Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

PURPOSE: The goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones. MATERIALS AND METHODS: For hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection. CONCLUSION: This study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production

PURPOSE: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies. MATERIALS AND METHODS: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone. CONCLUSION: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Alterations of Colonic Contractility in an Interleukin-10 Knockout Mouse Model of Inflammatory Bowel Disease

BACKGROUND/AIMS: Inflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10(-/-)) mice. METHODS: Prepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining. RESULTS: The spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10(-/-) mice compared to IL-10(+/+) mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10(-/-) mice compared to IL-10(+/+) mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10(-/-) mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10(-/-) mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased. CONCLUSIONS: These results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.

DNA vaccines targeting human papillomavirus-associated diseases: progresses in animal and clinical studies

Human papillomavirus (HPV) infection is a major cause of cervical cancer and its precancerous diseases. Cervical cancer is the second deadliest cancer killer among women worldwide. Moreover, HPV is also known to be a causative agent of oral, pharyngeal, anal and genital cancer. Recent application of HPV structural protein (L1)-targeted prophylactic vaccines (Gardasil(R) and Cervarix(R)) is expected to reduce the incidence of HPV infection and cervical cancer, and possibly other HPV-associated cancers. However, the benefit of the prophylactic vaccines for treating HPV-infected patients is unlikely, underscoring the importance of developing therapeutic vaccines against HPV infection. In this regard, numerous types of therapeutic vaccine approaches targeting the HPV regulatory proteins, E6 and E7, have been tested for their efficacy in animals and clinically. In this communication, we review HPV vaccine types, in particular DNA vaccines, their designs and delivery by electroporation and their immunologic and antitumor efficacy in animals and humans, along with the basics of HPV and its pathogenesis.

Overexpression of CD73 in epithelial ovarian carcinoma is associated with better prognosis, lower stage, better differentiation and lower regulatory T cell infiltration / 부인종양

OBJECTIVE: The purpose of the current study was to evaluate survival outcome according to the expression status of CD73 in patients with epithelial ovarian cancer. METHODS: A total of 167 patients with epithelial ovarian cancer were enrolled in the current study. For each patient, a retrospective review of medical records was conducted. Immunohistochemical staining for CD73, CD8, FoxP3, and CD68 was performed using tissue microarray made with paraffin embedded tissue block. RESULTS: Among the enrolled patients, 29.9% of patients (n=50) showed negative expression for CD73, whereas 70.1% of patients (n=117) showed positive expression for CD73. The CD73 positive group showed better prognosis compared to the CD73 negative group (5-year overall survival of CD73 positive group, 73.0%; that of CD73 negative group, 50.1%; p=0.023). CD73 was more frequently expressed in mucinous adenocarcinoma and clear cell carcinoma compared to serous or endometrioid adenocarcinoma. In addition, CD73 overexpressions were more frequently detected in patients with known good prognostic factors, i.e., low stage, well/moderate differentiation, negative peritoneal cytology, no lymphovascular involvement, and no macroscopic residual tumor after debulking surgery. There was significantly more infiltration of regulatory T cells in the CD73 negative group compared to the CD73 positive group. CONCLUSION: Good prognosis in patients with overexpression of CD73 may be due to that overexpression of CD73 was more frequently observed in epithelial ovarian cancer patients with known good prognostic factors. Therefore, this result means that favorable differentiation and stage have more influence on survival outcome than adverse effect of CD73 per se.

Overexpression of CD73 in epithelial ovarian carcinoma is associated with better prognosis, lower stage, better differentiation and lower regulatory T cell infiltration / 부인종양

OBJECTIVE: The purpose of the current study was to evaluate survival outcome according to the expression status of CD73 in patients with epithelial ovarian cancer. METHODS: A total of 167 patients with epithelial ovarian cancer were enrolled in the current study. For each patient, a retrospective review of medical records was conducted. Immunohistochemical staining for CD73, CD8, FoxP3, and CD68 was performed using tissue microarray made with paraffin embedded tissue block. RESULTS: Among the enrolled patients, 29.9% of patients (n=50) showed negative expression for CD73, whereas 70.1% of patients (n=117) showed positive expression for CD73. The CD73 positive group showed better prognosis compared to the CD73 negative group (5-year overall survival of CD73 positive group, 73.0%; that of CD73 negative group, 50.1%; p=0.023). CD73 was more frequently expressed in mucinous adenocarcinoma and clear cell carcinoma compared to serous or endometrioid adenocarcinoma. In addition, CD73 overexpressions were more frequently detected in patients with known good prognostic factors, i.e., low stage, well/moderate differentiation, negative peritoneal cytology, no lymphovascular involvement, and no macroscopic residual tumor after debulking surgery. There was significantly more infiltration of regulatory T cells in the CD73 negative group compared to the CD73 positive group. CONCLUSION: Good prognosis in patients with overexpression of CD73 may be due to that overexpression of CD73 was more frequently observed in epithelial ovarian cancer patients with known good prognostic factors. Therefore, this result means that favorable differentiation and stage have more influence on survival outcome than adverse effect of CD73 per se.

A Study for Verification of Hair Growth Effect of Azelaic Acid and Vitamin B6

PURPOSE: Interest in the augmentation of hair growth for functional and aesthetic purpose has increased dramatically in recent years. Many hair growth products have been released, but most of these have not been proven scientifically. This study aims to measure the hair growth effect of azelaic acid and vitamin B6, which have been known as hair growth materials, in animal models. METHODS: Six weeks old C57BL/6 mice were used in this study and hair of mice were removed by topical treatment. The mice were divided into five experimental groups according to the testing material such as saline (negative control), propylene glycol(vehicle control), azelaic acid, vitamin B6 and azelaic acid plus vitamin B6 in combination. Hair growth was documented photographically and histologically, and then analysed by the high quality hair analysis program system. The quantity of endocrine factors, IGF-I and TGF-beta1 in the skin of mice was measured by PCR analysis. RESULTS: The topical treatment of azelaic acid and vitamin B6 in combination for 2 weeks to dorsal skin accelerated hair regrowth more than other groups. The azelaic acid and vitamin B6-combined treatment also promoted hair follicle elongation and thickness compared to the others. Histologic studies showed increased number of basal cells in azelaic acid and vitamin B6-combined treatment. Furthermore, the azelaic acid and vitamin B6-combined group significantly increased the expression of IGF-I but decreased the expression of TGF-beta1 in the skin of mice compared to other groups. CONCLUSION: These results suggest that azelaic acid and vitamin B6, when used together, have an additive effect and might be used as hair growth materials.

Retraction: Comparison of As2O3 and As4O6 in the Detection of SiHa Cervical Cancer Cell Growth Inhibition Pathway / Journal of the Korean Cancer Association, 대한암학회지

No abstract available.

Survival Benefits of Neoadjuvant Chemotherapy Followed by Radical Surgery versus Radiotherapy in Locally Advanced Chemoresistant Cervical Cancer

The aim of this study was to analyze long-term survivals in patients with stage IB to IIA cervical cancer treated by neoadjuvant chemotherapy setting. Between February 1989 and January 1998, 94 women with previously untreated stage IB to IIA carcinoma of the uterine cervix who received cisplatin based neoadjuvant chemotherapy were enrolled in this study. All of patients with chemoresponse (complete response, n=15; partial response, n=47) and 16 patients with chemoresistance received radical surgery (RS group). The other 16 patients with chemoresistance received radiotherapy for definite treatment (RT group). In the RS group, the 10 yr survival estimation in patients with bulky tumors (diameter > or =4 cm, n=26) was similar to that with non-bulky tumors (83.3% vs. 89.3%, p=NS). In selected patients with chemoresistance, those treated by radiotherapy (n=16) showed significantly poorer survivals than those treated by radical surgery (n=16) [10 yr survival rates of RT (25%) vs. RS (76.4%), p=0.0111]. Our results support that a possible therapeutic benefit of neoadjuvant chemotherapy plus radical surgery is only in patients with bulky stage IB to IIA cervical cancer. In cases of chemoresistance, radical surgery might be a better definite treatment option.

