Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies.

BACKGROUND/AIM: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit karana ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells. MATERIALS AND METHODS: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma. RESULTS: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size. CONCLUSION: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

Post-transplant cyclophosphamide limits reactive donor T cells and delays the development of graft-versus-host disease in a humanized mouse model.

Graft-versus-host disease (GVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) that develops when donor T cells in the graft become reactive against the host. Post-transplant cyclophosphamide (PTCy) is increasingly used in mismatched allo-HSCT, but how PTCy impacts donor T cells and reduces GVHD is unclear. This study aimed to determine the effect of PTCy on reactive human donor T cells and GVHD development in a preclinical humanized mouse model. Immunodeficient NOD-scid-IL2Rγnull mice were injected intraperitoneally (i.p.) with 20 × 106 human peripheral blood mononuclear cells stained with carboxyfluorescein succinimidyl ester (CFSE) (day 0). Mice were subsequently injected (i.p.) with PTCy (33 mg kg-1 ) (PTCy-mice) or saline (saline-mice) (days 3 and 4). Mice were assessed for T-cell depletion on day 6 and monitored for GVHD for up to 10 weeks. Flow cytometric analysis of livers at day 6 revealed lower proportions of reactive (CFSElow ) human (h) CD3+ T cells in PTCy-mice compared with saline-mice. Over 10 weeks, PTCy-mice showed reduced weight loss and clinical GVHD, with prolonged survival and reduced histological liver GVHD compared with saline-mice. PTCy-mice also demonstrated increased splenic hCD4+ :hCD8+ T-cell ratios and reduced splenic Tregs (hCD4+  hCD25+  hCD127lo ) compared with saline-mice. This study demonstrates that PTCy reduces GVHD in a preclinical humanized mouse model. This corresponded to depletion of reactive human donor T cells, but fewer human Tregs.

Treatment with Subcritical Water-Hydrolyzed Citrus Pectin Ameliorated Cyclophosphamide-Induced Immunosuppression and Modulated Gut Microbiota Composition in ICR Mice.

Subcritical water can effectively hydrolyze pectin into smaller molecules while still maintaining its functional regions. Pectic heteropolysaccharide can mediate immune regulation; however, the possible effects of subcritical water-hydrolyzed citrus pectin (SCP) on the immune response remain unclear. Therefore, the effects of SCP on immunomodulatory functions and intestinal microbial dysbiosis were investigated using a cyclophosphamide-induced immunosuppressed mouse model. In this research, immunosuppressed ICR mice were administrated with SCP at dosages of 300/600/1200 mg/kg.bw by oral gavage, and body weight, immune organ indexes, cytokines, and gut microbiota were determined. The results showed that subcritical water treatment decreased the molecular mass and increased the content of galacturonic acid in citrus pectin hydrolysates. Meanwhile, the treatment with SCP improved immunoregulatory functional properties and bioactivities over the original citrus pectin. For example, SCP protected immune organs (accelerated recovery of immune organ indexes) and significantly enhanced the expression of immune-related cytokines (IL-2, IL-6, IFN-γ, and TNF-α). The results of the 16S rDNA sequencing analysis on an IlluminaMiSeq platform showed that SCP normalized Cy-induced gut dysbiosis. SCP ameliorated Cy-dependent changes in the relative abundance of several taxa, shifting the balance back to normal status (e.g., SCP increased beneficial Muribaculaceae, Ruminococcaceae, Bacteroidaceae, and Prevotellaceae while decreasing pathogenic Brevundimonas and Streptococcus). The results of this study suggest an innovative application of citrus pectin as an immunomodulator.

Silver nanoparticles as a therapeutic agent in experimental cyclosporiasis.

