Immunofilaments Are Well Tolerated after Local or Systemic Administration in Mice.

The invention of nanosized biomaterials has paved the way for novel therapeutics that can manipulate cells on a nanoscale. Nanosized immunofilaments (IFs) are synthetic filamentous polymers consisting out of polyisocyanopeptides, which have been recently established as a powerful platform to activate specific immune cells in vivo such that they raise an antitumor immune response. However, toxicological effects or immunogenicity toward the IFs have not yet been investigated. In this study, we evaluated potential toxic or immunogenic effects in C57BL/6 mice upon intravenous or subcutaneous injection of nonfunctionalized IFs or immunostimulatory IFs over 30 days. We here present a detailed analysis of the gross pathology, hematological parameters, blood biochemistry, histology, and antibody-response against the IF backbone. Our results demonstrate that IFs do not induce severe acute or chronic toxicity in mice. After 30 days, we only found elevated IgG-titers in intravenously injected but not subcutaneously injected mice. In summary, we demonstrate that IFs can be administered into a living organism without adverse side effects, thereby establishing the safety of IFs as a therapeutic intervention.

24 Hours Ex-vivo Hypothermic Acellular Perfusion of Porcine Forelimb: A 7-day Follow-up Study.

BACKGROUND: One of the limiting factors for vascularized composite allograft (VCA) storage is the short viable ischemic time (4-6 hours). Hypothermic machine perfusion (MP) enables near-physiological preservation, avoiding the deleterious effects of hypoxia and static cooling. This study aims to compare muscle injury after 24-hour acellular perfusion with static cold storage (SCS) in a porcine limb replantation model, examining outcomes for up to 7 days after reperfusion. METHODS: Sixteen procured porcine forelimbs were perfused hypothermic for 24 hours with Histidine-Tryptophan-Ketoglutarate (HTK, n=8) or preserved on ice for 4 hours (SCS, n=8) before orthotopic replantation. Muscle injury was assessed using biochemical markers and muscle biopsies were analyzed using the Histologic Injury Severity Score (HISS). RESULTS: During preservation, limb weight decreased by 2% in the SCS group and increased by 44% in the perfusion group (p<0.001). Twelve limbs (HTK, n=6; SCS, n=6) survived for 7 days. Three days after replantation, increased creatinine kinase levels were observed in the perfusion group (33781 mmol/liter versus 2163 mmol/liter; p<0.001). Mean endpoint HISS was 3.8 (SD 0.7) in the perfusion group and 1.8 (SD 0.7) in the SCS group (p=0.008), mostly due to increased edema (p=0.004). CONCLUSION: 24 hours of hypothermic MP and 4 hours of SCS of VCA demonstrated both minimal degenerated muscle tissue seven days after replantation.

Convolutional Neural Networks for the Evaluation of Chronic and Inflammatory Lesions in Kidney Transplant Biopsies.

In kidney transplant biopsies, both inflammation and chronic changes are important features that predict long-term graft survival. Quantitative scoring of these features is important for transplant diagnostics and kidney research. However, visual scoring is poorly reproducible and labor intensive. The goal of this study was to investigate the potential of convolutional neural networks (CNNs) to quantify inflammation and chronic features in kidney transplant biopsies. A structure segmentation CNN and a lymphocyte detection CNN were applied on 125 whole-slide image pairs of periodic acid-Schiff- and CD3-stained slides. The CNN results were used to quantify healthy and sclerotic glomeruli, interstitial fibrosis, tubular atrophy, and inflammation within both nonatrophic and atrophic tubuli, and in areas of interstitial fibrosis. The computed tissue features showed high correlation with Banff lesion scores of five pathologists (A.A., A.Dend., J.H.B., J.K., and T.N.). Analyses on a small subset showed a moderate correlation toward higher CD3+ cell density within scarred regions and higher CD3+ cell count inside atrophic tubuli correlated with long-term change of estimated glomerular filtration rate. The presented CNNs are valid tools to yield objective quantitative information on glomeruli number, fibrotic tissue, and inflammation within scarred and non-scarred kidney parenchyma in a reproducible manner. CNNs have the potential to improve kidney transplant diagnostics and will benefit the community as a novel method to generate surrogate end points for large-scale clinical studies.

