Validation of an on-chip p16ink4a/Ki-67 dual immunostaining cervical cytology system using microfluidic device technology.

More specific screening systems for cervical cancer may become necessary as the human papillomavirus (HPV) vaccine becomes more widespread. Although p16/Ki-67 dual-staining cytology has several advantages, it requires advanced diagnostic skills. Here, we developed an automated on-chip immunostaining method using a microfluidic device. An electroactive microwell array (EMA) microfluidic device with patterned thin-film electrodes at the bottom of each microwell was used for single-cell capture by dielectrophoresis. Immunostaining and dual staining for p16/Ki-67 were performed on diagnosed liquid cytology samples using the EMA device. The numbers of p16/Ki-67 dual-stained cells captured by the EMA device were determined and compared among the cervical intraepithelial neoplasia (CIN) lesion samples. Seven normal, fifteen CIN grade 3, and seven CIN grade 2 samples were examined. The percentage of dual-positive cells was 18.6% in the CIN grade 2 samples and 23.6% in the CIN grade 3 samples. The percentages of dual-positive staining increased significantly as the severity of the cervical lesions increased. p16/Ki67 dual immunostaining using the EMA device is as sensitive as the conventional method of confirming the histopathological diagnosis of cervical samples. This system enables a quantified parallel analysis at the individual cell level.

The TGF-ß/UCHL5/Smad2 Axis Contributes to the Pathogenesis of Placenta Accreta.

Placenta accreta is a high-risk condition causing obstetric crisis and hemorrhage; however, its pathogenesis remains unknown. We aimed to identify the factors contributing to trophoblast invasiveness and angiogenic potential, which in turn drive the pathogenesis of placenta accreta. We focused on the transforming growth factor (TGF)-ß1-Smad pathway and investigated the intrinsic relationship between the time- and dose-dependent inhibition of the ubiquitinating enzyme UCHL5 using bAP15, a deubiquitinase inhibitor, after TGF-ß1 stimulation and the invasive and angiogenic potential of two cell lines, gestational choriocarcinoma cell line JEG-3 and trophoblast cell line HTR-8/SVneo. UCHL5 inhibition negatively regulated TGF-ß1-induced Smad2 activation, decreasing extravillous trophoblast invasiveness. Smad1/5/9 and extracellular signal-regulated kinase (ERK) were simultaneously activated, and vascular endothelial growth factor was secreted into the trophoblast medium. However, extravillous trophoblast culture supernatant severely impaired the vasculogenic potential of human umbilical vein endothelial cells. These results suggest that the downstream ERK pathway and Smad1/5/9 potentially regulate the TGF-ß1-Smad pathway in extravillous trophoblasts, whereas Smad2 contributes to their invasiveness. The abnormal invasive and angiogenic capacities of extravillous cells, likely driven by the interaction between TGF-ß1-Smad and ERK pathways, underlie the pathogenesis of placenta accreta.

High-grade endometrial stromal sarcoma with YWHAE-NUTM2B fusion gene abnormality identified after 10 years of recurrent pulmonary metastases: A case report.

•Endometrial stromal sarcoma is the second most common type of uterine sarcoma.•Endometrial stromal sarcoma has undergone modifications since its proposal.•This case highlights the importance of accurately diagnosing endometrial stromal sarcoma.•Asymptomatic uterine fibroids may not be treated with therapeutic intervention or prompt regular check-ups.

Serum vascular endothelial growth factor associated with the progression of granulosa cell tumor: a report of two cases.

BACKGROUND: Granulosa cell tumors (GCTs) account for approximately 2% of ovarian malignancies and are considered a rare type of ovarian cancer. GCTs are characterized by irregular genital bleeding after menopause due to female hormone production as well as late recurrence around 5-10 years after initial treatment. In this study, we investigated two cases of GCTs to find a biomarker that can be used to evaluate the treatment and predict recurrence. CASE PRESENTATION: Case 1 was a 56-year-old woman who presented to our hospital with abdominal pain and distention. An abdominal tumor was found, and GCTs were diagnosed. Serum vascular endothelial growth factor (VEGF) levels decreased after surgery. Case 2 involved a 51-year-old woman with refractory GCTs. Carboplatin-paclitaxel combination therapy and bevacizumab were administered after the tumor resection. After chemotherapy, a decline in VEGF levels was observed, but serum VEGF levels increased again with disease progression. CONCLUSIONS: VEGF expression may be of clinical importance in GCTs as a clinical biomarker for disease progression, which may be used to determine the efficacy of bevacizumab against GCTs.

