Quantitative analysis of hemodynamics of congested island flaps under leech therapy

Background A congested flap is a good indication for leech therapy. However, determining the appropriate number of leeches as well as the appropriate application time in clinical cases is difficult. We analyzed hemodynamics in rabbit island flaps under leech therapy to find a suitable clinical procedure for determining the appropriate number of leeches to be used and the duration of therapy. Methods Island flaps were raised in 35 rabbit ears, and congestion was induced by venous clamping. Treatment involved use of 1 or 3 leeches and was begun 60 minutes after venous occlusion. Flaps were examined for area of surviving tissue, alterations in transcutaneous oxygen and carbon dioxide tension (TcPO2 and TcPCO2), and flow volume. Arteriole and venule diameters and flow velocities were examined microscopically. Results Flap survival area in the 3-leech therapy group was significantly larger than the 1-leech therapy group and the control group. With 3- leech therapy, TcPCO2 decreased significantly, as did arteriole and venule diameters. After clamp release, TcPCO2 and venule diameter continued to decrease in this group, and flow velocity increased significantly. Conclusions Leech therapy may salvage compromised flaps by replacing congested blood with new arterial blood and thus maintaining flap viability. TcPO2 and TcPCO2 monitoring may help evaluate the therapeutic effect and determine the appropriate number of leeches to apply and the duration of therapy

Quantitative analysis of hemodynamics of congested island flaps under leech therapy

Background A congested flap is a good indication for leech therapy. However, determining the appropriate number of leeches as well as the appropriate application time in clinical cases is difficult. We analyzed hemodynamics in rabbit island flaps under leech therapy to find a suitable clinical procedure for determining the appropriate number of leeches to be used and the duration of therapy. Methods Island flaps were raised in 35 rabbit ears, and congestion was induced by venous clamping. Treatment involved use of 1 or 3 leeches and was begun 60 minutes after venous occlusion. Flaps were examined for area of surviving tissue, alterations in transcutaneous oxygen and carbon dioxide tension (TcPO2 and TcPCO2), and flow volume. Arteriole and venule diameters and flow velocities were examined microscopically. Results Flap survival area in the 3-leech therapy group was significantly larger than the 1-leech therapy group and the control group. With 3- leech therapy, TcPCO2 decreased significantly, as did arteriole and venule diameters. After clamp release, TcPCO2 and venule diameter continued to decrease in this group, and flow velocity increased significantly. Conclusions Leech therapy may salvage compromised flaps by replacing congested blood with new arterial blood and thus maintaining flap viability. TcPO2 and TcPCO2 monitoring may help evaluate the therapeutic effect and determine the appropriate number of leeches to apply and the duration of therapy

Flap Monitoring by TcPCO2

Background: Measurement of transcutaneous oxygen pressure (TcPO2) and transcutaneous carbon dioxide pressure (TcPCO2) has been used for free flap monitoring. Because these values are obtained with sensor probes heated to 44°C, there is potential for low-temperature burns on skin flaps. We measured TcPO2 and TcPCO2 at 37°C in both animals and humans to determine the feasibility and safety of the procedure as a postoperative flap monitoring method. Methods: Twelve epigastric island flaps were elevated in rabbits, and TcPO2 and TcPCO2 were measured at 37°C before and after ligation of the pedicles. In addition, TcPO2 and TcPCO2 at 37°C were measured in healthy men. Subsequently, the method was applied to postoperative monitoring of free flaps in 49 clinical cases. Results: TcPO2 and TcPCO2 values were significantly affected by the experimental flap elevation. A rapid increase in TcPCO2 was observed with both arterial and venous occlusion. In the healthy men, TcPO2 and TcPCO2 were measurable at all skin surface sites. In the clinical cases of free flap transfer, TcPO2 values remained very low for at least 72 hours. TcPCO2 values ranged from 40 to 70 mmHg for 72 hours in more than 80% of cases. In 2 cases, TcPCO2 values increased to more than 90 mmHg, and exploration surgery was performed. These compromised flaps were saved by reanastomosis of the veins. Conclusions: Continuous monitoring of TcPCO2 at 37°C can provide objective information and alert doctors and nurses to the need for checking the free flap

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I) of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

Cathepsin C (CTSC) is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen) is localized exclusively in cortical rods (CRs) of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC) exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn