The effect of aminolevulinic acid hydrochloride topical powder photodynamic therapy versus nocardia rubra cell wall skeleton and observers group in the treatment of cervical low-grade squamous intraepithelial lesions with human papillomavirus infection: A non-randomized study, controlled pilot study.

There are insufficient studies comparing the efficacy of aminolevulinic acid hydrochloride topical powder (ALA) photodynamic therapy (PDT) against Nocardia rubra cell wall skeleton (Nr-CWS) therapy in the treatment of cervical low-grade squamous intraepithelial lesion (LSIL) with human papillomavirus (HPV), especially for long-term efficacy. Patients with cervical LSIL and HPV infection were divided into 3 treatment groups based on their own choice. All patients had a follow-up test including HPV testing, cytology and colposcopy at 4 to 6 months and 12 months after the treatment. Among142 patients, patients received 51 ALA PDT and 41 patients received Nr-CWS. Another 50 patients who refused treatment were included in the Observers group. Four to six months or 12 months after treatment, there was significant difference between 3 groups in the clearance rate of HR-HPV infection and the complete remission (CR) rates of cervical LSIL; the CR rates of cervical LSIL in the ALA PDT group was significantly higher than the Nr-CWS group; but there was no significant difference between 2 groups in the clearance rate of HPV infection. The CR rates of cervical LSIL and the clearance rate of HPV infection in the ALA PDT group was significantly higher than the Observers group; the CR rates of cervical LSIL and the clearance rate of HPV infection in the Nr-CWS group was significantly higher than the Observers group; there was no significant difference in the recurrence rates in ALA PDT and Nr-CWS group after 12 months. Both of ALA PDT and Nr-CWS group had lower recurrence rate than the Observers group. The effect of ALA PDT is similar to Nr-CWS in the clearance rate of HR-HPV infection. Compared to the Nr-CWS group, the CR rates of cervical LSIL were considerably greater in the ALA PDT group. The effect of ALA PDT in the clearance rate of HPV infection and CR rates of cervical LSIL was significantly higher than the follow-up group; Both of ALA PDT and Nr-CWS group had lower recurrence rate than the Observers group. For cervical LSIL with HPV infection, ALA PDT is a very successful therapeutic method that is noninvasive.

A retrospective study of immunotherapy using the cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guérin (BCG-CWS) for cervical cancer.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has the potential to promote adaptive immunity. We sought to examine the synergistic effect of BCG-CWS vaccination on cervical cancer patients undergoing standard treatments including surgery, chemotherapy, and/or radiation. We retrospectively analyzed 103 patients (13 cases administered with BCG-CWS vaccine and 90 controls without BCG-CWS) who underwent a standard treatment for cervical cancer from 2005 to 2021. The BCG-CWS group underwent repeated intradermal injections of the BCG-CWS vaccine before or immediately after the standard therapy start from 2011 to 2018. The vaccination was repeated weekly for 1 month, and then every 4 weeks thereafter. The effectiveness of the BCG-CWS vaccination on cervical cancer treatment was evaluated by determining the hazard ratios of overall survival between the BCG-CWS group and the control group with multivariate analysis using the Cox model. Hazard ratios between 2 groups were determined after adjustment by clinical parameters including surgery, chemotherapy, radiation, age, clinical stage, presence of human papillomavirus, and pathology. Long-term follow-up revealed a significantly better prognosis (hazard ratio: 0.2108, P = .008 by the Cox model) for patients with cervical cancer in the BCG-CWS group compared to patients in the control group. Among patients with advanced cancer worse than stage IB2, some completely cleared the disease, whereas the others showed long-term survival with recurrence. BCG-CWS therapy appears to be an effective immune adjuvant therapy for cervical cancer, although randomized control studies are needed to confirm this. We also need to clarify the underlying mechanisms slowing the progression of cervical cancer in those receiving this vaccination. This study sheds light on the potential of immunostimulatory drugs such as BCG-CWS and suggests the important role of immunity for cancer elimination in combination therapy.

Immune adjuvant therapy using Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) in advanced malignancies: A phase 1 study of safety and immunogenicity assessments.

The cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) is a bioactive component that is a strong immune adjuvant for cancer immunotherapy. BCG-CWS activates the innate immune system through various pattern recognition receptors and is expected to elicit antigen-specific cellular immune responses when co-administered with tumor antigens. To determine the recommended dose (RD) of BCG-CWS based on its safety profile, we conducted a phase I dose-escalation study of BCG-CWS in combination with WT1 peptide for patients with advanced cancer.The primary endpoint was the proportion of treatment-related adverse events (AEs) at each BCG-CWS dose. The secondary endpoints were immune responses and clinical effects. A BCG-CWS dose of 50, 100, or 200 µg/body was administered intradermally on days 0, 7, 21, and 42, followed by 2 mg of WT1 peptide on the next day. For the escalation of a dose level, 3 + 3 design was used.Study subjects were 18 patients with advanced WT1-expressing cancers refractory to standard anti-cancer therapies (7 melanoma, 5 colorectal, 4 hepatobiliary, 1 ovarian, and 1 lung). Dose-limiting toxicity occurred in the form of local skin reactions in 2 patients at a dose of 200 µg although no serious treatment-related systemic AEs were observed. Neutrophils and monocytes transiently increased in response to BCG-CWS. Some patients demonstrated the induction of the CD4 T cell subset and its differentiation from the naïve to memory phenotype, resulting in a tumor response.The RD of BCG-CWS was determined to be 100 µg/body. This dose was well tolerated and showed promising clinical effects with the induction of an appropriate immune response.

Nocardia rubra cell wall skeleton accelerates cutaneous wound healing by enhancing macrophage activation and angiogenesis.

Objective This study was performed to investigate the effect of Nocardia rubra cell wall skeleton (N-CWS) on wound healing of full-thickness skin defects. Methods Two 2- × 2-cm full-thickness wounds, one on each side of the midline, were made on the back of 12 rats. One wound was covered with Vaseline gauze soaked in normal saline, whereas the other was covered with Vaseline gauze and N-CWS. Wound dressings were changed every other day from day 0 (wound creation) to day 11. Four of the 12 rats were killed on day 7, and biopsy samples were obtained for biochemical and histopathological analyses. The expression levels of CD31, CD68, and F4/80 in the tissues were examined immunohistologically. The expression of transforming growth factor (TGF)-ß1 in the wound was determined by western blot. Results N-CWS increased the wound healing rate, reduced the complete wound healing time, and increased the expression levels of CD31, CD68, and F4/80 on day 7. The TGF-ß1 expression level in the wound was significantly higher in the N-CWS group than in the control group on day 7. Conclusions N-CWS can activate macrophages, increase TGF-ß1 expression, and enhance angiogenesis and thus accelerate cutaneous wound healing.

Comparison between three adjuvants for a vaccine against canine leishmaniasis: In vitro evaluation of macrophage killing ability.

The aim of this study was to evaluate, in terms of dog macrophage killing ability in vitro, a vaccine based on Leishmania infantum promastigote soluble antigen (LSA) formulated with three different adjuvants (BCG, AdjuPrime, MPL/TDM/CWS). A significant increase of the macrophage killing ability was observed in dogs vaccinated with LSA+MPL/TDM/CWS after 1 month from vaccination. A similar increase of macrophage parasitocidal ability was present only after 5 months in dogs vaccinated with LSA+BCG or LSA+AdjuPrime. In all dogs the augmented killing percentage was still present after 12 months from vaccination. Therefore, in particular LSA+MPL/TDM/CWS vaccine seems promising for further studies in dogs.

Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy.

The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guerin (BCG/CWS) is an effective antitumor immunotherapy agent. Here, we demonstrate that BCG/CWS has a radiosensitizing effect on colon cancer cells through the induction of autophagic cell death. Exposure of HCT116 colon cancer cells to BCG/CWS before ionizing radiation (IR) resulted in increased cell death in a caspase-independent manner. Treatment with BCG/CWS plus IR resulted in the induction of autophagy in colon cancer cells. Either the autophagy inhibitor 3-methyladenine or knockdown of beclin 1 or Atg7 significantly reduced tumor cell death induced by BCG/CWS plus IR, whereas the caspase inhibitor z-VAD-fmk failed to do so. BCG/CWS plus IR-mediated autophagy and cell death was mediated predominantly by the generation of reactive oxygen species (ROS). The c-Jun NH(2)-terminal kinase pathway functioned upstream of ROS generation in the induction of autophagy and cell death in HCT116 cells after co-treatment with BCG/CWS and IR. Furthermore, toll-like receptor (TLR) 2, and in part, TLR4, were responsible for BCG/CWS-induced radiosensitization. In vivo studies revealed that BCG/CWS-mediated radiosensitization of HCT116 xenograft growth is accompanied predominantly by autophagy. Our data suggest that BCG/CWS in combination with IR is a promising therapeutic strategy for enhancing radiation therapy in colon cancer cells through the induction of autophagy.

Innate immune therapy with a Bacillus Calmette-Guérin cell wall skeleton after radical surgery for non-small cell lung cancer: a case-control study.

