Transplantation of pancreatic beta-cell spheroids in mice via non-swellable hydrogel microwells composed of poly(HEMA-co-GelMA).

Pancreatic islet transplantation is an effective treatment for type I diabetes mellitus. However, many problems associated with pancreatic islet engraftment remain unresolved. In this study, we developed a hydrogel microwell device for islet implantation, fabricated by crosslinking gelatin-methacryloyl (GelMA) and 2-hydroxyethyl methacrylate (HEMA) in appropriate proportions. The fabricated hydrogel microwell device could be freeze-dried and restored by immersion in the culture medium at any time, allowing long-term storage and transport of the device for ready-to-use applications. In addition, due to its non-swelling properties, the shape of the wells of the device was maintained. Thus, the device allowed pancreatic ß cell lines to form spheroids and increase insulin secretion. Intraperitoneal implantation of the ß cell line-seeded GelMA/HEMA hydrogel microwell device reduced blood glucose levels in diabetic mice. In addition, they were easy to handle during transplantation and were removed from the transplant site without peritoneal adhesions or infiltration by inflammatory cells. These results suggest that the GelMA/HEMA hydrogel microwell device can go from spheroid and/or organoid fabrication to transplantation in a single step.

Near-infrared photoimmunotherapy targeting PD-L1: Improved efficacy by preconditioning the tumor microenvironment.

Near-infrared photoimmunotherapy (NIR-PIT) is a new type of cancer therapy that employs antibody-IRDye700DX conjugates (AbPCs) and near-infrared (NIR) light at a wavelength of 689 nm, the excitation wavelength of IR700. Administered intravenously, injected AbPCs bind specifically to cells expressing the target antigen, whereupon NIR light exposure causes rapid, selective killing. This process induces an anticancer T cell response, leading to sustained anticancer host immune response. Programmed cell death ligand-1 (PD-L1) is a major inhibitory immune checkpoint molecule expressed in various cancers. In this study, we first assessed the efficacy of PD-L1-targeted NIR-PIT (αPD-L1-PIT) in immune-competent tumor mouse models. αPD-L1-PIT showed a significant therapeutic effect on the tumor models with high PD-L1 expression. Furthermore, αPD-L1-PIT induced an abscopal effect on distant tumors and long-term immunological memory. In contrast, αPD-L1-PIT was not as effective for tumor models with low PD-L1 expression. To improve the efficacy of PD-L1-targeted NIR-PIT, PEGylated interferon-gamma (IFNγ) was administered with αPD-L1-PIT. The combination therapy improved the treatment efficacy by increasing PD-L1 expression leading to more efficient cell killing by αPD-L1-PIT. Furthermore, the PEGylated IFNγ led to a CD8+ T cell-dominant tumor microenvironment (TME) with an enhanced anticancer T cell response after αPD-L1-PIT. As a result, even so-called cold tumors exhibited complete responses after αPD-L1-PIT. Thus, combination therapy of PEGylated IFNγ and PD-L1-targeted NIR-PIT has the potential to be an important future strategy for cancer immunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) of bone metastases.

The bones are a common site for metastasis arising from solid tumors such as breast and prostate cancer. Chemotherapy, including immunotherapy, is rarely curative. Radiotherapy with pain palliation can temporize bone metastases but is generally considered a short-term solution and retreatment is difficult. Surgery is often necessary, yet recovery times might exceed life expectancy. Therefore, there is a need to develop new approaches to bone metastases that are effective but minimally invasive. Near-infrared photoimmunotherapy (NIR-PIT) uses antibodies labeled with IRDye700DX (IR700) which is activated by NIR light, resulting in rapid cell membrane damage and immunogenic cell death. NIR-PIT using an anti-epidermal growth factor receptor (EGFR) antibody-IR700 conjugate in patients with recurrent head and neck cancer received qualified approval in Japan in 2020 and is now widely used there. However, no bone metastases have yet been treated. In this study, the efficacy of NIR-PIT for bone metastases was investigated using a bone metastases mouse model successfully established by caudal artery injection of a human triple-negative breast cancer cell line, MDAMB468-GFP/luc. The bone metastatic lesions were treated with NIR-PIT using the anti-EGFR antibody, panitumumab-IR700 conjugate. Bioluminescence imaging and histological evaluation showed that EGFR-targeted NIR-PIT has a therapeutic effect on bone metastatic lesions in mice. In addition, micro-CT showed that repeated NIR-PIT led to repair of metastasis-induced bone destruction and restored bone cortex continuity consistent with healing. These data suggest that NIR-PIT has the potential for clinical application in the treatment of bone metastases.

Liver resection in a patient with persistent positive PCR test for coronavirus disease 2019 (COVID-19): a case report.

