Study of association between antibodies to non-HLA kidney self-antigens and progression to chronic immune injury after kidney transplantation.

BACKGROUND: Immune response to several kidney self-antigens (KSAg) such as Collagen IV (Col-IV), Perlecan (PL), and Fibronectin (FN) have been associated with antibody-mediated damage and poor allograft survival. Thus, the aim of this study was to determine if humoral immune responses to KSAg correlates with progression of chronic immune injury (CII) changes at 1 year or 2 years. METHODS: Kidney transplant recipients who underwent 1- or 2-year biopsies, with chronic interstitial inflammation (ci > 1) and/or glomerular membrane double contouring (cg > 0) were analyzed with matched controls. Sera were analyzed retrospectively for antibodies against KSAg using ELISA. The presence of antibodies to KSAg were compared at 0, 4, 12, and 24 months using logistic regression. RESULTS: We identified a cohort of 214 kidney transplant recipients. Of these, we identified 33 cases and matched 66 controls. Logistical regression showed an odds ratio of 1 with the confidence interval crossing 1 for the presence of response to KSAg at all the time points. CONCLUSIONS: Humoral immune responses to either KSAg alone or in combination with donor-specific anti-HLA antibodies are not associated with progression to CII at 1 and 2 years after kidney transplantation.

Circulating exosomes from brain death and cardiac death donors have distinct molecular and immunologic properties: A pilot study.

BACKGROUND AND AIMS: Comparison of donation after brain death (DBD) and donation after cardiac death (DCD) lung tissue before transplantation have demonstrated activation of pro-inflammatory cytokine pathway in DBD donors. The molecular and immunological properties of circulating exosomes from DBD and DCD donors were not previously described. METHODS: We collected plasma from 18 deceased donors (12 DBD and six DCD). Cytokines were analyzed by 30-Plex luminex Panels. Exosomes were analyzed for liver self-antigen (SAg), Transcription Factors and HLA class II (HLA-DR/DQ) using western blot. C57BL/6 animals were immunized with isolated exosomes to determine strength and magnitude of immune responses. Interferon (IFN)-γ and tumor necrosis factor-α producing cells were quantified by ELISPOT, specific antibodies to HLA class II antigens were measured by ELISA RESULTS: We demonstrate increased plasma levels of IFNγ, EGF, EOTAXIN, IP-10, MCP-1, RANTES, MIP-ß, VEGF, and interleukins - 6/8 in DBD plasma versus DCD. MiRNA isolated from exosome of DBD donors demonstrated significant increase in miR-421, which has been reported to correlate with higher level of Interleukin-6. Higher levels of liver SAg Collagen III (p = .008), pro-inflammatory transcription factors (NF-κB, p < .05; HIF1α, p = .021), CIITA (p = .011), and HLA class II (HLA-DR, p = .0003 and HLA-DQ, p = .013) were detected in exosomes from DBD versus DCD plasma. The circulating exosomes isolated from DBD donors were immunogenic in mice and led to the development of Abs to HLA-DR/DQ. CONCLUSIONS: This study provides potential new mechanisms by which DBD organs release exosomes that can activate immune pathways leading to cytokine release and allo-immune response.

Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

Accumulation of senescent cells contributes to age-related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) regulate many biological processes; however, the functional contributions of IGFBP2 in lung fibrosis remain largely unclear. Here, we report that intranasal delivery of recombinant IGFBP2 protects aged mice from weight loss and demonstrated antifibrotic effects after bleomycin lung injury. Notably, aged human-Igfbp2 transgenic mice reveal reduced senescence and senescent-associated secretory phenotype factors in alveolar epithelial type 2 (AEC2) cells and they ameliorated bleomycin-induced lung fibrosis. Finally, we demonstrate that IGFBP2 expression is significantly suppressed in AEC2 cells isolated from fibrotic lung regions of patients with IPF and/or pulmonary hypertension compared with patients with hypersensitivity pneumonitis and/or chronic obstructive pulmonary disease. Altogether, our study provides insights into how IGFBP2 regulates AEC2-cell-specific senescence and that restoring IGFBP2 levels in fibrotic lungs can prove effective for patients with IPF.

Association Between Antibodies Against Lung Self-Antigens and Gastroesophageal Reflux in Lung Transplant Candidates.

