[Regulatory effects of the Nocardia rubra cell wall skeleton on the biological function of human neutrophils].

Objective: To investigate the regulatory effects and mechanism of Nocardia rubra cell wall skeleton (Nr-CWS) on the biological function of human neutrophils. Methods: The experimental research method was used. Fifteen healthy adult volunteers (7 males and 8 females, aged 24 to 45 years) were recruited from Suzhou Physical Examination Center for physical examination from May to October 2022, the peripheral venous blood was collected, and neutrophils were extracted by immunomagnetic bead sorting. The cells were divided into normal control group without any treatment, Nr-CWS alone group treated with Nr-CWS of final mass concentration 60 ng/mL alone, endotoxin/lipopolysaccharide (LPS) alone group stimulated with LPS of final mass concentration 1 µg/mL alone, and LPS+Nr-CWS group stimulated with LPS first and then treated with Nr-CWS as before. After 1 h of culture, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of neutrophils were detected using the modified agarose chemotactic model; the proportion and fluorescence intensity of phagocytosis cells, the level of reactive oxygen species (ROS), the protein expression levels of granular protein CD35, CD66b, and CD63, and the concentrations of inflammatory cytokines of interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor alpha (TNF-α), and interferon-γ in cell culture supernatant were detected by flow cytometry. The number of samples in each group in the above experiments was 15. Data were statistically analyzed with analysis of variance for factorial design and independent sample t test. Results: After 1 h of culture, the chemotactic function score of cells in normal control group, Nr-CWS alone group, LPS alone group, and LPS+Nr-CWS group were 15.0, 14.5±0.5, 1.5±0.5, 12.0±1.5, respectively. Compared with those in normal control group, the chemotaxis distance, chemotactic cell percentage, chemotactic index, maximum chemotactic speed, and chemotactic function score of cells were significantly decreased in LPS alone group and LPS+Nr-CWS group (with t values of 18.36, 18.88, 54.28, 18.36, 46.77, 10.58, 14.74, 6.84, 10.58, and 4.24, respectively, P<0.05); compared with those in LPS alone group, the five chemotactic function indexes as above in LPS+Nr-CWS group were significantly increased (with t values of 11.47, 14.65, 11.62, 11.47, and 13.75, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the proportion and fluorescence intensity of phagocytosis cells were significantly increased in Nr-CWS alone group (with t values of 6.86 and 6.73, respectively, P<0.05), and the above two indexes were significantly decreased in LPS alone group (with t values of 7.35 and 22.72, respectively, P<0.05) and LPS+Nr-CWS group (with t values of 21.37 and 13.10, respectively, P<0.05). After 1 h of culture, compared with that in normal control group, the level of ROS of cells in LPS alone group was significantly increased (t=6.64, P<0.05); compared with that in LPS alone group, the level of ROS of cells in LPS+Nr-CWS group was significantly decreased (t=5.46, P<0.05). After 1 h of culture, compared with those in normal control group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly increased in LPS alone group and LPS+Nr-CWS group (with t values of 16.75, 17.45, 10.82, 5.70, 19.35, and 15.37, respectively, P<0.05); compared with those in LPS alone group, the protein expressions of CD35, CD66b, and CD63 of cells were significantly decreased in LPS+Nr-CWS group (with t values of 4.92, 5.72, and 3.18, respectively, P<0.05). After 1 h of culture, compared with those in normal control group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS alone group (with t values of 22.10, 9.50, 7.21, 10.22, 24.88, 8.43, and 47.48, respectively, P<0.05), and the concentrations of IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly increased in LPS+Nr-CWS group (with t values of 4.68, 5.12, 8.02, 5.58, and 7.13, respectively, P<0.05); compared with those in LPS alone group, the concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, and interferon-γ in cell culture supernatant were significantly decreased in LPS+Nr-CWS group (with t values of 5.39, 2.83, 5.79, 2.90, 5.87, 4.88, and 39.64, respectively, P<0.05). Conclusions: Nr-CWS can enhance the phagocytosis ability of neutrophils in normal condition and improve the chemotactic function, ROS level, degranulation protein level, and inflammatory factor level of human neutrophils in infectious condition. Nr-CWS can enhance the anti-infection ability of human neutrophils by regulating its biological behavior in innate immunity.

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.

[Evaluation of the efficacy and safety of Nocardia rubra cell wall skeleton immunotherapy for cervical high-risk HPV persistent infection].