Ex Vivo Machine Thrombolysis Reduces Rethrombosis Rates in Salvaged Thrombosed Myocutaneous Flaps in Swine.

BACKGROUND: There is a risk for thrombotic complications (2 to 5 percent) associated with microsurgical reconstruction. Current thrombolytic therapy has a salvage rate between 60 and 70 percent, but it is afflicted by bleeding complications (2 to 6 percent). The use of machine perfusion for delivering thrombolytic agents is a new method that could potentially reduce these complications. In this article, the authors compared flap salvage outcomes comparing machine thrombolysis versus a manual flush with tissue plasminogen activator. METHODS: Sixteen bilateral flaps (12 × 9 cm) were dissected from eight female Dutch Landrace pigs (70 kg). Thrombosis was induced in free rectus abdominis flaps by clamping the pedicle's veins for 2 hours. Flaps were either thrombolysed with 2 mg tissue plasminogen activator (1 mg/ml) during 2 hours of machine perfusion (perfusion group; n = 8) or injected intraarterially (manual group; n = 8) before replantation. Near-infrared fluorescence angiography was used to confirm thrombus formation and to assess tissue perfusion; muscle biopsy specimens were analyzed for ischemia/reperfusion injury directly after thrombolysis and 15 hours after replantation. RESULTS: A higher incidence of secondary thrombosis was seen in the manual group compared to the perfusion group ( n = 6 versus n = 0, respectively; p < 0.001), resulting in two complete flap failures. Fifteen hours after replantation, mean fluorescence intensities were 13.0 (95 percent CI, 10.1 to 15.8) and 24.6 (95 percent CI, 22.0 to 27.2) in the perfusion and manual group, respectively ( p < 0.001), and mean muscle injury scores were comparable, measuring 7.5 ± 1.5. CONCLUSION: Two hours of machine thrombolysis of compromised flaps in a porcine model showed higher salvage rates compared to a manual injection with tissue plasminogen activator and reduced the incidence of secondary thrombosis. CLINICAL RELEVANCE STATEMENT: Using machine perfusion systems for ex vivo thrombolysis provides the benefits of local treatment of a composite tissue without the risk of systemic complications and may improve salvage rates and reduce the incidence of secondary thrombosis.

Atypical fibroxanthoma and pleomorphic dermal sarcoma: Is superficial infiltration in subcutaneous tissue acceptable in AFX?

BACKGROUND: Atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS) are rare cutaneous neoplasms forming a spectrum. Case reports with recurrences and metastasis have been published despite the current view that AFX is benign. The aim of this study was to identify clinical and histopathological features that predict tumor recurrence. METHODS: A retrospective review of AFX and PDS cases was performed. Clinical characteristics were obtained from patient records. RESULTS: A total of 29 AFX and 23 PDS cases were identified. Review led to re-classification of 12 cases (18%). In 14/50 (26.9%) cases a recurrence occurred. Recurrences were significantly more likely to occur when the tumor showed any infiltration in the subcutaneous fat (100% vs 43.2%, p = 0.000) or when the tumor diameter exceeded 2 cm (46.2% vs 16.2%, p = 0.030). CONCLUSIONS: This study shows that histopathological distinction between AFX and PDS remains difficult with reclassification in 12 out of 52 (18%) cases upon review. All AFX cases solely confined to the dermis behaved benign. We therefore advocate to classify all cases with any form of subcutaneous extension as PDS, and only lesions without as AFX. This contrasts with the current general opinion in which superficial subcutaneous invasion is still accepted in AFX.

24-hour Perfusion of Porcine Myocutaneous Flaps Mitigates Reperfusion Injury: A 7-day Follow-up Study.

