Search Results (22)

Diabetic foot syndrome is often associated with inflammation. The aim of this study was to evaluate the impact of improved blood supply on the change in the clinical status and culturable bacteriota of chronic wounds. Patients with diabetic foot and peripheral arterial disease with a Rutherford score of 5 or 6 were included (n = 23). The blood supply to the limb was assessed with laboratory tests and two time-point qualitative cultures using a wound biopsy. The baseline parameters of the blood supply to the limb were Transcutaneous Oxygen Perfusion (TCPO2) of 15.0 mmHg, an Ankle Brachial Index (ABI) of 0.7, and a Toe Brachial Index (TBI) of 0.1, with an average Wound, Infection, Inflammation (WIfI) score of 5.7 (high). The most frequently isolated pathogens were Staphylococcus aureus (26.1%), followed by the Enterobacteriaceae family and Pseudomonas spp. (13.0%, each). Negative cultures were present in 47.8% (n = 11). The control parameters of blood supply improved; TCPO2 was 38.5 mmHg, the ABI was 0.9, and the TBI was 0.3, with a reduction in the average WIfI score to 3.7 (mild), while total colony-forming units (CFUs) increased by 13.5%. No cases of reocclusion or restenosis were observed during the study; however, small amputations were performed in two patients (8.7%). Five (21.7%) ulcers were significantly reduced and two (8.7%) progressed, while a negative culture at follow-up was obtained in five fewer patients than at baseline and nine patients presented growth despite having an initial negative result. Quantitative reduction was obtained in four (17.4%) cases. Pathogen distribution at follow-up resembled baseline findings. Optimizing clinical environments (enhancing blood flow and controlling inflammation) in general over focusing singularly on microbiota composition or revascularization seems to be crucial and arguably outweighed the impact of microbial change alone; in particular, reperfusion may increase the conditions to bacterial growth at the first stage. Full article

Objective: Monocytes are innate immune cells that play a central role in inflammation, an essential component during neovascularization. Our recent publication demonstrated that ischemia training by 24 h unilateral occlusion of the femoral artery (FA) can modify bone marrow-derived monocytes (BM-Mono), allowing them to improve collateral remodeling in a mouse model of hindlimb ischemia. Here, we expand on our previous findings, investigating a potential systemic effect of ischemia training and how this training can impact BM-Mono. Methods and Results: BM-Mono from mice exposed to ischemia training (24 h) or Sham (same surgical procedure without femoral artery occlusion–ischemia training) procedures were used as donors in adoptive transfer experiments where recipients were subjected to hindlimb ischemia. Donor cells were divided corresponding to the limb from which they were isolated (left—limb previously subjected to 24 h ischemia and right—contralateral limb). Recipients who received 24 h ischemic-trained monocytes isolated from either limb had remarkable blood flow recovery compared to recipients with Sham monocytes (monocytes isolated from Sham group—no ischemia training). Since these data suggested a systemic effect of ischemic training, circulating extracellular vesicles (EVs) were investigated as potential players. EVs were isolated from both groups, 24 h-trained and Sham, and the former showed increased expression of histone deacetylase 1 (HDAC1), which is known to downregulate 24-dehydrocholesterol reductase (Dhcr24) gene expression. Since we previously revealed that ischemia training downregulates Dhcr24 in BM-Mono, we incubated EVs from 24 h-trained and Sham groups with wild-type (WT) BM-Mono and demonstrated that WT BM-Mono incubated with 24 h-trained EVs had lower gene expression of Dhcr24 and an HDAC1 inhibitor blunted this effect. Next, we repeated the adoptive transfer experiment using Dhcr24 KO mice as donors of BM-Mono for WT mice subjected to hindlimb ischemia. Recipients who received Dhcr24 KO BM-Mono had greater limb perfusion than those who received WT BM-Mono. Further, we focused on the 24 h-trained monocytes (which previously showed downregulation of Dhcr24 gene expression and higher desmosterol) to test the expression of a few genes downstream of the desmosterol pathway, confirm the Dhcr24 protein level and assess its differentiation in M2-like macrophage phenotype. We found that 24 h-trained BM-Mono had greater expression of key genes in the desmosterol pathway, such as liver X receptors (LXRs) and ATP-binding cassette transporter (ABCA1), and we confirmed low protein expression of Dhcr24. Further, we demonstrated that ischemic-trained BM-Mono polarized towards an anti-inflammatory M2 macrophage phenotype. Finally, we demonstrated that 24 h-trained monocytes adhere less to endothelial cells, and the same pattern was shown by WT BM-Mono treated with Dhcr24 inhibitor. Conclusions: Ischemia training leads to a systemic effect that, at least in part, involves circulating EVs and potential epigenetic modification in BM-Mono. These ischemic-trained BM-Mono demonstrated an anti-inflammatory phenotype towards M2 macrophage differentiation and less ability to adhere to endothelial cells, which is associated with the downregulation of Dhcr24 in those cells. These data together suggest that Dhcr24 might be an important target within monocytes to improve the outcomes of hindlimb ischemia. Full article

