Cellular and physical microenvironments regulate the aggressiveness and sunitinib chemosensitivity of clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is the most predominant type of kidney cancer in adults and is responsible for approximately 85% of clinical cases. The tumor-specific microenvironment includes both cellular and physical factors, and it regulates the homeostasis and function of cancer cells. Perirenal adipose tissue and tumor-associated macrophages are the major cellular components of the RCC microenvironment. The RCC microvasculature network generates interstitial fluid flow, which is the movement of fluid through the extracellular compartments of tissues. This fluid flow is a specific physical characteristic of the microenvironment of RCC. We hypothesized that there may be an interaction between the cellular and physical microenvironments and that these two factors may play an important role in regulating the behavior of RCC. To elucidate the effects of adipose tissue, macrophages, and fluid flow stimulation on RCC and to investigate the relationships between these factors, we used a collagen gel culture method to generate cancer-stroma interactions and a gyratory shaker to create fluid flow stimulation. Adipose-related cells, monocytes, and fluid flow influenced the proliferative potential and invasive capacity of RCC cells. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by both fluid flow and cellular interactions between RCC and adipose tissue fragments or macrophages. Fluid flow stimulation synergistically enhanced the anti-proliferative effect of sunitinib on RCC cells, but macrophages abolished the synergistic anti-proliferative effect related to fluid flow stimulation. In conclusion, we established a reconstructed model to investigate the cellular and physical microenvironments of RCC in vitro. Our alternative culture model may provide a promising tool for further therapeutic investigations into many types of cancer. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro.

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT in vitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

High-density collagen patch prevents stricture after endoscopic circumferential submucosal dissection of the esophagus: a porcine model.

BACKGROUND AND AIMS: Extensive excision of the esophageal mucosa by endoscopic submucosal dissection (ESD) frequently evokes a luminal stricture. This study aimed to determine the efficacy of a high-density collagen patch for the prevention of esophageal stricture in extensive ESD. METHODS: Six pigs underwent circumferential esophageal ESD under general anesthesia. In 3 pigs, artificial ulcers were covered by 2 collagen patches. The other 3 pigs underwent circumferential ESD only. RESULTS: The 2 collagen patches were settled onto the ulcer surface using a general endoscope and instruments. The collagen patch-treated group showed significantly better patency rates on both the oral and anal sides of the wound area compared with the control group at day 14. The mucosal re-epithelization ratio was significantly promoted, and the extent of mucosal inflammation and fibrosis was significantly decreased with the collagen patch treatment in the wound area. The frequency of cells positive α-smooth muscle actin was significantly reduced in the collagen patch-treated group compared with the control group. CONCLUSIONS: We have established a high-density collagen device that can reduce the esophageal stricture associated with extensive ESD. This easy-to-handle device would be useful during superficial esophageal cancer treatment by ESD.

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073.

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl ß-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.

Damage to histaminergic tuberomammillary neurons and other hypothalamic neurons with traumatic brain injury.

The need for increased sleep after traumatic brain injury is a common and disabling complaint, yet its etiology is unknown. Previous studies have demonstrated diffuse damage to various hypothalamic systems, but the integrity of the histaminergic tuberomammillary nucleus, a major arousal-promoting system located in the posterior hypothalamus, has never been examined in head trauma patients. Here, we demonstrate that severe head trauma is associated with a marked loss (41%) of histaminergic neurons. Reduced histamine signaling may contribute to increased sleep need, and therapies that enhance histaminergic tone may improve arousal after head trauma or other conditions.

Interaction between thyrocytes and adipose tissue in vitro.

Adipose tissue (AT)-thyrocyte interaction is largely unknown. Here we described the interaction in a co-culture system, in which thyrocytes were cultured on AT fragment (ATF)-embedded collagen gel, using electron microscopy, immunocytochemistry, real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). ATFs promoted the hypertrophy, polarization and lipid accumulation of thyrocytes. ATFs did not affect the growth of thyroyctes, and inhibited their apoptosis. ATFs increased the protein expression of thyroglobulin (Tg) and paired box gene 8 (PAX8) in thyrocytes. In turn, thyrocytes decreased the concentration of leptin and adiponectin, and increased the expression of these mRNAs in ATFs. Thyrotropin (TSH) enhanced the ATF-induced nuclear hypertrophy and Tg protein expression in thyrocytes, while TSH enhanced the thyrocyte-induced expression of leptin and adiponectin mRNAs in ATFs. Finally, leptin promoted the hypertrophy and Tg protein expression in thyrocytes. TSH enhanced these leptin-induced effects. The data indicate an active interaction between thyrocytes and AT, suggesting that (i) ATFs may serve to regulate the morphology, survival and differentiation of thyrocytes probably through lipid accumulation partly in a TSH-synergistic way; (ii) thyrocytes may affect adipokine production from ATFs in a TSH-independent manner; and (3) leptin may be related to the hypertrophy and differentiation of thyrocytes in a TSH-synergistic way.