Gastroesophageal reflux (GER) and pretransplant antibodies against lung self-antigens (SAbs) collagen-V and/or k-alpha 1 tubulin are both independently associated with allograft dysfunction after lung transplantation (LTx). The role of GER in inducing lung injury and SAbs is unknown. We aimed to study the association between pre-LTx GER and SAbs. After IRB approval, we retrieved SAb assays conducted between 2015 and 2019 and collected 24 hour GER data for these patients. Patients were divided into 2 groups: no reflux (GER-) and pathologic reflux (GER+) to compare the prevalence of SAbs. Multivariate analysis was used to study the association between GER and SAbs in the whole cohort and in restrictive lung disease (RLD) and obstructive lung disease (OLD) subsets. Proximal esophageal reflux (PER) events ≥5 was considered abnormal. Patients (n = 134; 73 men) were divided into groups: GER- (54.5%, n = 73) and GER+ (45.5%, n = 61). The prevalence of GER was higher in the RLD than in the OLD subset (p < 0.001). The overall prevalence of SAbs was 53.7% (n = 72), higher in the GER+ than the GER- group (65.6% vs 43.8%, p = 0.012), but comparable between RLD and OLD subsets. Overall, SAbs were associated with GER (p = 0.012) and abnormal PER (p = 0.017). GER and abnormal PER increased the odds of SAbs in the RLD subset (OR [95% CI]: 2.825 [1.033-7.725], p = 0.040 and OR [95% CI]: 3.551 [1.271-9.925], p = 0.014, respectively) but not in the OLD subset. LTx candidates have a high prevalence of SAbs, which are significantly associated with GER and abnormal PER in patients with RLD.

A potential mechanism by which aspiration of duodenogastric fluid augments the risk for bronchiolitis obliterans syndrome after lung transplantation.

OBJECTIVE: Aspiration of duodenogastric refluxate may damage the respiratory epithelium of lung allografts in transplant recipients. We sought to define a mechanism by which aspiration of duodenogastric fluid augments the risk of bronchiolitis obliterans syndrome after lung transplant in a murine model. METHODS: We analyzed the immunological effects of acute aspiration of duodenogastric fluid (0.5 mL/kg) on transplant naive (strain DBA/2J) and transplanted mice (strain B6D2F1/J to strain DBA/2J). Serum antibodies to the lung self-antigens (SAgs) K-alpha1 tubulin and collagen-V were determined by enzyme-linked immunosorbent assay. Exosomes were isolated from serum, and immunoblot membranes were probed for antibodies to lung SAgs. Lung sections were assessed for fibrotic burden and obliterative bronchiolitis lesions by histologic and immunohistochemical analyses, including trichrome staining. RESULTS: Transplanted mice that received duodenogastric fluid developed higher levels of antibodies to the lung SAgs K-alpha1 tubulin and collagen-V and exosomes with lung SAgs on posttransplant days 14 and 28 than transplanted mice with sham aspiration or transplant naive mice (with and without aspiration). All lung allografts demonstrated severe grade A4 rejection on posttransplant day 14, with the highest mean fibrotic burden and mean number of obliterative bronchiolitis-like lesions per microscopic field on day 28 in recipients with aspiration. CONCLUSIONS: This study links aspiration of duodenogastric fluid after lung transplant to higher autoimmune responses to lung SAgs and the release of circulating exosomes with lung SAgs, which together promote sustained immune responses leading to extensive lung parenchymal damage and, ultimately, severe obliterative bronchiolitis-the histologic hallmark of bronchiolitis obliterans syndrome.

Vitamin D Supplementation: Association With Serum Cytokines in Pediatric Hematopoietic Stem Cell Transplantation.

Vitamin D deficiency is prevalent in pediatric patients presenting for hematopoietic stem cell transplantation (HSCT) and has been linked to poor clinical outcomes. Using the data from a randomized control trial, in this paper we explore the effects of vitamin D supplementation on circulating cytokine levels during pediatric HSCT (www.clinicaltrials.gov as NCT03176849). A total of 41 children, 20 received Stoss therapy and 21 children received standard of care vitamin D supplementation. Levels of 25(OH)D and 20 cytokines were assessed at baseline and day +30. Significantly (P < 0.05) higher levels of mostly proinflammatory cytokines, FGF, GCSF, TNFα, IL-2, IL-6, IP10 were detected pre-transplant for patients with low compared to those with normal vitamin D levels. In sex stratified models that compare changes in cytokines between Stoss vs. standard of care, females in the Stoss group show greater changes in mostly pro -inflammatory cytokines- IP-10 (P = 0.0047), MIG (P = 0.009), and RANTES (P = 0.0047), IL-2R (P = 0.07) and IL-6(P = 0.069). Despite a small sample size, these findings suggest vitamin D deficiency affects the pre-transplant cytokine milieu and higher doses of vitamin D (Stoss therapy) appears to influence proinflammatory cytokine responses in a sex specific manner during pediatric HSCT. Larger clinical trials are warranted to validate these results.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial-mesenchymal transition leading to chronic lung allograft dysfunction.

