T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation.

Limb transplantation is a life-changing procedure for amputees. However, limb recipients have a 6-fold greater rejection rate than solid organ transplant recipients, related in part to greater immunogenicity of the skin. Here, we report a detailed immunological and molecular characterization of individuals who underwent bilateral limb transplantation at our institution. Circulating Th17 cells are increased in limb transplant recipients over time. Molecular characterization of 770 genes in skin biopsies reveals upregulation of T cell effector immune molecules and chemokines, particularly CCL18. Skin antigen-presenting cells primarily express the chemokine CCL18, which binds to the CCR8 receptor. CCL18 treatment recruits more allo-T cells to the skin xenograft in a humanized skin transplantation model, leading to signs of accelerated graft rejection. Blockade of CCR8 remarkedly decreases CCL18-induced allo-T cell infiltration. Our results suggest that targeting the CCL18:CCR8 pathway could be a promising immunosuppressive approach in transplantation.

T cell depletion increases humoral response by favoring T follicular helper cells expansion.

Antibody-mediated rejection is a major cause of long-term graft loss in kidney transplant patients. T follicular helper (Tfh) cells are crucial for assisting B cell differentiation and are required for an efficient antibody response. Anti-thymocyte globulin (ATG) is a widely used lymphocyte-depleting induction therapy. However, less is known about how ATG affects Tfh cell development and donor-specific antibody (DSA) formation. We observed an increase in circulating Tfh cells at 6 months after kidney transplant in patients who received ATG. Using an NP-OVA immunization model, we found that ATG-treated mice had a higher percentage of Tfh cells, germinal center B cells, and higher titers of antigen-specific antibodies compared to controls. ATG-treated animals had lower levels of IL-2, a known Bcl-6 repressor, but higher levels of IL-21, pSTAT3 and Bcl-6, favoring Tfh differentiation. In a mouse kidney transplant model, ATG-treated recipients showed an increase in Tfh cells, DSA and C4d staining in the allograft. Although ATG was effective in depleting T cells, it favored the expansion of Tfh cells following depletion. Concomitant use of IL-2, tacrolimus, or rapamycin with ATG was essential to control Tfh cell expansion. In summary, ATG depletion favors Tfh expansion, enhancing antibody-mediated response.

Shockwave Therapy Plus Eccentric Exercises Versus Isolated Eccentric Exercises for Achilles Insertional Tendinopathy: A Double-Blinded Randomized Clinical Trial.

BACKGROUND: There remains a lack of consensus regarding the treatment of Achilles insertional tendinopathy. The condition is typically treated with eccentric exercises despite the absence of satisfactory and sustained results. Shockwave therapy was presented as an alternative, but there is a paucity of literature, with good outcomes, supporting its use. The purpose of the present single-center, double-blinded, placebo-controlled, randomized trial was to determine if the use of shockwave therapy in combination with eccentric exercises improves pain and function in patients with Achilles insertional tendinopathy. METHODS: A total of 119 patients with Achilles insertional tendinopathy were evaluated and enrolled in the study from February 2017 to February 2019. Patients were allocated to 1 of 2 treatment groups, eccentric exercises with extracorporeal shockwave therapy (SWT group) and eccentric exercises with sham shockwave therapy (control group). Three sessions of radial shockwaves (or sham treatment) were performed every 2 weeks and eccentric exercises were undertaken for 3 months. The primary outcome was the Victorian Institute of Sport Assessment-Achilles questionnaire (VISA-A) at 24 weeks. Secondary outcomes included the visual analogue scale, algometry, the Foot and Ankle Outcome Score, and the 12-Item Short Form Health Survey. RESULTS: Both groups showed significant improvement during the study period; however, there were no between-group differences in any of the outcomes (all p >0.05). At the 24-week evaluation, the SWT group exhibited a mean VISA-A of 63.2 (95% confidence interval, 8.0) compared with 62.3 (95% confidence interval, 6.9) in the control group (p = 0.876). There was a higher rate of failure (38.3%) but a lower rate of recurrence (17.0%) in the SWT group compared with the control group (11.5% and 34.6%, respectively; p = 0.002 and p = 0.047). There were no complications reported for either group. CONCLUSIONS: Extracorporeal shockwave therapy does not potentiate the effects of eccentric strengthening in the management of Achilles insertional tendinopathy. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection.

BACKGROUND: Developing a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells' proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection. METHODS: Using 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets. RESULTS: An exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature's negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell-mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature's negative predictive value was 90.6% and its positive predictive value was 77.8%. CONCLUSIONS: Our findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.

MMP3 Is a Non-invasive Biomarker of Rejection in Skin-Bearing Vascularized Composite Allotransplantation: A Multicenter Validation Study.

