Human leucocytes processed by fast-rate inertial microfluidics retain conventional functional characteristics.

The manufacturing of clinical cellular therapies is a complex process frequently requiring manipulation of cells, exchange of buffers and volume reduction. Current manufacturing processes rely on either low throughput open centrifugation-based devices, or expensive closed-process alternatives. Inertial focusing (IF) microfluidic devices offer the potential for high-throughput, inexpensive equipment which can be integrated into a closed system, but to date no IF devices have been approved for use in cell therapy manufacturing, and there is limited evidence for the effects that IF processing has on human cells. The IF device described in this study was designed to simultaneously separate leucocytes, perform buffer exchange and provide a volume reduction to the cell suspension, using high flow rates with high Reynolds numbers. The performance and effects of the IF device were characterized using peripheral blood mononuclear cells and isolated monocytes. Post-processing cell effects were investigated using multi-parameter flow cytometry to track cell viability, functional changes and fate. The IF device was highly efficient at separating CD14+ monocytes (approx. 97% to one outlet, approx. 60% buffer exchange, 15 ml min-1) and leucocyte processing was well tolerated with no significant differences in downstream viability, immunophenotype or metabolic activity when compared with leucocytes processed with conventional processing techniques. This detailed approach provides robust evidence that IF devices could offer significant benefits to clinical cell therapy manufacture.

Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

IL-6 and TGF-ß-Secreting Adoptively-Transferred Murine Mesenchymal Stromal Cells Accelerate Healing of Psoriasis-like Skin Inflammation and Upregulate IL-17A and TGF-ß.

Mesenchymal stromal cells (MSC) show promise as cellular therapeutics. Psoriasis is a chronic inflammatory disease affecting the skin and the joints. Injury, trauma, infection and medications can trigger psoriasis by disrupting epidermal keratinocyte proliferation and differentiation, which activates the innate immune system. Pro-inflammatory cytokine secretion drives a T helper 17 response and an imbalance of regulatory T cells. We hypothesized that MSC adoptive cellular therapy could immunomodulate and suppress the effector T cell hyperactivation that underlies the disease. We used the imiquimod-induced psoriasis-like skin inflammation model to study the therapeutic potential of bone marrow and adipose tissue-derived MSC in vivo. We compared the secretome and the in vivo therapeutic potential of MSC with and without cytokine pre-challenge ("licensing"). The infusion of both unlicensed and licensed MSC accelerated the healing of psoriatic lesions, and reduced epidermal thickness and CD3+ T cell infiltration while promoting the upregulation of IL-17A and TGF-ß. Concomitantly, the expression of keratinocyte differentiation markers in the skin was decreased. However, unlicensed MSC promoted the resolution of skin inflammation more efficiently. We show that MSC adoptive therapy upregulates the transcription and secretion of pro-regenerative and immunomodulatory molecules in the psoriatic lesion. Accelerated healing is associated with the secretion of TGF-ß and IL-6 in the skin and MSC drives the production of IL-17A and restrains T-cell-mediated pathology.

Meniscal and Articular Cartilage Predictors of Outcome After Revision ACL Reconstruction: A 6-Year Follow-up Cohort Study.

BACKGROUND: Meniscal and chondral damage is common in the patient undergoing revision anterior cruciate ligament (ACL) reconstruction. PURPOSE: To determine if meniscal and/or articular cartilage pathology at the time of revision ACL surgery significantly influences a patient's outcome at 6-year follow-up. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Patients undergoing revision ACL reconstruction were prospectively enrolled between 2006 and 2011. Data collection included baseline demographics, surgical technique, pathology, treatment, and scores from 4 validated patient-reported outcome instruments: International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Marx Activity Rating Scale. Patients were followed up at 6 years and asked to complete the identical set of outcome instruments. Regression analysis assessed the meniscal and articular cartilage pathology risk factors for clinical outcomes 6 years after revision ACL reconstruction. RESULTS: An overall 1234 patients were enrolled (716 males, 58%; median age, 26 years). Surgeons reported the pathology at the time of revision surgery in the medial meniscus (45%), lateral meniscus (36%), medial femoral condyle (43%), lateral femoral condyle (29%), medial tibial plateau (11%), lateral tibial plateau (17%), patella (30%), and trochlea (21%). Six-year follow-up was obtained on 79% of the sample (980/1234). Meniscal pathology and articular cartilage pathology (medial femoral condyle, lateral femoral condyle, lateral tibial plateau, trochlea, and patella) were significant drivers of poorer patient-reported outcomes at 6 years (IKDC, KOOS, WOMAC, and Marx). The most consistent factors driving outcomes were having a medial meniscal excision (either before or at the time of revision surgery) and patellofemoral articular cartilage pathology. Six-year Marx activity levels were negatively affected by having either a repair/excision of the medial meniscus (odds ratio range, 1.45-1.72; P≤ .04) or grade 3-4 patellar chondrosis (odds ratio, 1.72; P = .04). Meniscal pathology occurring before the index revision surgery negatively affected scores on all KOOS subscales except for sports/recreation (P < .05). Articular cartilage pathology significantly impaired all KOOS subscale scores (P < .05). Lower baseline outcome scores, higher body mass index, being a smoker, and incurring subsequent surgery all significantly increased the odds of reporting poorer clinical outcomes at 6 years. CONCLUSION: Meniscal and chondral pathology at the time of revision ACL reconstruction has continued significant detrimental effects on patient-reported outcomes at 6 years after revision surgery.