BACKGROUND: The perioperative mortality rate is high in patients with coronavirus disease 2019 (COVID-19), and infection control measures for medical care providers must be considered. Therefore, the timing for surgery in patients recovering from COVID-19 is difficult. CASE PRESENTATION: A 65-year-old man was admitted to a hospital with a diagnosis of moderate COVID-19. He was transferred to our hospital because of risk factors, including heavy smoking history, type 2 diabetes mellitus, and obesity (BMI 34). Vital signs on admission were a temperature of 36.1 °C, oxygen saturation > 95% at rest, and 94% on exertion with 3 L/min of oxygen. Chest computed tomography (CT) showed bilateral ground-glass opacities, predominantly in the lower lungs. Contrast-enhanced abdominal CT incidentally revealed a liver tumor with a diameter of 80 mm adjacent to the middle hepatic vein, which was diagnosed as hepatocellular carcinoma (HCC). After being administered baricitinib, remdesivir, dexamethasone, and heparin, the patient's COVID-19 pneumonia improved, his oxygen demand resolved, and he was discharged on day 13. Furthermore, the patient was initially scheduled for hepatectomy 8 weeks after the onset of COVID-19 following a discussion with the infection control team. However, 8 weeks after the onset of illness, a polymerase chain reaction (PCR) test was performed on nasopharyngeal swab fluid, which was observed to be positive. The positive results persisted till 10 and 11 weeks after onset. Both Ct values were high (≥ 31) out of 45 cycles, with no subjective symptoms. Since we determined that he was no longer contagious, surgery was performed 12 weeks after the onset of COVID-19. Notably, medical staff wearing personal protective equipment performed extended anatomical resection of the liver segment 8 ventral area in a negative-pressure room. The patient had a good postoperative course, with no major complications, including respiratory complications, and was discharged on postoperative day 14. Finally, none of the staff members was infected with COVID-19. CONCLUSIONS: We reported a case regarding the timing of surgery on a patient with persistently positive PCR test results after COVID-19, along with a literature review.

Disialoganglioside GD2-Targeted Near-Infrared Photoimmunotherapy (NIR-PIT) in Tumors of Neuroectodermal Origin.

Disialoganglioside (GD2) is a subtype of glycolipids that is highly expressed in tumors of neuroectodermal origins, such as neuroblastoma and osteosarcoma. Its limited expression in normal tissues makes GD2 a potential target for precision therapy. Several anti-GD2 monoclonal antibodies are currently in clinical use and have had moderate success. Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that arms antibodies with IRDye700DX (IR700) and then exposes this antibody-dye conjugate (ADC) to NIR light at a wavelength of 690 nm. NIR light irradiation induces a profound photochemical response in IR700, resulting in protein aggregates that lead to cell membrane damage and death. In this study, we examined the feasibility of GD2-targeted NIR-PIT. Although GD2, like other glycolipids, is only located in the outer leaflet of the cell membrane, the aggregates formation exerted sufficient physical force to disrupt the cell membrane and kill target cells in vitro. In in vivo studies, tumor growth was significantly inhibited after GD2-targeted NIR-PIT, resulting in prolonged survival. Following GD2-targeted NIR-PIT, activation of host immunity was observed. In conclusion, GD2-targeted NIR-PIT was similarly effective to the conventional protein-targeted NIR-PIT. This study demonstrates that membrane glycolipid can be a new target of NIR-PIT.

Diagnostic imaging in near-infrared photoimmunotherapy using a commercially available camera for indocyanine green.

Near-infrared photoimmunotherapy (NIR-PIT) is a new type of cancer treatment, which was recently approved in Japan for patients with inoperable head and neck cancer. NIR-PIT utilizes antibody-IRDye700DX (IR700) conjugates and NIR light at a wavelength of 690 nm. NIR light exposure leads to physicochemical changes in the antibody-IR700 conjugate cell receptor complex, inducing rapid necrotic cell death. Just as fluorescence guided surgery is useful for surgeons to resect tumors completely, real-time information of tumor locations would help clinicians irradiate NIR light more precisely. IR700 is a fluorescence dye that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for tumor detection. We hypothesized that irradiation with even low-power 690-nm laser light, attenuated by 99% with a neutral-density filter, could be detected with LIGHTVISION without fluorescence decay or therapeutic effect because of the long emission tail of IR700 beyond 800 nm (within the detection range of LIGHTVISION). We demonstrated that the LIGHTVISION camera, originally designed for ICG detection, can detect the tail of IR700 fluorescence in real time, thus enabling the visualization of target tumors.

