS was also recognized by DHAV-1-positive duck serum, suggesting the importance of these six amino acids of the epitope in antibody-epitope binding reactivity. Moreover, the competitive inhibition assay of mAb 2D10 binding to synthetic LPAPTS and to truncated VP1 protein fragments, as detected by Western blotting, verify that LPAPTS was the epitope of the VP1 protein. The location of the epitope 173 LPAPTS 178 in the C-terminus of VP1 of DHAV-1 is consistent with recent studies of FMDV, CAV-9, EV-9, HPeV-1 and HPeV-2 viruses [9,[11][12][13][14][15][16][17]. Further experiments are necessary to confirm the functional role of this epitope.

Conclusions
In summary, a highly conserved neutralizing linear B-cell epitope on the VP1 protein of DHAV-1 was identified. This conserved epitope may have potential use in the development of DHAV-1-specific diagnostic assays and epitope-based marker vaccines.
Supporting Information S1


s2fieldsofstudy: Biology Chemistry

text:
Catch Me If You Can: The Link between Autophagy and Viruses

Autophagy is a process that mediates the degradation of cytoplasmic material, such as damaged organelles and protein aggregates, to maintain cellular homeostasis (Fig. 1) [1]. The autophagic pathway begins with the sequestration of organelles and portions of the cytoplasm via a double-membrane termed the isolation membrane (or phagophore), which can be derived from several cellular compartments (including the endoplasmic reticulum [ER], Golgi complex, ERGolgi intermediate compartment [ERGIC], mitochondria, or ER-mitochondria associated membranes [MAMs], as well as the plasma membrane) [2]. The isolation membrane expands to completely envelop the isolated contents in a double-membrane vesicle called the autophagosome, which then undergoes maturation through fusion with lysosomes to form autolyosomes [3]. A hallmark of canonical autophagy (or “macroautophagy”) is autophagic flux, in which lysosomal enzymes degrade the contents within the autolysome. Alternatively, early/late endosomes can fuse with autophagosomes, forming amphisomes that can then mature to autolysosomes, in which both endosomal and autophagosomal contents are degraded.