Background/Objectives: α-1 antitrypsin (AAT) deficiency is an inherited, genetic condition characterized by reduced serum levels of AAT and increased risk of developing emphysema and liver disease. AAT is normally synthesized primarily in the liver, but muscle-targeting with a recombinant adeno-associated virus (rAAV) vector for α-1 antitrypsin (AAT) gene therapy has been used to minimize liver exposure to the virus and hepatotoxicity. Clinical trials of direct intramuscular (IM) administration of rAAV1-hAAT have demonstrated its overall safety and transgene expression for 5 years. However, the failure to reach the therapeutic target level after 100 large-volume (1.5 mL) IM injections of maximally concentrated vector led us to pursue a muscle-targeting approach using isolated limb perfusion. This targets the rAAV to a greater muscle mass and allows for a higher total volume (and thereby a higher dose) than is tolerable by multiple direct IM injections. Limb perfusion has been shown to be feasible in non-human primates using the rAAV1 serotype and a ubiquitous promoter expressing an epitope-tagged AAT matched to the host species. Methods: In this study, we performed a biodistribution and preclinical safety study in non-human primates with a clinical candidate rAAV1-human AAT (hAAT) vector at doses ranging from 3.0 × 10¹² to 1.3 × 10¹³ vg/kg, bracketing those used in our clinical trials. Results: We found that limb perfusion delivery of rAAV1-hAAT was safe and showed a biodistribution pattern similar to previous studies. However, serum levels of AAT obtained with high-dose limb perfusion still reached only ~50% of the target serum levels. Conclusions: Our results suggest that clinically effective AAT gene therapy may ultimately require delivery at doses between 3.5 × 10¹³–1 × 10¹⁴ vg/kg, which is within the dose range used for approved rAAV gene therapies. Muscle-targeting strategies could be incorporated when delivering systemic administration of high-dose rAAV gene therapies to increase transduction of muscle tissues and reduce the burden on the liver, especially in diseases that can present with hepatotoxicity such as AAT deficiency. Full article

In-transit metastases (ITM) in melanoma present a significant therapeutic challenge due to their advanced stage and complex clinical nature. From traditional management with surgical resection, ITM treatment has evolved with the advent of systemic therapies such as immune checkpoint inhibitors and targeted therapies, which have markedly improved survival outcomes. This study aims to review and highlight the efficacy of both systemic and locoregional treatment approaches for ITM. Methods include a comprehensive review of clinical studies examining the impact of treatments like immune checkpoint inhibitors, targeted therapies, Isolated Limb Perfusion, and electrochemotherapy. The results indicate that combining systemic therapies with locoregional treatments enhances both local disease control and overall survival rates. The introduction of modern immunotherapies has not diminished the effectiveness of locoregional therapies but rather improved patient outcomes when used in conjunction. The conclusions emphasize that a multidisciplinary approach integrating systemic and locoregional therapies offers a promising strategy for optimizing the management of ITM in melanoma patients. This integrated treatment model not only improves survival rates but also enhances the quality of life for patients, suggesting a shift in standard care practices toward more comprehensive therapeutic regimens. Full article