Effect of Different Educational Interventions on Knowledge of HPV Vaccination and Cervical Cancer among Young Women: A Preliminary Report.

The incidence and mortality rates of cervical cancer are rising among young women in Japan. In November 2021, the Japanese Ministry of Health, Labour, and Welfare reinstated the active recommendation for the human papillomavirus (HPV) vaccine, which was discontinued in June 2013 due to reports of adverse reactions, including chronic pain and motor dysfunction, following vaccination. However, vaccine hesitancy among the younger generation remains, and it is essential to identify the barriers in vaccination uptake. Therefore, we aimed to conduct a randomized study using different methods of providing educational contents to improve health literacy regarding cervical cancer and HPV vaccination among female students in Japan. Here, we present the results of our preliminary report and discuss current topics related to HPV vaccination in Japan. Data were collected from 27 female students-divided into three groups: no intervention, print-based intervention, and social networking service-based intervention-using the health literacy scale and communicative and critical health literacy scale. Our primary results indicate that participants' knowledge and health literacy improved post-intervention. Therefore, medical professionals must provide accurate scientific knowledge regarding routine HPV vaccination and the risk of cervical cancer to young women to improve their health literacy and subsequently increase the HPV vaccination rates.

Ulipristal acetate simultaneously provokes antiproliferative and proinflammatory responses in endometrial cancer cells.

Ulipristal acetate (UPA), a selective progesterone receptor modulator, is used for the treatment of uterine fibroids and selectively inhibits the proliferation and inflammation of leiomyoma cells. As few studies have focused on the molecular biological mechanism of UPA in Ishikawa endometrial cancer cells, we aimed to identify the effects of UPA on these cells. Ishikawa cells were treated with different concentrations of UPA. Cell viability and colony formation assays were performed to assess the growth of cancer cells, whereas invasion and migration assays were used to measure cell motility and invasiveness. Western blotting, caspase 3/7 assay, TUNEL assay, and flow cytometry were performed to analyze apoptosis. Moreover, expression levels of the proinflammatory cytokines oncostatin M, its receptor, interleukin 6, and interleukin 8 were examined using quantitative real-time PCR. UPA decreased cell viability and growth, thereby inhibiting cell migration and invasion via induction of apoptosis. Contrary to expectation, stand-alone application of UPA increased the expression of the proinflammatory cytokines but concomitant use of UPA and the estrogen receptor antagonist ICI 182,720 decreased it. These data revealed a novel dual role of UPA: It could attenuate cell growth via activation of apoptosis while simultaneously provoking the activation of proinflammatory cytokines in endometrial cancer cells. These indicate that the combination of UPA and an estrogen receptor antagonist may be useful in suppressing the secretion of proinflammatory cytokines by UPA alone.

Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response.

BACKGROUND: Xenotransplantation is associated with an inflammatory response. The proinflammatory cytokine, TNF-α, downregulates the expression of thrombomodulin (TBM), and induces coagulation dysfunction. Although human (h) TBM-transgenic pigs (p) have been developed to reduce coagulation dysfunction, the effect of TNF-α on the expression of hTBM and its functional activity has not been fully investigated. The aims of this study were to investigate (i) whether the expression of hTBM on pig (p) cells is down-regulated during TNF-α stimulation, and (ii) whether cells from hTBM pigs regulate the inflammatory response. METHODS: TNF-α-producing T, B, and natural killer cells in blood from baboons with pig heart or kidney xenografts were investigated by flow cytometry. TNF-α staining in the grafts was detected by immunohistochemistry. Aortic endothelial cells (AECs) from GTKO/CD46 and GTKO/CD46/hTBM pigs were stimulated by hTNF-α, and the expression of the inflammatory/coagulation regulatory protein, TBM, was investigated. RESULTS: After pig organ xenotransplantation, there was a trend to increases in TNF-α-producing T and natural killer cells in the blood of baboons. In vitro observations demonstrated that after hTNF-α stimulation, there was a significant reduction in the expression of endogenous pTBM on pAECs, and a significant increase in the expression of inflammatory molecules. Blocking of NF-κB signaling significantly up-regulated pTBM expression, and suppressed the inflammatory response induced by hTNF-α in pAECs. Whereas the expression of pTBM mRNA was significantly reduced by hTNF-α stimulation, hTBM expression on the GTKO/CD46/hTBM pAECs was not affected. Furthermore, after hTNF-α stimulation, there was significant suppression of expression of inflammatory molecules on GTKO/CD46/hTBM pAECs compared to GTKO/CD46 pAECs. CONCLUSIONS: The stable expression of hTBM in pig cells may locally regulate the inflammatory response. This will help suppress the inflammatory response and prevent coagulation dysregulation after xenotransplantation.

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-like Cells using the Three-dimensional Co-culture System, NICO-1.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Effect of murine double-minute 2 inhibitors in preclinical models of advanced clear cell carcinomas originating from ovaries and kidneys.

Advanced clear cell carcinomas originating from both ovaries and kidneys with cancerous peritonitis have poor prognoses. Murine double-minute 2 (MDM2) is a potential therapeutic target for clear cell ovarian carcinomas with WT TP53. Herein, we characterized the antiangiogenic and antitumor effects of the MDM2 inhibitors DS-3032b and DS-5272 in 6 clear cell ovarian carcinoma cell lines and 2 clear cell renal carcinoma cell lines, as well as in clear cell ovarian carcinomas s.c. xenograft and ID8 (murine ovarian cancer cells with WT TP53) cancer peritonitis mouse models. In clear cell ovarian carcinoma s.c. xenograft mouse models, DS-3032b significantly reduced WT TP53 clear cell ovarian carcinoma- and clear cell renal carcinoma-derived tumor volumes. In ID8 mouse models, DS-5272 significantly inhibited ascites production, reduced body weight, and significantly improved overall survival. Additionally, DS-5272 reduced the tumor burden of peritoneal dissemination and decreased CD31+ cells in a dose-dependent manner. Furthermore, DS-5272 significantly decreased vascular endothelial growth factor concentrations in both sera and ascites. Combined therapy with MDM2 inhibitors and everolimus showed synergistic, and dose-reduction potential, for clear cell carcinoma treatment. Our findings suggest that MDM2 inhibitors represent promising molecular targeted therapy for clear cell carcinomas, thereby warranting further studies to evaluate the efficacy and safety of dual MDM2/mTOR inhibitors in clear cell carcinoma patients.

Rikkunshito attenuates induction of epithelial-mesenchymal switch via activation of Sirtuin1 in ovarian cancer cells.

Rikkunshito, a traditional Japanese herbal medicine, improves appetite via activation of gastrointestinal hormone ghrelin pathway. The function of ghrelin is mediated by growth hormone secretagogue receptor (GHSR1a), and ghrelin has been known to possess diverse physiological functions including growth suppression of some cancer cells. Considering that increased ghrelin signaling by Rikkunshito could enhance sirtuin1 (SIRT1) activity in nervous system, we aimed to investigate the effect of Rikkunshito in ovarian cancer cells. Ovarian cancer cell lines were treated with Rikkunshito, and cellular viability, gene expressions and epithelial-mesenchymal transition (EMT) status were investigated. To investigate the involvement of SIRT1 by Rikkunshito in SKOV3 cancer cells, endogenous expression of SIRT1 was depleted using small interfering RNA (siRNA). Treatment with Rikkunshito elevated ghrelin, GHSR1a and SIRT1, while cellular viability was decreased. The treatment of Rikkunshito also inhibited cellular migration and invasion status in a dose-dependent manner, and these effects were translated to the enhanced EMT status, although the role of SIRT1 was not determined. Our study revealed a novel function of Rikkunshito in enhancing EMT status of ovarian cancer cells. Therefore, we would like to propose that Rikkunshito may be used as a novel adjunctive therapy in chemotherapy of ovarian cancer because platinum-based chemotherapy frequently used for the treatment of ovarian cancer inevitably impairs appetite.