Objective: To evaluate the efficacy and safety of Nocardia rubra cell wall skeleton (Nr-CWS) in the treatment of persistent cervical high-risk human papillomavirus (HR-HPV) infection. Methods: A randomized, double blind, multi-center trial was conducted. A total of 688 patients with clinically and pathologically confirmed HR-HPV infection of the cervix diagnosed in 13 hispital nationwide were recruited and divided into: (1) patients with simple HR-HPV infection lasting for 12 months or more; (2) patients with cervical intraepithelial neoplasia (CIN) Ⅰ and HR-HPV infection lasting for 12 months or more; (3) patients with the same HR-HPV subtype with no CINⅡ and more lesions after treatment with CINⅡ or CIN Ⅲ (CINⅡ/CIN Ⅲ). All participants were randomly divided into the test group and the control group at a ratio of 2∶1. The test group was locally treated with Nr-CWS freeze-dried powder and the control group was treated with freeze-dried powder without Nr-CWS. The efficacy and negative conversion rate of various subtypes of HR-HPV were evaluated at 1, 4, 8, and 12 months after treatment. The safety indicators of initial diagnosis and treatment were observed. Results: (1) This study included 555 patients with HR-HPV infection in the cervix (included 368 in the test group and 187 in the control group), with an age of (44.1±10.0) years. The baseline characteristics of the two groups of subjects, including age, proportion of Han people, weight, composition of HR-HPV subtypes, and proportion of each subgroup, were compared with no statistically significant differences (all P>0.05). (2) After 12 months of treatment, the effective rates of the test group and the control group were 91.0% (335/368) and 44.9% (84/187), respectively. The difference between the two groups was statistically significant (χ2=142.520, P<0.001). After 12 months of treatment, the negative conversion rates of HPV 16, 18, 52, and 58 infection in the test group were 79.2% (84/106), 73.3% (22/30), 83.1% (54/65), and 77.4% (48/62), respectively. The control group were 21.6% (11/51), 1/9, 35.1% (13/37), and 20.0% (8/40), respectively. The differences between the two groups were statistically significant (all P<0.001). (3) There were no statistically significant differences in vital signs (body weight, body temperature, respiration, pulse rate, systolic blood pressure, diastolic blood pressure, etc.) and laboratory routine indicators (blood cell analysis, urine routine examination) between the test group and the control group before treatment and at 1, 4, 8, and 12 months after treatment (all P>0.05); there was no statistically significant difference in the incidence of adverse reactions related to the investigational drug between the two groups of subjects [8.7% (32/368) vs 8.0% (15/187), respectively; χ2=0.073, P=0.787]. Conclusion: External use of Nr-CWS has good efficacy and safety in the treatment of high-risk HPV persistent infection in the cervix.

Exosomes derived from Nr-CWS pretreated MSCs facilitate diabetic wound healing by promoting angiogenesis via the circIARS1/miR-4782-5p/VEGFA axis.

Mesenchymal stem cell (MSC)-derived exosomes (Exos) were reported to a prospective candidate in accelerating diabetic wound healing due to their pro-angiogenic effect. MSCs pretreated with chemistry or biology factors were reported to advance the biological activities of MSC-derived exosomes. Hence, this study was designed to explore whether exosomes derived from human umbilical cord MSCs (hucMSCs) preconditioned with Nocardia rubra cell wall skeleton (Nr-CWS) exhibited superior proangiogenic effect on diabetic wound repair and its underlying molecular mechanisms. The results showed that Nr-CWS-Exos facilitated the proliferation, migration and tube formation of endothelial cells in vitro. In vivo, Nr-CWS-Exos exerted great effect on advancing wound healing by facilitating the angiogenesis of wound tissues compared with Exos. Furthermore, the expression of circIARS1 increased after HUVECs were treated with Nr-CWS-Exos. CircIARS1 promoted the pro-angiogenic effects of Nr-CWS-Exos on endothelial cellsvia the miR-4782-5p/VEGFA axis. Taken together, those data reveal that exosomes derived from Nr-CWS-pretreated MSCs might serve as an underlying strategy for diabetic wound treatment through advancing the biological function of endothelial cells via the circIARS1/miR-4782-5p/VEGFA axis.

Nocardia rubra cell-wall skeleton activates an immune response in cervical tissue via stimulating FPR3 to enhance dendritic cell-mediated Th1 differentiation.

