Live imaging of avian epiblast and anterior mesendoderm grafting reveals the complexity of cell dynamics during early brain development.

Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations.

Gastric Burkitt's monomorphic post-transplant lymphoproliferative disorder after kidney transplantation: A case report.

We report on a 53-year-old Japanese man diagnosed with gastric Burkitt's monomorphic post-transplant lymphoproliferative disorder (B-PTLD) after endoscopy for gastric discomfort 28 months after the patient underwent renal transplantation in Ethiopia. Serum Epstein-Barr virus (EBV) tests were negative before transplantation, but the tumor cells collected from a gastric biopsy showed positive EBV-encoded small RNAs (EBER) at B-PTLD onset. Intensive treatment started with R(rituximab)-CHOP therapy and continued with DA-EPOCH-R therapy has been effective, and relapse has not yet occurred. Burkitt lymphoma has a poor prognosis, but B-PTLD may be effectively treated with high-dose chemotherapy. This is a rare case of gastric B-PTLD in a Japanese patient.

Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.

Endoderm precursors expressing FoxA2 and Sox17 develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm-generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with Foxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate Foxa2 expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g., Eomes, Gsc, and Sox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E-cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin-rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E-cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post-gastrulation stage embryos: (1) definitive endoderm co-expressing high SOX17, GATA4, and E-cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E-cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E-cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin-rich matrix models the gastrulation-dependent endoderm precursor development.

Intrinsic lens potential of neural retina inhibited by Notch signaling as the cause of lens transdifferentiation.

Embryonic neural retinas of avians produce lenses under spreading culture conditions. This phenomenon has been regarded as a paradigm of transdifferentiation due to the overt change in cell type. Here we elucidated the underlying mechanisms. Retina-to-lens transdifferentiation occurs in spreading cultures, suggesting that it is triggered by altered cell-cell interactions. Thus, we tested the involvement of Notch signaling based on its role in retinal neurogenesis. Starting from E8 retina, a small number of crystallin-expressing lens cells began to develop after 20 days in control spreading cultures. By contrast, addition of Notch signal inhibitors to cultures after day 2 strongly promoted lens development beginning at day 11, and a 10-fold increase in δ-crystallin expression level. After Notch signal inhibition, transcription factor genes that regulate the early stage of eye development, Prox1 and Pitx3, were sequentially activated. These observations indicate that the lens differentiation potential is intrinsic to the neural retina, and this potential is repressed by Notch signaling during normal embryogenesis. Therefore, Notch suppression leads to lens transdifferentiation by disinhibiting the neural retina-intrinsic program of lens development.

Autosomal dominant form of type IV collagen nephropathy exists among patients with hereditary nephritis difficult to diagnose clinicopathologically.

AIM: Type IV collagen nephropathies include Alport Syndrome and thin basement membrane nephropathy (TBMN), which are caused by mutations in COL4A3/A4/A5 genes. Recently, reports of patients with heterozygous mutations in COL4A3/A4 have been increasing. The clinical course of these patients has a wide variety, and they are diagnosed as TBMN, autosomal dominant Alport syndrome (ADAS), or familial focal segmental glomerular sclerosis. However, diagnosis, frequency and clinicopathological manifestation of them remains unclear. We tested COL4A3/A4/A5 genes in patients with hereditary nephritis that was difficult to diagnose clinicopathologically, and investigated who should undergo such testing. METHODS: We performed immunostaining for α5 chain of type IV collagen [α5 (IV)] in 27 patients from 21 families who fitted the following criteria: (i) haematuria and proteinuria (± renal dysfunction); (ii) family history of haematuria, proteinuria, and/or renal dysfunction (autosomal dominant inheritance); (iii) no specific glomerulonephritis; and (iv) thinning, splitting, or lamellation of the glomerular basement membrane (GBM) on electron microscopy. Then we performed genetic testing in 19 patients from 16 families who showed normal α5 (IV) patterns. We conducted a retrospective analysis of their clinicopathological findings. RESULTS: Among 16 families, 69% were detected heterozygous mutations in COL4A3/A4, suggesting the diagnosis of TBMN/ADAS. Twenty-one percent of patients developed end stage renal disease. All patients showed thinning of GBM, which was accompanied by splitting or lamellation in seven patients. CONCLUSION: A considerable fraction of patients with hereditary nephritis that is difficult to diagnose clinicopathologically have TBMN/ADAS. It is important to recognize TBMN/ADAS and perform genetic testing in appropriate patients.