Autophagy during Viral Infections: A Blessing in Disguise?
Autophagy is thought to be an ancient process that may have evolved to combat infection by a number of intracellular pathogens [4]. Work from our laboratory has shown that autophagy is induced by placental-derived microRNAs (miRNAs) carried in exosomes to attenuate viral infections in non-placental cells [5]. Furthermore, human placental trophoblasts, the specialized cells that comprise the placenta, exhibit high levels of autophagy themselves, which might contribute to their resistance to viral infections [5]. Autophagy is often a constitutive process that occurs at basal levels to maintain cellular homeostasis. However, autophagy can also be induced in response to cellular stresses such as nutrient deprivation, the unfolded protein response (UPR), or oxidative stress [6]. Given the amount of stress a viral infection elicits within a host cell, it is not surprising that this event often triggers autophagy, which can function as either a proviral or antiviral pathway, depending on the viral inducer.
Autophagosome formation requires extensive membrane remodeling, which is also induced during the replication of positive-strand RNA viruses. Indeed, many positive strand RNA viruses including picornaviruses and flaviviruses induce the autophagic process during their replicative life cycles to generate the membranes necessary for the biogenesis of their replication organelles. In addition, a diverse array of other viruses also induce autophagy (reviewed in [4]), including members of the paramyxoviridae, orthomyxoviridae, togaviridae, and herpesviridae. During infection, viruses (and/or virally-encoded proteins) can be targeted for degradation by induction of the autophagic pathway as a means to control their replication. For example, Sindbis virus capsid protein is targeted to autophagosomes and degraded during the process of autophagic flux, which functions to suppress new virion formation [7]. Interestingly, several herpesviruses express proteins that directly inhibit the formation of autophagosomes, indicating that these viruses may have evolved strategies to evade the degradative nature of the autophagic pathway [4]. Indeed, decreased neurovirulence is observed in mice infected with a mutant herpes simplex virus-1 that is unable to block autophagosome formation [8].
Host cells induce the formation of autophagosomes through a variety of mechanisms and in response to several events during the viral life cycle. During antiviral signaling, engagement of vesicular stomatitis virus by the pattern recognition receptor Toll-7 at the cell surface induces an autophagy-dependent innate immune response mediated by phosphatidylinositol 3-kinase (PI3K)-Akt-signaling in Drosophila that limits viral replication [9,10]. In addition, autophagy controls Rift Valley fever virus (RVFV) replication in both flies and mammals via toll-like receptor signaling [11]. Some viruses induce autophagy at the earliest stages of their life cyclesmeasles virus (MeV) induces autophagy through binding to CD46, a cell surface receptor required for MeV entry [12]. At later stages of infection, expression of the MeV C protein is sufficient to induce a second wave of autophagy via interaction with immunity-associated GTPase family M (IRGM), a known regulator of autophagy [13]. Consistent with this, several other viruses have been shown to induce the formation of autophagosomes at late stages of their replicative life cycles, often as a consequence of the dramatic increases in protein production resulting from viral gene expression. For example, it has been reported that hepatitis C virus (HCV) induces autophagy through interaction with IRGM and ER stress by triggering the UPR [13,14]. The autophagic machinery has been shown to be involved in the initial translation of HCV RNA, but not maintenance of viral replication [15]. Several other viruses benefit from the induction of autophagy and have evolved strategies to directly manipulate the autophagic machinery in order to enhance their replication and/or egress. How Do Viruses Benefit from Autophagy?
Enteroviruses have been extensively studied for the beneficial role that autophagy plays in their replicative life cycles. Replication of several members of the enterovirus family including poliovirus (PV) and coxsackievirus B (CVB) is enhanced by virus-induced autophagy [16,17]. Both PV and CVB infection results in the formation of autophagosome-like double-membraned vesicles, which serve as scaffolding for viral RNA replication [16,18]. Thus, these viruses usurp the autophagic pathway to provide the membranes necessary for these replication sites. It has been suggested that acidification of vesicles, potentially maturing autophagosomes, during PV infection promotes maturation of virions to the infectious particles [19]. However, autophagydependent degradation is not required for PV replication [19]; thus, maturation of virions does not depend on the degradative capacity of autolysosomes. Furthermore, a recent report suggests that PV takes advantage of autophagy-dependent exocytosis to aid in the release of virions into the extracellular space prior to viral-mediated lysis of the host cell in a process termed autophagosome-mediated exit without lysis (AWOL) [20]. Consistent with this finding, CVB can also be released in microvesicles containing the autophagosomal marker, microtubuleassociated protein 1A/1B-light chain 3 (LC3-II) [21]. Unlike PV, CVB infection has been suggested to block the formation of autolysosomes, which has been hypothesized to be a mechanism to evade lysosomal degradation [17]. However, the role autophagic flux might play in CVB replication remains unclear. Although the precise mechanism(s) by which PV and CVB induce the autophagic pathway and manipulate select aspects of autophagy require further delineation, it is clear that autophagy plays an important role in the enhancement of enterovirus infection, most likely independent of autophagic flux.
In direct contrast to enteroviruses, MeV replication benefits from autophagic flux. Despite the completion of the autophagic maturation process, MeV proteins are not targeted for degradation by autophagosome-lysosome fusion [22]. Rather, induction of autophagy during MeV infection serves to prevent the induction of cell death. Given the role of autophagy in maintaining cellular homeostasis, it may not be surprising that infection activates this pro-survival pathway to prolong the life of the cell in order to generate a maximal number of progeny virions.
Similar to PV, autophagy also benefits the maturation of dengue virus (DENV) particles [23]. After replication in ER-associated vesicles, DENV particles are assembled in the ER and transported through the secretory pathway prior to their release into the extracelluar space. Within the Golgi complex, one of the viral surface glycoproteins, prM, is cleaved by the resident trans-Golgi protease furin, which results in the generation of infectious viral particles [24]. Inhibition of autophagy decreases the specific infectivity of extracellular virions, which corresponds with the observed decrease in prM cleavage on virions released from the cell.
Unlike enteroviruses, DENV directly benefits from a specific autophagy-dependent process termed "lipophagy" to increase its replication. Lipophagy represents an alternative pathway of lipid metabolism and is mediated by the degradative nature of autophagic flux. During DENV infection, lipid droplets colocalize with autolysosomes, which correlates with a decrease in cellular triglycerides [25]. Consequently, the released free fatty acids from lipid droplets are processed in the mitochondria via β-oxidation, resulting in an increase in cellular ATP. It has been proposed that this increase in ATP provides the source of energy required to facilitate the various processes involved in DENV replication. Thus, DENV infection induces autophagydependent lipid metabolism ("lipophagy") to facilitate its replication. In another study, it was proposed that inhibition of autophagosome formation dramatically limits, but does not completely abolish, DENV replication [26]. Thus, either DENV does not absolutely require autophagy for replication or there are additional, yet to be defined pathways that regulate autophagosome formation during infection.