Nocardia rubra cell wall skeleton (Nr-CWS) has proven to be a successful medicine for therapy of cervical human papillomavirus infection. The mechanism of action of Nr-CWS is unclear but may involve a stimulatory effect on the host immune system. We previously found that CD4+ T cells were increased in cervical tissue after Nr-CWS treatment. Microarray data from these cervical tissues revealed the significant upregulation of formylated peptide receptor 3 (FPR3). This study aimed to explore the role of Nr-CWS in immunomodulatory based on these findings. Examination of CD4+ T cell subsets in cervical tissue from patients who received Nr-CWS revealed substantial increases in Th1 cytokines and transcription factors. The regulatory effects of Nr-CWS on the function and phenotype of dendritic cells (DCs) were assessed in comparison with the traditional DC maturation inducer lipopolysaccharide (LPS). Similar to LPS, Nr-CWS potently induced DC maturation and interleukin-12 (IL-12) secretion. Differentiation of T cells induced by Nr-CWS stimulated DCs was assessed using the mixed lymphocyte reaction assay. Significant differentiation towards Th1 was evident. Finally, FPR3 expression in DCs in response to Nr-CWS and LPS was measured. Nr-CWS potently upregulated FPR3 expression, while the LPS did not. Silencing FPR3 in DCs reduced Nr-CWS-induced IL-12 production and Th1 cell polarization in co-cultured T cells. The collective findings indicate that Nr-CWS may target FPR3 on the surface of DC cells and activate a Th1-type immune response. The findings clarify the basis of the antiviral immune effects of Nr-CWS on human papillomavirus.

Nocardia rubra cell wall skeleton regulates tumour-associated macrophage polarization by reprogramming M2 macrophages into M1 macrophages via STAT1/STAT6 pathways.

Targeted therapy with tumour-associated macrophages (TAMs) has emerged as a new paradigm for immunotherapy of cervical cancer. Nocardia rubra cell wall skeleton (Nr-CWS) for external use is an immunotherapeutic agent. In this study, we aimed to explore the effects of Nr-CWS on TAMs and the potential mechanisms. Cervical tissue samples were collected before and after Nr-CWS treatment from patients with high-risk HPV infection and cervical intraepithelial neoplasia (CIN). The effect of Nr-CWS on macrophages in vivo was examined by immunohistochemistry and double-labeling immunofluorescence histochemistry. In vitro experiments were performed using a TAM model established by THP-1 cells under Nr-CWS treatment. We found that Nr-CWS treatment significantly reduced the numbers of total macrophages and M2 macrophages, increased the proportion of M1 macrophages and decreased the proportion of M2 macrophages in cervical tissues. After Nr-CWS treatment in vitro, the expression levels of the M1 macrophage markers were increased, while the expression levels of the M2 macrophage markers were decreased. Nr-CWS treatment also activated STAT1 pathways but inhibited STAT6 pathways. These results indicated that Nr-CWS may improve local immune response and reverse immunosuppression by regulating the M2 to M1 polarization of TAMs via STAT1/STAT6 pathways.

Th1 immune maturation effects of Nocardia rubra cell-wall skeleton via PI3K/Akt/PAX8 regulatory axis.

Nocardia rubra cell-wall skeleton (Nr-CWS) is reported as an external immunotherapeutic enhancer with the advantage of antitumor effect on human cancers. However, the immune regulatory role of Nr-CWS is not fully illustrated. We studied mouse CD4+ T lymphocytes isolated from mice spleen were induced by Nr-CWS and observed that the differentiation of Th1 CD4+ T cells and the cytokines of IL-2, TNF-α, IFN-γ were all enhanced by Nr-CWS. Furthermore, RNA sequencing was conducted to investigate the different mRNA profiling induced by Nr-CWS. We observed that paired box 8 (PAX8) was significantly up-regulated in Nr-CWS-treated Th1 cells compared to control. As a transcription factor, chromatin immunoprecipitation sequencing was carried out to study the genome-wide distribution of PAX8. Interestingly, we found that the binding domain of PAX8 was elevated by Nr-CWS, and the target genes associated with these binding sites showed a positive correlation between their transcription and PAX8 binding strength. Finally, we determined that Nr-CWS could enhance the activity of the PI3 K/Akt signaling pathway. Akt agonist could mimic the effect of Nr-CWS for PAX8 up-regulation, while Akt inhibitor compromised the expression of PAX8. Taken together, we determined a novel role of Nr-CWS in boosting the activity of Th1 maturation via the PI3 K/Akt/PAX8 axis.

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.

[Efficacy and safety of Nocardia rubra cell wall skeleton for the treatment of erosive oral lichen planus].