PURPOSE: We investigated whether adjuvant immunotherapy with Bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) and surgical resection was better than resection, with or without other adjuvant therapy, for patients with non-small cell lung cancer (NSCLC). METHODS: The case group comprised 71 patients who underwent radical surgery for NSCLC, followed by BCG-CWS immunotherapy, with follow-up data available. The case-control study was designed with one control selected for each case-group patient. Each control was matched by pathological stage and year of birth (+/-5 years). BCG-CWS 200 microg was inoculated intracutaneously in the upper arm four times per week (sensitization phase); then at 4-week intervals (therapeutic phase). RESULTS: The case-group patients received 45 +/- 22.6 (average +/- SD) cycles of BCG-CWS inoculation. Overall 5-year and 10-year survival rates were 71% and 61% for the case-group patients, and 63% and 43% for the control-group patients. The survival rate of the case group was better than that of the control group (not significant; P = 0.114). The same trend was seen in the patients with stage III or N+ NSCLC (not significant; P = 0.114, P = 0.168). There were no life-threatening adverse events. CONCLUSIONS: BCG-CWS immunotherapy seemed to improve survival after resection of NSCLC, especially locally advanced NSCLC. Moreover, this immunotherapy did not compromise quality of life during treatment.

[Effect of Nocardia rubra cell wall skeleton on the growth of HeLa cell line infected with HPV].

OBJECTIVE: To investigate the effects of Nocardia rubra cell wall skeleton (Nr-CWS) on the HeLa cell line, one of the cell lines of human cervical cancer, infected with HPV. METHODS: HPV-infected HeLa (HPV 18-positive cells) cultured in vitro were divided into two groups: the experiment group and control group. Nr-CWS was added to the experiment group and PBS to the control. The growth and proliferation of HeLa cells were detected with MTT and flow cytometry technology. Inhibitive effect of HeLa transplanted tumor was investigated in Scid mice. RESULTS: The growth of HeLa cells in the experimental group was apparently decreased compared with that of the control. The results of flow cytometry demonstrated that more HeLa cells were transferred into quiescent phase in the experimental group than that in the control. While less in the proliferative phase, both of the volume and weight of HeLa transplanted tumor with drug-added group were less than those of control group. CONCLUSION: The Nocardia rubra cell wall skeleton is a potiental growth inhibitor and inducer of apoptosis of cervical cancer cells in vitro and may provide a new way in prevention or supplementary management of anti-human papilloma virus.

[Effect of Nocardia rubra cell wall skeleton (Nr-CWS) on oncogenicity of TC-1 cells and anti-human papillomavirus effect of Nr-CWS in lower genital tract of women].

OBJECTIVE: To detect the effect of Nocardia rubra cell wall skeleton (Nr-CWS) on tumorigenicity induced by TC-1 cells and to clinically study anti-human papillomavirus effect of Nr-CWS in lower genital tract of women. METHODS: Tumor model was established by injecting TC-1 cells subcutaneously in SCID mice, then divided them into 3 groups randomly and injected with isovolumetric physiological saline, 60 micrograms/ml Nr-CWS and 120 micrograms/ml Nr-CWS respectively, the growth of tumors was measured one week later. Nr-CWS was applied on 45 HPV positive women whose TCT test was normal and without cervical erosion 2-3 days after menstruation. HPV was detected again 3 months later to explore the effect of Nr-CWS on HPV infection in female lower genital tract. RESULTS: The animal experiment showed the weight of transplanted tumors in treated group was less than that of control group (chi2=12.5, P= 0.002). The tumor inhibition rate was 59.1 percent and 84.2 percent in the groups treated with Nr-CWS 60 and 120 micrograms/ml Nr-CWS; the results of HPV detection in 23 out of the 45 cases (51.1 percent) became negative after the 3-month treatment; the viral load was reduced in 9, and there was no change in viral load in 13 cases. Significant difference was found between the rates of undetectable viral load and the natural viral disappearance rate (P less than 0.05). CONCLUSION: Nr-CWS has an inhibitory effect to TC-1 cell tumorigenesis and clinical application of Nr-CWS may eliminate the HPV infection in lower genital tract of a considerable proportion of women with HPV infection.

Immunostimulation in the urinary bladder by local application of Nocardia rubra cell-wall skeletons (Rubratin) and bacillus Calmette-Guérin as therapy for superficial bladder cancer: a comparative study.