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p = .0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

Stoss therapy is safe for treatment of vitamin D deficiency in pediatric patients undergoing HSCT.

Vitamin D deficiency remains common among pediatric patients undergoing hematopoietic stem cell transplant (HSCT) despite both aggressive and standard of care strategies. This study examined the safety and efficacy of single high-dose oral vitamin D therapy (Stoss therapy) for treatment of vitamin D deficiency in HSCT recipients. Patients ages 1-21 years presenting for HSCT were randomized to receive either Stoss regimen plus weekly/daily supplementation or standard of care, per US Endocrine Society guidelines. Among the total 48 subjects, 22 (46%) were randomized to Stoss and 26 (54%) to control arms. Baseline 25-hydroxyvitamin D (25-OHD) levels were insufficient/deficient in total of 34 (71%) patients, without difference between treatment groups. The Stoss regimen was well tolerated and no toxicity was observed. At Day +30, mean 25-OHD levels were significantly higher (P = 0.04) with Stoss (42.3 ± 12 µg/l) compared to controls (35.6 ± 14.3 µg/l), and a higher proportion of Stoss patients had adequate vitamin D levels than controls (85% vs 65%). Stoss therapy is a safe and efficacious treatment option for vitamin D deficiency in children undergoing HSCT and may achieve sufficient levels more rapidly than standard of care. This trial was registered at www.clinicaltrials.gov as NCT03176849.

Residual endotoxin induces primary graft dysfunction through ischemia/reperfusion-primed alveolar macrophages.

Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAMs) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor NCMs. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAMs. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or coreceptor CD14 on donor TRAMs prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia/reperfusion-primed donor TRAMs.

Chronic Lung Allograft Dysfunction: Immune Responses Induced by Circulating Exosomes with Lung-Associated Self-Antigens.

Exosomes, nanovesicles shown to regulate physiological processes in vivo, have been implicated in pathological conditions including cancer, autoimmune disease, infectious disease, neurodegenerative disease, and allograft rejection. Studies of lung transplant recipients with primary graft dysfunction, respiratory viral infection, and (acute) rejection have demonstrated circulating exosomes containing donor-mismatched human leukocyte antigen and lung-associated self-antigens, K-alpha 1 tubulin and collagen V, indicating that exosomes are originating from the transplanted organ. These circulating exosomes likely play a role in activating immune responses that lead to increased risk of chronic lung allograft dysfunction, as exosomes efficiently present their antigens to the immune system by all known pathways of antigen recognition (i.e., direct, indirect, and semidirect pathways). Here, we discuss exosome biogenesis, describe their contents, and address the mechanism of exosome-mediated activation of immune responses that lead to allograft rejection, especially after lung transplantation.

A novel mechanism for immune regulation after human lung transplantation.

