Intraoperative bladder visualization by indocyanine green filling and subsequent washout in endoscopic hysterectomy: ICG-Washout method.

INTRODUCTION: Despite a potential risk of bladder injury in laparoscopic hysterectomy (LH) and robot-assisted LH (RaLH), an intraoperative method for delineating the entire bladder with indocyanine green (ICG) has not been established. METHODS: We conducted a preliminary experiment using porcine bladders to verify the appropriate amount of ICG for intraoperative bladder visualization. Afterward, intraoperative bladder visualization was tried in LH and RaLH in two patients suspected of having adhesions around the bladder after previous abdominal surgery. RESULTS: Although near-infrared (NIR) fluorescence was well observed through the wall of the porcine bladder filled with ICG solution at a concentration of 0.024 mg/mL, the subsequent replacement of the ICG solution with saline made the NIR fluorescence brighter. In both patients, the bladder was successfully delineated by NIR fluorescence after filling the bladder with ICG solution and the subsequent washout with saline. CONCLUSION: The ICG-Washout method for locating the bladder by NIR fluorescence could be useful in LH and RaLH.

Outcomes of Cessation of Nucleos(t)ide Analog Administration on Hepatitis B Virus Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation: A Nationwide Retrospective Study.

Monitoring of hepatitis B virus (HBV)-DNA and HBV-DNA-guided preemptive therapy using nucleos(t)ide analogs (NAs) are recommended to prevent the development of hepatitis due to HBV reactivation after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in recipients with resolved HBV infection. However, little is known about the appropriate duration of NA treatment and the effect of NA cessation on the recurrence of HBV reactivation. This study aimed to clarify the consequences of NA cessation in allo-HSCT recipients with resolved HBV infection who experienced HBV reactivation following transplantation. We retrospectively reviewed the clinical records of recipients with resolved HBV infection (hepatitis B surface antigen [HBsAg]-negative, anti-HBc-positive) before allo-HSCT who had been diagnosed with HBV reactivation (HBsAg-positive and/or HBV-DNA detectable) after allo-HSCT between January 2010 and December 2020. A total of 72 patients from 16 institutions were registered (median age, 60 years; age range, 27 to 73 years; 42 males and 30 females). The day of initial HBV reactivation ranged from day 10 to day 3034 after allo-HSCT (median, 513 days). Anti-HBs were lost in >80% of the patients at the time of HBV reactivation. All 72 patients received preemptive NAs, and no fatal HBV reactivation-related hepatitis was observed. HBV-DNA without hepatitis was continuously detected in 5 patients during the follow-up period. Administration of NAs was discontinued in 24 of 72 patients (33%) by physician decision. Second HBV reactivation occurred in 11 of the 24 patients (46%) in whom administration of NAs was discontinued. The duration of NA treatment did not differ significantly between patients with or without second HBV reactivation. The frequency of further HBV reactivation tended to be lower in patients with an anti-HBs titer of >10 mIU/mL at the time of NA cessation. Multiple reactivations of HBV after NA cessation was common in patients with HBV reactivation who underwent allo-HSCT despite the long duration of NAs. Careful monitoring of HBV-DNA is important even after the discontinuation of NAs in the case with HBV reactivation after allo-HSCT, because multiple reactivations could occur. Active immunization by HB vaccine might be effective for suppressing further HBV reactivation after cessation of NAs.

A novel Foley catheter made of high-intensity near-infrared fluorescent silicone rubber for image-guided surgery of lower rectal cancer.