Locoregionally advanced and metastatic melanoma represent a challenging clinical problem, but in the era of immune checkpoint blockade and intralesional and infusional therapies, more options are available for use. Isolated limb infusion (ILI) was first introduced in the 1990s for the management of advanced melanoma, followed by the utilization of isolated extremity perfusion (ILP). Following this, intralesional oncolytic viruses, xanthene dyes, and cytokines were introduced for the management of in-transit metastases as well as unresectable, advanced melanoma. In 2015, the Food and Drug Administration (FDA) approved the first oncolytic intralesional therapy, talimogene laherparepvec (T-VEC), for the treatment of advanced melanoma. Additionally, immune checkpoint inhibition has demonstrated efficacy in the management of advanced melanomas, and this improvement in outcomes has been extrapolated to aid in the management of in-transit metastatic disease. Finally, percutaneous hepatic perfusion (PHP), also approved by the FDA, has been reported to have a significant impact on the treatment of hepatic disease in uveal melanoma. While some of these treatments have less utility due to inferior outcomes as well as higher toxicity profiles, there are selective patient profiles for which these therapies carry a role. This review highlights intralesional and infusional therapies for the management of metastatic melanoma. Full article

Background: Isolated limb hyperthermic-antiblastic perfusion (ILP) was the most effective local treatment for advanced in-transit melanoma, but the advent of modern effective immunotherapy (IT), such as immune checkpoint inhibitors, has changed the treatment landscape. Methods: This study evaluated the role of the association between ILP and IT in the treatment of locally advanced unresectable melanoma, particularly in relation to modern systemic therapies. We analyzed 187 consecutive patients who were treated with ILP (melphalan or melphalan associated with TNF-alpha) for advanced melanoma at the Veneto Institute of Oncology of Padua (Italy) and the Padua University Hospital (Italy) between June 1989 and September 2021. Overall survival (OS), disease-specific survival (DSS), local disease-free survival (local DFS) and distant disease-free survival (distant DFS) were evaluated. Local toxicity was classified according to the Wieberdink scale and surgical complications according to the Clavien–Dindo classification. Response to locoregional therapy was evaluated during follow-up according to the RECIST 1.1 criteria (Response Evaluation Criteria in Solid Tumor). Results: A total of 99 patients were treated with ILP and 88 with IT + ILP. The overall response rate was 67% in both groups. At 36 months, OS was 43% in the ILP group and 61% in the ILP + IT group (p = 0.02); DSS was 43% in the ILP group and 64% in the ILP + IT group (p = 0.02); local DFS was the 37% in ILP group and 53% in the ILP + IT group (p = 0.04); and distant DFS was 33% in the ILP group and 35% in the ILP + IT group (p = 0.40). Adjusting for age and lymph node involvement, receiving ILP + IT was associated with improved OS (p = 0.01) and DSS (p = 0.007) but not local DFS (p = 0.13) and distant DFS (p = 0.21). Conclusions: Our findings confirm the synergy between ILP and IT. ILP remains a valuable loco-regional treatment option in the era of effective systemic treatments. Further studies are needed to establish the optimal combination of loco-regional and systemic treatments and address the best timing of this combination to obtain the highest local response rate. Full article

Isolated limb perfusion (ILP) involves the local administration of high doses of anticancer drugs into a limb affected by unresectable locally advanced tumors (with special regard to in-transit melanoma metastases), minimizing systemic side effects. Tumor response to anticancer drugs may depend on the expression of apoptosis-related genes, such as SURVIVIN and MDM2. This retrospective cohort study investigated the association between tumor SURVIVIN and MDM2 expression levels and treatment response or clinical outcomes in patients undergoing ILP for in-transit melanoma metastases. The study cohort consisted of 62 patients with in-transit metastases who underwent ILP with tumor necrosis factor (TNF) and melphalan. Tissue samples were taken from the in-transit metastases, and RNA was extracted for gene expression analysis. Patients’ response to treatment was assessed using clinical and radiological criteria two months after ILP, and disease response was classified as complete, partial, or stable/progressive disease. Disease-free survival (DFS) and overall survival (OS) were also analyzed. Expression of SURVIVIN and/or MDM2 was observed in 48% of patients; in these cases, complete response to ILP occurred in 40% of cases, with the overall response rate (complete + partial) being 85%. Patients with expression of MDM2 alone had a lower complete response rate (28%), while patients with expression of SURVIVIN alone had a higher complete response rate (50%). The combined expression of MDM2 and SURVIVIN resulted in a complete response rate of 30%. Patients without expression (of SURVIVIN or MDM2) had the highest complete response rate (58%). Survival analysis showed that high MDM2 expression was independently associated with a lower probability of a complete response to ILP. In addition, patients with MDM2 expression were three times more likely to have an incomplete response to ILP. This study highlights the importance of considering SURVIVIN and MDM2 expression in patients undergoing ILP for in-transit cutaneous melanoma metastases. High MDM2 expression was found to be an independent factor associated with a reduced likelihood of achieving a complete response to ILP, suggesting potential mechanisms of chemoresistance. These data support further research to explore the role of already available targeted therapies (i.e., MDM2 inhibitors) in improving tumor response to ILP in patients with in-transit melanoma metastases. Full article

Background: Isolated limb perfusion (ILP) for soft tissue sarcomas (STS) is usually performed with tumor necrosis factor alpha (TNF-α) and melphalan. ILP regularly leads to a total blood loss (BLt) of 1.5–2 L/patient. Blood inflow from the central blood circulation to the limb is influenced by unstable pressure gradients and pain reactions after the administration of melphalan. With perioperative regional anesthesia (RA), pain levels can be reduced, and the pressure gradient stabilized resulting in a reduced BLt. The aim of this study was to compare the BLt with and without RA in patients with ILP during circulation of drugs. Methods: Patients were treated according to the following protocol: After the establishment of limb circulation, ILP was started with the administration of TNF-α. Half the dose of melphalan was given as a bolus after 30 min, and the remaining dose was continuously administered in the following 30 min. The extremity was washed out after 90 min. ILP with perioperative RA (supraclavicular plexus block/peridural catheter) was performed prospectively in 17 patients and compared to a matched retrospective control group of 17 patients without RA. BLt was documented and perioperative anesthesiological data were analyzed for response rates after the application of melphalan (RaM). Results: BLt and RaM tended to be lower for the intervention group with RA if compared to the control group without RA in all analyses. The trend of lower BLt and RaM in ILP with RA was more pronounced for the upper extremity compared to the lower extremity. Results were not statistically significant. Conclusion: These findings indicate that the use of RA can help to stabilize hemodynamic anesthetic management and reduce the BLt in ILP, especially during perfusion of the upper extremities. Full article

Isolated limb perfusion (ILP) is an effective locoregional treatment for melanoma in-transit metastasis, but the advent of modern effective immunotherapy, such as ICI (immune checkpoint inhibitors), has changed the treatment landscape. The primary aims of this study were to compare the characteristics of the patient population receiving ILP before and after the introduction of modern systemic treatments and to assess if outcomes after ILP were influenced by previous immunotherapy treatment. A single-centre analysis of patients that underwent ILP for melanoma in-transit metastasis between 2010 and 2021 was conducted, with patients grouped and compared by treatment time period: pre-ICI era (2010–2014) and ICI era (2017–2021). 218 patients were included. Patients undergoing ILP in the ICI era were slightly older (median age 73 vs. 68 years) compared to the pre-ICI era, with no other difference found. The overall response rate (ORR) was 83% vs. 84% and the complete response (CR) rate was 52% vs. 47% for the pre-ICI era and the ICI era, respectively. For patients that had received and failed immunotherapy prior to ILP (n = 20), the ORR was 75% and the CR rate was 50%. Melanoma-specific survival has improved, with a 3-year survival rate of 54% in the pre-ICI era vs. 86% in the ICI era. The patient population undergoing ILP for in-transit melanoma is largely unchanged in the current era of effective systemic treatments. Response rates have not decreased, and prior ICI treatment did not affect response rates, making ILP still a valid treatment option for this patient group. Full article

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU⁺) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68⁺/MRC-1⁺ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth. Full article

The interleukin-21 receptor (IL-21R) can be upregulated in endothelial cells (EC) from ischemic muscles in mice following hind-limb ischemia (HLI), an experimental peripheral arterial disease (PAD) model, blocking this ligand–receptor pathway-impaired STAT3 activation, angiogenesis, and perfusion recovery. We sought to identify mRNA and microRNA transcripts that were differentially regulated following HLI, based on the ischemic muscle having intact, or reduced, IL-21/IL21R signaling. In this comparison, 200 mRNAs were differentially expressed but only six microRNA (miR)/miR clusters (and among these only miR-30b) were upregulated in EC isolated from ischemic muscle. Next, myoglobin-overexpressing transgenic (MgTG) C57BL/6 mice examined following HLI and IL-21 overexpression displayed greater angiogenesis, better perfusion recovery, and less tissue necrosis, with increased miR-30b expression. In EC cultured under hypoxia serum starvation, knock-down of miR-30b reduced, while overexpression of miR-30b increased IL-21-mediated EC survival and angiogenesis. In Il21r^−/− mice following HLI, miR-30b overexpression vs. control improved perfusion recovery, with a reduction of suppressor of cytokine signaling 3, a miR-30b target and negative regulator of STAT3. Together, miR-30b appears both necessary and sufficient for IL21/IL-21R-mediated angiogenesis and may present a new therapeutic option to treat PAD if the IL21R is not available for activation. Full article

Sarcomas are characterized by a high metastatic potential and aggressive growth. Despite surgery, chemotherapy plays an important role in the treatment of these tumors. Optimal anti-cancer therapy with maximized local efficacy and minimized systemic side effects has been the object of many studies for a long time. To improve the local efficacy of anti-tumor therapy, isolated limb perfusion with high-dose cytostatic agents has been introduced in surgical oncology. In order to control the local distribution of substances, radiolabeled cytostatic drugs or perfusion solutions have been applied but often require the presence of specialized personnel and result in a certain exposure to radiation. In this study, we present a novel strategy using indocyanine green to track tumor perfusion with high-dose cytostatic therapy. In a rat cadaver model, the femoral vessels were cannulated and connected to a peristaltic pump to provide circulation within the selected limb. The perfusion solution contained indocyanine green and high-dose doxorubicin. An infrared camera enabled the visualization of indocyanine green during limb perfusion, and subsequent leakage control was successfully performed. Histologic analysis of sections derived proximally from the injection site excluded systemic drug dispersion. In this study, the application of indocyanine green was proven to be a safe and cost- and time-efficient method for precise leakage control in isolated limb perfusion with a high-dose cytostatic agent. Full article

Background. Isolated limb perfusion (ILP) is a locoregional procedure indicated by the unresectable melanoma of the limbs. Its complexity and highly demanding multidisciplinary approach means that it is a technique only implemented in a few referral centers around the globe. This report aims to examine its potential role in the era of targeted therapies and immunotherapy by conducting a systematic review of the literature on ILP. Methods. PubMed, Embase and Cochrane Library were searched. The eligibility criteria included publications from 2000–2020 providing valid data o effectiveness, survival or toxicity. Studies in which the perfusion methodology was not clearly described, letters to the editor, non-systematic reviews and studies that applied outdated clinical guidelines were excluded. To rule out studies of a low methodological quality and assess the risk of bias, the following aspects were also required: a detailed description of the applied ILP regimen, the clinical context, follow-up periods, analyzed clinical endpoints, and the number of analyzed ILPs. The disagreements were resolved by consensus. The results are presented in tables and figures. Results. Twenty-seven studies including 2637 ILPs were selected. The median overall response rate was 85%, with a median complete response rate of 58.5%. The median overall survival was 38 months, with a 5-year overall survival of 35%. The toxicity was generally mild according to Wieberdink toxicity criteria. Discussion. ILP still offer a high efficacy in selected patients. The main limitation of our review is the heterogeneity and age of most of the articles, as well as the absence of clinical trials comparing ILP with other procedures, making it difficult to transfer its results to the current era. Conclusions. ILP is still an effective and safe procedure for selected patients with unresectable melanoma of the limbs. In the era of targeted therapies and immunotherapy, ILP remains an acceptable and reasonable palliative treatment alternative, especially to avoid limb amputations. The ongoing clinical trials combining systemic therapies and ILP will provide more valuable information in the future to clarify the potential synergism of both strategies. Full article

Background: To evaluate the potential of simultaneously acquired 18F-FDG PET- and MR-derived quantitative imaging data sets of primary soft-tissue sarcomas for the prediction of neoadjuvant treatment response, the metastatic status and tumor grade. Methods: A total of 52 patients with a high-risk soft-tissue sarcoma underwent a 18F-FDG PET/MR examination within one week before the start of neoadjuvant treatment. For each patient, the maximum tumor size, metabolic activity (SUVs), and diffusion-restriction (ADC values) of the tumor manifestations were determined. A Mann–Whitney-U test was used, and ROC analysis was performed to evaluate the potential to predict histopathological treatment response, the metastatic status or tumor grade. The results from the histopathological analysis served as reference standard. Results: Soft-tissue sarcomas with a histopathological treatment response revealed a significantly higher metabolic activity than tumors in the non-responder group. In addition, grade 3 tumors showed a significant higher 18F-FDG uptake than grade 2 tumors. Furthermore, no significant correlation between the different outcome variables and tumor size or calculated ADC-values could be identified. Conclusion: Measurements of the metabolic activity of primary and untreated soft-tissue sarcomas could non-invasively deliver relevant information that may be used for treatment planning and risk-stratification of high-risk sarcoma patients in a pretherapeutic setting. Full article

For decades, isolated limb infusion (ILI) and hyperthermic isolated limb perfusion (HILP) have been used to treat melanoma in-transit metastases and unresectable sarcoma confined to the limb utilizing the effect of loco-regional high-dose chemotherapy to the isolated limb. Both procedures are able to provide high response rates in patients with numerous or bulky lesions in whom other loco-regional treatments are becoming ineffective. In comparison to systemic therapies, on the other hand, ILI and HILP have the advantage of not being associated with systemic side-effects. Although in principle ILI and HILP are similar procedures, ILI is technically simpler to perform and differs from HILP in that it takes advantage of the hypoxic and acidotic environment that develops in the isolated limb, potentiating anti-tumour activity of the cytotoxic agents melphalan +/− actinomycin-D. Due to its simplicity, ILI can be used in both preclinical and clinical studies to test new cytotoxic regimens and combinations with the aim to overcome tumour resistance. In the future, administration of cytotoxic agents by ILI, in combination with systemic treatments such as BRAF/MEK/KIT inhibitors, immunotherapy (CTLA-4 blockade), and/or programmed death (PD-1) pathway inhibitors, has the potential to improve responses further by inducing increased tumour cell death while limiting the ability of the tumour to suppress the immune response. Full article