Anti-Tumor Effect of Inhibition of DNA Damage Response Proteins, ATM and ATR, in Endometrial Cancer Cells.

: While the incidence of endometrial cancer continues to rise, the therapeutic options remain limited for advanced or recurrent cases, and most cases are resistant to therapy. The anti-tumor effect of many chemotherapeutic drugs and radiotherapy depends on the induction of DNA damage in cancer cells; thus, activation of DNA damage response (DDR) pathways is considered an important factor affecting resistance to therapy. When some DDR pathways are inactivated, inhibition of other DDR pathways can induce cancer-specific synthetic lethality. Therefore, DDR pathways are considered as promising candidates for molecular-targeted therapy for cancer. The crosstalking ataxia telangiectasia mutated and Rad3 related and checkpoint kinase 1 (ATR-Chk1) and ataxia telangiectasia mutated and Rad3 related and checkpoint kinase 2 (ATM-Chk2) pathways are the main pathways of DNA damage response. In this study, we investigated the anti-tumor effect of inhibitors of these pathways in vitro by assessing the effect of the combination of ATM or ATR inhibitors and conventional DNA-damaging therapy (doxorubicin (DXR), cisplatin (CDDP), and irradiation) on endometrial cancer cells. Both the inhibitors enhanced the sensitivity of cells to DXR, CDDP, and irradiation. Moreover, the combination of ATR and Chk1 inhibitors induced DNA damage in endometrial cancer cells and inhibited cell proliferation synergistically. Therefore, these molecular therapies targeting DNA damage response pathways are promising new treatment strategies for endometrial cancer.

The proteasome deubiquitinase inhibitor bAP15 downregulates TGF-ß/Smad signaling and induces apoptosis via UCHL5 inhibition in ovarian cancer.

The ubiquitin-proteasome pathway plays an important role in the regulation of cellular proteins. As an alternative to the proteasome itself, recent research has focused on methods to modulate the regulation of deubiquitinating enzymes (DUBs) upstream of the proteasome, identifying DUBs as novel therapeutic targets in breast, endometrial, and prostate cancers, along with multiple myeloma. bAP15, an inhibitor of the 19S proteasome DUBs UCHL5 and USP14, results in cell growth inhibition in several human cancers; however, the mechanism remains poorly understood in ovarian cancer. Here, we found that aberrant UCHL5 expression predicted shorter progression-free survival (PFS) in a cohort of 1435 patients with ovarian cancer described in the Gene Expression Omnibus and The Cancer Genome Atlas databases. The subgroup of patients with TP53 mutations was significantly more likely to exhibit poor PFS (p <0.001). Moreover, we found bAP15 could suppress TP53-mutant ovarian cancer cell survival by regulating TGF-ß signaling through inhibiting UCHL5 expression and dephosphorylating Smad2, consequently inducing apoptosis. bAP15 (2.5 and 5.0 mg/kg) also exerted significant anti-tumor effect on nude mice bearing subcutaneous SKOV3 xenografts. As activated TGF-ß signaling is involved in ovarian cancer progression, these findings suggest that UCHL5 inhibition offers potential opportunities for a novel targeted therapy against TGF-ß-activated ovarian cancer.

On-chip immunofluorescence analysis of single cervical cells using an electroactive microwell array with barrier for cervical screening.

Several specific tests for cervical screening have been developed recently, including p16/Ki67 dual immunostaining for diagnosing high-risk human papillomavirus positive squamous intraepithelial lesion in the cervix. However, manual screening of cells in an entire glass slide is currently a standard clinical procedure for quantification and interpretation of immunocytochemical features of the cells. Here, we developed a microfluidic device containing an electroactive microwell array with barriers (EMAB) for highly efficient single-cell trapping followed by on-chip immunofluorescence analysis with minimum loss of the sample. EMAB utilizes patterned electrodes at the bottom of cell-sized microwells to trap single cells using dielectrophoresis (DEP) and cell-holding structures behind the microwells to stabilize the position of trapped cells even without DEP. Using the device, we evaluated the performance of p16/Ki67 dual immunostaining of HeLa cells on the chip. The device shows 98% cell-trapping efficiency as well as 92% cell-holding efficiency against the fixed HeLa cells, and we successfully demonstrated high-efficiency on-chip immunofluorescence analysis with minimal loss of sample. p16/Ki67 dual immunostaining using EMAB may be useful for complementary tests for cervical screening in confirming the histopathological diagnosis.