BACKGROUND: Urethral injury occurs in 1-6 % of male cases during minimally invasive surgery of lower rectal cancer. A Foley catheter emitting near-infrared (NIR) fluorescence of sufficient intensity has been expected to locate the urethra during image-guided surgery. Although it has been difficult to impart NIR fluorescent properties to biocompatible thermosetting polymers, we have recently succeeded in developing a NIR fluorescent compound for silicone rubber and a NIR fluorescent Foley catheter (HICARL). Here, we evaluated its NIR fluorescence properties and visibility performance using porcine anorectal isolation specimens. METHODS: The HICARL catheter was made of a mixture of solid silicone rubber and a NIR fluorescent compound that emits fluorescence with a wavelength of 820-880 nm, while a conventional transparent Foley catheter was made of solid silicone rubber only. As a standard for comparison of the intensity of NIR fluorescence, a transparent Foley catheter the lumen of which was filled with a mixture of indocyanine green (ICG) and human plasma was used. As a comparison to assess the visibility performance of the HICARL catheter, a transparent Foley catheter into which a commercially available NIR fluorescent polyurethane ureteral catheter (NIRC) was placed was used. RESULTS: A NIR fluorescence quantitative imaging analysis revealed that the Foley-NIRC catheter and the HICARL catheter emitted 3.42 ± 0.42 and 6.43 ± 0.07 times more fluorescence than the Foley-ICG catheter, respectively. The location of the HICARL catheter placed in the anorectum with a wall thickness of 3.8 ± 0.1 mm was clearly delineated in its entirety by NIR fluorescence, while that of the Foley-NIRC catheter was faintly or only partially visible. CONCLUSIONS: The HICARL catheter emitting NIR fluorescence of sufficient intensity is a promising and easy-to-use tool for urethral visualization during image-guided surgery of lower rectal cancer.

Dual action of serotonin on local excitatory and inhibitory neural circuits regulating the corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus.

Serotonergic neurons originating from the raphe nuclei have been proposed to regulate corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVH). Since glutamate- and γ-aminobutyric acid (GABA)-containing neurons, constituting the hypothalamic local circuits, innervate PVH CRF neurons, we examined whether they mediate the actions of serotonin (5-hydroxytryptamine [5-HT]) on CRF neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in PVH CRF neurons, under whole cell patch-clamp, using the CRF-modified yellow fluorescent protein (Venus) ΔNeo mouse. Serotonin elicited an increase in the frequency of sEPSCs in 77% of the cells and a decrease in the frequency of sIPSCs in 71% of the cells, tested in normal medium. Neither the amplitude nor decay time of sEPSC and sIPSC was affected, thus the site(s) of action of serotonin may be presynaptic. In the presence of tetrodotoxin (TTX), serotonin had no significant effects on either parameter of sEPSC or sIPSC, indicating that the effects of serotonin are action potential-dependent, and that the presynaptic interneurons are largely intact within the slice; distant neurons may exist, though, since some 20%-30% of neurons did not respond to serotonin without TTX. We next examined through what receptor subtype(s) serotonin exerts its effects on presynaptic interneurons. DOI (5-HT2A/2C agonist) mimicked the action of serotonin on the sIPSCs, and the serotonin-induced decrease in sIPSC frequency was inhibited by a selective 5-HT2C antagonist RS102221. 8-OH-DPAT (5-HT1A/7 agonist) mimicked the action of serotonin on the sEPSCs, and the serotonin-induced increase in sEPSC frequency was inhibited by a selective 5-HT7 antagonist SB269970. Thus, serotonin showed a dual action on PVH CRF neurons, by upregulating glutamatergic- and downregulating GABAergic interneurons; the former may partly be mediated by 5-HT7 receptors, whereas the latter by 5-HT2C receptors. The CRF-Venus ΔNeo mouse was useful for the electrophysiological examination.

Efficacy and Safety of Gastrointestinal Tumour Site Marking with da Vinci-Compatible Near-Infrared Fluorescent Clips: A Case Series.

