Peritoneal Expression of Membrane Complement Regulators Is Decreased in Peritoneal Dialysis Patients with Infected Peritonitis.

In peritoneal dialysis (PD) patients, fungi and Pseudomonas aeruginosa are considered important causative microorganisms for peritonitis with poor prognosis. Our objective was to explore expressions of membrane complement (C) regulators (CRegs) and tissue injuries in the peritoneum of patients with PD-related peritonitis, including fungal and Pseudomonas aeruginosa peritonitis. In peritoneal biopsy tissues obtained at PD catheter removal, we investigated the severity of peritonitis-associated peritoneal injuries and the expression of CRegs, CD46, CD55, and CD59 against peritoneal tissues without any episode of peritonitis. In addition, we evaluated peritoneal injuries among fungal and Pseudomonas aeruginosa-peritonitis (P1) and Gram-positive bacterial peritonitis (P2). We also observed deposition of C activation products such as activated C and C5b-9 and measured sC5b-9 in the PD fluid of patients. As a result, the severity of peritoneal injuries correlated inversely with the expression of peritoneal CRegs. Peritoneal CReg expression in peritonitis was significantly reduced compared to no peritonitis. Peritoneal injuries were more severe in P1 than in P2. CReg expression was further decreased and C5b-9 further increased in P1 than in P2. In conclusion, severe peritoneal injuries due to fungal and Pseudomonas aeruginosa-peritonitis decreased CReg expression and increased deposition of activated C3 and C5b-9 in the peritoneum, suggesting that peritonitis, particularly fungal and Pseudomonas aeruginosa-peritonitis, might induce susceptibility to further peritoneal injuries due to excessive C activation.

Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer.

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8+ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung injury.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.

Establishment of human airway epithelial cells with doxycycline-inducible cell growth and fluorescence reporters.

We previously reported the successful establishment of multiple immortalized cell lines that preserved the original nature of the primary cells via co-expression of R24C mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). However, as these genes are kind of oncogenes, tools to control their expression levels are favorable. In this study, we describe a new polycistronic lentiviral vector expressing proliferation factors, CDK4R24C and Cyclin D1 along with enhanced green fluorescence protein (EGFP) under the control of doxycycline (Dox)-dependent transactivator (rtTA) and tetracycline response element (TRE). By introducing the Dox-inducible lentiviral vector into human airway epithelial cells, we established a novel human airway epithelial cell line harboring polycistronic Dox-inducible CDK4R24C and Cyclin D1, referred to as Tet-on K4D cells. We showed that the cell growth of Tet-on K4D cells could be controlled by Dox. Furthermore, expression of K4D genes and rtTA gene can be independently monitored by fluorescent imaging. Cultured airway epithelial cells are useful as a tool for studying the pathogenesis of lung disorders. Altogether, our established human airway epithelial cells could be used for a variety of studies such as lung pathology and biology underlying the differentiation process to form the complex pseudostratified multicellular layers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00477-0.

Proenkephalin+ regulatory T cells expanded by ultraviolet B exposure maintain skin homeostasis with a healing function.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

Long-term culture of human lung adenocarcinoma A549 cells enhances the replication of human influenza A viruses.

Long-term culture of the human lung adenocarcinoma cell line A549 promotes the differentiation of these cells toward an alveolar type II cell phenotype. Here, we evaluated the susceptibility of long-term cultured A549 cells to human influenza viruses. A549 cells were cultured continuously for 25 days (D25-A549) or 1 day (D1-A549) in Ham's F12K medium. Six human influenza A viruses grew much faster in D25-A549 cells than in D1-A549 cells; however, two influenza B viruses replicated poorly in both cell types. Two avian influenza viruses replicated efficiently in both cell types, with similar titres. Expression levels of human virus receptors were higher in D25-A549 cells than in D1-A549 cells. D25-A549 cells thus more efficiently support the replication of human influenza A viruses compared with D1-A549 cells. Our data suggest that long-term cultured A549 cells will be useful for influenza A virus research.

Regulatory T cells expressing abundant CTLA-4 on the cell surface with a proliferative gene profile are key features of human head and neck cancer.