BACKGROUND: Static cold storage is the gold standard of preservation in vascularized composite allotransplantation and allows a preservation time of 4-6 hours. Machine preservation is a promising technique for prolonged preservation; however, studies on extended preservation that compare different preservatives are scarce. This study aims to assess the feasibility of 24-hour acellular perfusion and compares different preservation solutions in a porcine myocutaneous flap replantation model. METHODS: Six harvested bilateral myocutaneous flaps of three Dutch Landrace pigs were perfused hypothermically for 24 hours with University of Wisconsin machine perfusion solution (UW-MPS; n = 2) or histidine-tryptophan-ketoglutarate solution (HTK; n = 2) or preserved on ice for 4 hours (n = 2) before orthotopic replantation. Animals were observed for 7 days after replantation. Skeletal muscle injury was assessed by biochemical markers during perfusion, and muscle biopsies were analyzed for ischemia reperfusion injury directly after preservation and at 1, 3, and 7 days after replantation. RESULTS: Markers of muscle damage varied during perfusion, but decreased overall in both perfusion groups. Flap weight increased 60% and 97% in the HTK-perfused flaps, compared with -6% and -7% in the UW-MPS-perfused flaps after 24 hours. Histopathologic evaluation demonstrated decreased muscle damage in flaps perfused with HTK compared with the UW-MPS-perfused flaps at 1 week after replantation. CONCLUSIONS: Machine perfusion of myocutaneous flaps for 24 hours with subsequent replantation is feasible, but warrants further research. Perfusion with HTK solution seemed to result in better histological outcomes 7 days after reperfusion compared with UW-MPS.

Rectus Abdominis Flap Replantation after 18 h Hypothermic Extracorporeal Perfusion-A Porcine Model.

Cold storage remains the clinical standard for composite tissue preservation but is time-limited. A long ischemia time during surgery will adversely affect postoperative outcomes due to ischemia-reperfusion injury. Extracorporeal perfusion (ECP) seems to be a promising alternative for prolonged preservation, but more evidence is needed to support its use and to identify optimal perfusion fluids. This article assessed musculocutaneous flap vitality after prolonged ECP and compared outcomes after replantation to short static cold storage (SCS). Unilateral musculocutaneous rectus abdominis flaps were raised from 15 pigs and preserved by 4 h SCS (n = 5), 18 h mid-thermic ECP with Histidine-Tryptophan-Ketoglutarate (HTK, n = 5) or University of Wisconsin solution (UW, n = 5). Flaps were replanted and observed for 12 h. Skeletal muscle histology was assessed (score 0-12; high scores equal more damage), blood and perfusate samples were collected and weight was recorded as a marker for oedema. Mean histological scores were 4.0 after HTK preservation, 5.6 after UW perfusion and 5.0 after SCS (p = 0.366). Creatinine kinase (CK) was higher after ECP compared to SCS (p < 0.001). No weight increase was observed during UW perfusion, but increased 56% during HTK perfusion. Following 12 h reperfusion, mean weight gain reduced 39% in the HTK group and increased 24% in the UW group and 17% in the SCS group. To conclude, skeletal muscle seemed well preserved after 18 h ECP with HTK or UW perfusion, with comparable histological results to 4 h SCS upon short reperfusion. The high oedema rate during HTK perfusion remains a challenge that needs to be further addressed.

Successful 18-h acellular extracorporeal perfusion and replantation of porcine limbs - Histology versus nerve stimulation.

The current standard for composite tissue preservation is static cold storage (SCS) and is limited to 6 h until irreversible muscle damage occurs. Extracorporeal perfusion (ECP) is a promising technique for prolonged preservation, however, functional results have been scarcely researched. This article assessed neuromuscular function and compared results to histological alterations to predict muscle damage after ECP. Forelimbs of twelve Dutch landrace pigs were amputated and preserved by 4 h SCS at 4-6 °C (n = 6) or 18 h mid-thermic ECP with University of Wisconsin solution (n = 6). Limbs were replanted and observed for 12 h. Sham surgery was performed on contralateral forelimbs (n = 12). Histology analysis scored four subgroups representing different alterations (higher score equals more damage). Muscle contraction after median nerve stimulation was comparable between ECP, SCS, and sham limbs (P = 0.193). Histology scores were higher in ECP limbs compared to SCS limbs (4.8 vs. 1.5, P = 0.013). This was mainly based on more oedema in these limbs. In-vivo muscle contraction was well preserved after 18 h ECP compared to short SCS, although histology seemed inferior in this group. Histology, therefore, did not correlate to muscle function at 12 h after replantation. This leads to the question whether histology or neuromuscular function is the best predictor for transplant success.