Fluorescence Imaging of Tumor-Accumulating Antibody-IR700 Conjugates Prior to Near-Infrared Photoimmunotherapy (NIR-PIT) Using a Commercially Available Camera Designed for Indocyanine Green.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses antibody-IRDye700DX (IR700) conjugates and was recently approved in Japan for patients with inoperable head and neck cancer. Exposure of the tumor with NIR light at a wavelength of 690 nm leads to physicochemical changes in the antibody-IR700 conjugate-cell receptor complex, resulting in increased hydrophobicity and damage to the integrity of the cell membrane. However, it is important that the tumor be completely exposed to light during NIR-PIT, and thus, a method to provide real-time information on tumor location would help clinicians direct light more accurately. IR700 is a fluorophore that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use in operating rooms. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for NIR-PIT target tumor detection. Due to the limited benefits of adding IR700 molecules, the additional conjugation of IRDye800CW (IR800) or ICG-EG4-Sulfo-OSu (ICG-EG4), which has an overlapping spectrum with ICG, to trastuzumab-IR700 conjugates was performed. Conjugation of second NIR dyes did not interfere the efficacy of NIR-PIT. The dual conjugation of IR800 and IR700 to trastuzumab clearly visualized target tumors with LIGHTVISION by detecting emission light of IR800. We demonstrated that the conjugation of second NIR dyes enables us to provide a real-time feedback of tumor locations prior to NIR-PIT.

Intraoperative indocyanine green fluorescence navigation facilitates complete removal of lymph node metastases from hepatocellular carcinoma.

Indocyanine green (ICG) is a fluorescent dye that selectively accumulates in primary hepatocellular carcinoma (HCC) as well as in extrahepatic metastases of HCC. Reported here is a case of metachronous lymph node (LN) metastases from HCC that were resected using ICG fluorescence navigation. A man in his 70s was referred to this department for suspected LN metastasis from HCC. Computed tomography revealed an enlarged suprapancreatic LN. After a laparotomy, an ICG fluorescence imaging system intraoperatively revealed strong fluorescence of this LN, which was then easily resected. An examination after the removal of the LN revealed fluorescence from the adjacent lymphatic tissue as well, so an additional resection was performed. Pathologically, both LNs were confirmed to be metastases from HCC. In this case, some lymphatic tissue metastases from HCC could not be identified prior to surgery, but intraoperative use of ICG fluorescence navigation facilitated their complete removal.

Effect of Short PEG on Near-Infrared BODIPY-Based Activatable Optical Probes.

Targeted near-infrared (NIR) fluorescence probes are playing a significant role in biomedical imaging because NIR penetrates deeper into tissues and is associated with reduced autofluorescence compared to visible light fluorescence probes. Long-wavelength emitting 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is an attractive platform for synthesizing NIR fluorophores because of its high photostability, high molar absorption coefficient, and sharp absorption and emission spectra. However, its lipophilicity hampers the conjugation chemistry necessary to add targeting moieties. In this study, we synthesized a novel NIR BODIPY derivative, NMP14. Substitutions of ethylene-bridged pyrrole units at the 3- or 5-position of the parent BODIPY chromophore result in a red shift of more than 200 nm. However, NMP14 cannot be conjugated to antibodies because of its hydrophobicity. Therefore, we synthesized NMP13 by adding short poly(ethylene glycol) to NMP14 and successfully conjugated NMP13 to cetuximab and trastuzumab. In vitro microscopic studies showed that NMP13 conjugated antibodies were activated after internalization and lysosomal processing, which means that NMP13 acts as an activatable probe only turning on after cellular internalization. After the administration of NMP13 conjugated antibodies, mice tumors were detected with high tumor to background ratios for a long period. These results suggest that NMP13 has potential as an activatable fluorescence probe for further clinical applications.

Generation of mesothelial progenitor-like cells from mouse-induced pluripotent stem cells.

Mesothelial cells, which cover the surface of visceral organs and serous cavities in mammals, play a crucial role in preventing adhesion. We previously reported that primary mesothelial progenitor cells (MPCs) can not only prevent postoperative adhesion but also promote liver regeneration after hepatectomy. Induced pluripotent stem cells (iPSCs) have the potential to be used for regenerative medicine. Here, we have established a differentiation protocol for mouse iPSC-derived MPCs (miMPCs) via the exposure to defined factors, as well as purification using MPC-specific cell surface antigens. Furthermore, the miMPCs had the ability to suppress postoperative adhesion and facilitate liver regeneration. This is the first report highlighting the generation of functional miMPCs, which may offer potential for de novo cell therapy.

Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree.

Cholangiocarcinoma (CC) is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC) at the peribiliary gland (PBG) of large bile ducts and liver stem/progenitor cell (LPC) at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC) can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5) is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. In vitro experiments using siRNA for SAMD5 revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines. CONCLUSION: SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.

Acute and chronic acalculous cholecystitis associated with aortic dissection.