FOXP3+ regulatory T (Treg) cells suppress anti-tumor immunity. The suppression of Treg cells is regulated by cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose expression on the cell surface is tightly regulated. Here we found that Treg cells expressing abundant CTLA-4 on the cell surface (surface-CTLA-4+ Treg) were expanded in human head and neck cancer tissues. RNA sequencing of surface-CTLA-4+ and surface-CTLA-4- Treg cells infiltrating human head and neck cancer tissues revealed that surface-CTLA-4+ Treg cells have a previously undescribed gene expression profile correlating to cell cycle, cell proliferation, and DNA replication. Moreover, surface-CTLA-4+ Treg cells were PD-1+ , actively proliferated and associated with CD45RA- FOXP3high Treg cells with strong suppressive function. Thus, surface-CTLA-4+ Treg cells with a proliferative gene expression signature and phenotype are key features of head and neck cancer. Targeting surface-CTLA-4+ Treg cells might be new strategies to evoke effective immune responses to head and neck cancer.

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

C5a inhibitor protects against ischemia/reperfusion injury in rat small intestine.

Acute mesenteric ischemia (AMI) is caused by considerable intestinal injury, which is associated with intestinal ischemia followed by reperfusion. To elucidate the mechanisms of ischemia/reperfusion injuries, a C5a inhibitory peptide termed AcPepA was used to examine the role of C5a anaphylatoxin, induction of inflammatory cells, and cell proliferation of the intestinal epithelial cells in an experimental AMI model. In this rat model, the superior mesenteric artery was occluded and subsequently reperfused (Induce-I/R). Other groups were treated with AcPepA before ischemia or reperfusion. Induce-I/R induced injuries in the intestine and AcPepA significantly decreased the proportion of severely injured villi. Induce-I/R induced secondary receptor for C5a-positive polymorphonuclear leukocytes in the vessels and CD204-positive macrophages near the injured site; this was correlated with hypoxia-induced factor 1-alpha-positive cells. Induction of these inflammatory cells was attenuated by AcPepA. In addition, AcPepA increased proliferation of epithelial cells in the villi, possibly preventing further damage. Therefore, Induce-I/R activates C5a followed by the accumulation of polymorphonuclear leukocyte and hypoxia-induced factor 1-alpha-producing macrophages, leading to villus injury. AcPepA, a C5a inhibitory peptide, blocks the deleterious effects of C5a, indicating it has a therapeutic effect on the inflammatory consequences of experimental AMI.

Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells.

In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

Expression of membrane complement regulators, CD46, CD55 and CD59, in mesothelial cells of patients on peritoneal dialysis therapy.

We investigated the expression of membrane complement regulators (CRegs), CD46, CD55 and CD59 in human mesothelial cells, and correlated with clinical background and level of complement (C) activation products in peritoneal dialysis (PD) fluids (PDF) to clarify influence of the C activation system in PD patients. Expression of CRegs was assessed on primary cultures of mesothelial cells (HPMC) harvested from PD fluid of 31 PD patients. Because expression of CD55 but not CD46 and CD59 in mesothelial cells was significantly correlated to value of dialysate-to-plasma creatinine concentration ratio (D/P Cre) (p<0.005) as an indicator of peritoneal function, we focused on analysis of CD55 expression of HPMCs in comparison with levels of C activation products in the PDF of the PD patients, and their background factors. When comparing expression of the CRegs between systemic neutrophils and HPMC, no correlation was observed, supporting that change of CRegs' expression in HPMC was independently occurring in the peritoneum. Expression of CD55 protein in HPMC was closely correlated with expression at the mRNA level (p<0.0001) and was inversely correlated with levels of sC5b-9 (p<0.05), but not C3, C4, IL6 and CA125 in the PDF. Complications of diabetes, usage of icodextrin and residual renal function were not correlated with change of CD55 expression in HPMCs. Our data show that the process of PD therapy modifies expression of CD55 on peritoneal mesothelium and triggers local C activation. These findings support efforts to modify PD therapy to limit effects on activation and regulation of the C system.