OBJECTIVE: To observe the efficacy and safety of Nocardia rubra cell wall skeleton (Nr-CWS) for the treatment of erosive oral lichen planus (EOLP). METHODS: Sixty patients with clinically and pathologically diagnosed EOLP were randomly divided into the experimental group and control group according to the random number. Patients in the experimental group were treated with lyophilized powder containing Nr-CWS combined with normal saline. Patients in the control group received topical placebo without Nr-CWS combined with normal saline. Changes in the EOLP lesion area and the patient's pain level were recorded at the timepoints of weeks 1, 2, and 4 after the two different treatments, respectively. The changes of the patient's REU scoring system (reticulation, erythema, ulceration), the visual analogue scale and the oral health impact score (OHIP-14) were compared between the experimental group and control group after treatment, and the safety indicators of the two groups at the initial diagnosis and after 4 weeks' treatment were also observed, respectively. RESULTS: Totally, 62 patients with clinically and pathologically diagnosed EOLP were enrolled, 2 of whom were lost to the follow-up, with 31 in the experimental group, and 29 in the control group. The mean age of the experimental group and control group were (52.9±12.4) years and (54.07±12.40) years, respectively. There was no significant difference in the oral periodontal index between the experimental group and control group. In the experimental group, the erosive area of oral lichen planus was significantly reduced 1, 2, and 4 weeks after the Nr-CWS's treatment (P < 0.05), the reduction rate was 81.75%, the patient's pain index was also decreased (P < 0.05), and in addition, the OHIP-14 was reduced (P < 0.05). The changes of the REU scoring system, the visual analogue scale and the OHIP-14 were significantly different between the experimental group and control group after treatment. There was no significant difference in the safety index between the two groups. CONCLUSION: The priliminary data show that the Nr-CWS is effective and safe to treat EOLP.

Distribution of BCG-CWS-Loaded Nanoparticles in the Spleen After Intravenous Injection Affects Cytotoxic T Lymphocyte Activity.

Interest has developed in the bacillus Calmette-Guerin (BCG) cell wall skeleton (BCG-CWS) as a noninfectious adjuvant. Although BCG-CWS readily undergoes aggregation, in a previous study, we applied it to cancer immunotherapy via intravenous administration by encapsulating the BCG-CWS into nanoparticles (CWS-NPs). The CWS-NPs were taken up by major histocompatibility complex (MHC) class II+ (MHC-II+) cells and induced antigen-specific cytotoxic T lymphocyte (CTL) activity. However, the nature of the contribution of MHC-II+ cells to the CTL response continues to be unclear. In this study, we investigated the relationship between the distribution of CWS-NPs in the spleen and CTL activity. The main MHC-II+ cells that internalized the CWS-NPs were B cells. Decreasing the level of polyethylene glycol modification increased the uptake of CWS-NPs by B cells, leading to an increased CTL activity. A comparison of CWS-NPs with different uptake efficiencies into dendritic cells and B cells suggested that the DCs with internalized CWS-NPs may contribute to CTL activation compared with B cells. We succeeded in enhancing CTL activity by the CWS-NPs, and the findings reported herein should provide important information regarding target cells for the development of CWS-NP.

Nocardia Rubra Cell Wall Skeleton Up-Regulates T Cell Subsets and Inhibits PD-1/PD-L1 Pathway to Promote Local Immune Status of Patients With High-Risk Human Papillomavirus Infection and Cervical Intraepithelial Neoplasia.

The Nocardia rubra cell wall skeleton (Nr-CWS) for external use is an immune enhancer, which has been widely used in human cervix diseases such as cervical erosion, but the mechanism of Nr-CWS enhancing immunity is still unclear. The purpose of this study was to explore the effect and mechanism of Nr-CWS on the local immune status of cervical tissue in patients with high-risk human papillomavirus (HR-HPV) infection and cervical precancerous lesion, cervical intraepithelial neoplasia (CIN). The recruited patients with HR-HPV infection and CIN were treated with Nr-CWS. The specimens were taken from these patients before and after local application of Nr-CWS respectively. The normal control specimens were tested simultaneously. Serial section analysis of immunohistochemistry and co-expression analysis were performed to characterize populations of T cells and the expressions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1). The levels of cytokines in local cervical tissue were also detected. Nr-CWS significantly increased T cells including CD4+, CD8+ T cells, and reduced the expression of PD-L1 in the patients' local cervical tissues. Co-expression analyses showed that the proportions of PD-1+CD4+ cells in CD4+ T cells and PD-1+CD8+ cells in CD8+ T cells decreased after Nr-CWS application. Furthermore, the increase in the number of immune cells was accompanied by increased pro-inflammatory cytokines interleukin-12 (IL-12), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and decreased suppressive cytokine IL-10. The results indicate that Nr-CWS, as an immunotherapeutic agent for HR-HPV infection and CIN, plays an immune promoting role related to the upregulation of T cell subsets and the inhibition of PD-1/PD-L1 pathway.