Cytokeratin immunohistochemistry improves interobserver variability between unskilled pathologists in the evaluation of tumor budding in T1 colorectal cancer.

Tumor budding is a major risk factor for T1 colorectal cancer. Quality control of the pathological diagnosis of budding is crucial, irrespective of the pathologist's experience. This study examines the interobserver variability according to pathologists' experience and evaluates the influence of cytokeratin (CK) immunostaining in the assessment of budding. Hematoxylin-eosin (HE) and CK-immunostained slides of 40 cases with T1 primary colorectal cancer were examined. Budding grades were individually evaluated by 12 pathologists who we categorized into three groups by their experience (expert, with >10 years of experience (n = 4), senior, with 5-10 years (n = 4), and junior, < 5 years (n = 4)). The results revealed a tendency for the more experienced pathologists to assign higher budding grades compared to the less-experienced pathologists. In the junior group, the interobserver variability obtained with HE slides was poor, but it was markedly improved in the evaluation using CK-immunostained slides. The benefit of CK immunostaining was only slight in the expert group. CK immunostaining would be useful when a pathologist is not experienced enough or does not have enough confidence in the assessment of budding.

Differential effects of adipose tissue stromal cells on the apoptosis, growth and invasion of bladder urothelial carcinoma between the superficial and invasive types.

OBJECTIVES: To clarify the interaction between adipose tissue stromal cells and bladder cancer cells. METHODS: Superficial (RT4) and invasive (EJ) urothelial carcinoma cells were cultured on adipose tissue stromal cell-embedded or non-embedded collagen gel. Cells were analyzed by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. RESULTS: Adipose tissue stromal cells inhibited growth of RT4, while they promoted the apoptosis. In contrast, adipose tissue stromal cells promoted growth of EJ, but they did not affect the apoptosis. Adipose tissue stromal cells slightly promoted expression of mitogen-activated protein kinase cascade in RT4 and EJ. Adipose tissue stromal cells promoted display of the molecular-targeted agent human epidermal growth factor receptor-2 in only RT4. In turn, RT4 and EJ enhanced α-smooth muscle actin (myofibroblast marker) and S-100 protein (adipocyte marker) expression of adipose tissue stromal cells, respectively. CONCLUSIONS: These findings suggest that: (i) adipose tissue stromal cells might suppress the progression of superficial-type cancer, whereas they might promote that of invasive type; (ii) adipose tissue stromal cell-activated mitogen-activated protein kinase pathway might play differential roles in both types of bladder cancer; (iii) human epidermal growth factor receptor-2 could represent a critical therapeutic agent for the superficial type under adipose tissue stromal cells-cancer interaction; and (iv) superficial bladder cancer might promote myofibroblast differentiation of adipose tissue stromal cells as a cancer-associate phenotype, whereas invasive bladder cancer might promote their adipocyte differentiation.

Low Counts of Plasmacytoid Dendritic Cells after Engraftment Are Associated with High Early Mortality after Allogeneic Stem Cell Transplantation.

Dendritic cells (DCs) are antigen-presenting cells that drive immune responses and tolerance and are divided in different subsets: myeloid DCs (mDCs: lineage-; HLA-DR+, 11c+), plasmacytoid dendritic cells (pDCs: HLA-DR+, CD123+), and monocyte-derived DCs (moDC: lineage-, 11c+, 16+). After hematopoietic stem cell transplantation (HSCT), low DC counts in the recipients' peripheral blood (PB) have been associated with worse outcomes, but the relevance of DC graft content remains unclear, and there are few data in the setting of unrelated donor HSCT. We evaluated the DC graft content and monitored DC recovery in PB from 111 HSCT recipients (median age, 17 years; range 1 to 74), who received bone marrow (46%), umbilical cord blood (32%), or PB (22%) from unrelated (81%) or related donors (19%). In 86 patients with sustained allogeneic recovery, patients with higher counts of all DC subsets (pDC, mDC, and moDC) 3 weeks after engraftment had lower incidence of nonrelapse mortality (NMR) and acute graft-versus-host disease (aGVHD) and better survival. pDC counts were associated with more striking results: patients with higher pDC counts had much lower incidences of NRM (3% versus 47%, P < .0001), lower incidence of aGVHD (24% versus 67%, P < .0001), and better overall survival (92% versus 45%, P < .0001). In contrast, higher pDC counts in the graft was associated with an increased risk of aGVHD (55% versus 26%, P = .02). Our results indicate that DC counts are closely correlated with HSCT outcomes and warrant further prospective evaluation and possible early therapeutic interventions to ameliorate severe aGVHD and decrease mortality.

Molecular Basis of KELnull Phenotype in Brazilians.