OBJECTIVE: Lung transplantation is therapeutic for end-stage lung disease, but survival is limited due to bronchiolitis obliterans syndrome and restrictive chronic lung allograft dysfunction. We sought a common denominator in lung transplant recipients, analyzing risk factors that trigger immune responses that lead to bronchiolitis obliterans syndrome. METHODS: We collected blood from patients who underwent lung transplant at our institution. Exosomes were isolated from the sera of recipients with risk factors for chronic rejection and from stable recipients. Exosomes were analyzed with western blot, using antibodies to lung self-antigens K alpha 1 tubulin and collagen-V, costimulatory molecules (costimulatory molecule 80, costimulatory molecule 86), transcription factors (nuclear factor kappa-light-chain-enhancer of activated B cells, hypoxia-inducible factor 1α, Class II Major Histocompatibility Complex Transactivator), and 20S proteasome. RESULTS: Of the 90 patients included, we identified 5 with grade 3 primary graft dysfunction, 5 without, 15 with respiratory viral infection, 10 with acute rejection, 10 with donor-specific antibodies (DSA), 5 without DSA, and 10 who were stable for exosome isolation. Recipients with grade 3 primary graft dysfunction, respiratory viral infection, acute rejection, and DSA had exosomes containing self-antigens; exosomes from stable recipients did not. Exosomes from recipients with grade 3 primary graft dysfunction, acute rejection, and DSA also demonstrated costimulatory molecule 80, costimulatory molecule 86, major histocompatibility complex class II, transcription factor, and 20S proteasome. CONCLUSIONS: Transplanted lungs with grade 3 primary graft dysfunction, symptomatic respiratory viral infection, acute rejection, and immune responses induce exosomes that contain self-antigens, costimulatory molecules, major histocompatibility complex class II, transcription factors, and 20S proteasome. Release of circulating exosomes post-transplant from the aforementioned stress-inducing insults augment immunity and may play an important role in the pathogenesis of bronchiolitis obliterans syndrome.

Lung Retransplantation for Chronic Rejection: A Single-Center Experience.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is nonreversible and remains the biggest obstacle to long-term survival after lung transplantation (LTx). Retransplantation is the sole definitive therapeutic option for CLAD. We analyzed our single-center experience with retransplantation as a treatment option for CLAD. METHODS: From March 1, 2010, to May 31, 2016, 419 consecutive patients underwent LTx at our institution; 29 of these procedures were retransplantations for CLAD. We analyzed demographic characteristics, lung allocation score, operation type, length of stay, and perioperative outcomes. Actuarial survival was estimated using Kaplan-Meier survival curves. RESULTS: In total, 29 of 419 patients (6.9%) underwent retransplantation for CLAD. Median time from primary LTx to retransplantation was 1,163 days (range: 304 to 3,971 days). Patients undergoing retransplantation were younger and had higher lung allocation scores than primary transplantation patients. Most LTx procedures were bilateral (93% of retransplantations, 95% of primary LTx). Rates of cardiopulmonary bypass, extracorporeal membrane oxygenation support for severe primary graft dysfunction, and re-exploration for bleeding were higher in retransplantation patients (p = 0.010, p = 0.019, and p = 0.029, respectively). One- and 5-year survival rates in the retransplantation group were similar to those of the primary LTx group (89.2% and 64.3% versus 89.7% and 58.2%, respectively; p = 0.79). CONCLUSIONS: Lung retransplantation is a viable treatment option for CLAD after LTx. In this study, retransplantation patients were younger, had higher lung allocation scores, and were more likely to require cardiopulmonary bypass and postoperative extracorporeal membrane oxygenation support than primary LTx patients. Postoperative length of stay and short- and mid-term survival were comparable with those of primary LTx patients.

Significant role for microRNA-21 affecting toll-like receptor pathway in primary graft dysfunction after human lung transplantation.

BACKGROUND: MicroRNAs (miRNAs) were recently identified as modulators of immune responses after human lung transplantation (LTx). This study was undertaken to assess the contribution of miRNAs to the pathogenesis of primary graft dysfunction (PGD) after LTx. METHODS: Of the 39 recipients, 14 (35.9%) developed Grade 3 PGD (i.e., severe PGD) within the first 72 hours of LTx. The remaining 25 recipients (64.1%) had Grade 2 or less PGD, and served as the control group. miRNAs were isolated from cells purified by bronchoalveolar lavage (BAL). Bioinformatic prediction and validation by luciferase reporter assays were performed to identify targets regulated by miR-21. Transfection of human monocytic cell line (THP-1) was conducted to determine miR-21's cellular function. RESULTS: Pilot miRNA profiling of donor BAL samples before implantation in PGD (n = 6) revealed significant upregulation in 44 miRNAs and downregulation in 80 miRNAs compared with control (n = 6). Validation using a separate cohort demonstrated significant underexpression of miR-21 in patients with severe PGD. Furthermore, underexpression of miR-21 levels was negatively correlated with clinical PGD grades (Grade 2 PGD vs Grade 0 PGD: p = 0.042; Grade 3 PGD vs Grade 0 PGD: p = 0.004). Molecular analysis demonstrated that miR-21 targeted key components in the toll-like receptor (TLR) signaling pathway, including TLR4, IRAK3 and CXCL10. Further, incubation of THP-1 cells with cell-free BAL from severe PGD resulted in transactivation of inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). In contrast, increased expression of miR-21 resulted in marked suppression of IL-1-ß and TNF-α production. CONCLUSIONS: Underexpression of miR-21 may lead to the development of severe PGD by activating key components of the TLR pathway.