Functional modulation of CD8+ T cell by approved novel immune enhancer: Nocardia rubra Cell-Wall Skeletons (Nr-CWS).

Nocardia rubra cell wall skeleton (Nr-CWS) has been reported to have innate immunostimulating and anti-tumor activities. However, the immunomodulatory effects of Nr-CWS on CD8+ T cells and their related mechanisms are still unknown. In this work, our team purified CD8+T cells from spleen cells and explored the phenotype and function of NR-CWS in vitro on CD8+T cells. We observed that Nr-CWS can significantly up-regulate the expression of CD69 and CD25 on CD8+T cells, with no significant effect on apoptosis or cell death of CD8+T cells that occurs in vitro during culture. In addition, the effect of perforin and granzyme B was increased after Nr-CWS treatment, but did not substantially alter the expression of TRAIL and FasL. A variety of cytokine analyses have shown that of the cytokines examined (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6 and IL-10), only IFN-γ and TNF The increase in -α was more pronounced, and the effect of Nr-CWS in CD8+T cell culture medium on CD8+ T cells was independent of Th cells. Our results demonstrated that Nr-CWS could up-regulate CD69 and CD25 expression on CD8+T cells, promoting IFN-γ and TNF-α secretion, and enhancing perforin and granzyme B production. Thus Nr-CWS may have Immunoaugmenting therapeutic activity via an increase in CD8+T cells response.

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.

Application of BCG-CWS as a Systemic Adjuvant by Using Nanoparticulation Technology.

The intravesical instillation of live Bacillus Calmette-Guerin (BCG) for treating bladder cancer is a powerful cancer immunotherapy. The BCG cell wall skeleton (BCG-CWS) is the main component of the adjuvant, leading to the induction of antitumor immunity. However, the use of live BCG and BCG-CWS is currently limited to local administration because of the infectiousness of live BCG and the insolubility of BCG-CWS. We previously developed a water-dispersible nanoparticle (NP) formulation of BCG-CWS (CWS-NP), which could be used to apply BCG components for use as a systemically injected adjuvant for the treatment of cancers other than bladder cancer. In the present study, we examined the possible use of CWS-NP for cancer immunotherapy, when intravenously administered. The CWS-NP was a highly uniform dispersion and showed no aggregation in serum. The intravenously injected CWS-NP accumulated in the spleen and was efficiently taken up by dendritic cells, leading to their maturation. The coadministration of CWS-NP and ovalbumin (OVA) loaded NP resulted in the generation of OVA-specific cytotoxic T cells and inhibited the growth of E.G7-OVA tumors. These results represent the first findings related to the use of systemically injected CWS-NP as an adjuvant for cancer immunotherapy.

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth.

In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

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.

Nocardia rubra cell-wall skeleton promotes CD4+ T cell activation and drives Th1 immune response.

Several lines of evidences have shown that Nocardia rubra cell wall skeleton (Nr-CWS) has immunoregulatory and anti-tumor activities. However, there is no information about the effect of Nr-CWS on CD4+ T cells. The aim of this study was to explore the effect of Nr-CWS on the phenotype and function of CD4+ T cells. Our results of in vitro experiments showed that Nr-CWS could significantly up-regulate the expression of CD69 and CD25 on CD4+ T cells, promote the proliferation of CD4+ T cells, increase the production of IFN-γ, TNF-α and IL-2 in the supernatants, but has no significant effect on the apoptosis and death of CD4+ T cells. Results of in vivo experiments showed that Nr-CWS could promote the proliferation of CD4+ T cells, and increase the production of IL-2, IFN-γ and TNF-α (Th1 type cytokines). These data suggest that Nr-CWS can enhance the activation of CD4+ T cells, promote the proliferation of CD4+ T cells and the differentiation of CD4+ T cells to Th1 cells.

Development of a Drug Delivery System for Cancer Immunotherapy.

