On-chip erythrocyte deformability test under optical pressure.

This paper presents a novel method for an on-chip erythrocyte deformability test under optical pressure, especially to enhance the level of sensitivity with respect to the detection of cancerous diseases. To demonstrate the performance and sensitivity of the combined method, we introduce the concept of transit velocity, a modified elongation index, and shape recovery time of individual erythrocytes in a strictly confined region (2 microm deep, 4 microm wide, and 100 microm long). Finally, we investigate a synergy or convergence effect due to the combination of these parameters for in situ detection of cancerous diseases under optical pressure.

Expansion channel for microchip flow cytometers.

This paper presents a novel way of designing a flow focusing channel for microchip flow cytometers. With this method we increased throughput and sensitivity of particle detection at the same time. Generally, to increase the detection throughput of a flow cytometer, the speed of the flow inside the focusing channel needs to be increased, hence reducing the time of exposure to laser beam. With the shorter exposure time, both the fluorescence and scatter signal from the target particles become dimmer. To increase the sensitivity of signal detection, however, the speed of the flow should be decreased so as to decrease throughput of detection. To overcome this dilemmatic problem, we integrated an expansion channel inside a focusing channel. Signals from particles in an expansion channel were about 10 times brighter than those in a normal channel. With this enhanced sensitivity, we could also speed up the inlet flow, which in turn increases the overall throughput of detection.

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model.

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

Immunization with adenoviral vectors carrying recombinant IL-12 and E7 enhanced the antitumor immunity to human papillomavirus 16-associated tumor.

OBJECTIVE: Human papillomavirus (HPV) infection play a significant role in cervical carcinogenesis, and HPV oncoprotein E7 has important functions in the formation and maintenance of cervical cancers. Interleukin-12 (IL-12) has been reported to induce cellular immune responses, and has also been demonstrated to suppress the growth of tumors and the expression of E7. Here, we investigate the utility of adenovirus E7 (AdE7) and adenovirus IL-12 (AdIL-12) for protection against TC-1 tumor using an animal model. METHODS: The antitumor effects induced by AdIL-12 and/or E7 were assessed by measurements of tumor size. E7-specific antibody and INF-gamma production in sera were measured, as were T-helper cell proliferative responses. Cytotoxic T-lymphocytes (CTL) and T cell subset depletion studies were also performed. RESULTS: Infection of tumor sites with a combination of AdIL-12 and AdE7 resulted in an antitumor effect which was significantly more profound than that which resulted from singular infections with either AdIL-12 or AdE7. Combined infection resulted in regression of 9-mm-sized tumors in approximately 80% of our experimental animals as compared to the PBS group. Serum levels of E7-specific antibody and INF-gamma production, as well as T-helper cell proliferative responses, were found to be significantly higher in coinfected with AdIL-12 and AdE7 group than in single infection with either AdIL-12 or AdE7 group. CTL responses only exhibited by the AdIL-12 and AdE7 coinjected group suggested that these tumor suppression effects were mediated primarily by CD8+ and, to a lesser degree, by CD4+ T cells. CONCLUSION: Combined injection with adenovirus carrying IL-12 and E7 induced significant antitumor immunity against TC-1 tumors. They may prove useful in clinical applications for the treatment of HPV-associated tumors.

Immunization with adenoviral vectors carrying recombinant IL-12 and E7 enhanced the antitumor immunity against human papillomavirus 16-associated tumor.

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.

Interferon regulatory factor-1 mediates interferon-gamma-induced apoptosis in ovarian carcinoma cells.

Interferon-gamma (IFN-gamma), as one of interferon family that regulates antiviral, antiproliferative, and immunomodulatory responses, has been implicated for the growth regulation of ovarian cancer cells. However, the molecular mechanisms are not yet fully defined. To analyze detailed mechanisms, the ovarian cancer cell lines (2774, PA-1, OVCAR-3, and SKOV-3) were treated with IFN-gamma. The growth of 2774 was most effectively suppressed than that of other cells in both time-course and dose-dependent experiments. The order of sensitivity in other cells was PA-1 >> OVCAR-3 > SKOV-3 (not responded at all). The DNA fragmentation and DAPI staining assays suggested that the IFN-gamma-mediated cytotoxicity could be triggered by apoptosis. The treatment induced IFN regulatory factor-1 (IRF-1) in two IFN-gamma-sensitive cells (2774, PA-1), whereas IRF-1 was not induced in two IFN-gamma-resistant cells (OVCAR-3, SKOV-3). The levels of p53 and p21WAF1 were not strikingly changed in all four cells. Interestingly, the expression of interleukin-converting enzyme (ICE, or caspase-1) was increased by the treatment in a kinetically consistent manner to the induction of IRF-1. However, CD95 (Fas/APO-1) was not changed. Apoptosis was greatly induced, when IRF-1 was transiently expressed in PA-1 without the treatment of IFN-gamma. However, it was repressed when IRF-1 together with IRF-2, an antagonist of IRF-1, were coexpressed. In addition, the effect of IFN-gamma was reduced in the 2774 and PA-1 cells stably expressing either IRF-1 antisense or IRF-2 sense, as shown by the cytotoxicity and FACS analysis. Furthermore, the IFN-gamma-induced apoptosis was greatly reduced, when inhibitors of ICE were treated into PA-1 cells. Taken together, these results suggest that IRF-1 directly mediates the IFN-gamma-induced apoptosis via the activation of caspase-1 gene expression in IFN-gamma-sensitive ovarian cancer cells.

Photodynamic effects of Radachlorin on cervical cancer cells.

PURPOSE: Photodynamic therapy (PDT) is a novel treatment modality, which produces local tissue necrosis with laser light following the prior administration of a photosensitizing agent. Radachlorin has recently been shown to be a promising PDT sensitizer. In order to elucidate the antitumor effects of PDT using Radachlorin on cervical cancer, growth inhibition studies on a HPV-associated tumor cell line, TC-1 cells in vitro and animals with an established TC-1 tumor in vivo were determined. MATERIALS AND METHODS: TC-1 tumor cells were exposed to various concentrations of Radachlorin and PDT, with irradiation of 12.5 or 25 J/cm(2) at an irradiance of 20 mW/cm(2) using a Won-PDT D662 laser at 662 nm in vitro. C57BL/6 mice with TC-1 tumor were injected with Radachlorin via different routes and treated with PDT in vivo. A growth suppression study was then used to evaluate the effects at various time points after PDT. RESULTS: The results showed that irradiation of TC-1 tumor cells in the presence of Radachlorin induced significant cell growth inhibition. Animals with established TC-1 tumors exhibited significantly smaller tumor sizes over time when treated with Radachlorin and irradiation. CONCLUSION: PDT after the application of Radachlorin appears to be effective against TC-1 tumors both in vitro and in vivo.