Acalculous cholecystitis is a rare but life-threatening disease, but its pathogenesis is not fully revealed yet. We experienced two acalculous cholecystitis cases associated with aortic dissection. In Case 1, acalculous cholecystitis occurred just after the exacerbation of the aortic dissection. Laparotomy showed necrotized cholecystitis with fresh thrombi formation. Case 2 developed acalculous cholecystitis on the 65th hospital day of aortic dissection. Laparotomy revealed the perforation of the gallbladder. Histological study revealed fibrosis and hemosiderosis in the subserosal layer. The histological findings of these two patients are quite different: Case 1 is acute ischemic and Case 2 is chronic ischemic. While a few cases of acute ischemic cholecystitis have been reported previously, chronic acalculous cholecystitis (CAC) has not been documented. History of aortic dissection could be a risk factor of acute and CAC due to relatively decreased splanchnic blood flow.

Cell-based therapy for preventing postoperative adhesion and promoting regeneration after hepatectomy.

Postoperative adhesion is a critical clinical issue after almost all abdominal or pelvic surgeries including liver surgery. Postoperative adhesion causes several complications, such as small bowel obstruction and chronic abdominal pain. Furthermore, it makes reoperation much more difficult, leading to increased mortality and morbidity rate. Postoperative adhesion is particularly problematic for repeated hepatectomy, since hepatic malignant neoplasm recurs frequently and repeated hepatectomy is widely used as one of the most curative treatments. Several treatments to reduce postoperative adhesion have been developed, which include laparoscopic surgery, administration of pharmacological agents and use of prophylactic barrier materials. However, none of them are optimal. We have proposed a novel treatment using a cell sheet of fetal liver mesothelial cells (FL-MCs) to prevent postoperative adhesion in a novel mouse model. Besides adhesion, repeated hepatectomy has another serious problem; although the liver has a remarkable ability to regenerate, the recovery of liver mass and function of the remnant liver after multiple repeated hepatectomy is limited. The FL-MC cell sheet enhances proliferation of hepatocytes after hepatectomy by providing growth factors for hepatocytes. Thus the FL-MC sheet could simultaneously solve the two problems associated with repeated hepatectomy.

Use of mouse liver mesothelial cells to prevent postoperative adhesion and promote liver regeneration after hepatectomy.

BACKGROUND & AIMS: Repeated hepatectomy is widely accepted as one of the most effective curative treatment for recurrent hepatocellular carcinoma or liver metastasis from colorectal cancer. It has, however, two critical issues; postoperative adhesion and decrease of liver regenerative capacity. Postoperative adhesion makes surgical operations technically more demanding, leading to increased mortality and morbidity rates. Although the liver has a remarkable regenerative ability, volume and functional restoration after multiple repeated hepatectomy is not generally complete. So a new procedure that overcomes these two issues is required. We examined if a fetal liver mesothelial cells (FL-MCs) sheet could solve these two clinical issues simultaneously. METHODS: We established a novel mouse hepatectomy model that reproduces postoperative adhesion on the resected liver surface. We isolated FL-MCs from mouse fetal liver and prepared a cell sheet. The FL-MCs sheet was then transplanted to the resected liver surface. RESULTS: The FL-MCs sheet effectively prevented postoperative adhesion by expressing PCLP1, one of the transmembrane sialomucin family proteins and by activating the fibrinolytic system. Furthermore, the FL-MCs sheet facilitated liver regeneration by providing growth factors for hepatocytes, allowing quick recovery of liver weight and function. Additionally, we showed that an allogeneic FL-MCs sheet was as effective as a syngeneic cell sheet. CONCLUSIONS: We demonstrate that the FL-MCs sheet is able to not only prevent postoperative adhesion but also promote liver regeneration in both syngeneic and allogeneic transplantation, and hence FL-MCs may serve as a potentially useful cell source for regenerative medicine after hepatectomy.

Nephronectin is upregulated in acute and chronic hepatitis and aggravates liver injury by recruiting CD4 positive cells.

Nephronectin (Npnt) is an extracellular matrix protein known to play a critical role in kidney development; however, its physiological role in the liver remains elusive. Here we show that Npnt expression is upregulated in mouse models of both acute and chronic hepatitis induced by Concanavalin A (Con A) and 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC), respectively. In both models, Npnt was localized in inflammatory foci and was mainly secreted from mesenchymal cells and in part by cholangiocytes. Interestingly, ectopic expression of Npnt in hepatocytes induced the development of granuloma-like cell clusters mainly composed of CD4(+) T cells or NKT cells but did not induce apparent hepatitis. Furthermore, we found that Npnt exacerbated the Con A-induced acute hepatitis. These results indicate that Npnt plays an important role in the initiation of hepatitis by recruiting CD4(+) T cells or NKT cells into the foci of inflammation. In addition, we reveal that Npnt expression is also upregulated in human hepatitis. Therefore, Npnt may be a potential therapeutic target for acute and chronic hepatitis.