Delivery systems are a powerful technology for enhancing the effect of cancer immunotherapy. We have been in the process of developing lipid-based delivery systems for controlling the physical properties and dynamics of immunofunctional molecules such as antigens and adjuvants. The lipid nanoparticulation of these molecules improves their physical properties, resulting in a good water dispensability, greater stability, and small size. The cell wall skeleton of bacille Calmette-Guerin (BCG-CWS) could be used to replace live BCG as a drug for treating bladder cancer, but problems associated with the physical properties of BCG-CWS have prevented its use. To overcome such problems, we developed a novel packaging method that permits BCG-CWS to be encapsulated into lipid nanoparticles, which induce antitumor responses against bladder cancer. Lipid nanoparticulation also improves the intracellular trafficking and biodistribution of immunofunctional molecules. Cyclic di-GMP (c-di-GMP) is an adjuvant that is recognized by the cytosolic sensor. However, c-di-GMP cannot pass through the cell membrane. We encapsulated c-di-GMP into lipid nanoparticles containing a pH-responsive lipid that was developed in our laboratory and achieved efficient cytosolic delivery and the induction of antitumor immunity. Furthermore, we are attempting to control the functions of immune cells by RNA interference. We have recently succeeded in the efficient delivery of small interfering RNA into mouse dendritic cells (DCs), which led to the enhancement of antitumor activity of DCs. In this review, our recent efforts regarding cancer immunotherapy using lipid-based nanoparticles are reviewed.

Inferring Gene Networks for Strains of Dehalococcoides Highlights Conserved Relationships between Genes Encoding Core Catabolic and Cell-Wall Structural Proteins.

The interpretation of high-throughput gene expression data for non-model microorganisms remains obscured because of the high fraction of hypothetical genes and the limited number of methods for the robust inference of gene networks. Therefore, to elucidate gene-gene and gene-condition linkages in the bioremediation-important genus Dehalococcoides, we applied a Bayesian inference strategy called Reverse Engineering/Forward Simulation (REFS™) on transcriptomic data collected from two organohalide-respiring communities containing different Dehalococcoides mccartyi strains: the Cornell University mixed community D2 and the commercially available KB-1® bioaugmentation culture. In total, 49 and 24 microarray datasets were included in the REFS™ analysis to generate an ensemble of 1,000 networks for the Dehalococcoides population in the Cornell D2 and KB-1® culture, respectively. Considering only linkages that appeared in the consensus network for each culture (exceeding the determined frequency cutoff of ≥ 60%), the resulting Cornell D2 and KB-1® consensus networks maintained 1,105 nodes (genes or conditions) with 974 edges and 1,714 nodes with 1,455 edges, respectively. These consensus networks captured multiple strong and biologically informative relationships. One of the main highlighted relationships shared between these two cultures was a direct edge between the transcript encoding for the major reductive dehalogenase (tceA (D2) or vcrA (KB-1®)) and the transcript for the putative S-layer cell wall protein (DET1407 (D2) or KB1_1396 (KB-1®)). Additionally, transcripts for two key oxidoreductases (a [Ni Fe] hydrogenase, Hup, and a protein with similarity to a formate dehydrogenase, "Fdh") were strongly linked, generalizing a strong relationship noted previously for Dehalococcoides mccartyi strain 195 to multiple strains of Dehalococcoides. Notably, the pangenome array utilized when monitoring the KB-1® culture was capable of resolving signals from multiple strains, and the network inference engine was able to reconstruct gene networks in the distinct strain populations.

Mechanism responsible for the antitumor effect of BCG-CWS using the LEEL method in a mouse bladder cancer model.

We previously reported on the development of a water soluble formulation of the cell wall skeleton of BCG (BCG-CWS), a major immune active center of BCG, by encapsulating it into a nanoparticle (CWS-NP). The CWS-NP allowed us to clarify the machinery associated with the BCG mediated anti-bladder tumor effect, especially the roles of bladder cancer cells and dendritic cells (DCs) in the initial step, which remains poorly understood. We show herein that the internalization of BCG-CWS by bladder cancer cells, but not DCs, is indispensable for the induction of an antitumor effect against bladder cancer. Tumor growth was significantly inhibited in mice that had been inoculated with mouse bladder cancer (MBT-2) cells containing internalized BCG-CWS. On the other hand, the internalization of BCG-CWS by DCs had only a minor effect on inducing an antitumor effect against MBT-2 tumors. This was clarified for the first time by using the CWS-NP. This finding provides insights into our understanding of the role of bladder cancer cells and DCs in BCG therapy against bladder cancer.