The efficacy and safety of anti-interleukin-6 receptor monoclonal blockade in a renal transplant patient with Castleman disease: early post-transplant outcome.

BACKGROUND: Multicentric Castleman disease (MCD) is an uncommon lymphoproliferative disease characterized by systemic inflammatory reactions associated with the dysregulated production of interleukin-6 (IL-6). In patients with MCD, renal involvement is uncommon, with only one report published regarding kidney transplantation (KTx) to treat end-stage renal disease (ESRD) secondary to MCD. Recent clinical observations have shown that IL-6 production is implicated in allograft rejection, while IL-6 receptor blockade (with tocilizumab [TCZ]) reduces alloantibody generation and thereby improves graft survival; however, the efficacy and safety of TCZ in MCD patients undergoing KTx is still unknown. CASE PRESENTATION: Herein, we describe the case of a 50-year-old man with MCD who received living-donor KTx for ESRD. Post-operative immunosuppression consisted of a triple-drug regimen (tacrolimus, mycophenolate mofetil and methylprednisolone) with TCZ that was administered intravenously every 2 weeks. At 17 months post-transplantation, the patient remains asymptomatic, and the allograft pathology has shown no evidence of rejection and no development of de novo donor-specific antibody (DSA). CONCLUSIONS: To our knowledge, this is the second reported case of an MCD patient with ESRD who underwent successful KTx. TCZ safely supported the patient during the perioperative period, and this drug may be useful for blocking the generation of donor-specific antibodies and reducing the risk of rejection episodes. KTx in combination with TCZ is thus considered a viable treatment option for ESRD due to MCD.

Chx10 functions as a regulator of molecular pathways controlling the regional identity in the primordial retina.

The light-sensitive neural retina (NR) and the retinal pigmented epithelium (RPE) develop from a common primordium, the optic vesicle, raising the question of how they acquire and maintain distinct identities. Here, we demonstrate that sustained misexpression of the Chx10 homeobox gene in the presumptive RPE in chick suppresses accumulation of melanin pigments and promotes ectopic NR-like neural differentiation. This phenotypic change involved ectopic expression of NR transcription factor genes, Sox2, Six3, Rx1 and Optx2, which, when misexpressed, counteracted RPE development without upregulating Chx10. These results suggest that Chx10 can function as a cell autonomous regulator of the regional identity in the primordial retina, presumably through a downstream transcriptional cascade.

Four-dimensional phase-contrast vastly undersampled isotropic projection reconstruction (4D PC-VIPR) MR evaluation of the renal arteries in transplant recipients: Preliminary results.

PURPOSE: To assess the performance of four-dimensional phase-contrast vastly undersampled isotropic projection reconstruction (4D PC-VIPR) at 3.0T in depicting intrarenal arteries compared with computed tomography angiography (CTA), and its correlation with arterial flowmetry in comparison with Doppler ultrasonography (DUS). MATERIALS AND METHODS: In our prospective single-arm study, subjects were 25 patients who underwent renal transplant-related surgery at our hospital between July 2011 and June 2015. In the morphological study, depictions of renal artery branches delineated by magnetic resonance angiography (MRA)/4D PC-VIPR without gadolinium contrast agent were compared in seven living transplant recipients with the same kidney delineated by CTA in seven living transplant donors. In the flowmetric study, flow velocities in the renal (main stem), segmental, and interlobar arteries during systole and diastole were measured in 12 recipients using noncontrast MRA/4D PC-VIPR, and were compared with those obtained from DUS. RESULTS: Concerning MRA, average confidence levels of delineation rated by six observers for secondary to third level renal artery branches were 82.9-100% and for the fourth to fifth branches were 60.8-89.7% (average kappa value of 0.588 [95% confidence interval: 0.522-0.653]). Total flow velocities measured using 4D PC-VIPR and DUS demonstrated significant correlations during both systole and diastole with acceptable bias (r = 0.902; P < 0.001 in systole and r = 0.734; P < 0.001 in diastole). CONCLUSION: 4D PC-VIPR was useful in generating both morphological and hemodynamic information for evaluation of transplant intrarenal arteries without the need for contrast media. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:595-603.

Temporal dissociation of developmental events in the chick eye under low temperature conditions.