Opposite Roles of B7.1 and CD28 Costimulatory Molecules for Protective Immunity against HSV-2 Challenge in a gD DNA Vaccine Model

BACKGROUND: Costimulation is a critical process in Ag-specific immune responses. Both B7.1 and CD28 molecules have been reported to stimulate T cell responses during antigen presentation. Therefore, we tested whether Ag-specific immune responses as well as protective immunity are influenced by coinjecting with B7.1 and CD28 cDNAs in a mouse HSV-2 challenge model system. METHODS: ELISA was used to detect levels of antibodies, cytokines and chemokines while thymidine incorporation assay was used to evaluate T cell proliferation levels. RESULTS: Ag-specific antibody responses were enhanced by CD28 coinjection but not by B7.1 coinjection. Furthermore, CD28 coinjection increased IgG1 production to a significant level, as compared to pgD+pcDNA3, suggesting that CD28 drives Th2 type responses. In contrast, B7.1 coinjection showed the opposite, suggesting a Th1 bias. B7.1 coinjection also enhanced Ag-specific Th cell proliferative responses as well as production of Th1 type cytokines and chemokines significantly higher than pgD+pcDNA3. However, CD28 coinjection decreased Ag-specific Th cell proliferative responses as well as production of Th1 types of cytokines and chemokine significantly lower than pgD+pcDNA3. Only MCP-1 production was enhanced by CD28. B7.1 coimmunized animals exhibited an enhanced survival rate as well as decreased herpetic lesion formation, as compared to pgD+pcDNA3. In contrast, CD28 vaccinated animals exhibited decreased survival from lethal challenge. CONCLUSION: This study shows that B7.1 enhances protective Th1 type cellular immunity against HSV-2 challenge while CD28 drives a more detrimental Th2 type immunity against HSV-2 challenge, supporting an opposite role of B7.1 and CD28 in Ag-specific immune responses to a Th1 vs Th2 type.

Immunization with Adenoviral Vectors Carrying Recombinant IL-12 and E7 Enhanced the Antitumor Immunity against Human Papillomavirus 16-associated Tumor / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Human papillomavirus (HPV) infection has a significant role in cervical carcinogenesis, and HPV oncoprotein E7 plays an important part in the formation and maintenance of cervical cancer. Interleukin-12 (IL-12) has been reported to induce a cellular immune response, and to suppress the tumor growth and the E7 production. Here we describe the use of adenoviral delivery of the HPV 16 E7 subunit (AdE7) along with adenoviral delivery of IL-12 (AdIL-12) in mice with HPV-associated tumors. MATERIALS AND METHODS: Mice were injected with TC-1 cells to establish TC-1 tumor, and then they were immunized with AdIL-12 and/or AdE7 intratumorally. The anti tumor effects induced by AdIL-12 and/or E7 were evaluated by measuring the size of the tumor. E7-specific antibody and INF-gamma production in sera, and the T-helper cell proliferative responses were then measured. Cytotoxic T-lymphocyte (CTL) and T cell subset depletion studies were also performed. RESULTS: Combined AdIL-12 and AdE7 infection at the tumor sites significantly enhanced the antitumor effects more than that of AdIL-12 or AdE7 single infection. This combined infection resulted in regression of the 9 mm sized tumors in 80% of animals as compare to the PBS group. E7-specific antibody and INF-gamma production in the sera, and the T-helper cell proliferative responses were significantly higher with coinfection of AdIL-12 and AdE7 than with AdIL-12 or AdE7 alone. CTL response induced by AdIL-12 and AdE7 in the coinjected group suggested that tumor suppression was mediated by mostly CD8+ and only a little by the CD4+ T cells. CONCLUSION: IL-12 and E7 application using adenovirus vector showed antitumor immunity effects against TC-1 tumor, and this system could be use in clinical applications for HPV-associated cancer.

A Case of Classic Kaposi's Sarcoma Developing on the Face / 대한피부과학회지

Classic Kaposi's sarcoma is a human herpesvirus-8 associated with a multicentric lymphoangioproliferative tumor primarily arising in the lower extremities, but rarely in the head and neck. We herein report a 63-year-old man with primary classic Kaposi's sarcoma on the face. He presented with asymptomatic, erythematous papules on the nasal ala which had been noticed 2 months earlier. Histopathologic examination and nested polymerase chain reaction analysis in the tissue disclosed typical features of Kaposi's sarcoma.