Array comparative genomic hybridization analysis discloses chromosome copy number alterations as indicators of patient outcome in lymph node-negative breast cancer.

BACKGROUND: Patients with lymph node metastasis-negative (pN0) invasive breast cancer have favorable outcomes following initial treatment. However, false negatives which occur during routine histologic examination of lymph nodes are reported to underestimate the clinical stage of disease. To identify a high-risk group in pN0 invasive breast cancer, we examined copy number alterations (CNAs) of 800 cancer-related genes. METHODS: Using array-based comparative genomic hybridization (CGH) in 51 pN0 cases (19 relapsed and 32 non-relapsed cases), the positivities of specific gene CNAs in the relapsed and non-relapsed groups were compared. An unsupervised hierarchical cluster analysis was then performed to identify case groups that were correlated with patient outcomes. RESULTS: The cluster analysis identified three distinct clusters of cases: groups 1, 2, and 3. The major component was triple-negative cases (69%, 9 of 13) in group 1, luminal B-like (57%, 13 of 23) and HER2-overexpressing (26%, 6 of 23) subtypes in group 2, and luminal A-like subtype (60%, 9 of 15) in group 3. Among all 51 cases, those in group 1 showed significantly worse overall survival (OS) than group 2 (p = 0.014), and 5q15 loss was correlated with worse OS (p = 0.017). Among 19 relapsed cases, both OS and relapse-free survival (RFS) rates were significantly lower in group 1 than in group 2 (p = 0.0083 and 0.0018, respectively), and 5q15 loss, 12p13.31 gain, and absence of 16p13.3 gain were significantly correlated with worse OS and RFS (p = 0.019 and 0.0027, respectively). CONCLUSIONS: As the target genes in these loci, NR2F1 (5q15), TNFRSF1A (12p13.31), and ABCA3 (16p13.3) were examined. 5q15 loss, 12p13.31 gain, and absence of 16q13.3 gain were potential indicators of high-risk recurrence and aggressive clinical behavior of pN0 invasive breast cancers.

Development of retrocorneal membrane following pig-to-monkey penetrating keratoplasty.

Recent reports of long-term survival after wild-type (WT) pig-to-monkey corneal xenotransplantation are encouraging. We experienced the rapid development of retrocorneal membranes, a rare complication after corneal allotransplantation (although seen in infants and young children). The original specific aim of the study was to determine the factors associated with successful (young) pig corneal transplantation in monkeys. However, when it was obvious that retrocorneal membranes rapidly developed, our aims became to determine the factors involved in its development after both WT and Genetically engineered (GE ) pig corneal xenotransplantation and to investigate the characteristics of the retrocorneal membrane. Rhesus monkeys were recipients of penetrating keratoplasty using WT and GE pigs (n=2, respectively, 1-3 months old). Local/systemic steroids were administered for 3 months. Grafts were evaluated by slit lamp for corneal transparency, edema, and neovascularization. Hematoxylin and eosin, Masson trichrome staining, and immunohistochemical analysis were performed. Gal staining was also carried out to distinguish the origin of the membrane. All penetrating keratoplasty recipients developed fibrous retrocorneal membranes in the early post-transplantation period, regardless of whether the graft was from a WT or GE pig. There were no features of rejection, with no cell infiltrate in the graft or anterior chamber during the three-month follow-up. There was no difference in the clinical course between the two groups (WT or GE corneas). Immunohistochemistry indicated that the retrocorneal membranes were CK negative, α-SMA positive, and vimentin positive, suggesting that they were of fibrous (keratocytic) origin. Also, the membrane was Gal positive, suggesting that it is derived from pig cornea. Following pig-to-monkey corneal xenotransplantation, we report that retrocorneal membranes are derived from donor pig keratocytes. Prevention of retrocorneal membranes will be necessary to achieve successful corneal xenotransplantation.