What Mechanisms Mediate Viral Manipulation of Autophagy?
A recent report has shown that human parainfluenza virus type 3 (HPIV3) infection induces the accumulation of cytoplasmic autophagosomes by the direct inhibition of autophagic flux [27]. Autolysosome formation is dependent on the interaction of three SNAREs. Fusion of autophagosomes with lysosomes is dependent on binding of the adaptor protein SNAP29 with syntaxin-17, located on the autophagosomal membrane, and the late endosome/lysosome membrane protein VAMP8 [28]. Expression of the HPIV3 phosphoprotein (P) inhibits the interaction of syntaxin-17 with SNAP29 by specifically interacting with both SNARE domains of SNAP29, thus directly inhibiting the ability of autophagosomes to fuse with lysosomes [27]. The inhibition of autolysosome formation results in an increase in extracellular virion production by a currently unknown mechanism [27].
Accumulation of autophagosomes is also observed during influenza A virus (IAV) infection [29]. The viral matrix 2 (M2) ion-channel protein contains a highly conserved LC3-interacting region (LIR) that mediates its interaction with the autophagosome-associated component LC3. Upon infection, LC3 relocalizes to the plasma membrane in an M2 LIR-dependent manner. Disruption of M2-LC3 interactions decreases filamentous virion budding and stability. Thus, in addition to modulating cell death pathways [30], IAV infection may subvert autophagy to enhance transmission to new cells and/or hosts by increasing virion stability.
Human immunodeficiency virus-1 (HIV-1) infection also results in the subversion of the autophagic pathway to facilitate new virion formation [31]. The HIV-1 precursor protein Gag interacts with LC3, which facilitates the processing of Gag into the virion core structural proteins. Furthermore, the HIV-1 accessory protein Nef blocks autophagosome maturation through its interaction with Beclin-1, a central regulator of autophagy. Deletion of Nef from the viral genome leads to autophagy-dependent degradation of the viral capsid protein p24. Therefore, HIV-1 infection requires the induction of autophagy for Gag processing, but inhibits the degradative capacity of this pathway to increase virion production.

Perspectives
Despite our appreciation that many viruses utilize autophagy in a proviral manner, relatively little is known regarding the specific mechanisms by which viruses induce autophagy and/or manipulate this pathway for their gain. In addition, the term "autophagy" most commonly refers to macroautophagy, a non-selective process that mediates bulk degradation, but there are many selective forms of autophagy that are named after the degradation targets, including mitophagy (mitochondria); pexophagy (peroxisomes); aggrephagy (protein aggregates); glycophagy (glycogens); lipophagy (lipids); and, recently, ER-phagy (endoplasmic reticulum) (reviewed in [32]). It will, therefore, be important to determine if these specialized autophagic processes are exploited by viruses to enhance their infection, as has been described for DENV and lipophagy. Recent work from our laboratory has identified a novel regulator of a noncanonical autophagic pathway that functions independent of the core initiation machinery to limit enterovirus infection [33], suggesting that enteroviruses might target noncanonical forms of autophagy to maximize their replication. In addition, the recent identification of specific regulators of autophagosome maturation, such as syntaxin-17, SNAP29, and VAMP8, presents an exciting opportunity to further define the role of this critical autophagic process in limiting and/or enhancing viral infections. The identification of specific regulators of the various stages of autophagy may also serve to clarify seeming inconsistencies in the literature, which were often published prior to the identification of these regulators. Although the field of autophagy is rapidly progressing, there is still much to be learned regarding the specific molecules that regulate this tightly controlled pathway and the mechanisms by which viruses target these molecules to facilitate their replication.