The chick embryonic eye is an excellent model for the study of vertebrate organogenesis. Key events in eye development involve thickening, invagination and cytodifferentiation of the lens primordium. While these events occur successively at different developmental stages, the extent to which these events are temporally related is largely unknown. Here we show that the lens invagination is highly sensitive to temperature. Lowering of incubation temperature to 29°C at embryonic day 2 delayed the onset of invagination of the lens, but not thickening and cytodifferentiation, leading to abnormal protrusion of the eye. The temperature shift also delayed the inward bending of the underlying retinal primordium, even in the absence of the lens. Taken together, our results suggest that lens invagination is initiated independently of thickening and cytodifferentiation, possibly by mechanisms associated with morphogenesis of the primordial retina.

BK Virus Nephropathy After Kidney Transplantation and Its Diagnosis Using Urinary Micro RNA.

BK virus-associated nephritis (BKVAN) is an important cause of graft loss in renal transplant recipients B K viremia occurs in up to 30% of renal transplant recipients. Since the discovery of BKV in 1971, effective prophylaxis and treatment have not been established, and it is not uncommon for a transplant kidney to be lost without cure of BKVAN. BK virus infection is reactivated when cellular immunity is suppressed, which is often during the first year after kidney transplantation when cellular immunity is most suppressed. Clinically, it is caused by reactivation of latent infection or new infection from the donor kidney, leading to viremia, viremia, and transplant nephropathy. BK virus nephropathy is currently diagnosed definitively by measuring the amount of BK virus DNA in the blood and proving SV40-positive cells in transplant kidney tissue obtained by transplant kidney biopsy, but the time required for diagnosis and the low sensitivity of immunohistochemistry using antibodies are problematic. Therefore, we investigated whether the diagnosis of BK virus nephropathy could be made earlier by searching for miRNAs in the urine of renal transplant recipients.

Early diagnosis of tuberculous peritonitis in peritoneal dialysis patients using the T-SPOT test.

A 49-year-old man was admitted with peritonitis nine months after starting continuous ambulatory peritoneal dialysis (CAPD) for kidney failure. Ceftazidime and cefazolin were started. Peritoneal dialysate culture was negative for bacteria, but antibiotic treatment was continued because peritonitis improved. Twenty days later, the patient was discharged with no signs of peritonitis. However, 40-day culture of the original peritoneal dialysate detected Mycobacterium tuberculosis, and peritonitis recurred, leading to readmission. A T-SPOT test was performed and was positive in 4 days. Anti-tuberculosis therapy was started, which cured the peritonitis. The T-SPOT test may enable early diagnosis of tuberculosis.

Renal cell carcinoma preceded by a rheumatoid­like paraneoplastic syndrome: A case report.

A man with polycystic kidney disease and a history of renal transplantation at the age of 55 years developed seronegative rheumatoid arthritis (RA) at the age of 68 years. Treatment with a biological derivative led to remission; however, the patient relapsed 2 years later. After being switched to baricitinib, the patient again achieved remission. After 2 years, when the patient was aged 72 years, RA recurred, and the right native kidney became enlarged due to the presence of a large tumor. Surgical nephrectomy was performed, and the tumor was classified as renal cell carcinoma (RCC), not otherwise specified. The cancer tissue comprised sarcomatoid and rhabdoid cells with marked neutrophil infiltration, and the tumor cells were positive for interleukin-6. The patient, aged 73 years, experienced a resolution of joint pain following surgical intervention; however, they died because of systemic metastases ~10 weeks post-operation. Based on the clinical course, the RA-like lesions and subsequent RCC were considered to represent a paraneoplastic syndrome.

De novo posttransplant membranous nephropathy after COVID-19 vaccination 9 years after renal transplantation in a patient with polycystic kidney disease.

A 63-year-old man with polycystic kidney disease underwent kidney transplantation from his wife. Nine years later, after the first and second doses of the COVID-19 vaccination, he developed proteinuria, hematuria, and elevated C-reactive protein. Kidney biopsy 7 months after the initial appearance of proteinuria showed immunoglobulin (Ig)-G granular stain, predominantly IgG1, and spike formation in the glomerular basement membrane. Electron microscopy revealed mainly subepithelial deposits, which corresponds to membranous nephropathy (MN) stage 3 of the Ehrenreich-Churg classification indicating chronic disease, but it also showed electron-dense deposits and endothelial damage. Because a kidney biopsy was performed 1 h after renal transplantation and a biopsy of the patient's native kidney showed intact glomeruli, atypical de novo posttransplant membranous nephropathy (MN) was diagnosed, and a close relationship with COVID-19 vaccination was assumed. Clinicians should consider the involvement of COVID-19 vaccination in de novo posttransplant MN with unclear pathogenesis.