Cyclosporiasis is an emerging worldwide infection caused by an obligate intracellular protozoan parasite, Cyclospora cayetanensis. In immunocompetent patients, it is mainly manifested by self-limited diarrhea, which is persistent and may be fatal in immunocompromised patients. The standard treatment for cyclosporiasis is a combination of two antibiotics, trimethoprim and sulfamethoxazole. Gastrointestinal, haematologic and renal side effects were reported with this combination. Moreover, sulfa allergy, foetal anomalies and recurrence were recorded with no alternative drug treatment option. In this study, silver nanoparticles were chemically synthesized to be evaluated for the first time for their anti-cyclospora effects in both immunocompetent and immunosuppressed experimental mice in comparison to the standard treatment. The effect of silver nanoparticles was assessed through studying stool oocyst load, oocyst viability, ultrastructural changes in oocysts, and estimation of serum gamma interferon. Toxic effect of the therapeutic agents was evaluated by measuring liver enzymes, urea and creatinine in mouse sera. Results showed that silver nanoparticles had promising anti-cyclospora potentials. The animals that received these nanoparticles showed a statistically significant decrease in the oocyst burden and number of viable oocysts in stool and a statistically significant increase in serum gamma interferon in comparison to the corresponding group receiving the standard treatment and to the infected non-treated control group. Scanning electron microscopic examination revealed mutilated oocysts with irregularities, poring and perforations. Biochemical results showed no evidence of toxicity of silver nanoparticles, as the sera of the mice showed a statistically non-significant decrease in liver enzymes in immunocompetent subgroups, and a statistically significant decrease in immunosuppressed subgroups. Furthermore, a statistically non-significant decrease in urea and creatinine was recorded in all subgroups. Thus, silver nanoparticles proved their effectiveness against Cyclospora infection, and this will draw the attention to its use as an alternative to the standard therapy.

Cyclophosphamide Enhances Cancer Antibody Immunotherapy in the Resistant Bone Marrow Niche by Modulating Macrophage FcγR Expression.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated cancer. Many hematologic and solid tumors are resistant to therapeutic antibodies in the bone marrow (BM), but not in the periphery (e.g., spleen). We previously showed that cyclophosphamide (CTX) sensitizes the BM niche to antibody therapeutics. Here, we show that (i) BM resistance was induced not only by the tumor but also by the intrinsic BM microenvironment; (ii) CTX treatment overcame both intrinsic and extrinsic resistance mechanisms by augmenting macrophage activation and phagocytosis, including significant upregulation of activating Fcγ receptors (FcγRIII and FcγRIV) and downregulation of the inhibitory receptor, FcγRIIB; and (iii) CTX synergized with cetuximab (anti-EGFR) and trastuzumab (anti-Her2) in eliminating metastatic breast cancer in the BM of humanized mice. These findings provide insights into the mechanisms by which CTX synergizes with antibody therapeutics in resistant niche-specific organs and its applicability in treating BM-resident tumors.

A high-specificity immunoassay for the therapeutic drug monitoring of cyclophosphamide.

Personalized medicine is pushing forward new diagnostic techniques to aid in controlling drug therapeutic levels and their toxic effects. This study aims to develop a high-throughput screening method for therapeutic drug monitoring (TDM) and occupational exposure of cyclophosphamide (CP), an alkylating agent used as a chemotherapeutic and immunosuppressive drug. In order to achieve this goal, an immunizing hapten that exposes the cyclophosphamide moiety has been designed for the first time. Antibodies produced against this hapten have been used to develop an indirect competitive ELISA for the quantification of CP with high specificity and low cross-reactivity with some metabolites and other anticancer drugs. The assay obtained showed a LOD of 22 ± 6 nM in serum samples, with concentrations much below the blood CP levels of patients treated with the drug. A new tool for the detection and quantification of CP is provided which could be relevant for future pharmacokinetic studies and for therapeutic index improvement.

Neem leaf glycoprotein generates superior tumor specific central memory CD8+ T cells than cyclophosphamide that averts post-surgery solid sarcoma recurrence.

The success of cancer vaccines is limited as most of them induce corrupted CD8+ T cell memory populations. We reported earlier that a natural immunomodulator, neem leaf glycoprotein (NLGP), therapeutically restricts tumor growth in a CD8+ T cell-dependent manner. Here, our objective is to study whether memory CD8+ T cell population is generated in sarcoma hosts after therapeutic NLGP treatment and their role in prevention of post-surgery tumor recurrence, in comparison to the immunostimulatory metronomic cyclophosphamide (CTX) treatment. We found that therapeutic NLGP and CTX treatment generates central memory CD8+ T (TCM) cells with characteristic CD44+CD62LhighCCR7highIL-2high phenotypes. But these TCM cells are functionally impaired to prevent re-appearance of tumors along with compromised proliferative, IL-2 secretive and cytotoxic status. This might be due to the presence of tumor load, even a small one in the host, which serves as a persistent source of tumor antigens thereby corrupting the TCM cells so generated. Surgical removal of the persisting tumors from the host restored the functional characteristics of memory CD8+ T cells, preventing tumor recurrence after surgery till end of the experiment. Moreover, we observed that generation of superior TCM cells in NLGP treated surgically removed tumor hosts is related to the activation of Wnt signalling in memory CD8+ T cells with concomitant inhibition of GSK-3ß and stabilisation of ß-catenin, which ultimately activates transcription of Wnt target genes, like, eomesodermin, a signature molecule of CD8+ TCM cells.