Background: There is unmet need for non-invasive immunomonitoring to improve diagnosis and treatment of acute rejection in vascularized composite allotransplantation (VCA). Circulating matrix metalloproteinase 3 (MMP3) was described as a candidate non-invasive biomarker to predict treatment response to acute rejection in clinical VCA. However, larger validation studies are yet to be reported to allow for more definitive conclusions. Methods: We retrospectively measured MMP3 levels using ELISA in a total of 140 longitudinal serum samples from six internal and three external face transplant recipients, as well as three internal and seven external upper extremity transplant recipients. The control groups comprised serum samples from 36 kidney transplant recipients, 14 healthy controls, and 38 patients with autoimmune skin disease. A linear mixed model was used to study the effect of rejection state (pre-transplant, no-rejection, non-severe rejection (NSR), and severe rejection) on MMP3 levels. Results: In VCA, MMP3 levels increased significantly (p < 0.001) between pre- and post-transplant no-rejection states. A further increase occurred during severe rejection (p < 0.001), while there was no difference in MMP3 levels between non-severe and no-rejection episodes. A threshold of 5-fold increase from pre-transplant levels could discriminate severe from NSR with 76% sensitivity and 81% specificity (AUC = 0.79, 95% CI = 0.65-0.92, p < 0.001). In kidney transplantation, the MMP3 levels were significantly (p < 0.001) elevated during antibody-mediated rejection but not during T-cell mediated rejection (TCMR) (p = 0.547). MMP3 levels in healthy controls and autoimmune skin disease patients were comparable with either pre-transplant or no-rejection/NSR episodes of VCA patients. Conclusion: The results of this study suggest that serum MMP3 protein is a promising marker for stratifying patients according to severity of rejection, complementary to biopsy findings.

Shockwave Therapy Associated With Eccentric Strengthening for Achilles Insertional Tendinopathy: A Prospective Study.

Background. The usual initial treatment for insertional Achilles tendinopathy is nonsurgical. Yet there is no standard conservative treatment for Achilles insertional tendinopathy. Shockwave therapy (SWT) has become a reliable option for the management of this illness over the past years. The aim of this study is to report the effectiveness of low-energy SWT associated with an eccentric strengthening protocol in 19 consecutive patients. Methods. This is a prospective study with 19 patients aged between 26 and 72 years diagnosed with insertional Achilles tendinopathy. The protocol consisted of SWT associated with eccentric exercises for 12 weeks. All patients were evaluated on the first day and after 24 weeks (final follow-up) with the Victorian Institute of Sports Assessment-Achilles (VISA-A) score, visual analogue scale (VAS), American Orthopaedic Foot and Ankle Society (AOFAS) questionnaire, and by algometry. At the last follow-up, patients were also assessed for adherence to the protocol, complications and final outcome (in their perception as success or fail). Results and Conclusion. Fifteen (79%) patients were fully adherent to the Alfredson protocol, and 13 (68%) patients considered the treatment protocol successful. At the last evaluation, patients demanded higher pressure on calcaneus to trigger pain (algometry 1), reported less pain when the algometer was applied with 3 kg (algometry 2), had less global pain (VAS), and had higher AOFAS and VISA-A scores. This study evidences that eccentric loading associated with SWT can dramatically improve patients' symptoms. We can conclude that eccentric loading associated with SWT is an effective treatment for Achilles insertional tendinopathy. Levels of Evidence: Therapeutic, Level III: Prospective cohort.

Shock wave therapy associated with eccentric strengthening versus isolated eccentric strengthening for Achilles insertional tendinopathy treatment: a double-blinded randomised clinical trial protocol.

BACKGROUND: There is no consensus regarding the treatment of Achilles insertional tendinopathies. Eccentric training remains the main choice in the conservative treatment of this illness; however, the good results in the management of non-insertional Achilles tendinopathy were not replicated in the insertional condition. Low energy shock wave therapy has been described as an alternative to these patients, but has yet to be empirically tested. HYPOTHESIS: Shock wave therapy, adjunctive to the eccentric strengthening protocol, will improve measures of pain and function. DESIGN: Double blind, placebo-controlled, parallel groups, randomised clinical trial. MATERIALS AND METHODS: 93 patients with a diagnosis of chronic insertional tendinopathy, referred from primary or secondary healthcare services, will be assessed and enrolled in this study. They will be divided into two groups (randomised by sequentially numbered identical envelopes, which will be administered serially to participants), one containing the combination of low energy shock wave and eccentric exercises, as treatment and the other comprehending the exercises and the placebo treatment (an apparatus placed in the therapeutic head). The assessments will occur in 2, 4, 6, 12 and 24 weeks. Patients will be evaluated primarily by the Victorian Institute of Sport Assessment-Achilles questionnaire and secondarily by the visual analogue scale, Algometry, the American Orthopedic Foot and Ankle Society scale, the Foot and Ankle Outcome Score and the 12-item Short Form Health Survey. We will use comparison of two proportions via relative frequency analysis, the Pearson Correlation the χ2 test and the analysis of variance for statistical analyses. DISCUSSION: This study intends to demonstrate if the association of the eccentric exercise programme with the shock wave therapy can produce good results regarding the treatment of the Achilles insertional tendinopathy. In an attempt to prevent the high costs and complications associated with the surgical intervention, we will try to prove this combination as a viable therapeutic option in the conservative management of this prevalent condition. The strengths of the study are the design and the novelty of the combination of methods. The main limitation is the short follow-up course. ETHICS AND DISSEMINATION: The study is registered in the Clinical Trials database (protocol number: 8094833648737701) and was approved by the University Ethics Committee (number: 1373481). TRIAL REGISTRATION NUMBER: 8094833648737701 (NCT02757664); Pre-results.