BACKGROUND: KELnull (K0) persons can produce clinically significant anti-KEL5 antibody after transfusion and/or pregnancy, requiring K0 blood transfusion when indicated. 37 K0 alleles have been reported in studies over different populations, but none in Amerindian-Caucasian descendants from South America. The aim of this study was to identify the molecular basis of K0 phenotype in Brazilians. METHODS: We investigated three K0 samples from different Brazilian blood banks (Recife, Manaus, and Vila Velha) in women with anti-KEL5. KEL antigen typing was performed by serologic techniques, and the K0 status was confirmed by flow cytometry. PCR-RFLP and DNA sequencing of the KEL coding and exon-intron regions were also performed. RESULTS: RBCs of the 3 patients were phenotyped as KEL:-1,-2,-3,-4,-7. The 3 patients had the same KEL*02/02 genotype and were negative for KEL*02.03 and KEL*02.06 alleles. The Recife K0 patient was homozygous for IVS16 + 1g>a mutation (KEL*02N.31 allele). The flow cytometry with anti-KEL1, anti-KEL2, anti-KEL3, anti-KEL4, and anti-CD238 confirmed the K0 phenotype. In addition, we found the c.10423C>T mutation (KEL*02N.04 allele) in both the Manaus K0 and the Vila Velha K0 patients. CONCLUSION: This report represents the first study of K0 molecular basis performed in Amerindian-Caucasian descendants from South America.

Increase of histaminergic tuberomammillary neurons in narcolepsy.

OBJECTIVE: Narcolepsy is caused by loss of the hypothalamic neurons producing the orexin/hypocretin neuropeptides. One key target of the orexin system is the histaminergic neurons of the tuberomammillary nucleus (TMN), an essential wake-promoting system. As cerebrospinal fluid histamine levels may be low in patients with narcolepsy, we examined histaminergic neurons in patients with narcolepsy and in 2 mouse models of narcolepsy. METHODS: We counted the number of hypothalamic neurons producing orexin, melanin-concentrating hormone, and histamine in 7 narcolepsy patients and 12 control subjects using stereological techniques. We identified histaminergic neurons using immunostaining for histidine decarboxylase. We also examined these systems in 6 wild-type mice, 6 orexin/ataxin-3 transgenic mice, and 5 orexin ligand knockout mice. RESULTS: Compared to controls, narcolepsy patients had 94% more histaminergic TMN neurons (233,572 ± 49,476 vs 120,455 ± 10,665, p < 0.001). This increase was higher in 5 narcolepsy patients with >90% orexin neuron loss than in 2 patients with ≤75% orexin neuron loss (252,279 ± 46,264 vs 186,804 ± 1,256, p = 0.03). Similarly, the number of histaminergic TMN neurons was increased 53% in orexin ligand knockout mice compared to wild-type mice, whereas orexin/ataxin-3 transgenic mice showed an intermediate 28% increase. INTERPRETATION: This surprising increase in histaminergic neurons in narcolepsy may be a compensatory response to loss of excitatory drive from the orexin neurons and may contribute to some of the symptoms of narcolepsy such as preserved consciousness during cataplexy and fragmented nighttime sleep. In addition, this finding may have therapeutic implications, as medications that enhance histamine signaling are now under development.

Histopathology of tenosynovium in trigger fingers.

Stenosing flexor tenosynovitis, trigger finger, is a common clinical disorder causing painful locking or contracture of the involved digits, and most instances are idiopathic. This problem is generally caused by a size mismatch between the swollen flexor tendon and the thickened first annular pulley. Although hypertrophic pulleys have been histologically and ultrasonographically detected, little is known about the histopathology of the tenosynovium covering the tendons of trigger fingers. We identified chondrocytoid cells that produced hyaluronic acid in 23 (61%) fingers and hypocellular collagen matrix in 32 (84%) fingers around the tenosynovium among 38 specimens of tenosynovium from patients with trigger fingers. These chondrocytoid cells expressed the synovial B cell marker CD44, but not the chondrocyte marker S-100 protein. The incidence of these findings was much higher than that of conventional findings of synovitis, such as inflammatory infiltrate (37%), increased vascularity (37%), hyperplasia of synovial lining cells (21%), or fibrin exudation (5%). We discovered the following distinctive histopathological features of trigger finger: hyaluronic acid-producing chondrocytoid cells originated from fibroblastic synovial B cells, and a hypocellular collagen matrix surrounding the tenosynovium. Thus, an edematous extracellular matrix with active hyaluronic acid synthesis might increase pressure under the pulley and contribute to the progression of stenosis.

Orexin receptor 2 expression in the posterior hypothalamus rescues sleepiness in narcoleptic mice.

Narcolepsy is caused by a loss of orexin/hypocretin signaling, resulting in chronic sleepiness, fragmented non-rapid eye movement sleep, and cataplexy. To identify the neuronal circuits underlying narcolepsy, we produced a mouse model in which a loxP-flanked gene cassette disrupts production of the orexin receptor type 2 (OX2R; also known as HCRTR2), but normal OX2R expression can be restored by Cre recombinase. Mice lacking OX2R signaling had poor maintenance of wakefulness indicative of sleepiness and fragmented sleep and lacked any electrophysiological response to orexin-A in the wake-promoting neurons of the tuberomammillary nucleus. These defects were completely recovered by crossing them with mice that express Cre in the female germline, thus globally deleting the transcription-disrupter cassette. Then, by using an adeno-associated viral vector coding for Cre recombinase, we found that focal restoration of OX2R in neurons of the tuberomammillary nucleus and adjacent parts of the posterior hypothalamus completely rescued the sleepiness of these mice, but their fragmented sleep was unimproved. These observations demonstrate that the tuberomammillary region plays an essential role in the wake-promoting effects of orexins, but orexins must stabilize sleep through other targets.

Natural killer cells 56bright16- have higher counts in the umbilical cord blood than in the adult peripheral blood.

INTRODUCTION AND HYPOTHESIS: Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplantation in the absence of a compatible donor. The UCB transplantation has a lower incidence of chronic graft versus host disease (GvHD), but is associated with slower engraftment and slower immune reconstitution, compared to other sources. Dendritic cells (DCs) and Natural Killer cells (NKs) play a central role in the development of GvHD and the graft versus leukemia (GvL) effect, as well as in the control of infectious complications. METHOD: We quantified by multiparametric flow cytometry monocytes, lymphocytes, NK cells, and DCs, including their subsets, in UCB samples from 54 healthy newborns and peripheral blood (PB) from 25 healthy adult volunteers. RESULTS: In the UCB samples, there were higher counts of NK cells 56bright16- (median 0.024 × 109/L), compared to the PB samples (0.012 × 109/L, p < 0.0001), NK 56dim16bright (median 0.446 × 109/L vs. 0.259 × 109/L for PB samples, p = 0.001) and plasmacytoid dendritic cells (pDCs, median 0.008 × 109/L for UCB samples vs. 0.006 × 109/L for PB samples, p = 0.03). Moreover, non-classic monocyte counts were lower in UCB than in PB (median 0.024 × 109/L vs. 0.051 × 109/L, respectively, p < 0.0001). CONCLUSION: In conclusion, there were higher counts of NK cells and pDCs and lower counts of non-classic monocytes in UCB than in PB from healthy individuals. These findings might explain the lower incidence and severity of chronic GvHD, although maintaining the GvL effect, in UCB transplant recipients, compared to other stem cell sources.

MAPK/ERK and PI3K/AKT signaling pathways are activated in adolescent and adult acute lymphoblastic leukemia.

BACKGROUND: The mitogen-activated protein kinase (MAPK)/ERK signaling cascade and the phosphoinosytol-3 phosphate/Akt (PI3K/Akt) pathways are involved in proliferation and differentiation of hematopoietic cells. The frequency of PI3K/Akt and MAPK pathway activation in adult acute lymphoblastic leukemia (ALL) still need to be elucidated. AIMS: To assess the activity and prognostic implications of MAPK/ERK and PI3K/Akt pathways in adult (ALL). METHODS: We examined 28 precursor-B-cell ALL and 6 T-cell primary ALL samples. Flow cytometry was employed to analyze the expression levels of phosphorylated ERK and phosphorylated Akt. RESULTS: Ten out of 15 (67%) ALL fresh samples (7 B-cell, 3 T-cell) showed constitutive p-ERK expression. The p-ERK mean fluorescent index ratio (MFI (R)) showed a tendency to be higher in ALL than in normal T lymphocytes (1.26 [0.74-3.10] vs. 1.08 [1.02-1.21], respectively [p = .069]) and was significantly lower than in leukemic cell lines (median MFI (R) 3.83 [3.71-5.97] [p < .001]). Expression of p-Akt was found in 35% (12/34) (10 B-cell, 2 T-cell). The median MFI (R) expression for p-Akt in primary blast cell was 1.13 (0.48-9.90) compared to 1.01 (1.00-1.20) in normal T lymphocytes (p = ns) and lower than in leukemic cell lines (median MFI (R) 2.10 [1.77-3.40] [p = .037]). Moreover, expression of p-ERK was negatively associated with the expression of CD34 (1.22 [0.74-1.33] vs. 1.52 [1.15-3.10] for CD34(+) and CD34(-) group, respectively, p = .009). CONCLUSION: Our findings suggest that both MAPK/ERK and PI3K/Akt are constitutively activated in adult ALL, indicating a targeted therapy potential for ALL by using inhibitors of these pathways.