BACKGROUND: The conventional near-infrared fluorescent clip (NIRFC) ZEOCLIP FS® has been used successfully in marking tumour sites during laparoscopic surgeries. However, this clip is difficult to observe with the Firefly imaging system equipped with the da Vinci® surgical system. We have been involved in the modification of ZEOCLIP FS® and development of da Vinci-compatible NIRFC. This is the first prospective single-centre case series study verifying the usefulness and safety of the da Vinci-compatible NIRFC. METHODS: Twenty-eight consecutive patients undergoing da Vinci®-assisted surgery for gastrointestinal cancer (16 gastric, 4 oesophageal, and 8 rectal cases) between May 2021 and May 2022 were enrolled. RESULTS: Tumour location was identified by the da Vinci-compatible NIRFCs in 21 of 28 (75%) patients, which involved 12 gastric (75%), 4 oesophageal (100%), and 5 rectal (62%) cancer cases. No adverse events were observed. CONCLUSION: Tumour site marking with da Vinci-compatible NIRFC was feasible in 28 patients enrolled in this study. Further studies are warranted to substantiate the safety and improve the recognition rate.

Risk factors for CAR-T cell manufacturing failure among DLBCL patients: A nationwide survey in Japan.

For successful chimeric antigen receptor T (CAR-T) cell therapy, CAR-T cells must be manufactured without failure caused by suboptimal expansion. In order to determine risk factors for CAR-T cell manufacturing failure, we performed a nationwide cohort study in Japan and analysed patients with diffuse large B-cell lymphoma (DLBCL) who underwent tisagenlecleucel production. We compared clinical factors between 30 cases that failed (7.4%) with those that succeeded (n = 378). Among the failures, the proportion of patients previously treated with bendamustine (43.3% vs. 14.8%; p < 0.001) was significantly higher, and their platelet counts (12.0 vs. 17.0 × 104 /µL; p = 0.01) and CD4/CD8 T-cell ratio (0.30 vs. 0.56; p < 0.01) in peripheral blood at apheresis were significantly lower than in the successful group. Multivariate analysis revealed that repeated bendamustine use with short washout periods prior to apheresis (odds ratio [OR], 5.52; p = 0.013 for ≥6 cycles with washout period of 3-24 months; OR, 57.09; p = 0.005 for ≥3 cycles with washout period of <3 months), low platelet counts (OR, 0.495 per 105 /µL; p = 0.022) or low CD4/CD8 ratios (

Delayed perforation after cold snare polypectomy for small colonic polyps in a patient receiving oral corticosteroids.

This case report describes a fatal outcome due to delayed perforation after cold snare polypectomy in a patient with bullous pemphigoid receiving oral corticosteroids. Cold snare polypectomy has become the standard treatment for small colorectal polyps because of the procedure's safety and simplicity. In this case, however, corticosteroid therapy and vasculitis may have caused local necrosis and tearing of the intestinal wall. Corticosteroids are widely used, and perforation after cold snare polypectomy is extremely rare. However, some patients on corticosteroid therapy may have special pathologies, such as in this case, and we advise physicians to use appropriate judgment and extreme caution in determining the indication for endoscopic therapy.

Complete Bone Marrow Necrosis with Charcot-Leyden Crystals Caused by Myeloid Neoplasm with Mutated NPM1 and TET2.

Bone marrow necrosis (BMN) has various underlying diseases. In hematological malignancies, both lymphoid and myeloid neoplasms have been shown to cause BMN. Charcot-Leyden crystals (CLCs) are bipyramidal crystals that have been found in patients with immune system diseases, tumors, skin diseases, asthma, infections, and intestinal diseases. Because the combination of CLCs and acute myeloid leukemia (AML) is rare, the relationship between BMN, CLCs, and AML remains largely unexplored. We herein report a suspected case of AML that was difficult to diagnose morphologically because of complete BMN with CLCs but achieved complete hematologic remission with treatment similar to that for AML.

The impact of the quantitative assessment procedure for coronary artery bypass graft evaluations using high-resolution near-infrared fluorescence angiography.