CpG 1018® adjuvant enhances Tdap immune responses against Bordetella pertussis in mice.

Bordetella pertussis is the causative agent of whooping cough (pertussis), a severe respiratory disease that can be fatal, particularly in infants. Despite high vaccine coverage, pertussis remains a problem because the currently used DTaP and Tdap vaccines do not completely prevent infection or transmission. It is well established that the alum adjuvant is a potential weakness of the acellular vaccines because the immunity provided by it is short-term. We aimed to evaluate the potential of CpG 1018® adjuvant to improve antibody responses and enhance protection against B. pertussis challenge in a murine model. A titrated range of Tdap vaccine doses were evaluated in order to best identify the adjuvant capability of CpG 1018. Antibody responses to pertussis toxin (PT), filamentous hemagglutinin (FHA), or the whole bacterium were increased due to the inclusion of CpG 1018. In B. pertussis intranasal challenge studies, we observed improved protection and bacterial clearance from the lower respiratory tract due to adding CpG 1018 to 1/20th the human dose of Tdap. Further, we determined that Tdap and Tdap + CpG 1018 were both capable of facilitating clearance of strains that do not express pertactin (PRN-), which are rising in prevalence. Functional phenotyping of antibodies revealed that the inclusion of CpG 1018 induced more bacterial opsonization and antibodies of the Th1 phenotype (IgG2a and IgG2b). This study demonstrates the potential of adding CpG 1018 to Tdap to improve immunogenicity and protection against B. pertussis compared to the conventional, alum-only adjuvanted Tdap vaccine.

A CpG 1018 adjuvanted neuraminidase vaccine provides robust protection from influenza virus challenge in mice.

Influenza virus infections pose a significant threat to global health. Vaccination is the main countermeasure against influenza virus spread, however, the effectiveness of vaccines is variable. Current seasonal influenza virus vaccines mostly rely on the immunodominant hemagglutinin (HA) glycoprotein on the viral surface, which usually leads to a narrow and strain-specific immune response. The HA undergoes constant antigenic drift, which can lead to a dramatic loss in vaccine effectiveness, requiring the annual reformulation and readministration of influenza virus vaccines. Recently, it has been demonstrated that the subdominant glycoprotein, neuraminidase (NA), is an attractive target for vaccine development. Here, we tested a newly developed recombinant influenza virus N1 neuraminidase vaccine candidate, named N1-MPP, adjuvanted with CpG 1018, a TLR9 agonist. Additionally, N2-MPP and B-NA-MPP vaccine constructs have been generated to cover the range of influenza viruses that are seasonally circulating in humans. These constructs have been characterized in vitro and in vivo regarding their functionality and protective potential. Furthermore, a trivalent NA-MPP mix was tested. No antigenic competition between the individual NA constructs was detected. By adjuvating the recombinant protein constructs with CpG 1018 it was possible to induce a strong and robust immune response against the NA, which provided full protection against morbidity and mortality after high lethal challenges in vivo. This study provides important insights for the development of a broadly protective NA-based influenza virus vaccine candidate.

Hamsters Protected from SARS-CoV-2 Delta Variant Challenge after Two Doses of Adjuvanted SARS-CoV-2 Recombinant Spike Protein (S-2P) and One Dose of Beta S-2P.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern negatively impact the effectiveness of vaccines. In this study, we challenge hamsters with the delta variant after 2- or 3-dose inoculations with SARS-CoV-2 vaccines constructed from stabilized prefusion spike proteins (S-2P) of Wuhan (W) and beta (B) variants. Compared to 3 doses of W S-2P, 2 doses of W S-2P followed by a third dose of B S-2P induced the highest neutralizing antibody titer against live SARS-CoV-2 virus and enhanced neutralization of omicron variant pseudovirus. Reduced lung live virus titer and pathology suggested that all vaccination regimens protect hamsters from SARS-CoV-2 delta variant challenge.

Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease.

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease.

Study protocol: a multicentre, open-label, parallel-group, phase 2, randomised controlled trial of autologous macrophage therapy for liver cirrhosis (MATCH).

INTRODUCTION: Liver cirrhosis is a growing global healthcare challenge. Cirrhosis is characterised by severe liver fibrosis, organ dysfunction and complications related to portal hypertension. There are no licensed antifibrotic or proregenerative medicines and liver transplantation is a scarce resource. Hepatic macrophages can promote both liver fibrogenesis and fibrosis regression. The safety and feasibility of peripheral infusion of ex vivo matured autologous monocyte-derived macrophages in patients with compensated cirrhosis has been demonstrated. METHODS AND ANALYSIS: The efficacy of autologous macrophage therapy, compared with standard medical care, will be investigated in a cohort of adult patients with compensated cirrhosis in a multicentre, open-label, parallel-group, phase 2, randomised controlled trial. The primary outcome is the change in Model for End-Stage Liver Disease score at 90 days. The trial will provide the first high-quality examination of the efficacy of autologous macrophage therapy in improving liver function, non-invasive fibrosis markers and other clinical outcomes in patients with compensated cirrhosis. ETHICS AND DISSEMINATION: The trial will be conducted according to the ethical principles of the Declaration of Helsinki 2013 and has been approved by Scotland A Research Ethics Committee (reference 15/SS/0121), National Health Service Lothian Research and Development department and the Medicine and Health Care Regulatory Agency-UK. Final results will be presented in peer-reviewed journals and at relevant conferences. TRIAL REGISTRATION NUMBERS: ISRCTN10368050 and EudraCT; reference 2015-000963-15.

Association Between Graft Choice and 6-Year Outcomes of Revision Anterior Cruciate Ligament Reconstruction in the MARS Cohort.

BACKGROUND: Although graft choice may be limited in the revision setting based on previously used grafts, most surgeons believe that graft choice for anterior cruciate ligament (ACL) reconstruction is an important factor related to outcome. HYPOTHESIS: In the ACL revision setting, there would be no difference between autograft and allograft in rerupture rate and patient-reported outcomes (PROs) at 6-year follow-up. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Patients who had revision surgery were identified and prospectively enrolled in this cohort study by 83 surgeons over 52 sites. Data collected included baseline characteristics, surgical technique and pathology, and a series of validated PRO measures. Patients were followed up at 6 years and asked to complete the identical set of PRO instruments. Incidence of additional surgery and reoperation because of graft failure were also recorded. Multivariable regression models were used to determine the predictors (risk factors) of PROs, graft rerupture, and reoperation at 6 years after revision surgery. RESULTS: A total of 1234 patients including 716 (58%) men were enrolled. A total of 325 (26%) underwent revision using a bone-patellar tendon-bone (BTB) autograft; 251 (20%), soft tissue autograft; 289 (23%), BTB allograft; 302 (25%), soft tissue allograft; and 67 (5%), other graft. Questionnaires and telephone follow-up for subsequent surgery information were obtained for 809 (66%) patients, while telephone follow-up was only obtained for an additional 128 patients for the total follow-up on 949 (77%) patients. Graft choice was a significant predictor of 6-year Marx Activity Rating Scale scores (P = .024). Specifically, patients who received a BTB autograft for revision reconstruction had higher activity levels than did patients who received a BTB allograft (odds ratio [OR], 1.92; 95% CI, 1.25-2.94). Graft rerupture was reported in 5.8% (55/949) of patients by their 6-year follow-up: 3.5% (16/455) of patients with autografts and 8.4% (37/441) of patients with allografts. Use of a BTB autograft for revision resulted in patients being 4.2 times less likely to sustain a subsequent graft rupture than if a BTB allograft were utilized (P = .011; 95% CI, 1.56-11.27). No significant differences were found in graft rerupture rates between BTB autograft and soft tissue autografts (P = .87) or between BTB autografts and soft tissue allografts (P = .36). Use of an autograft was found to be a significant predictor of having fewer reoperations within 6 years compared with using an allograft (P = .010; OR, 0.56; 95% CI, 0.36-0.87). CONCLUSION: BTB and soft tissue autografts had a decreased risk in graft rerupture compared with BTB allografts. BTB autografts were associated with higher activity level than were BTB allografts at 6 years after revision reconstruction. Surgeons and patients should consider this information when choosing a graft for revision ACL reconstruction.

Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy.

Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved good manufacturing practice (GMP)-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analysing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL:T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analysed using a t-stochastic neighbour embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using interleukin (IL)-2, tumour necrosis factor (TNF)-α and interferon (IFN)-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible, and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.

CpG-adjuvanted stable prefusion SARS-CoV-2 spike protein protected hamsters from SARS-CoV-2 challenge.

The COVID-19 pandemic presents an unprecedented challenge to global public health. Rapid development and deployment of safe and effective vaccines are imperative to control the pandemic. In the current study, we applied our adjuvanted stable prefusion SARS-CoV-2 spike (S-2P)-based vaccine, MVC-COV1901, to hamster models to demonstrate immunogenicity and protection from virus challenge. Golden Syrian hamsters immunized intramuscularly with two injections of 1 µg or 5 µg of S-2P adjuvanted with CpG 1018 and aluminum hydroxide (alum) were challenged intranasally with SARS-CoV-2. Prior to virus challenge, the vaccine induced high levels of neutralizing antibodies with 10,000-fold higher IgG level and an average of 50-fold higher pseudovirus neutralizing titers in either dose groups than vehicle or adjuvant control groups. Six days after infection, vaccinated hamsters did not display any weight loss associated with infection and had significantly reduced lung pathology and most importantly, lung viral load levels were reduced to lower than detection limit compared to unvaccinated animals. Vaccination with either 1 µg or 5 µg of adjuvanted S-2P produced comparable immunogenicity and protection from infection. This study builds upon our previous results to support the clinical development of MVC-COV1901 as a safe, highly immunogenic, and protective COVID-19 vaccine.

Chemokines and their receptors: predictors of the therapeutic potential of mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSCs) are promising cellular therapeutics for the treatment of inflammatory and degenerative disorders due to their anti-inflammatory, immunomodulatory and regenerative potentials. MSCs can be sourced from a variety of tissues within the body, but bone marrow is the most frequently used starting material for clinical use. The chemokine family contains many regulators of inflammation, cellular function and cellular migration-all critical factors in understanding the potential potency of a novel cellular therapeutic. In this review, we focus on expression of chemokine receptors and chemokine ligands by MSCs isolated from different tissues. We discuss the differential migratory, angiogenetic and immunomodulatory potential to understand the role that tissue source of MSC may play within a clinical context. Furthermore, this is strongly associated with leukocyte recruitment, immunomodulatory potential and T cell inhibition potential and we hypothesize that chemokine profiling can be used to predict the in vivo therapeutic potential of MSCs isolated from new sources and compare them to BM MSCs.

ACL Study Group survey reveals the evolution of anterior cruciate ligament reconstruction graft choice over the past three decades.

PURPOSE: Anterior cruciate ligament reconstruction (ACLR) aims to restore knee function and stability, allowing patients to return to the activities they enjoy and minimize further injury to the meniscus and cartilage and their ultimate progression to osteoarthritis. This study aims to present the evolution of graft choice over the last three decades according to members of the ACL Study Group (SG). METHODS: Prior to the January 2020 ACL SG biannual meeting, a survey was administered consisting of 87 questions and 16 categories, including ACLR graft choice. A similar questionnaire has been administered prior to each meeting and survey results from the past 14 meetings (1992 through 2020, excluding 1994) are included in this work. Survey responses are reported as frequencies in percentages to quantify changes in practice over the surgery period. RESULTS: In 1992, the most frequent graft choice for primary ACLR was bone-patellar tendon-bone (BTB) autograft, at nearly 90%. Hamstring tendon (HT) autografts have increased in popularity, currently over 50%, followed by just under 40% BTB autograft. Recently, quadriceps tendon (QT) autograft has increased in popularity since 2014. CONCLUSION: Autograft (HT, BTB, QT) is an overwhelming favorite for primary ACLR over allograft. The preference for HT autograft increased over the study period relative to BTB autograft, with QT autograft gaining in popularity in recent years. Graft selection should be individualized for each patient and understanding the global trends in graft choice can help orthopaedic surgeons discuss graft options with their patients and determine the appropriate graft for each case. LEVEL OF EVIDENCE: Level V, Expert Opinion.