Twelve patients with superficial bladder cancer were treated with intravesical instillations of Rubratin (ASTA Pharma AG, Frankfurt, Germany), a cell-wall preparation of Nocardia rubra. The objective was to compare the immunostimulating effect of Rubratin with that of bacillus Calmette-Guérin (BCG). Local immunostimulation was determined by cytokine induction in serially collected urine samples during the first 24 h after each instillation, leukocyte influx into the urine, and phenotypic analysis of the lymphocyte fraction. Levels of Rubratin-induced interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha were significantly elevated compared with pretherapy levels. Rubratin induced leukocyte influx into the urine. T-cell activation (IL-2 receptor and human leukocyte antigen-DR expression) can be induced, and CD4:CD8 cell ratios can be increased. All parameters indicated that Rubratin-induced immunostimulation was less than that associated with BCG. In conclusion, although local Rubratin-induced immunostimulation occurs in a limited number of patients, the amount of immunocompetent cells attracted to the bladder seems to be less than that associated with BCG therapy, thus resulting in lower levels of cytokine production (which may reflect less clinical efficacy).

[Long-term results of intravesical N-CWS instillation to prevent recurrence after surgery for bladder cancer].

OBJECTIVE: To evaluate the long term results of intravesical nocardia rubre cell wall skeleton instillation to prevent recurrence after surgery for bladder cancer. METHODS: A randomized and placedo-controlled trial on intravesical N-CWS instillation versus mitomycin-C(MMC) therapy for recurrence prevention in postopenative bladder cancer patients was carried out. RESULTS: Fourty-five patients were treated with N-CWS with 39 followed for 12 to 60 months. Thirteen patients developed recurrence. The 1-year tumor free survival rate was 87.2% and that at 5 years was 66.7%. Thirty patients as treated with MMC serving as controll with 25 followed for the same duration, twelve patients developed recurrence. The 1-year tumor free survival rate was 84% and that at 5 years was 52%. The results indicate that N-CWS gave better results with rare and milder side effects. CONCLUSION: N-CWS is believed to be one of the effective drugs in preventing bladder cancer recurrence after surgery.

Postoperative immunostimulation after complete resection improves survival of patients with stage I nonsmall cell lung carcinoma.

BACKGROUND: approximately 40% of primary lung carcinoma patients who die within 1 month after a complete resection have residual tumor in regional or distant organs, emphasizing the importance of postoperative adjuvant therapy. In this study, the effectiveness of transfer factor (TF) and nocardia rubra-cell wall skeleton (N-CWS) as adjuvant therapy for patients with primary, completely resected nonsmall cell carcinoma of the lung was evaluated in a randomized controlled trial METHODS: A total of 82 patients with Stage I disease who had a complete resection were allocated randomly into 2 groups: TF + N-CWS (n = 41) or control (surgery only) (n = 41). RESULTS: The distributions of age, sex, histology, differentiation, T classification, tumor size, visceral pleural invasion, and the site of origin, were similar in the two groups. The 5- and 10-year disease specific survival rates in the TF + N-CWS group were 85% and 85%, respectively, and those in the control group were 72% and 64%, respectively. There was a statistically significant difference between the two groups (P = 0.041). When the survival was analyzed according to clinical characteristics, significant differences were observed in patients with no visceral pleural invasion or with T1 disease. The frequency of distant metastasis was significantly less in the TF + N-CWS group than in the control group. CONCLUSIONS: These results indicate that TF + N-CWS is beneficial as adjuvant therapy after surgical treatment of Stage I nonsmall cell carcinoma of the lung.

Former poison gas workers and cancer: incidence and inhibition of tumor formation by treatment with biological response modifier N-CWS.

Mustard gas is known to have mutagenic and carcinogenic effects on animal and human cells. In this report, 1,632 male Japanese who worked in poison gas factories at some time between the years 1927 and 1945 were studied to determine comparative risk for development of cancer, the reference population being data on Japanese males overall. The standardized mortality ratio (SMR) for lung cancer in workers directly and indirectly involved in the production of mustard gas was significantly elevated. In addition, SMR for lung cancer in worker who had worked for more than 5 years was also significantly elevated. Thus, poison gas workers who had engaged in the production of mustard gas or related work for more than 5 years are a high-risk group for lung cancer. Under the cancer preventive program, Nocardia rubra cell-wall skeleton (N-CWS) was administered to 146 former poison gas workers. During a 4.5 year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of smoking level, a significant suppression of development of cancers was noted in the N-CWS-treated workers as compared to the untreated controls. Although the molecular mechanisms of carcinogenesis in former poison gas workers remains unclear, our study proposes the possible effect of biological response modifiers in the prevention of cancer development in high-risk human subjects.

Adoptive immunotherapy of poorly immunogenic tumors with in vitro sensitized cells generated by intratumoral administration of biological response modifiers.