PURPOSE: Near-infrared angiography (NIR) is used for on-site graft assessment during coronary artery bypass grafting. This study evaluated the results of a quantitative NIR assessment using a new high-resolution NIR device (h-NIR) for graft assessment. METHODS: Forty-three patients were enrolled in our study. Internal thoracic artery (ITA) grafts anastomosed to the left anterior descending artery and examined intraoperatively using h-NIR were included. The ITA grafts were divided into 2 groups for a comparative analysis: patent grafts (P group; n = 37) and failed grafts (F group; n = 6). The graft flow was evaluated by a "quantitative NIR assessment", and the fluorescence luminance intensity (FLI) was measured. Direct observation of the graft and anastomosis with h-NIR was also performed. RESULTS: The FLI was higher in the P group than in the F group. The receiver operating characteristic analysis revealed the following cut-off values for FLIs depending on imaging duration: 21.1% at 1 s, 35.5% at 2 s, 58.4% at 3 s, and 83.3% at 4 s. The sensitivity and specificity for detecting graft failure were 83.3% and 69.8-80.6%, respectively. Furthermore, h-NIR was also able to visualize arterial dissection in ITA grafts. CONCLUSIONS: A quantitative NIR assessment with an h-NIR device can improve the detectability of anastomotic stenosis, and h-NIR successfully detected arterial dissection of grafts.

Donepezil, an anti-Alzheimer's disease drug, promotes differentiation and regeneration in injured skeletal muscle through the elevation of the expression of myogenic regulatory factors.

We previously demonstrated that donepezil, an anti-Alzheimer's disease drug, improved skeletal muscle atrophy by enhancing the angiogenesis of endothelial cells and activating the proliferation of satellite cells in a mouse model of peripheral arterial disease. However, the effect of donepezil on muscle differentiation during regeneration remains unclear. Therefore, we measured the expressions of myogenic regulatory factors and late muscle differentiation markers in donepezil-treated C2C12 myoblast cells before and after the induction of cell differentiation. The results indicate that the expressions of myogenin, troponin T (TnT) and myosin heavy chain (MyHC) were significantly increased and myotube formation was accelerated in donepezil-treated cells under the differentiation condition. However, the promotive effect of donepezil on muscle differentiation could not be reproduced by the addition of acetylcholine (ACh) and was not disrupted after treatment with ACh receptor blockers. Moreover, other kinds of acetylcholinesterase inhibitors failed to promote muscle differentiation in C2C12 cells. These results indicate that the specific characteristics of donepezil in the promotion of muscle differentiation are independent of its acetylcholinesterase-inhibitory action. We further found that donepezil induced an incremental shift of the cross-sectional area of myofibers and elevated the expressions of myogenin, TnT and MyHC in a mouse model of cardiotoxin injury. These results suggest that donepezil promotes the differentiation of muscle regeneration upon injury via the elevation of the expressions of myogenic regulatory factors and late muscle differentiation markers. Our findings suggest that donepezil can be a useful therapeutic agent for injured skeletal muscle treatment.

Analytic and Integrative Framework for Understanding Human Sympathetic Arterial Baroreflex Function: Equilibrium Diagram of Arterial Pressure and Plasma Norepinephrine Level.

BACKGROUND: The sympathetic arterial baroreflex is a closed-loop feedback system for stabilizing arterial pressure (AP). Identification of unique functions of the closed system in humans is a challenge. Here we propose an analytic and integrative framework for identifying a static operating point and open-loop gain to characterize sympathetic arterial baroreflex in humans. METHODS AND RESULTS: An equilibrium diagram with two crossing functions of mechanoneural (MN) and neuromechanical (NM) arcs was analyzed during graded tilt maneuvers in seven healthy subjects. AP and plasma norepinephrine level (PNE), as a surrogate for sympathetic nerve activity, and were recorded after vagal modulation of heart function was blocked by atropine. The MN-arc curve was described as a locus of operating points during -7, 0, 15, and 60° head-up tilting (HUT) on a PNE-AP plane. The NM-arc curve was drawn as a line between operating points before and after ganglionic blockade (trimethaphan, 0.1 mg⋅ml-1⋅kg-1) during 0° or 15° HUT. Gain values were estimated from the slopes of these functional curves. Finally, an open-loop gain, which is a most important index for performance of arterial baroreflex, was given by a product of the gain values of MN (GMN) and NM arcs (GNM). Gain values of MN was 8.92 ± 3.07 pg⋅ml-1⋅mmHg-1; and GNM at 0° and 15° HUT were 0.61 ± 0.08 and 0.36 ± 0.05 mmHg⋅ml⋅pg-1, respectively. A postural change from supine to 15° HUT significantly reduced the open-loop gain from 5.62 ± 0.98 to 3.75 ± 0.62. The effects of HUT on the NM arc and open-loop gain seemed to be similar to those of blood loss observed in our previous animal studies. CONCLUSION: An equilibrium-diagram analysis contributes to a quantitative and integrative understanding of function of human sympathetic arterial baroreflex.