An anteroposterior wave of vascular inhibitor downregulation signals aortae fusion along the embryonic midline axis.

Paracrine signals, both positive and negative, regulate the positioning and remodeling of embryonic blood vessels. In the embryos of mammals and birds, the first major remodeling event is the fusion of bilateral dorsal aortae at the midline to form the dorsal aorta. Although the original bilaterality of the dorsal aortae occurs as the result of inhibitory factors (antagonists of BMP signaling) secreted from the midline by the notochord, it is unknown how fusion is later signaled. Here, we report that dorsal aortae fusion is tightly regulated by a change in signaling by the notochord along the anteroposterior axis. During aortae fusion, the notochord ceases to exert its negative influence on vessel formation. This is achieved by a transcriptional downregulation of negative regulators while positive regulators are maintained at pre-fusion levels. In particular, Chordin, the most abundant BMP antagonist expressed in the notochord prior to fusion, undergoes a dramatic downregulation in an anterior to posterior wave. With inhibitory signals diminished and sustained expression of the positive factors SHH and VEGF at the midline, fusion of the dorsal aortae is signaled. These results demonstrate a novel mechanism by which major modifications of the vascular pattern can occur through modulation of vascular inhibitors without changes in the levels of positive vascular regulators.

Nitric oxide induces vascular endothelial growth factor expression in the rat placenta in vivo and in vitro.

We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6-24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

[Salvage therapy for castration-refractory prostate cancer resistant to docetaxel].

OBJECTIVES: To evaluate the treatment for castration-refractory prostate cancer (CRPC) resistant to docetaxel MATERIALS AND METHODS: Among 45 patients with CRPC treated with docetaxel (70-75 mg/m2) every 3 to 4 weeks at Hamamatsu University Hospital from January 2004 to July 2012, 19 patients underwent salvage treatments. We retrospectively analyzed the medical records of 14 patients except for 5 patients who were enrolled in clinical trials. RESULTS: The median age and serum prostate-specific antigen (PSA) level at starting salvage treatments was 71 years (range 45 to 79) and 241.1 ng/mL (range 3.06 to 1,643.0), respectively. All patients maintained castration status. Salvage treatments include DTX (30 mg/m2) + cisplatin (CDDP) (70 mg/m2)/carboplatin (Area under the curve = 4), etoposide + CDDP, paclitaxel + CDDP, cyclophosphamide, S-l, tegaful-uracil. The reasons why 14 patients moved to salvage treatments after DTX were progressive disease in 12 patients and adverse events in 2. Eight patients had a PSA response, 3 patients>50% and 5 patients<50%. Six patients had a PSA progression. The median overall survival was 10.4 months (range 4.1 to 27.3). All patients died of cancer, 13 patients with prostate cancer and one patient with lung adenocarcinoma. Most adverse events were mild. Transitory grade 3 leukopenia was observed in 2 patients, and grade 3 anemia in 2. No grade 4 toxicities were noted. CONCLUSIONS: All salvage treatments without grade 4 toxicities described in this study may be acceptable in the patients with CRPC progressing after docetaxel although the effect would be limited.

[Bilateral adrenal hemorrhage due to adrenal metastasis of lung cancer].

A 58-year-old man presented with nausea and left flank pain. The patient was referred to our hospital based on clear detection of anemia and computed tomography findings of bilateral adrenal tumors with hemorrhage and a mass in the apex of the left lung. Right adrenal artery embolization had no effect on enlargement of the right adrenal hematoma or advanced anemia. Right adrenalectomy was then performed in an attempt to control hemorrhaging and make a definitive diagnosis, and the patient's anemia improved following the operation. Histopathological diagnosis suggested adrenal metastasis of lung adenocarcinoma, which was subsequently diagnosed given similarities in transbronchial biopsy findings to those in the right adrenal gland. Adrenal hemorrhage due to metastasis of lung cancer is an extremely rare condition; indeed, to our knowledge, the present case is only the 26th reported worldwide. However, prognosis for this mortal condition may be improved should patients receive adrenalectomy followed by an appropriate treatment regimen.

First Multicenter Clinical Trial on Machine Perfusion Preservation for Marginal Donor Kidney Transplantation in Japan.