Complement C5A antagonist treatment improves the acute circulatory and inflammatory consequences of experimental cardiac tamponade.

OBJECTIVE: Cardiogenic shock often leads to splanchnic macro- and microcirculatory complications, and these events are linked to local and systemic inflammatory activation. Our aim was to investigate the consequences of complement C5a antagonist treatment on the early circulatory and inflammatory changes in a clinically relevant large animal model of cardiac tamponade. DESIGN AND SETTING: A randomized, controlled in vivo animal study in a university research laboratory. SUBJECTS: Anesthetized, ventilated, and thoracotomized Vietnamese mini pigs (24 ± 3 kg). INTERVENTIONS: Group 1 (n = 6) served as sham-operated control. In group 2 (n = 7), cardiac tamponade was induced for 60 minutes by the administration of intrapericardial fluid, while the mean arterial pressure was kept in the interval 40 to 45 mm Hg. Group 3 (n = 6) was treated with a complement C5a antagonist compound (the peptide acetyl-peptide-A, 4 mg/kg) after 45 minutes of tamponade. MEASUREMENTS AND MAIN RESULTS: The macrohemodynamics, including the superior mesenteric artery flow, was monitored; the average red blood cell velocity in the small intestinal mucosa was determined by an intravital orthogonal polarization imaging technique. The whole blood superoxide production, the plasma level of high-mobility group box protein-1 and big-endothelin and the small intestinal myeloperoxidase activity were measured. One hundred eighty minutes after the relief of tamponade, the mean arterial pressure was decreased, while the plasma levels of superoxide, high-mobility group box protein-1, and big-endothelin, and the intestinal myeloperoxidase activity were increased. The administration of acetyl-peptide-A normalized the mean arterial pressure and preserved the cardiac output, while the superior mesenteric artery flow and mucosal average red blood cell velocity were increased significantly, and the plasma superoxide, high-mobility group box protein-1, big-endothelin, and intestinal myeloperoxidase levels were reduced. CONCLUSIONS: These results provide evidence that blockade of the C5a effects significantly influences the acute splanchnic macro- and microhemodynamic complications and decreases the potentially harmful inflammatory consequences of experimental cardiogenic shock.

Interleukin-17A-producing T lymphocytes in chronic active Epstein-Barr virus infection.

T helper (Th) 17 cells are reportedly effector T cells that produce interleukin (IL)-17A and play a significant role in the development of autoimmune diseases and immune responses for antimicrobial host defense. Production of IL-17A in chronic active Epstein-Barr virus infection (CAEBV) was studied to investigate its contribution to pathogenesis of this disease. Significantly more IL-17A-producing cells were detected in the peripheral blood of CAEBV patients than in that of healthy controls, although a significant difference in serum IL-17A production was not confirmed. Of the IL-17A-producing cells, 91.8% were cluster of differentiation (CD)4-positive Th17 cells. Moreover, there were significantly more IL-17A-producing cells among CD4(+) cells in peripheral blood of CAEBV patients than in that of controls (1.97 ± 0.69% vs. 1.09 ± 0.53%, P = 0.0073). These data suggest that IL-17A-producing cells may influence the pathophysiology of CAEBV.

Endothelin receptor antagonist attenuates oxidative stress in a neonatal sepsis piglet model.

BACKGROUND: Oxidative stress (oxidant-antioxidant imbalance) plays an important role in the pathophysiology of neonatal sepsis. This study evaluated whether an antisense peptide endothelin receptor antagonist, ETR-P1/fl, could attenuate oxidative stress in a neonatal sepsis model. METHODS: A total of 18 3-d-old piglets were anesthetized and mechanically ventilated. Six piglets received cecal ligation and perforation (CLP group) for induction of sepsis. Six piglets also received continuous infusion (0.05 mg/kg/h) of ETR-P1/fl 30 min after CLP (ETR-P1/fl group). Six piglets received a sham operation. Serum total hydroperoxide (TH), biological antioxidant potentials (BAPs), oxidative stress index (OSI, calculated as TH/BAP), interleukin (IL)-6, serum glutamic oxaloacetic transaminase (GOT), and creatinine were measured before CLP and at 1, 3, and 6 h after CLP. RESULTS: CLP evoked a state of shock resulting in elevated TH, OSI, and IL-6 levels. ETR-P1/fl administration after CLP resulted in lower serum TH at 1 and 3 h after CLP, OSI at 1 and 3 h after CLP, IL-6 at 1 and 3 h after CLP, and GOT at 3 and 6 h after CLP as compared with the CLP group. CONCLUSION: ETR-P1/fl treatment significantly attenuated the elevation of serum oxidative stress markers (TH and OSI), IL-6, and GOT in a progressive neonatal sepsis CLP model.

