The Preliminary Study Of The Effectivity And The Mechanism As Well As The Security Of The Low Dose CY In The Treatment Of CA

Part Ⅰ The study of the effectivity of the low dose CY in the treatment of CAObjectives:To investigate the therapeutic effect of low-dose CY in treating the CA.Methods:86patients with extensive large warts were recruited in this study and were all treated with laser therapy. Then the patients were separated into three groups randomly. One group (n=52) received cyclophosphamide(CY)50mg orally, once a day for one week. Another group (n=26) received placebo,A200mg dose was used in the third group (n=8) once a day for one week. The follow-up was conducted at different time points to observe the recurrence.Results:In the low dose CY group, the patients showed complete clearance and no recurrence in the first six weeks. However, nine recurrences were observed thereafter. But, following another week of low-dose CY treatment alone,7out of9patients resolved completely without second recurrence. In the placebo group,81%patients recurred, most within eight weeks. In contrast, the patients who were treated with laser therapy and high-dose CY,Six patients recurred within8weeks.Conclusions:Low-dose cyclophosphamide (CY) can effectively prevent the recurrence of large CA in clinical patients after laser therapy. Part ⅡThe investigation of the mechanism and security of low-dose CY in the treatment of CAObjectives:1.To investigate the immunologic mechanism of the treatment of CA using low-dose CY;2.To research the security of the treatment of CA using low-dose CY.Methods:1. Treg cells, T cells and NK cells were isolated from the peripheral blood from the low-dose CY group (n=8), high-dose CY group (n=8), placebo group (n=8) and the healthy volunteers (n-6) respectively to test the quantity.2. To examine the cell proliferation and the cell specific kill of T cells and HPV-specific T cells from the low-dose CY group (n=6) and the placebo group (n=6) respectively. To test the amount of IFN-γ secreted from the NK cells after the stimulation of IL-2and IL-12; to test the cytotoxicity of NK cells after the stimulation of IL-2to the K562cells and to the HPV-infected wart cells.3.36patients with large warts (type6HPV infection) were chosen. The superficial skin area around the laser treatment site was scraped carefully with a cell scraper after laser therapy. The HPV DNA copies were determined by real time PCR. The above laser-treated patients were separated to two groups randomly.24patients were treated with low-dose CY and12patients received placebo for seven days.14days later, the superficial skin area around the laser treatment site was scraped for HPV DNA detection.4. Patients were treated with low-dose CY (n=18) or placebo (n=14) for seven days after laser therapy. Three weeks later, the peripheral blood was collected. The serums anti ds-DNA antibodies and the serum rheumatoid factors (including the anti-IgM, anti-IgG and anti-IgA autoantibody) were determined. And the serum levels of glutamic-pyruvate transaminase and creatinine were detected.Results:1.The amount of CD3+CD4+FOXP3+Treg cells from the CA patients after laser therapy were almost as same as the healthy volunteers. The amount of Treg cells in percentage and in absolute number were both significantly decreased after the low-dose CY treatment group; the Treg cells were also decreased obviously in the high-dose CY treatment group. The number of CD3+T cells and CD3-CD56+NK cells in the peripheral blood were almost the same in low-dose CY group and the placebo group, but descended obviously in the high-dose CY group compared with placebo group.2. The proliferation of T cells was increased in response to anti-CD3and CD28stimulation in CY group compared to placebo group. Furthermore, the activity of HPV specific T cells was enhanced in CY group, evaluated by either proliferation or cytotoxicity. NK cells in CY group produced more IFN-y in response to IL-2and IL-12stimulation, compared to that in the placebo group. And low-dose CY treatment augmented the cytolysis to NK cell-sensitive K562cells and HPV-infected wart cells.3. A similar amount of virus DNA was detected in all36samples after laser therapy. However,14days after CY treatment (n=24) or placebo treatment (n=12), a very high copy number was only detected in placebo treated samples, but HPV DNA was almost undetected in21CY-treated samples and much lower in other three patients.4. The anti-dsDNA, rheumatoid factor (including the anti-IgM, anti-IgG and anti-IgA autoantibody) and the level of glutamic-pyruvate transaminase (GPT) and creatinine in patients’serum did not show any difference between the low-dose CY group for3weeks and the placebo group.Conclusions:Low-dose CY could selectivity depress the immunosuppressive Treg cells in CA patients and low-dose CY treatment is not associated with autoimmunity and has little harm to the liver and the kidney.