We investigated the efficacy of intratumoral administration of biological response modifiers (BRM) in induction of in vitro sensitized (IVS) cells for adoptive immunotherapy of the poorly immunogenic MCA 102 sarcoma and B16-BL6(BL6) melanoma. We used the bacterial immunoadjuvant Nocardia rubra cell wall skeleton (N-CWS), and a streptococcal preparation, OK-432, for MCA 102 and BL6, respectively. After C57BL/6(B6) mice were inoculated subcutaneously (s.c.) with viable MCA 102 or BL6 tumor cells in the foot-pad, mice were injected intratumorally (i.t.) with N-CWS ranging from 10 to 400 micrograms or OK-432 ranging from 1 to 100 micrograms. Draining popliteal lymph nodes (LN) were harvested 7 days after i.t. administration of BRM, and LN cells were cultured with irradiated tumor cells in the presence of IL-2 for 11 days. These IVS cells (7.5 x 10(6) or 2 x 10(6)) were transferred intravenously (i.v.) to B6 mice with 4 day pulmonary metastases established by i.v. injection of viable MCA 102 cells (1 x 10(6)) or viable BL6 cells (3 x 10(5)). The mice were also received intraperitoneally 4 x 10(4) IU/day of IL-2 for 4 days after adoptive transfer. The transfer of IVS cells from mice immunized by i.t. injection of 100 micrograms of N-CWS 1 week after inoculation of tumor cells significantly reduced MCA 102 pulmonary metastases, compared with control IVS cells without administration of N-CWS. Moreover, the transfer of IVS cells from mice immunized by i.t. injection of 10 micrograms of OK-432 3 days after inoculation of tumor cells significantly reduced BL6 pulmonary metastases compared with control IVS cells without administration of OK-432. The administration of N-CWS resulted in no enhancement of in vitro cytotoxicity. Although the administration of 10 micrograms of OK-432 augmented in vitro cytotoxicity of IVS cells against BL6, cytotoxic activity was lower than that of IVS cells immunized with N-CWS. The major phenotype was CD8+ cells in IVS cells immunized with N-CWS or OK-432. These results suggest that i.t. administration of N-CWS and OK-432 facilitates the production of sensitized T-cells, and this administration route of BRM may be useful in the adoptive immunotherapy of human cancer.

Effect of DETOX as an adjuvant for melanoma vaccine.

The identification of effective adjuvants is critical for tumor vaccine development. Towards this end, we examined whether the immunogenicity of a melanoma vaccine could be potentiated by DETOX, an adjuvant consisting of monophosphoryl lipid A (MPL) and purified mycobacterial cell-wall skeleton (CWS). Nineteen patients with resected stage III melanoma were immunized with a polyvalent melanoma antigen vaccine (40 micrograms) admixed with DETOX, q3 wks x 4. Seven patients received vaccine + low-dose DETOX (10 micrograms MPL + 100 micrograms CWS) and 12 received vaccine + high-dose DETOX (20 micrograms MPL + 200 micrograms CWS). A non-randomized control group of 35 patients was treated similarly with 40 micrograms vaccine + alum. One week after the fourth vaccine immunization, melanoma antibodies were increased over baseline in 7/7 (100%) patients treated with vaccine + low-dose DETOX, 8/12 (67%) patients treated with vaccine + high-dose DETOX, and in 4/19 (21%) of vaccine + alum patients. For the entire DETOX group, the antibody response rate was 15/19 (79%) compared 4/19 (21%) in the alum group (p < 0.001). In contrast, a strong delayed-type hypersensitivity (DTH) response (> or = 15 mm increase in DTH response over baseline) was induced in 50% of the entire DETOX group versus in 47% of the alum group. Median disease-free (DF) survival for the entire DETOX group was 17.8 months compared with 32.1 months in the alum group (p < 0.05). In conclusion, DETOX markedly potentiated antibody but had little effect on DTH responses to melanoma vaccine immunization. It did not appear to improve disease-free survival in comparison to alum in this non-randomized study.

Clinical usefulness of continuous administration of Nocardia rubra cell wall skeleton (N-CWS) in diffuse panbronchiolitis (DPB).

Eight patients with diffuse panbronchiolitis (DPB) who had repeated intractable airway infections were continuously treated with Nocardia rubra cell wall skeleton (N-CWS), a biological response modifier. As a result, subjective symptoms were reduced in 6 patients. Antibiotics therapy could be discontinued completely in two patients and the dose of antibiotics could be reduced considerably in two other patients. No adverse reactions in relation to N-CWS were observed. These results suggest that N-CWS is effective in treating erythromycin-resistant DPB.