Comparison of As2O3 and As4O6 in the Detection of SiHa Cervical Cancer Cell Growth Inhibition Pathway / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: An arsenical compound, As2O3, has been reported to be effective for treating acute leukemia and inducing apoptosis in many different tumor cells. In this study, the ability of As4O6 to suppress cell growth and induce gene expression patterns was tested using a cDNA microarray in HPV16 immortalized cervical carcinoma cells, SiHa cells, along with As2O3. MATERIALS AND METHODS: A novel arsenical compound, As4O6, was designed and its ability to induce cell growth inhibition as well as gene expression profiles along with As2O3 in HPV16 infected SiHa cervical cancer cells was compared. Both As2O3 and As4O6 induced apoptosis in SiHa cells, as determined by DNA ladder formation. To further compare the gene expression profiles between these two drugs, a 384 cDNA microarray system was employed. Also, the gene expression profiles were classified into the Gene Ontology (GO) to investigate apoptosis-related cellular processes. RESULTS: As4O6 was more effective i suppressing the growth of SiHa cells in vitro compared to As2O3. In the case of treatment with As2O3, 41 genes were up- or down- regulated at least 2 fold compared to non-treatment. However, 65 genes were up- or down-regulated by As4O6 treatment. In particular, 27 genes were commonly regulated by both arsenic compounds. Also, the GO analysis indicated that down-regulation of cell-regulatory functions, such as cell cycle, protein kinase activity and DNA repair, induced anti-tumor effect. CONCLUSION: These data support that As4O6 could be more effective than As2O3 in inhibiting the growth of HPV16 infected cervical cancer cells. This appears to be mediated through a unique, but overlapping regulatory mechanism(s), suggesting that the regulated genes and cellular processes could be further used as a new potential drug approach for treating cervical cancer in clinical settings.

The Effects of Green Tea Extracts (Polyphenon E, EGCG) in Human Papilloma Virus Positive Cervical Lesion / 대한산부인과학회잡지

OBJECTIVE: We tried to confirm the effects of green tea extracts (polyphenon E, EGCG) in patients with human papilloma virus (HPV) positive cervical lesion. METHODS: We divided 51 HPV positive cervical lesion patients (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) into 4 group and 37 patient as placebo control. We applied poly E ointment two times per week (27 patients), poly E ointment plus poly E capsule (8 patients), poly E capsule (6 patients), EGCG capsule (10 patients) 200 mg each for 8 to 12 weeks. RESULTS: Among 27 patients with poly E ointment group, 20 patients responded (74%), such as chronic cervicitis (12/18), mild dysplasia (4/5), moderate dysplasia (2/2) and severe dysplasia (1/2). Among 8 patients with poly E ointment and poly E capsule group, 6 patients responded (75%), 6 patients poly E capsule group responded 3 patients (50%). 10 EGCG capsule patients group responded 6 patients (60%). Overall responsive rate is 69% (35/51) in case of green tea extracted treated group and 10% (4/39) in placebo controlled group (P<0.05). CONCLUSION: The effects of green tea extract in HPV positive cervical lesion were statistically significant (P<0.05). This result suggests that green tea extract has highly potential of new treatment agent for HPV infected cervical lesion.

A Case of Glassy Cell Carcinoma of the Uterine Cervix with Photodynamic Therapy prior to Radical Surgery / 대한산부인과학회잡지

It has been known that glassy cell carcinoma (GCC) of uterine cervix is rare and rapidly progressive, and has a poor prognosis. Here we describe a case of GCC in which photodynamic therapy (PDT) was performed prior to radical hysterectomy. The patient, a 42 year-old woman at the stage of FIGO Ib2 underwent interstitial PDT (Photogem, 2 mg/kg; light, p=100 mV, W=100 J/cm2, 15 min/each 4 direction). The cervical lesion displayed inflammation and necrosis at 48 h following PDT. At 2 weeks post PDT, inflammatory reaction was disappeared and the tumor volume was decreased. No side effects of PDT were also observed. Subsequently, the patient underwent radical hysterectomy and pelvic lymph node resection. This case suggests that PDT prior to radical surgery might be an effective way to reduce tumor size without any side effects. More cycles of PDT might be beneficial for treating GCC of the uterine cervix.