Polyomavirus Reactivation and Immune Responses to Kidney-Specific Self-Antigens in Transplantation.

Humoral immune responses against donor antigens are important determinants of long-term transplant outcomes. Reactivation of the polyomavirus BK has been associated with de novo antibodies against mismatched donor HLA antigens in kidney transplantation. The effect of polyomavirus reactivation (BK viremia or JC viruria) on antibodies to kidney-specific self-antigens is unknown. We previously reported excellent 5-year outcomes after minimization of immunosuppression for BK viremia and after no intervention for JC viruria. Here, we report the 10-year results of this trial (n=193) along with a nested case-control study (n=40) to explore associations between polyomavirus reactivation and immune responses to the self-antigens fibronectin (FN) and collagen type-IV (Col-IV). Consistent with 5-year findings, subjects taking tacrolimus, compared with those taking cyclosporin, had less acute rejection (11% versus 22%, P=0.05) and graft loss (9% versus 22%, P=0.01) along with better transplant function (eGFR 65±19 versus 50±24 ml/min per 1.73 m2, P<0.001) at 10 years. Subjects undergoing immunosuppression reduction for BK viremia had 10-year outcomes similar to those without viremia. In the case-control study, antibodies to FN/Col-IV were more prevalent during year 1 in subjects with polyomavirus reactivation than in those without reactivation (48% versus 11%, P=0.04). Subjects with antibodies to FN/Col-IV had more acute rejection than did those without these antibodies (38% versus 8%, P=0.02). These data demonstrate the long-term safety and effectiveness of minimizing immunosuppression to treat BK viremia. Furthermore, these results indicate that polyomavirus reactivation associates with immune responses to kidney-specific self-antigens that may increase the risk for acute rejection through unclear mechanisms.

Lung Injury Combined with Loss of Regulatory T Cells Leads to De Novo Lung-Restricted Autoimmunity.

More than one third of patients with chronic lung disease undergoing lung transplantation have pre-existing Abs against lung-restricted self-Ags, collagen type V (ColV), and k-α1 tubulin (KAT). These Abs can also develop de novo after lung transplantation and mediate allograft rejection. However, the mechanisms leading to lung-restricted autoimmunity remain unknown. Because these self-Ags are normally sequestered, tissue injury is required to expose them to the immune system. We previously showed that respiratory viruses can induce apoptosis in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), the key mediators of self-tolerance. Therefore, we hypothesized that lung-tissue injury can lead to lung-restricted immunity if it occurs in a setting when Tregs are impaired. We found that human lung recipients who suffer respiratory viral infections experienced a decrease in peripheral Tregs. Pre-existing lung allograft injury from donor-directed Abs or gastroesophageal reflux led to new ColV and KAT Abs post respiratory viral infection. Similarly, murine parainfluenza (Sendai) respiratory viral infection caused a decrease in Tregs. Intratracheal instillation of anti-MHC class I Abs, but not isotype control, followed by murine Sendai virus infection led to development of Abs against ColV and KAT, but not collagen type II (ColII), a cartilaginous protein. This was associated with expansion of IFN-γ-producing CD4(+) T cells specific to ColV and KAT, but not ColII. Intratracheal anti-MHC class I Abs or hydrochloric acid in Foxp3-DTR mice induced ColV and KAT, but not ColII, immunity, only if Tregs were depleted using diphtheria toxin. We conclude that tissue injury combined with loss of Tregs can lead to lung-tissue-restricted immunity.

Maternal T-Cell Engraftment Interferes With Human Leukocyte Antigen Typing in Severe Combined Immunodeficiency.