Effect of Rho-kinase Inhibitor, Y27632, on Porcine Corneal Endothelial Cell Culture, Inflammation and Immune Regulation.

PURPOSE: To investigate the effect of the Rho-kinase inhibitor, Y27632, on pig corneal endothelial cell (pCEC) culture, and on inflammation and immune regulation of the responses of human cells to pCECs. METHODS: pCECs were cultured with/without Y27632 to assess cell proliferation and in vitro wound healing assay. The level of MCP-1 and VEGF in pCECs stimulated with human TNF-α were measured. Proliferation of human PBMCs stimulated with pCECs, and cytokine production in human T cells, and monocyte migration after stimulation were investigated. RESULTS: Y27632 promoted pCEC proliferation, prevented pCEC death, and enhanced in vitro wound healing. After stimulation, there were significantly lower levels of MCP-1 and VEGF measured in pCECs cultured with Y27632, and significantly reduced human PBMC proliferation, cytokine production, and monocyte migration. CONCLUSIONS: The application of the Rho-kinase inhibitor will be beneficial when culturing pCECs, and may provide a novel therapy to reduce inflammation after corneal xenotransplantation.

Expression of NeuGc on Pig Corneas and Its Potential Significance in Pig Corneal Xenotransplantation.

PURPOSE: Pigs expressing neither galactose-α1,3-galactose (Gal) nor N-glycolylneuraminic acid (NeuGc) take xenotransplantation one step closer to the clinic. Our aims were (1) to document the lack of NeuGc expression on corneas and aortas and cultured endothelial cells [aortic endothelial cells (AECs); corneal (CECs)] of GTKO/NeuGcKO pigs, and (2) to investigate whether the absence of NeuGc reduced human antibody binding to the tissues and cells. METHODS: Wild-type (WT), GTKO, and GTKO/NeuGcKO pigs were used for the study. Human tissues and cultured cells were negative controls. Immunofluorescence staining was performed using anti-Gal and anti-NeuGc antibodies, and human IgM and IgG binding to tissues was determined. Flow cytometric analysis was used to determine Gal and NeuGc expression on cultured CECs and AECs and to measure human IgM/IgG binding to these cells. RESULTS: Both Gal and NeuGc were detected on WT pig corneas and aortas. Although GTKO pigs expressed NeuGc, neither humans nor GTKO/NeuGcKO pigs expressed Gal or NeuGc. Human IgM/IgG binding to corneas and aortas from GTKO and GTKO/NeuGcKO pigs was reduced compared with binding to WT pigs. Human antibody binding to GTKO/NeuGcKO AECs was significantly less than that to GTKO AECs, but there was no significant difference in binding between GTKO and GTKO/NeuGcKO CECs. CONCLUSIONS: The absence of NeuGc on GTKO aortic tissue and AECs is associated with reduced human antibody binding, and possibly will provide a better outcome in clinical xenotransplantation using vascularized organs. For clinical corneal xenotransplantation, the absence of NeuGc expression on GTKO/NeuGcKO pig corneas may not prove an advantage over GTKO corneas.

In vitro testing of an anti-CD40 monoclonal antibody, clone 2C10, in primates and pigs.