Phase I clinical trial of a five-peptide cancer vaccine combined with cyclophosphamide in advanced solid tumors.

We designed a phase I trial to investigate the safety, immune responses and clinical benefits of a five-peptide cancer vaccine in combination with chemotherapy. Study subjects were patients positive for HLA-A2402 with locally advanced, metastatic, and/or recurrent gastrointestinal, lung or cervical cancer. Eighteen patients including nine cases of colorectal cancer were treated with escalating doses of cyclophosphamide 4days before vaccination. Five HLA-A2402-restricted, tumor-associated antigen (TAA) epitope peptides from KOC1, TTK, URLC10, DEPDC1 and MPHOSPH1 were injected weekly for 4weeks. Treatment was well tolerated without any adverse events above grade 3. Analysis of peripheral blood lymphocytes showed that the number of regulatory T cells dropped from baseline after administration of cyclophosphamide and confirmed that TAA-specific T cell responses were associated significantly with longer overall survival. This phase I clinical trial demonstrated safety and promising immune responses that correlated with vaccine-induced T-cell responses. Therefore, this approach warrants further clinical studies.

Extended Tumor Control after Dendritic Cell Vaccination with Low-Dose Cyclophosphamide as Adjuvant Treatment in Patients with Malignant Pleural Mesothelioma.

RATIONALE: We demonstrated previously that autologous tumor lysate-pulsed dendritic cell-based immunotherapy in patients with malignant pleural mesothelioma is feasible, well-tolerated, and capable of inducing immunologic responses against tumor cells. In our murine model, we found that reduction of regulatory T cells with metronomic cyclophosphamide increased the efficacy of immunotherapy. OBJECTIVES: To assess the decrease in number of peripheral blood regulatory T cells during combination therapy of low-dose cyclophosphamide and dendritic cell immunotherapy and determine the induction of immunologic responses with this treatment in patients with mesothelioma. METHODS: Ten patients with malignant pleural mesothelioma received metronomic cyclophosphamide and dendritic cell-based immunotherapy. During the treatment, peripheral blood mononuclear cells were analyzed for regulatory T cells and immunologic responses. MEASUREMENTS AND MAIN RESULTS: Administration of dendritic cells pulsed with autologous tumor lysate combined with cyclophosphamide in patients with mesothelioma was safe, the only side effect being moderate fever. Dendritic cell vaccination combined with cyclophosphamide resulted in radiographic disease control in 8 of the 10 patients. Overall survival was promising, with 7 out of 10 patients having a survival of greater than or equal to 24 months and two patients still alive after 50 and 66 months. Low-dose cyclophosphamide reduced the percentage of regulatory T cells of total CD4 cells in peripheral blood from 9.43 (range, 4.34-26.10) to 4.51 (range, 0.27-10.30) after 7 days of cyclophosphamide treatment (P = 0.02). CONCLUSIONS: Consolidation therapy with autologous tumor lysate-pulsed dendritic cell-based therapy and simultaneously reducing the tumor-induced immune suppression is well-tolerated and shows signs of clinical activity in patients with mesothelioma. Clinical trial registered with www.clinicaltrials.gov (NCT 01241682).

Chronodependent effect of interleukin-2 on mouse spleen cells in the model of cyclophosphamide immunosuppression.

We studied the chronodependent effect of IL-2 in the experimental model of immunodeficiency, cyclophosphamide-induced immunosuppression in mice. IL-2 in a dose of 100 U/ mouse was administered at 10.00 and 16.00 for 3 days after injection of cyclophosphamide. In contrast to the morning treatment with the cytokine, evening administration produced antiapoptotic effect on splenocytes and stimulated proliferation to a greater extent. This was accompanied by an increase in the number of CD4(+), CD25(+) and CD4(+)25(+) cells in the spleen to a level of intact mice. More pronounced effect of the evening mode of IL-2 administration on the proliferation and subpopulation composition of mouse spleen cells in the studied model can be associated with high blood level of CD25(+) cells at this time of the day.

The design of propolis flavone microemulsion and its effect on enhancing the immunity and antioxidant activity in mice.

The objective of the present study was to formulate a microemulsion system for improving the activity of propolis flavone (PF). Pseudo-ternary phase diagrams were constructed to evaluate the existence area of PF microemulsion (PFM). The formulation was characterized by particle size, zeta potential, morphology and stability. The results showed that the optimal PFM formulation consists of 5.3% ethyl acetate, 14% RH-40, 7% ethanol and 73.7% water (w/w), with a solubility of PF up to 3.0 mg mL(-1). The immune-enhancing and antioxidant activity of PFM in vitro and in vivo were performed. The results showed that PFM could significantly promote the splenocyte proliferation and the secretion of IL-2 and IFN-γ in vitro. In vivo, PFM at high and medium doses was able to significantly increase the thymus and spleen indices, enhance splenocyte activity and improve the contents of IgG and IgM in serum, it could also improve the antioxidant activity, significantly increase the levels of superoxidase dismutase and glutathione peroxidase, and decrease the malondialdehyde levels compared with PF. These results indicated that microemulsion could be used as an effective formulation for enhancing the activity of PF. Therefore, microemulsion would be expected to exploit into a new-type preparation of PF.

Chemical composition and antioxidant activities in immumosuppressed mice of polysaccharides isolated from Mosla chinensis Maxim cv. jiangxiangru.

Polysaccharide MP was isolated from Mosla chinensis Maxim cv. jiangxiangru. It was composed of rhamnose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 5.364:12.260:3.448:12.260:32.567:30.651, with 11.00%±0.24% uronic acid and 9.046%±0.04% protein. Its antioxidant activity on the cyclophosphamide-induced immunosuppressed mice was investigated. The spleen and the thymus indices were investigated, and the biochemical parameters were evaluated in three organs (liver, heart and kidney). MP was able to overcome the cyclophosphamide-induced immunosuppression and can significantly raise the T-AOC, CAT, SOD and GSH-PX level. It also raised the spleen and thymus indices and decreased the MDA level in mice. MP could play an important role during the prevention process of oxidative damage in immunological system.

T-cell-replete HLA-haploidentical hematopoietic transplantation for hematologic malignancies using post-transplantation cyclophosphamide results in outcomes equivalent to those of contemporaneous HLA-matched related and unrelated donor transplantation.

PURPOSE: T-cell-replete grafts from haploidentical donors using post-transplantation cyclophosphamide may represent a solution for patients who require allogeneic hematopoietic cell transplantation (alloHCT) but lack a conventional donor. We compared outcomes of alloHCT using haploidentical donors with those of transplantation using conventional HLA-matched sibling donors (MRDs) and HLA-matched unrelated donors (MUDs). PATIENTS AND METHODS: Outcomes of 271 consecutive patients undergoing T-cell-replete first alloHCT for hematologic malignancies performed contemporaneously at a single center (53 using haploidentical donors; 117, MRDs; 101, MUDs) were compared. Overall and disease-free survival (DFS) were adjusted for effects of significant patient-, disease-, and transplantation-related covariates using a stratified Cox model. RESULTS: Patient characteristics were similar between the three donor groups. For patients undergoing MRD, MUD, and haploidentical transplantation, 24-month cumulative incidences of nonrelapse mortality were 13%, 16%, and 7% and of relapse were 34%, 34%, and 33%, respectively (P not significant [NS]). Cumulative incidences of grades 3 to 4 acute graft-versus-host disease (GVHD) at 6 months were 8%, 11%, and 11%, respectively (P NS); extensive chronic GVHD occurred in 54%, 54%, and 38% of patients, respectively (P < .05 for those undergoing haploidentical donor v MRD or MUD transplantation). Adjusted 24-month probabilities of survival were 76%, 67%, and 64% and of DFS were 53%, 52%, and 60%, respectively; these were not significantly different among the three donor groups. CONCLUSION: Haploidentical transplantation performed using T-cell-replete grafts and post-transplantation cyclophosphamide achieves outcomes equivalent to those of contemporaneous transplantation performed using MRDs and MUDs. Such transplantation represents a valid alternative for patients who lack a conventional donor.

Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?

Chemotherapy can cause immunosuppression, which may trigger latent intestinal parasitic infections in stools to emerge. This study investigated whether intestinal parasites can emerge as opportunistic infections in breast and colorectal cancer patients (n=46 and n=15, respectively) undergoing chemotherapy treatment. Breast cancer patients were receiving a 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen (6 chemotherapy cycles), and colorectal cancer patients were receiving either an oxaliplatin/5-fluorouracil/folinic acid (FOLFOX) regimen (12 cycles) or a 5-fluorouracil/folinic acid (Mayo) regimen (6 cycles). Patients had Blastocystis hominis and microsporidia infections that were only present during the intermediate chemotherapy cycles. Thus, cancer patients undergoing chemotherapy should be screened repeatedly for intestinal parasites, namely B. hominis and microsporidia, as they may reduce the efficacy of chemotherapy treatments.