Novel complementary peptides to target molecules.

We generated an evolutionary computer program that generates complementary peptide (C-pep) sequences, with the potential to interact with a target peptide, by comparing several physico-chemical parameters of each pair of the complementary peptides being analyzed. We generated C-peps to target several molecules. About 30% of synthesized C-peps interfered with the function of their targets. C5a stimulates generation of TNFα and other inflammatory cytokines. Inhibition of C5a should be effective against sepsis, which impairs the status of cancer-bearing patients. One of the inhibitory C-peps of C5a, termed AcPepA, was effective in Cynomolgus monkeys intravenously infused with a lethal dose of bacterial LPS (4 mg/kg) destined to die. The monkeys were rescued by intravenous administration of 2 mg/kg/h of AcPepA. The excellent therapeutic effect of AcPepA is likely to be due to restriction of high mobility group box 1 (HMGB1) surge induced by the effect of C5a on C5L2, which is the second C5a receptor, since the released HMGB1 has the capacity to stimulate TLR4 as an endogeneous ligand resulting in further activation of inflammatory cells to release inflammatory cytokines forming a positive feedback circuit of inflammation.

Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner.

Ebolavirus (EBOV) is an enveloped, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever with mortality rates of up to 90% in humans and nonhuman primates. Previous studies suggest roles for clathrin- or caveolae-mediated endocytosis in EBOV entry; however, ebolavirus virions are long, filamentous particles that are larger than the plasma membrane invaginations that characterize clathrin- or caveolae-mediated endocytosis. The mechanism of EBOV entry remains, therefore, poorly understood. To better understand Ebolavirus entry, we carried out internalization studies with fluorescently labeled, biologically contained Ebolavirus and Ebolavirus-like particles (Ebola VLPs), both of which resemble authentic Ebolavirus in their morphology. We examined the mechanism of Ebolavirus internalization by real-time analysis of these fluorescently labeled Ebolavirus particles and found that their internalization was independent of clathrin- or caveolae-mediated endocytosis, but that they co-localized with sorting nexin (SNX) 5, a marker of macropinocytosis-specific endosomes (macropinosomes). Moreover, the internalization of Ebolavirus virions accelerated the uptake of a macropinocytosis-specific cargo, was associated with plasma membrane ruffling, and was dependent on cellular GTPases and kinases involved in macropinocytosis. A pseudotyped vesicular stomatitis virus possessing the Ebolavirus glycoprotein (GP) also co-localized with SNX5 and its internalization and infectivity were affected by macropinocytosis inhibitors. Taken together, our data suggest that Ebolavirus is internalized into cells by stimulating macropinocytosis in a GP-dependent manner. These findings provide new insights into the lifecycle of Ebolavirus and may aid in the development of therapeutics for Ebolavirus infection.

Endothelin receptor antagonist attenuates inflammatory response and prolongs the survival time in a neonatal sepsis model.

PURPOSE: To evaluate effects of endothelin receptor antagonist ETR-P1/fl in a neonatal sepsis model. METHOD: Eighteen anesthetized and mechanically ventilated 3-day-old piglets were divided into three groups. Six piglets received cecal ligation and perforation (CLP group). Six piglets were administrated a continuous infusion of ETR-P1/fl (0.05 mg/kg/h), an antisense homology box-derived peptide with an endothelin A receptor antagonist effect, starting 30 min after CLP (ETR-P1/fl group). Six piglets acted as the sham group. Mean arterial pressure (MAP), heart rate, cardiac output, arterial blood gas, body temp (BT), serum nitrite and nitrate (NOx), tumor necrosis factor (TNF)-α, and high-mobility group box 1 (HMGB-1) were measured before CLP and at 1, 3, 6, and 9 h after CLP. RESULTS: Cecal ligation and perforation exposure evoked a state of shock and showed deteriorated cardiac output, pulmonary hypertension, decreased MAP, low oxygen saturation, and base excess (BE) with elevated TNF-α, NOx, and HMGB1. ETR-P1/fl administration resulted in higher MAP at 6 and 9 h after CLP, less negative BE, lower mean pulmonary arterial pressure (mPAP)/MAP ratio at 9 h after CLP, and lower TNF-α, NOx, and HMGB-1 compared to the CLP group. BT showed no differences between the groups. Survival time in the ETR-P1/fl group was longer than in the CLP group (18.9 ± 2.3 h vs. 9.0 ± 0.8 h, p < 0.01). CONCLUSIONS: ETR-P1/fl treatment significantly attenuated the elevation of NOx, TNF-α, and HMGB-1, which improved the systemic hypotension, pulmonary hypertension, and blood gases, thereby causing improvement of survival time in a progressive neonatal sepsis CLP model.

Connective tissue growth factor (CTGF/CCN2) is increased in peritoneal dialysis patients with high peritoneal solute transport rate.

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis (PD) therapy that often occurs in association with peritoneal high transport rate and ultrafiltration failure (UFF). To study the possible pathogenic role of connective tissue growth factor (CTGF) in the relationship of PF and UFF, dialysate CTGF contents (n = 178) and tissue CTGF expression (n = 61) were investigated by ELISA, real-time PCR, immunohistochemistry, and in situ hybridization. CTGF production with and without TGF-beta1 stimulation in human peritoneal mesothelial cells (HPMC) from the spent patients' peritoneal dialysate (n = 32) was studied in vitro. The dialysate-to-plasma ratio for creatinine (D/P Cr) was positively correlated to dialysate CTGF concentration and estimated local peritoneal production of CTGF. CTGF mRNA expression was 11.4-fold higher in peritoneal membranes with UFF than in pre-PD renal failure peritoneum and was correlated with thickness of the peritoneum. CTGF protein and mRNA were detected in mesothelium and in fibroblast-like cells. In cultured HPMC, TGF-beta(1)-induced expression of CTGF mRNA was increased at 12 and 24 h and was correlated with D/P Cr. In contrast, bone morphogenic protein-4 mRNA expression was inversely correlated with D/P Cr. Our results suggest that high peritoneal transport state is associated with fibrosis and increased peritoneal CTGF expression and production by mesothelial cells, which can be stimulated by TGF-beta1. Dialysate CTGF concentration could be a biomarker for both peritoneal fibrosis and membrane function. Functional alteration of mesothelial cells may be involved in progression of peritoneal fibrosis in high transport state.

Effect of the antibody immunotherapy by the anti-MUC1 monoclonal antibody to the oral squamous cell carcinoma in vitro.

To conserve both form and function in the oral area, effective and selective drugs against oral cancer will be required. We focused on MUC1, a transmembrane glycoprotein, that is considered tumor-associated antigens (TAAs) for cancer therapy. Recently, studies were done to evaluate the patterns of MUC1 expression in oral squamous cell carcinomas (OSCCs) and it was found that higher MUC1 expression correlates with tumor invasion and metastasis. Using oral squamous cell carcinoma cell lines, we demonstrate here that tumor-specific targeting of MUC1 with the specific monoclonal antibody C595 has functional consequences with regard to complement deposition on MUC1-expressing oral cancer cell lines. Anti-MUC1 monoclonal antibody (mAb) also induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) to OSCC cells, and these effects were strongly correlated with MUC1 expression. Thus, these results indicate that anti-MUC1 mAb could provide a useful tool against OSCCs, and may provide insight into the development of low side-effect targeting therapy for this malignant disease.