BACKGROUND: Machine perfusion has not been widely used because of its low demand in Japan; however, we believe its advantages may increase the number of organ transplants. METHODS: Here, we report the first clinical trial of machine perfusion for kidney transplantation in Japan. We used the CMP-X08 perfusion device (Chuo-Seiko Co, Ltd, Asahikawa, Hokkaido, Japan) to preserve the donated organs. The flow rate, perfusion pressure, renal resistance, and temperature were monitored during continuous hypothermic perfusion. RESULTS: From August 2020 to the present, 13 cases of perfusion-preserved kidney transplantation have been performed. Of these, ten and 3 cases were performed using organs donated after brain death (DBD) and cardiac death (DCD), respectively. The average age of the recipients was 55.9 ± 7.3 (45-66) years. The average dialysis period was 14.8 ± 8.4 (0-26) years. The donor's final creatinine level before organ retrieval was 1.58 ± 1.0 (0.46-3.07) mg/dL. The warm ischemic times of the 3 DCD donors were 3, 12, and 18 minutes. The average total ischemic time was 12.0 ± 3.7 (7.17-19.88) hours. The average MP time was 140 (60-240) minutes. A total of 7 cases had delayed graft function. The best creatinine level during hospitalization was 1.17 ± 0.43 (0.71-1.85) mg/dL. There were no primary non-functional cases, and perfusion preservation was safely performed in all cases. CONCLUSIONS: Therefore, we present this report as the first clinical trial on machine perfusion for kidney transplantation from marginal donors with DBD and DCD in Japan.

Wnt signal-dependent antero-posterior specification of early-stage CNS primordia modeled in EpiSC-derived neural stem cells.

The specification of the embryonic central nervous system (CNS) into future brain (forebrain, midbrain, or hindbrain) and spinal cord (SC) regions is a critical step of CNS development. A previous chicken embryo study indicated that anterior epiblast cells marked by Sox2 N2 enhancer activity are specified to the respective brain regions during the transition phase of the epiblast to the neural plate-forming neural primordium. The present study showed that the SC precursors positioned posterior to the hindbrain precursors in the anterior epiblast migrated posteriorly in contrast to the anterior migration of brain precursors. The anteroposterior specification of the CNS precursors occurs at an analogous time (∼E7.5) in mouse embryos, in which an anterior-to-posterior incremental gradient of Wnt signal strength was observed. To examine the possible Wnt signal contribution to the anteroposterior CNS primordium specification, we utilized mouse epiblast stem cell (EpiSC)-derived neurogenesis in culture. EpiSCs maintained in an activin- and FGF2-containing medium start neural development after the removal of activin, following a day in a transitory state. We placed activin-free EpiSCs in EGF- and FGF2-containing medium to arrest neural development and expand the cells into neural stem cells (NSCs). Simultaneously, a Wnt antagonist or agonist was added to the culture, with the anticipation that different levels of Wnt signals would act on the transitory cells to specify CNS regionality; then, the Wnt-treated cells were expanded as NSCs. Gene expression profiles of six NSC lines were analyzed using microarrays and single-cell RNA-seq. The NSC lines demonstrated anteroposterior regional specification in response to increasing Wnt signal input levels: forebrain-midbrain-, hindbrain-, cervical SC-, and thoracic SC-like lines. The regional coverage of these NSC lines had a range; for instance, the XN1 line expressed Otx2 and En2, indicating midbrain characteristics, but additionally expressed the SC-characteristic Hoxa5. The ranges in the anteroposterior specification of neural primordia may be narrowed as neural development proceeds. The thoracic SC is presumably the posterior limit of the contribution by anterior epiblast-derived neural progenitors, as the characteristics of more posterior SC regions were not displayed.

Antiphospholipid Syndrome Nephropathy with Acute Thrombotic Microangiopathy after Renal Transplantation.

We experienced a 36-year-old man with lupus nephritis and antiphospholipid syndrome (APS) who received a donor kidney from his father. Twenty-two months after transplantation, at a time of poor adherence to immunosuppressants and warfarin, the patient developed sudden graft loss due to hemolytic uremic syndrome with rapid deterioration of renal function, thrombocytopenia, and hemolytic anemia. A kidney biopsy showed thrombotic microangiopathy (TMA) related to platelet thrombus formation; however, there was no recurrence of lupus and no findings suggestive of post-transplant rejection, so acute TMA associated with APS was thought to be the cause of the graft loss. This case highlights the importance of instructing patients with lupus nephritis to adhere to treatment with warfarin, a therapeutic drug for APS.