OBJECTIVES: To report the laboratory investigation of a case of severe combined immunodeficiency (SCID) with maternal T-cell engraftment, focusing on the interference of human leukocyte antigen (HLA) typing by blood chimerism. METHODS: HLA typing was performed with three different methods, including sequence-specific primer (SSP), sequence-specific oligonucleotide, and Sanger sequencing on peripheral blood leukocytes and buccal cells, from a 3-month-old boy and peripheral blood leukocytes from his parents. Short tandem repeat (STR) testing was performed in parallel. RESULTS: HLA typing of the patient's peripheral blood leukocytes using the SSP method demonstrated three different alleles for each of the HLA-B and HLA-C loci, with both maternal alleles present at each locus. Typing results from the patient's buccal cells showed a normal pattern of inheritance for paternal and maternal haplotypes. STR enrichment testing of the patient's CD3+ T lymphocytes and CD15+ myeloid cells confirmed maternal T-cell engraftment, while the myeloid cell profile matched the patient's buccal cells. CONCLUSIONS: Maternal T-cell engraftment may interfere with HLA typing in patients with SCID. Selection of the appropriate typing methods and specimens is critical for accurate HLA typing and immunologic assessment before allogeneic hematopoietic stem cell transplantation.

Normothermic extracorporeal liver perfusion for donation after cardiac death (DCD) livers.

BACKGROUND: The susceptibility of extended criteria livers to ischemia reperfusion injury is a major obstacle in organ cold preservation. Normothermic extracorporeal liver perfusion (NELP) has been investigated to reduce ischemic damage, restore physiologic function, and assess viability of the liver prior to transplant. The goal of this study is to compare physiological parameters of livers maintained continuously on NELP to those preserved in cold solution. METHODS: Livers from 9 female landrace pigs were subjected to either 20 minutes (W20-NELP), 40 minutes (W40-NELP), or 60 minutes (W60-NELP) of warm ischemia followed by 6 hours of NELP followed by a 2-hour NELP evaluation phase. This was compared with 3 livers subjected to 40 minutes of warm ischemia time followed by 6 hours of cold storage (W40-Cold) and a 2-hour NELP evaluation phase. Groups were compared with the 2-way analysis of variance test. RESULTS: NELP stabilized transaminases accompanied by significant improvement in bile production and decline in lactate and INR values in all W-NELP groups. Histologic analysis demonstrated significant improvement from 0 hour (mild-to-moderate sinusoidal dilation and zone 3 necrosis) to the end of the NELP run (minimal necrosis and mild IRI). Comparison of W40-NELP and W40-Cold revealed greater bile production and oxygen extraction ratio in W40-NELP. In contrast, markers of cellular and functional damage were increased in the W40-Cold group. CONCLUSION: NELP improves metabolic and functional parameters of livers with either short or extended warm ischemia times compared with livers subjected to comparable cold ischemia times.

MicroRNA-144 dysregulates the transforming growth factor-ß signaling cascade and contributes to the development of bronchiolitis obliterans syndrome after human lung transplantation.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS), chronic lung allograft rejection, remains an impediment for the function of the transplanted organ. In this study, we defined the role of the microRNA (miRNA) miR-144 in fibroproliferation leading to BOS. METHODS: Biopsy specimens were obtained from 20 lung transplant recipients with BOS((+)) and 19 without BOS((-)). Expression of miR-144 and its target, transforming growth factor-ß (TGF-ß)-induced factor homeobox 1(TGIF1), were analyzed by real-time polymerase chain reaction and Western blot. Overexpression of miR-144 and luciferase reporter genes were performed to elucidate miRNA-target interactions. The function of miR-144 was evaluated by transfecting fibroblasts and determining the response to TGF-ß by analyzing Sma- and Mad-related family (Smads), fibroblast growth factor, TGF-ß, and vascular endothelial growth factor. Smooth muscle actin-α-positive stress fibers and F-actin filaments in lung fibroblasts were analyzed by immunofluorescence. RESULTS: Analysis of miR-144 in the biopsy specimens demonstrated 4.1 ± 0.8-fold increases in BOS(+) compared with BOS(-) patients, with a significant reduction in TGIF1 (3.6 ± 1.2-fold), a corepressor of Smads. In vitro transfection confirmed that over-expression of miR-144 results in a reduction in TGIF1 and an increase in SMAD2, SMAD4, fibroblast growth factor-6, TGF-ß, and vascular endothelial growth factor. Increasing miR-144 by transfecting, increased smooth muscle actin-α and fibronectin, and knockdown of miR-144 diminished fibrogenesis in MRC-5 fibroblasts. CONCLUSIONS: miR-144 is a critical regulator of the TGF-ß signaling cascade and is over-expressed in lungs with BOS. Therefore, miR-144 is a potential target toward preventing fibrosis leading to BOS after lung transplant.

Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.