Rate of infection following revision anterior cruciate ligament reconstruction and associated patient- and surgeon-dependent risk factors: Retrospective results from MOON and MARS data collected from 2002 to 2011.

Infection is a rare occurrence after revision anterior cruciate ligament reconstruction (rACLR). Because of the low rates of infection, it has been difficult to identify risk factors for infection in this patient population. The purpose of this study was to report the rate of infection following rACLR and assess whether infection is associated with patient- and surgeon-dependent risk factors. We reviewed two large prospective cohorts to identify patients with postoperative infections following rACLR. Age, sex, body mass index (BMI), smoking status, history of diabetes, and graft choice were recorded for each patient. The association of these factors with postoperative infection following rACLR was assessed. There were 1423 rACLR cases in the combined cohort, with 9 (0.6%) reporting postoperative infections. Allografts had a higher risk of infection than autografts (odds ratio, 6.8; 95% CI, 0.9-54.5; p = .045). Diabetes (odds ratio, 28.6; 95% CI, 5.5-149.9; p = .004) was a risk factor for infection. Patient age, sex, BMI, and smoking status were not associated with risk of infection after rACLR.

Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19.

The COVID-19 pandemic is a worldwide health emergency which calls for an unprecedented race for vaccines and treatment. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein, S-2P. To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of neutralizing antibodies in sera of immunized mice against pseudotyped lentivirus reporter or live wild-type SARS-CoV-2. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018 and alum as a candidate vaccine to prevent COVID-19 disease.

The use of mesenchymal stromal cells in the treatment of coronavirus disease 2019.

More than seven months into the coronavirus disease -19 (COVID-19) pandemic, infection from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to over 21.2 million cases and resulted in over 760,000 deaths worldwide so far. As a result, COVID-19 has changed all our lives as we battle to curtail the spread of the infection in the absence of specific therapies against coronaviruses and in anticipation of a proven safe and efficacious vaccine. Common with previous outbreaks of coronavirus infections, SARS and Middle East respiratory syndrome, COVID-19 can lead to acute respiratory distress syndrome (ARDS) that arises due to an imbalanced immune response. While several repurposed antiviral and host-response drugs are under examination as potential treatments, other novel therapeutics are also being explored to alleviate the effects on critically ill patients. The use of mesenchymal stromal cells (MSCs) for COVID-19 has become an attractive avenue down which almost 70 different clinical trial teams have ventured. Successfully trialled for the treatment of other conditions such as multiple sclerosis, osteoarthritis and graft versus host disease, MSCs possess both regenerative and immunomodulatory properties, the latter of which can be harnessed to reduce the severity and longevity of ARDS in patients under intensive care due to SARS-CoV-2 infection.

Meniscal Repair in the Setting of Revision Anterior Cruciate Ligament Reconstruction: Results From the MARS Cohort.

BACKGROUND: Meniscal preservation has been demonstrated to contribute to long-term knee health. This has been a successful intervention in patients with isolated tears and tears associated with anterior cruciate ligament (ACL) reconstruction. However, the results of meniscal repair in the setting of revision ACL reconstruction have not been documented. PURPOSE: To examine the prevalence and 2-year operative success rate of meniscal repairs in the revision ACL setting. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: All cases of revision ACL reconstruction with concomitant meniscal repair from a multicenter group between 2006 and 2011 were selected. Two-year follow-up was obtained by phone and email to determine whether any subsequent surgery had occurred to either knee since the initial revision ACL reconstruction. If so, operative reports were obtained, whenever possible, to verify the pathologic condition and subsequent treatment. RESULTS: In total, 218 patients (18%) from 1205 revision ACL reconstructions underwent concurrent meniscal repairs. There were 235 repairs performed: 153 medial, 48 lateral, and 17 medial and lateral. The majority of these repairs (n = 178; 76%) were performed with all-inside techniques. Two-year surgical follow-up was obtained on 90% (197/218) of the cohort. Overall, the meniscal repair failure rate was 8.6% (17/197) at 2 years. Of the 17 failures, 15 were medial (13 all-inside, 2 inside-out) and 2 were lateral (both all-inside). Four medial failures were treated in conjunction with a subsequent repeat revision ACL reconstruction. CONCLUSION: Meniscal repair in the revision ACL reconstruction setting does not have a high failure rate at 2-year follow-up. Failure rates for medial and lateral repairs were both <10% and consistent with success rates of primary ACL reconstruction meniscal repair. Medial tears underwent reoperation for failure at a significantly higher rate than lateral tears.

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). METHODS: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. RESULTS: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. CONCLUSIONS: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization.

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes.