A minimally destructive protocol for DNA extraction from ancient teeth.

Ancient DNA sampling methods-although optimized for efficient DNA extraction-are destructive, relying on drilling or cutting and powdering (parts of) bones and teeth. As the field of ancient DNA has grown, so have concerns about the impact of destructive sampling of the skeletal remains from which ancient DNA is obtained. Due to a particularly high concentration of endogenous DNA, the cementum of tooth roots is often targeted for ancient DNA sampling, but destructive sampling methods of the cementum often result in the loss of at least one entire root. Here, we present a minimally destructive method for extracting ancient DNA from dental cementum present on the surface of tooth roots. This method does not require destructive drilling or grinding, and, following extraction, the tooth remains safe to handle and suitable for most morphological studies, as well as other biochemical studies, such as radiocarbon dating. We extracted and sequenced ancient DNA from 30 teeth (and nine corresponding petrous bones) using this minimally destructive extraction method in addition to a typical tooth sampling method. We find that the minimally destructive method can provide ancient DNA that is of comparable quality to extracts produced from teeth that have undergone destructive sampling processes. Further, we find that a rigorous cleaning of the tooth surface combining diluted bleach and UV light irradiation seems sufficient to minimize external contaminants usually removed through the physical removal of a superficial layer when sampling through regular powdering methods.

A novel technique for laser-assisted revascularization: an in vitro pilot study.

The common limitation of surgical revascularization procedures for severe tissue ischemia due to cardiovascular diseases is the need to interrupt blood flow during the intervention. We aim to introduce a new technique that allows a sutureless, non-occlusive revascularization. A 3-step technique was developed using rabbit's aorta to simulate a side-to-side anastomosis model. It enables the creation of a bypass circuit for revascularization. The first step was the soldering of 2 vessels in a side-to-side fashion based on the laser-assisted vascular anastomosis (LAVA) principle using a diode laser emitting irradiation at 810 nm with an albumin-based solder patch between them, followed by the creation of a channel within the patch using either a holmium-doped yttrium aluminum garnet laser (Ho:YAG) at λ = 2100 nm or a xenon-chloride excimer laser (XeCl) at λ = 308 nm. Thereby, a bypass circuit was created, thus allowing a non-ischemic revascularization. The system was deemed functional when a flow was observed across the anastomosis. The highest average tensile strength recorded after side-to-side LAVA using a diode laser power of 3.2 W for 60 s was 2278.6 ± 800 mN (n = 20). The Ho:YAG laser created the channels with less tension on the anastomosis than the excimer laser. Histological analysis showed limited thermal damage and good patch-tissue adaptation. The preliminary results of this feasibility study outline the foundations for an entirely sutureless laser-assisted revascularization procedure. The next studies will evaluate the rheological parameters across the bypass circuit to optimize the post-anastomotic flow.

Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA-E/human CD46 double-transgenic pig limbs with human blood.

BACKGROUND: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. METHODS: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. RESULTS: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. CONCLUSIONS: Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.

Genetic affinities among the historical provinces of Romania and Central Europe as revealed by an mtDNA analysis.

BACKGROUND: As a major crossroads between Asia and Europe, Romania has experienced continuous migration and invasion episodes. The precise routes may have been shaped by the topology of the territory and had diverse impacts on the genetic structure of mitochondrial DNA (mtDNA) in historical Romanian provinces. We studied 714 Romanians from all historical provinces, Wallachia, Dobrudja, Moldavia, and Transylvania, by analyzing the mtDNA control region and coding markers to encompass the complete landscape of mtDNA haplogroups. RESULTS: We observed a homogenous distribution of the majority of haplogroups among the Romanian provinces and a clear association with the European populations. A principal component analysis and multidimensional scaling analysis supported the genetic similarity of the Wallachia, Moldavia, and Dobrudja groups with the Balkans, while the Transylvania population was closely related to Central European groups. These findings could be explained by the topology of the Romanian territory, where the Carpathian Arch played an important role in migration patterns. Signals of Asian maternal lineages were observed in all Romanian historical provinces, indicating gene flow along the migration routes through East Asia and Europe. CONCLUSIONS: Our current findings based on the mtDNA analysis of populations in historical provinces of Romania suggest similarity between populations in Transylvania and Central Europe, supported both by the observed clines in haplogroup frequencies for several European and Asian maternal lineages and MDS analyses.

Intra-graft injection of tacrolimus promotes survival of vascularized composite allotransplantation.

BACKGROUND: Immunosuppressive therapies derived from solid organ transplantation are effective in promoting survival of vascularized composite allotransplantation (VCA), but they cause serious side effects that are difficult to justify for this non-life-saving procedure. Unlike solid organ transplantation, hand and face transplants offer the possibility of site-specific immunosuppression for reducing systemic exposure while increasing intra-graft concentrations of the drug. Therefore, in this study, we tested whether a single intra-graft injection tacrolimus could promote VCA survival. METHODS: Brown Norway-to-Lewis hind limb transplantations were performed, and animals were left untreated (group I), treated with a daily injection of 1-mg/kg tacrolimus for 21 days (group 2) or injected with 7-mg tacrolimus directly into the transplanted limb on day 1 (group III). Graft rejection was monitored, and animals were sacrificed at grade 3 rejection or 200 days after transplantation. RESULTS: Intra-graft injection of tacrolimus significantly prolonged allograft survival as compared to untreated animals or animals treated with systemic tacrolimus. Half of the intra-graft-treated rats rejected their graft on average at day 70.5. Interestingly, the other half remained rejection-free for more than 200 days without signs of kidney or liver toxicity. In these animals, tacrolimus was detected in the VCA skin but not in the blood until day 200. Long-term survival was not linked to induction of donor-specific tolerance but to a higher level of lymphocyte chimerism. CONCLUSIONS: Intra-graft delivery of tacrolimus may promote VCA survival by increasing tissue drug availability and promoting the establishment of transient chimerism and thus long-term graft acceptance.

Binding of indocyanine green in polycaprolactone fibers using blend electrospinning for in vivo laser-assisted vascular anastomosis.

BACKGROUND AND OBJECTIVE: The clinical application of laser-assisted vascular anastomosis is afflicted by unreliable and low bonding strengths as well as tedious handling during microvascular surgery. The challenge to be met arises from the flow-off of the chromophore during soldering that changes the absorption and stains the surrounding tissue, leading to an uncontrollable thermal damage zone. In this study, we investigated the feasibility to produce an indocyanine green (ICG)-loaded patch by electrospinning and tested its applicability to both in vitro and in vivo microvascular laser soldering. MATERIALS AND METHODS: A blend of polycaprolactone and ICG was electrospun to produce a pliable patch. Prior to soldering, the patch was soaked in 40% wt. bovine serum albumin solution. The solder patch was wrapped in vitro around blood vessel stumps of rabbit aortas. An intraluminal balloon catheter enabled an easy alignment and held the setup in place. The soldering energy was delivered via a diffusor fiber from the vessel lumen using a diode laser at 810 nm. During the procedure, the surface temperature was observed with an infrared camera. Afterward, samples were embedded in methylmethacrylate and epon to study thermal damage. The quality of the fusion was assessed by measuring the tensile strength. After in vitro tests with rabbit aortas, eight large white pigs were subjected to an acute in vivo experiment, and the artery of the latissimus dorsi flap was anastomosed to the distal femoral artery. RESULTS: The ICG-loaded patch, produced by electrospinning, has a thickness of 279 ± 62 µm, a fiber diameter of 1.20 ± 0.19 µm, and an attenuation coefficient of 1,119 ± 183 cm-1 at a wavelength of 790 nm. The patch was pliable and easy to handle during surgery. No leakage of the chromophore was observed. Thermal damage was restricted to the Tunica adventitia and Tunica media and the area of the vessel wall that was covered with the patch. Six pigs were successfully treated, without any bleeding and with a continuous blood flow. The in vivo flap model yielded a similar tensile strength compared to in vitro laser-assisted vascular anastomoses (138 ± 52 vs. 117 ± 30 mN/mm2 ). CONCLUSION: Our study demonstrated the applicability of the ICG-loaded patch for laser-assisted vascular anastomosis. By using electrospinning, ICG could be bound to polymer fibers, avoiding its flow-off and the staining of the surrounding tissue. This patch demonstrated several advantages over liquid solder as it was easier to apply, ensured a high and reliable bonding strength while maintaining a constant concentration of ICG concentration during the surgery. Lasers Surg. Med. 49:928-939, 2017. © 2017 Wiley Periodicals, Inc.

Endoluminal laser-assisted vascular anastomosis-an in vivo study in a pig model.

Microvascular surgery is time consuming and requires high expertise. Laser-assisted vascular anastomosis (LAVA) is a promising sutureless technique that has the potential to facilitate this procedure. In this study, we evaluate the handling of our soldering material and the 1-week patency rate in a porcine model. Six pigs were subjected to LAVA. For each pig, the saphenous artery on one side was transected while the contralateral side was used as control. A porous polycaprolactone scaffold soaked in 40% (w/w) bovine serum albumin solution in combination with 0.1% (w/w) indocyanine green was wrapped at the anastomosis site and at the control site. Both sides were then soldered with a diode laser coupled into a light diffuser fiber emitting radiation with a wavelength of 808 nm and a power of 2-2.2 W. Vessels were successfully soldered with a 100% immediate patency rate. The 1-week patency rate was 83% for the anastomoses versus 67% for the control side. Vessels irradiated for 80 to 90 s tended to maintain the highest patency rate. Macroscopically, there was no difference between the two sides. The patch was easy to handle provided that the environment could be kept dry. This study shows the potential and the limitations of endoluminal LAVA as a one-step procedure without the use of stay sutures. Further studies are needed to improve the soldering material, the long-term patency rate, and standardized irradiation parameters. The long-term effects of laser soldering on the vessel wall remain to be determined.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND: Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS: α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS: No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1ß, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION: Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Sonographic wrist measurements and detection of anatomical features in carpal tunnel syndrome.

INTRODUCTION: This study compares anatomical findings at wrist level in patients with known carpal tunnel syndrome (CTS) and controls by ultrasonography (US). MATERIAL AND METHODS: Wrist-US investigations of 28 consecutive patients with 38 diagnosed, idiopathic CTS were compared to 49 healthy volunteers without history of CTS. Internal wrists dimensions, the presence of flexor muscle bellies in the carpal tunnel, and cross-sectional area of the median nerve were analyzed. The findings were correlated to gender, age, and BMI. RESULTS: US demonstrated a square internal carpal tunnel configuration in CTS patients compared to controls (P < 0.001). Patients with CTS showed a trend towards the presence of flexor muscles bellies in the carpal tunnel (odds ratio 1.77, 95% CI 0.337-8.33). CTS was present in women with higher BMI (P = 0.015). CONCLUSION: US allowed detection of specific anatomical features at wrist level in CTS patients. This observation may enable--following confirmation in larger prospective studies--risk evaluation for CTS development.

A quantitative analysis of microcirculation in sore-prone pressure areas on conventional and pressure relief hospital mattresses using laser Doppler flowmetry and tissue spectrophotometry.

BACKGROUND: Pressure ulcers are associated with severe impairment for the patients and high economic load. With this study we wanted to gain more insight to the skin perfusion dynamics due to external loading. Furthermore, we evaluated the effect of different types of pressure relief mattresses. METHODS: A total of 25 healthy volunteers were enrolled in the study. Perfusion dynamics of the sacral and the heel area were assessed using the O2C-device, which combines a laser light, to determine blood flow, and white light to determine the relative amount of hemoglobin. Three mattresses were evaluated compared to a hard surface: a standard hospital foam mattress bed, a visco-elastic foam mattress, and an air-fluidized bed. RESULTS: In the heel area, only the air-fluidized bed was able to maintain the blood circulation (mean blood flow of 13.6 ± 6 versus 3.9 ± 3 AU and mean relative amount of hemoglobin of 44.0 ± 14 versus 32.7 ± 12 AU.) In the sacral area, all used mattresses revealed an improvement of blood circulation compared to the hard surface. CONCLUSION: The results of this study form a more precise pattern of perfusion changes due to external loading on various pressure relief mattresses. This knowledge may reduce the incidence of pressure ulcers and may be an influencing factor in pressure relief mattress selection.

[The risk-reducing mastectomy: unnecessary hysteria or life-saving prophylaxis?]. / Die prophylaktische Mastektomie: unnötige Hysterie oder nötige Vorsorge.

The prophylactic (risk-reducing) mastectomy is a world-wide recognized method for specifically treating the increased breast cancer risk in patients showing a BRCA1 and/or BRCA2 mutation as well as other patient groups at increased breast cancer risk. This option should be offered to all patients having the pertinent risk profile. Breast reconstruction is an integral part of the risk-reducing mastectomy procedure and all possible methods of breast reconstruction, especially autologous tissue reconstruction should be offered to all patients having a medical indication and desiring this surgical treatment. These patients are best managed in certified Breast Care Centres where the different medical and surgical specialists can address interdisciplinary all aspects of genetic counselling, preoperative counselling, mastectomy and reconstructive techniques as well as the necessary postoperative surveillance.

Sentinel Lymph Node Detection in Cutaneous Melanoma Using Indocyanine Green-Based Near-Infrared Fluorescence Imaging: A Systematic Review and Meta-Analysis.

The standard of care approach to identify sentinel lymph nodes (SLNs) in clinically non-metastatic cutaneous melanoma patients is technetium (Tc)-based lymphoscintigraphy. This technique is associated with radiation exposure, a long intervention time, high costs, and limited availability. Indocyanine green (ICG)-based near-infrared fluorescence imaging offers a potential alternative if proven to be of comparable diagnostic accuracy. While several clinical cohorts have compared these modalities, no systematic review exists that provides a quantitative analysis of their results. Hence, a systematic literature review was conducted in December 2023 considering clinical studies comparing the diagnostic accuracy of ICG and Tc for sentinel lymph node biopsy in cutaneous melanoma patients. Three hundred nineteen studies were identified and further screened in accordance with the PRISMA 2020 guidelines, resulting in seven studies being included in the final meta-analysis. Tc identified a significantly higher number of SLNs and metastatic SLNs in prospective studies only. However, in the overall meta-analysis of all included comparative studies, no significant differences were found regarding the identification of metastatic patients or the false negative rate (FNR). ICG may be a non-inferior alternative to Tc for intraoperative guidance in sentinel lymph node biopsy in cutaneous melanoma patients. Future randomized controlled trials are needed, especially regarding the preoperative, transcutaneous identification of the affected lymph node basin.

Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model.

Background: Vascularized composite allotransplantation (VCA) offers the potential for a biological, functional reconstruction in individuals with limb loss or facial disfigurement. Yet, it faces substantial challenges due to heightened immune rejection rates compared to solid organ transplants. A deep understanding of the genetic and immunological drivers of VCA rejection is essential to improve VCA outcomes. Methods: Heterotopic porcine hindlimb VCA models were established and followed until reaching the endpoint. Skin and muscle samples were obtained from VCA transplant recipient pigs for histological assessments and RNA sequencing analysis. The rejection groups included recipients with moderate pathological rejection, treated locally with tacrolimus encapsulated in triglycerol-monostearate gel (TGMS-TAC), as well as recipients with severe end-stage rejection presenting evident necrosis. Healthy donor tissue served as controls. Bioinformatics analysis, immunofluorescence, and electron microscopy were utilized to examine gene expression patterns and the expression of immune response markers. Results: Our comprehensive analyses encompassed differentially expressed genes, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways, spanning various composite tissues including skin and muscle, in comparison to the healthy control group. The analysis revealed a consistency and reproducibility in alignment with the pathological rejection grading. Genes and pathways associated with innate immunity, notably pattern recognition receptors (PRRs), damage-associated molecular patterns (DAMPs), and antigen processing and presentation pathways, exhibited upregulation in the VCA rejection groups compared to the healthy controls. Our investigation identified significant shifts in gene expression related to cytokines, chemokines, complement pathways, and diverse immune cell types, with CD8 T cells and macrophages notably enriched in the VCA rejection tissues. Mechanisms of cell death, such as apoptosis, necroptosis and ferroptosis were observed and coexisted in rejected tissues. Conclusion: Our study provides insights into the genetic profile of tissue rejection in the porcine VCA model. We comprehensively analyze the molecular landscape of immune rejection mechanisms, from innate immunity activation to critical stages such as antigen recognition, cytotoxic rejection, and cell death. This research advances our understanding of graft rejection mechanisms and offers potential for improving diagnostic and therapeutic strategies to enhance the long-term success of VCA.

Bioglass/ceria nanoparticle hybrids for the treatment of seroma: a comparative long-term study in rats.

Background: Seroma formation is a common postoperative complication. Fibrin-based glues are typically employed in an attempt to seal the cavity. Recently, the first nanoparticle (NP)-based treatment approaches have emerged. Nanoparticle dispersions can be used as tissue glues, capitalizing on a phenomenon known as 'nanobridging'. In this process, macromolecules such as proteins physically adsorb onto the NP surface, leading to macroscopic adhesion. Although significant early seroma reduction has been shown, little is known about long-term efficacy of NPs. The aim of this study was to assess the long-term effects of NPs in reducing seroma formation, and to understand their underlying mechanism. Methods: Seroma was surgically induced bilaterally in 20 Lewis rats. On postoperative day (POD) 7, seromas were aspirated on both sides. In 10 rats, one side was treated with NPs, while the contralateral side received only NP carrier solution. In the other 10 rats, one side was treated with fibrin glue, while the other was left untreated. Seroma fluid, blood and tissue samples were obtained at defined time points. Biochemical, histopathological and immunohistochemical assessments were made. Results: NP-treated sides showed no macroscopically visible seroma formation after application on POD 7, in stark contrast to the fibrin-treated sides, where 60% of the rats had seromas on POD 14, and 50% on POD 21. At the endpoint (POD 42), sides treated with nanoparticles (NPs) exhibited significant macroscopic differences compared to other groups, including the absence of a cavity, and increased fibrous adhesions. Histologically, there were more macrophage groupings and collagen type 1 (COL1) deposits in the superficial capsule on NP-treated sides. Conclusion: NPs not only significantly reduced early manifestations of seroma and demonstrated an anti-inflammatory response, but they also led to increased adhesion formation over the long term, suggesting a decreased risk of seroma recurrence. These findings highlight both the adhesive properties of NPs and their potential for clinical therapy.

[Reconstruction of Oncological Defects in the Pelvic-perineal Region: Report on the Consensus Workshop at the 44th Annual Meeting of the DAM 2023 in Bern, CH]. / Rekonstruktion onkologischer Defekte im Becken-Perinealbereich ­ Bericht zum Konsensus-Workshop im Rahmen der 44. Jahrestagung der DAM 2023 in Bern, CH.

The surgical-oncological treatment of pelvic and perineal malignancies is associated with a high complication rate and morbidity for patients. Modern multimodal treatment modalities, such as neoadjuvant radio-chemotherapy for anal or rectal cancer, increase the long-term survival rate while reducing the risk of local recurrence. Simultaneously, the increasing surgical radicality and higher oncological safety with wide resection margins is inevitably associated with larger and, due to radiation, more complex tissue defects in the perineal and sacral parts of the pelvic floor. Therefore, the plastic-surgical reconstruction of complex pelvic-perineal defects following oncological resection remains challenging. The reconstructive armamentarium, and thus the treatment of such defects, is broad and ranges from local, regional and muscle-based flaps to microvascular and perforator-based procedures. While the use of flaps is associated with a significant, well-documented reduction in postoperative complications compared to primary closure, there is still a lack of reliable data directly comparing the postoperative results of different reconstructive approaches. Additionaly, the current data shows that the quality of life of these patients is rarely recorded in a standardised manner. In a consensus workshop at the 44th annual meeting of the German-speaking Association for Microsurgery on the topic of "Reconstruction of oncological defects in the pelvic-perineal area", the current literature was discussed and recommendations for the reconstruction of complex defects in this area were developed. The aim of this workshop was to identify knowledge gaps and establish an expert consensus to ensure and continuously improve the quality of reconstruction in this challenging area. In addition, the importance of the "patient-reported outcome measures" in pelvic reconstruction was highlighted, and the commitment to its widespread use in the era of value-based healthcare was affirmed.

A local drug delivery system prolongs graft survival by dampening T cell infiltration and neutrophil extracellular trap formation in vascularized composite allografts.

Introduction: The standard treatment for preventing rejection in vascularized composite allotransplantation (VCA) currently relies on systemic immunosuppression, which exposes the host to well-known side effects. Locally administered immunosuppression strategies have shown promising results to bypass this hurdle. Nevertheless, their progress has been slow, partially attributed to a limited understanding of the essential mechanisms underlying graft rejection. Recent discoveries highlight the crucial involvement of innate immune components, such as neutrophil extracellular traps (NETs), in organ transplantation. Here we aimed to prolong graft survival through a tacrolimus-based drug delivery system and to understand the role of NETs in VCA graft rejection. Methods: To prevent off-target toxicity and promote graft survival, we tested a locally administered tacrolimus-loaded on-demand drug delivery system (TGMS-TAC) in a multiple MHC-mismatched porcine VCA model. Off-target toxicity was assessed in tissue and blood. Graft rejection was evaluated macroscopically while the complement system, T cells, neutrophils and NETs were analyzed in graft tissues by immunofluorescence and/or western blot. Plasmatic levels of inflammatory cytokines were measured using a Luminex magnetic-bead porcine panel, and NETs were measured in plasma and tissue using DNA-MPO ELISA. Lastly, to evaluate the effect of tacrolimus on NET formation, NETs were induced in-vitro in porcine and human peripheral neutrophils following incubation with tacrolimus. Results: Repeated intra-graft administrations of TGMS-TAC minimized systemic toxicity and prolonged graft survival. Nevertheless, signs of rejection were observed at endpoint. Systemically, there were no increases in cytokine levels, complement anaphylatoxins, T-cell subpopulations, or neutrophils during rejection. Yet, tissue analysis showed local infiltration of T cells and neutrophils, together with neutrophil extracellular traps (NETs) in rejected grafts. Interestingly, intra-graft administration of tacrolimus contributed to a reduction in both T-cellular infiltration and NETs. In fact, in-vitro NETosis assessment showed a 62-84% reduction in NETs after stimulated neutrophils were treated with tacrolimus. Conclusion: Our data indicate that the proposed local delivery of immunosuppression avoids off-target toxicity while prolonging graft survival in a multiple MHC-mismatch VCA model. Furthermore, NETs are found to play a role in graft rejection and could therefore be a potential innovative therapeutic target.

The Genetic Origin of the Indo-Europeans.

The Yamnaya archaeological complex appeared around 3300BCE across the steppes north of the Black and Caspian Seas, and by 3000BCE reached its maximal extent from Hungary in the west to Kazakhstan in the east. To localize the ancestral and geographical origins of the Yamnaya among the diverse Eneolithic people that preceded them, we studied ancient DNA data from 428 individuals of which 299 are reported for the first time, demonstrating three previously unknown Eneolithic genetic clines. First, a "Caucasus-Lower Volga" (CLV) Cline suffused with Caucasus hunter-gatherer (CHG) ancestry extended between a Caucasus Neolithic southern end in Neolithic Armenia, and a steppe northern end in Berezhnovka in the Lower Volga. Bidirectional gene flow across the CLV cline created admixed intermediate populations in both the north Caucasus, such as the Maikop people, and on the steppe, such as those at the site of Remontnoye north of the Manych depression. CLV people also helped form two major riverine clines by admixing with distinct groups of European hunter-gatherers. A "Volga Cline" was formed as Lower Volga people mixed with upriver populations that had more Eastern hunter-gatherer (EHG) ancestry, creating genetically hyper-variable populations as at Khvalynsk in the Middle Volga. A "Dnipro Cline" was formed as CLV people bearing both Caucasus Neolithic and Lower Volga ancestry moved west and acquired Ukraine Neolithic hunter-gatherer (UNHG) ancestry to establish the population of the Serednii Stih culture from which the direct ancestors of the Yamnaya themselves were formed around 4000BCE. This population grew rapidly after 3750-3350BCE, precipitating the expansion of people of the Yamnaya culture who totally displaced previous groups on the Volga and further east, while admixing with more sedentary groups in the west. CLV cline people with Lower Volga ancestry contributed four fifths of the ancestry of the Yamnaya, but also, entering Anatolia from the east, contributed at least a tenth of the ancestry of Bronze Age Central Anatolians, where the Hittite language, related to the Indo-European languages spread by the Yamnaya, was spoken. We thus propose that the final unity of the speakers of the "Proto-Indo-Anatolian" ancestral language of both Anatolian and Indo-European languages can be traced to CLV cline people sometime between 4400-4000 BCE.

Ischemia/reperfusion injury of porcine limbs after extracorporeal perfusion.

BACKGROUND: Revascularization of amputated extremities after prolonged ischemia is complicated by reperfusion injury. We assessed ischemia/reperfusion (I/R) injury of porcine extremities after prolonged preservation using extracorporeal circulation (ECC). METHODS: Forelimbs of 32 pigs were divided into four groups based on ischemia times: group I: 6 h, group II: 12 h, group III: 0 h plus replantation, and group IV: 6 h plus replantation. Limbs were perfused with autologous blood using ECC for 12 h except group II with only 5 h perfusion. Limbs from groups III and IV were heterotopically replanted with a 7-d follow-up. Contralateral limbs served as controls in all groups. Tissue, plasma, and serum were analyzed for the extent of I/R injury. RESULTS: No significant differences in tissue wet/dry ratios were found within or between groups. This finding was confirmed by histology, except for an increased damage in group IV muscles compared with baseline (P = 0.016). Complement C3 deposition was only increased in group IV muscle (P = 0.031), group II nerves (P = 0.046), and group II vessels (P = 0.037). Group IV muscle and nerve tissues were the only ones with significant IgM antibody deposition (P = 0.031) at end of perfusion. Values were normal again after replantation. Reduced complement activity and elevated IL-6, IL-8, MCP-1, VEGF, PDGF-bb, bFGF, and complement split products were found during perfusion but were normal again after replantation. Staining for heparin sulfate proteoglycans and von Willebrand factor confirmed minimal activation of endothelial cells. CONCLUSION: The results demonstrate that prolonged limb preservation using ECC has minimal impact on I/R-induced tissue injury. Extracorporeal perfusion is a potential limb-preserving technique encouraging further studies for use in limb revascularization.

Transition from nerve stimulator to sonographically guided axillary brachial plexus anesthesia in hand surgery: block quality and patient satisfaction during the transition period.

OBJECTIVES: Sonographic guidance for peripheral nerve anesthesia has proven increasingly successful in clinical practice; however, fears that a change to sonographically guided regional anesthesia may impair the block quality and operating room work flow persist in certain units. In this retrospective cohort study, block quality and patient satisfaction during the transition period from nerve stimulator to sonographic guidance for axillary brachial plexus anesthesia in a tertiary referral center were investigated. METHODS: Anesthesia records of all patients who had elective surgery of the wrist or hand during the transition time (September 1, 2006-August 25, 2007) were reviewed for block success, placement time, anesthesiologist training level, local anesthetic volume, and requirement of additional analgesics. Postoperative records were reviewed, and patient satisfaction was assessed by telephone interviews in matched subgroups. RESULTS: Of 415 blocks, 341 were sonographically guided, and 74 were nerve stimulator guided. Sonographically guided blocks were mostly performed by novices, whereas nerve stimulator-guided blocks were performed by advanced users (72.3% versus 14%; P < .001). Block performance times and success rates were similar in both groups. In sonographically guided blocks, significantly less local anesthetics were applied compared to nerve stimulator-guided blocks (mean ± SD, 36.1 ± 7.1 versus 43.9 ± 6.1 mL; P< .001), and less opioids were required (fentanyl, 66.1 ± 30 versus 90 ± 62 µg; P< .001). Interviewed patients reported significantly less procedure-related discomfort, pain, and prolonged procedure time when block placement was sonographically guided (2% versus 20%; P = .002). CONCLUSIONS: Transition from nerve stimulator to sonographic guidance for axillary brachial plexus blocks did not change block performance times or success rates. Patient satisfaction was improved even during the early institutional transition period.

Evaluation of a porcine whole-limb heterotopic autotransplantation model.

BACKGROUND: Recently performed vascularized composite tissue allotransplantations (CTAs) stimulate the ongoing research in the area of whole-limb transplantation. A reliable in vivo animal model is required for investigations in vascularized whole-limb CTA. The model should allow in vivo assessment in whole-limb preservation, allograft and xenograft response, and host immunomodulation. The goal of this study is to describe and evaluate the in vivo feasibility and reproducibility of a whole-limb porcine model as a basis for future research in this field. MATERIALS AND METHODS: In seven large white pigs, one forelimb was amputated under anesthesia and autotransplanted heterotopically with an arc of rotation of 180° and partially placed in a subcutaneous pocket. Clinical parameters were monitored and muscle biopsies were analyzed using ultrastructural morphological assessment of mitochondria quality after an observation period of 7 days. RESULTS: All animals could fully mobilize postoperatively without restrictions. At sacrifice, the anastomosed pedicle vessels of the limb were patent in six animals. In one pig, venous thrombosis could be observed. Muscle response was triggered following direct electrostimulation in six replanted limbs. The replanted extremities gained 12.97% weight within 7 days postreplantation compared with the amputation baseline values (P = 0.464 while maintaining normal compartment pressures at sacrifice (8.25 ± 5.31 cmH(2)O, P = 0.60). The ultrastructural evaluation of mitochondria morphology revealed intact mitochondria without signs of ischemia/reperfusion damage. CONCLUSION: This porcine model proved feasible, reliable, and reproducible for whole-limb autotransplantation. It presents significant potential in future preclinical research of whole-limb CTA transplantation.