View-tuned and view-invariant face encoding in IT cortex is explained by selected natural image fragments.

Humans recognize individual faces regardless of variation in the facial view. The view-tuned face neurons in the inferior temporal (IT) cortex are regarded as the neural substrate for view-invariant face recognition. This study approximated visual features encoded by these neurons as combinations of local orientations and colors, originated from natural image fragments. The resultant features reproduced the preference of these neurons to particular facial views. We also found that faces of one identity were separable from the faces of other identities in a space where each axis represented one of these features. These results suggested that view-invariant face representation was established by combining view sensitive visual features. The face representation with these features suggested that, with respect to view-invariant face representation, the seemingly complex and deeply layered ventral visual pathway can be approximated via a shallow network, comprised of layers of low-level processing for local orientations and colors (V1/V2-level) and the layers which detect particular sets of low-level elements derived from natural image fragments (IT-level).

A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.

Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.

Innovative marking method using novel endoscopic clip equipped with fluorescent resin to locate gastric cancer.

An asymptomatic 76-year-old man presented to our department for the treatment of gastric cancer. Esophagogastroduodenoscopy revealed a superficial elevated lesion with an irregular central depression in the lower third of the stomach; this was confirmed to be adenocarcinoma by biopsy, while abdominal contrast-enhanced CT revealed no abnormal lesions. Based on the patient's clinical diagnosis of early gastric cancer, we planned laparoscopic gastrectomy with preoperative placement of four endoscopic marking clips equipped with indocyanine green-conjugated resin to determine the resection margin. During surgery, a dedicated laparoscopic system was used to detect indocyanine green fluorescence emitted by the clips and determine their precise position. The clips helped to identify an accurate resection line for the stomach, enabling accurate laparoscopic distal gastrectomy with regional lymphadenectomy. We successfully demonstrated the usefulness of clips with fluorescent resin for detecting gastric cancer in patients. We report the first case using the clips to accurately locate a site of interest.

Extracorporeal membrane oxygenation in Stenotrophomonas maltophilia pneumonia during acute myeloid leukemia: A case report.

Stenotrophomonas maltophilia (S. maltophilia) is a Gram-negative, multidrug-resistant organism that both opportunistically infects the bloodstream and leads to pneumonia in immunosuppressed patients, including those with hematologic malignancies. In patients with severe respiratory failure, venovenous extracorporeal membrane oxygenation (VV ECMO) can stabilize the respiratory status. However, whether ECMO in patients with hematologic malignancies improves the clinical outcomes is still controversial because ECMO increases the risk of the exacerbation of sepsis and bleeding. We report a case of a 46-year-old man with Stenotrophomonas maltophilia hemorrhagic pneumonia acquired during consolidation chemotherapy for acute myeloid leukemia in whom VV ECMO lead to a good clinical outcome. The stabilization of his respiratory status achieved with VV ECMO allowed time for trimethoprim-sulfamethoxazole antibiotic therapy to improve the pneumonia. We suggest the background of patients, including comorbidities and general conditions, should be taken into account when considering the clinical indications of ECMO.

Effectiveness and safety of tumor site marking with near-infrared fluorescent clips in colorectal laparoscopic surgery: A case series study.