BACKGROUND: The CD40/CD154 and CD28/B7 pathways are important in allo- and xeno-transplantation. Owing to the thrombotic complications of anti-CD154mAb, anti-CD40mAb has emerged as a promising inhibitor of costimulation. Various clones of anti-CD40mAb have been developed against primate species, e.g., clone 2C10 against rhesus monkeys. We have compared the in vitro efficacy of 2C10 to prevent a T cell response in primates and pigs. METHODS: The binding of 2C10 to antigen-presenting cells (PBMCs [B cells]) of humans, rhesus and cynomolgus monkeys, baboons, and pigs was measured by flow cytometry, and was also tested indirectly by a blocking assay. The functional capacity of 2C10 was tested by mixed lymphocyte reaction (MLR) with polyclonal stimulation by phytohemagglutinin (PHA) and also with wild-type pig aortic endothelial cells (pAECs) as stimulators. RESULTS: There was a significant reduction in binding of 2C10 to baboon PBMCs compared to rhesus, cynomolgus, and human PBMCs, and minimal binding to pig PBMCs. The blocking assay confirmed that the binding of 2C10 was significantly lower to baboon PBMCs when compared to the other primate species tested. The functional assay with PHA showed significantly reduced inhibition of PBMC proliferation in humans, cynomolgus monkeys, and baboons compared to rhesus monkeys, which was confirmed on MLR with pAECs. CONCLUSIONS: Since both the binding and functional activity of 2C10 in the baboon is lower than in rhesus monkeys, in vivo treatment using 2C10 in the baboon might require a higher dose or more frequent administration in comparison to rhesus monkeys. It may also be beneficial to develop species-specific clones of anti-CD40mAb.

A comparison of three methods of decellularization of pig corneas to reduce immunogenicity.

AIM: To investigate whether decellularization using different techniques can reduce immunogenicity of the cornea, and to explore the decellularized cornea as a scaffold for cultured corneal endothelial cells (CECs). Transplantation of decellularized porcine corneas increases graft transparency and survival for longer periods compared with fresh grafts. METHODS: Six-month-old wild-type pig corneas were cut into 100-200 µm thickness, and then decellularized by three different methods: 1) 0.1% sodium dodecyl sulfate (SDS); 2) hypoxic nitrogen (N2); and 3) hypertonic NaCl. Thickness and transparency were assessed visually. Fresh and decellularized corneas were stained with hematoxylin/eosin (H&E), and for the presence of galactose-α1,3-galactose (Gal) and N-glycolylneuraminic acid (NeuGc, a nonGal antigen). Also, a human IgM/IgG binding assay was performed. Cultured porcine CECs were seeded on the surface of the decellularized cornea and examined after H&E staining. RESULTS: All three methods of decellularization reduced the number of keratocytes in the stromal tissue by >80% while the collagen structure remained preserved. No remaining nuclei stained positive for Gal or NeuGc, and expression of these oligosaccharides on collagen was also greatly decreased compared to expression on fresh corneas. Human IgM/IgG binding to decellularized corneal tissue was considerably reduced compared to fresh corneal tissue. The cultured CECs formed a confluent monolayer on the surface of decellularized tissue. CONCLUSION: Though incomplete, the significant reduction in the cellular component of the decellularized cornea should be associated with a significantly reduced in vivo immune response compared to fresh corneas.

Distribution of non-gal antigens in pig cornea: relevance to corneal xenotransplantation.

PURPOSE: The aim of this study was to investigate the distribution of antigens other than galactose-α-1,3-galactose (Gal) (non-Gal) recognized by human and rhesus monkey serum antibodies in the α-1,3-galactosyltransferase gene-knockout (GTKO) pig cornea. METHODS: The distribution of non-Gal, specifically N-glycolylneuraminic acid (NeuGc), in the corneas from wild-type (WT) and GTKO pigs was identified. Corneal sections from WT and GTKO pigs were incubated with human or rhesus monkey serum to determine immunoglobulin (Ig)M and IgG binding to corneal tissue by means of fluorescent microscopy. RESULTS: Strong expression of NeuGc was found in all layers of both WT and GTKO pig corneas. In both humans and monkeys, antibody binding (IgG > IgM) to GTKO was found to be weaker than that to entire WT pig corneas, but in both, most antibody binding, especially IgG, was to the epithelium. There was weak diffuse antibody binding, especially of IgG, to the corneal stroma, suggesting binding to antigens expressed on collagen. There was no or minimal binding of IgM/IgG to the corneal endothelium. CONCLUSIONS: Although the cornea is avascular, antibodies in primate serum can bind to pig antigens, especially on epithelial cells and stromal collagen. Although the binding to entire GTKO corneas was weaker than that to WT corneas, deletion of the expression of NeuGc and expression of human complement-regulatory proteins in the pig cornea will be important if prolonged clinical corneal xenograft survival is to be achieved.