BACKGROUND: In colorectal laparoscopic surgery, accuracy of tumor marking has been an important but not fully resolved issue. The tattoo marking technique or intraoperative endoscopy have been used but they either carry the risk of accidental intestinal puncture or require either longer operation times, a skilled endoscopist and/or intraoperative colon insufflation. We supposed that tumor site marking with the near-infrared fluorescent clips, ZEOCLIP FS clips (Zeon Medical Co., Ltd., Tokyo, Japan) might overcome disadvantages of both tattoo marking and intraoperative endoscopy-based tumor localization methods. This is the first report on the case series using near-infrared fluorescent marking clip. We summarize the early results in 30 patients, who underwent colorectal laparoscopic surgery; we focus particularly on effectiveness and safety of the method. MATERIALS AND METHODS: Thirty consecutive patients, who underwent laparoscopic surgery for colorectal cancer after previous endoscopic ZEOCLIP FS placement were enrolled from May 2019 till October 2019. The primary endpoint was the rate of intraoperative clip detection and the secondary endpoints were: the rate of adverse effects, percentage of slipped clips and usefulness of plain abdominal radiography to preoperatively confirm the clip retention. Locations of fluorescent clips were identified with a full-color fluorescence laparoscope. All operations and clip placements were performed by the same senior surgeon with sufficient experience in both procedures. RESULTS: Fluorescent clips could be detected in 94.1% of tumor lesions. Three (2.1%) clips dropped before surgery. Plain abdominal radiography was sufficient to assess clip retention in all cases. No adverse effects related to either clip placement or clip detection were observed. CONCLUSION: The ZEOCLIP FS could be easily detected from the serosal side of the intestinal tract when placed 1-2 days before surgery. Fluorescent clip-guided laparoscopy may be considered a safe and effective method for localization of colorectal tumor sites. The Research Registry UIN: researchregistry5400.

Novel endoscopic marking clip equipped with resin-conjugated fluorescent indocyanine green during laparoscopic surgery for gastrointestinal cancer.

PURPOSE: Intraoperative identification of the cancer location is often difficult to conduct during laparoscopic surgery, especially in early-stage cancers. This study aimed to investigate the feasibility and accuracy of a novel endoscopic clip resin-conjugated fluorescent indocyanine green during laparoscopic surgery for gastrointestinal cancer. METHODS: Preoperative placement of endoscopic marking clips equipped with resin-conjugated fluorescent indocyanine green was performed to determine the resection margin in eight patients with gastrointestinal cancer. During laparoscopic surgery, a dedicated laparoscopic system with a xenon light source was used to detect fluorescence. The evaluation determined whether the fluorescent from the clips was visualized during laparoscopic surgery. RESULTS: Fluorescent signal emitted from ICG in the resin of the clips was detected in six patients from the outer layer of the serosal surfaces of the gastrointestinal tract, and the clips aided in accurate resection line of the organ. There were no significant differences of age, gender, and BMI between the patients in whom we could and could not detect ICG fluorescence. CONCLUSIONS: The results demonstrated the usefulness of a novel clip-equipped fluorescent resin, which is a promising diagnostic tool to detect accurate tumor location during laparoscopic surgery.

Near-infrared fluorescent solid material for visualizing indwelling devices implanted for medical use.

BACKGROUND: In clinical practice, various devices are implanted into the body for medical reasons. As X-ray fluoroscopy is necessary to visualize medical devices implanted into the body, the development of a less-invasive visualization method is highly desired. This study aimed to investigate the clinical applicability of our novel solid material that emits near-infrared fluorescence. METHODS: We developed a solid resin material that emits near-infrared fluorescence. This material incorporates a near-infrared fluorescent pigment, with quantum yield ≥ 20 times than that of indocyanine green. It can be sterilized for medical treatment. This resin material is designed to be molded into a catheter and inserted into the body with an endoscope clip. In this preclinical experiment using a swine model, the resin material was embedded into the body of the swine and visualized with a near-infrared fluorescence camera system. RESULTS: Endoscopic clips were placed in the mucosa of the stomach, esophagus, and large intestine, and the indwelling ureteral catheters were successfully visualized by near-infrared fluorescence laparoscopy. CONCLUSIONS: We confirmed the tissue permeability of the fluorescence emitted by our novel near-infrared fluorescent material and the possibility of its clinical application. This material may allow visualization of devices embedded in the body.