Evaluation of the acute and 28-day sub-acute intravenous toxicity of α-l-guluronic acid (ALG; G2013) in mice.

α-l-Guluronic acid (ALG; G2013) has been previously introduced as a new anti-inflammatory agent with promising therapeutic effects. Thus, in the present study, we aimed to evaluate the acute and sub-acute toxicity of ALG through intravenous (i.v.) administration in Balb/C mice. ALG was administrated i.v. to the mice with doses of 300, 600, and 1000 mg/kg of body weight to investigate acute toxicity (single dose) and with doses of 25, 50, and 100 mg/kg of body weight to sub-acute toxicity study (daily injections for a period of 28 days). The mortality rate, food and water intake, behavior, body weight, gross necropsy, hematological and biochemical parameters as well as histopathological presentations of the vital organs (kidneys, liver, lungs, spleen, and heart) were examined in treated groups and compared to the healthy controls. The results of both acute and sub-acute studies showed that i.v. administrations of ALG did not affect the investigated parameters in both sexes, indicating that the LD50 of ALG was higher than 1000 mg/kg of body weight. As no difference was observed in toxicity profiles of investigated doses, no-observed-adverse-effect-level for i.v. administration of ALG in the sub-acute study was greater than 100 mg/kg body weight in both female and male mice. According to the finding, i.v. administration of ALG did not lead to any clinical sign in abovementioned doses, suggesting that ALG was well tolerated up to 1000 mg/kg. These pre-clinical findings support the application of ALG in the future clinical trials.

Protective and anti-inflammatory effect of selenium nano-particles against bleomycin-induced pulmonary injury in male rats.

Pulmonary fibrosis (PF) is an interstitial lung disease, in which the exact pathologic mechanisms are not fully understood. Drug trials for the treatment of PF have shown disappointing results and controversial. Recently, selenium nanoparticles (SeNPs) have received great attention for potential use in treatments, due to high bioactivity features and lower toxicity. This study evaluated the protective effect of SeNPs against pulmonary injury induced by bleomycin (single dose, 4 mg/kg, intratracheal) in male rats in early and late phases of the disease. The rats were treated with SeNPs by intraperitoneal injection (0.5 mg SeNP/kg) for five consecutive days in the early phase (a day after injection of bleomycin) and late phase (a week after injection of bleomycin). The results showed that injection of SeNPs in the early phase improved the degree of alveolitis and inflammation and lung structure damage. Also, led to significant decreases in density of transforming growth factor- ß1 (TGF-ß1) in the lung and tumor necrosis factor-α (TNF-α) levels in the serum and lung homogenates compared with bleomycin-administrated group. Notably, treatment with the SeNP during the late phase did not show any ameliorative effects. Thus, the data suggest that SeNP has a protective effect against bleomycin-induced pulmonary injury in rats in the early phase of the disease. This might mean that SeNPs may be a new therapeutic agent for the improvement of this disease in the early phases.

Gold nanoparticles and polyethylene glycol alleviate clinical symptoms and alter cytokine secretion in a mouse model of experimental autoimmune encephalomyelitis.

Gold nanoparticles (GNPs) are attractive nanoparticles with unique electronic and optical properties in the nanotechnology field and are widely used in various biomedical fields. Studies have shown that these particles also exhibit antioxidant and anti-inflammatory properties. On the other hand, polyethylene glycol (PEG) that used to stabilize GNPs also exhibits antioxidant and anti-inflammatory properties due to their membrane resealing properties. The aim of this study was to evaluate the ameliorative effect of GNPs and PEG in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). EAE was induced in female C57BL/6 mice with injection of an emulsion of myelin oligodendrocyte glycoprotein (MOG35-55) peptide and Freund's adjuvant. GNPs measuring 25 nm were prepared, and their size was determined using transmission electron microscopy (TEM), then intraperitoneal injection of GNPs and PEG (MW 1500; 30% w/v) was initiated after immunization and continued until the day 27 postimmunization (13 injections in total). The EAE clinical scores and body weights were evaluated. We analyzed cental nervous system's cell infiltration and demyelinated lesions using hematoxylin and eosin and luxol fast blue staining, respectively. Also, interleukin-23 and interleukin-27 were examined using the ELISA technique. The severity of MS symptoms was significantly decreased in the treated groups with GNPs and PEG. Histological examination of the spinal cord showed that the number and severity of cells' infiltration and demyelinated lesions decreased significantly, and also the cytokine levels of IL-23 and IL-27 altered in treated groups. These results show that GNPs and PEG ameliorate the clinical course of EAE in mice. Our findings demonstrate proof of principle for potential of GNPs and PEG as novel agents for therapeutic approaches in the alleviated clinical symptoms of MS. © 2019 IUBMB Life, 71(9):1313-1321, 2019.

Effects of Chlorella vulgaris on tumor growth in mammary tumor-bearing Balb/c mice: discussing association of an immune-suppressed protumor microenvironment with serum IFNγ and IgG decrease and spleen IgG potentiation.

PURPOSE: Chlorella vulgaris (CV) has exhibited immune-enhancing and protective activities against cancer and infections. However, there is an increasing concern about the use of Chlorella species in human, regarding its various molecules with antigenic features found in infectious microorganisms. Our goal was to investigate the impact of higher concentrations of CV on tumor growth in spontaneous mouse mammary tumor (SMMT) models. METHODS: Balb/c mice were daily given CV powder at doses of 0, 200, or 300 mg/kg for 42 days (CONTROL, CV200, and CV300 groups, respectively; n = 6/group). On day 14, the SMMT was inoculated. Tumor volume (TV) and body weight (BW) were monitored on 5-day intervals following tumor challenge. On day 43, blood, spleen, lungs, and tumor tissues were collected. Histopathological examinations on lungs and tumor tissues were performed following hematoxylin-eosin staining. Intratumor expression of 27 genes was assessed by real-time PCR. Total IgG, IFNγ, and IL-4 levels in serum and spleen culture supernatant were measured by ELISA. RESULTS: The TV/BW index showed significant increase in the CV200 group compared to the CONTROL (p = 0.047). The CV200 tumors exhibited more malignant phenotype, higher angiogenesis rate, and lower peritumoral neutrophil and macrophage-to-lymphocyte infiltration ratio compared to the CONTROL. Serum concentrations of IFNγ, IL-4, and IgG were declined, and the spleen IFNγ and IgG production was higher in the CV200 compared to the CONTROL. The IL-1ß, IL-10, TGFß1, FOXP3, HO-1, Gr1, CD11b, PCNA, LCN2, iNOS2, VEGFR2, CD31, and CD105L expressions were markedly increased in the CV200 tumors compared to the CONTROL (p = 0.001, 0.002, 0.006, 0.021, 0.004, 0.030, 0.016, 0.031, 0.025, 0.008, 0.014, 0.022, and 0.037, respectively). The changes in cytokine, IgG and gene expression values considerably correlated with tumor size, as well as with each other. CONCLUSIONS: Our data provided evidence that C. vulgaris at a specific dose (200 mg/kg) promoted tumor growth in a mammary tumor model. This consequence might reflect an immune derangement in favor of developing a protumor microenvironment. However, this hypothesis needs to be further investigated in future.

Reduction toxicity of Amphotericin B through loading into a novel nanoformulation of anionic linear globular dendrimer for improve treatment of leishmania major.

Amphotericin B (A) as an antileishmanial drug has limited clinical application owing to severe side-effects and low-water solubility. This is the first study reported using Anionic Linear Globular Dendrimer (ALGD) as A carrier for the increase of A solubility rate, decrease its toxicity, and improve its therapeutic effects. ALGD was synthesized and A was loaded into nanoparticles for the first time with the drug-loading efficiency of 82%. Drug loading was confirmed using characterization methods. The drug solubility rate was increased by 478-folds. The results of the study showed that the A toxicity was significantly decreased by 95% in vitro and in vivo environments, which was confirmed by pathology findings and enzymatic evaluation. Furthermore, the nanodrug caused that mortality rate was reached to zero. Moreover, the nanodrug was as potent as the free drug and glucantime (GUL) in reducing the parasite burden and parasite number. These findings indicated the potency of ALGD to decrease the drug side-effects, increase the drug solubility rate, and improve the drug efficacy. Moreover, the nanoformulation was a non-toxic and cost-effective formulation. The conformity between in vitro and in vivo results suggested that the A-loaded ALGD could be considered as a promising candidate in reducing the side-effects of A in leishmaniasis treatment.

Diagnostic investigations of canine prostatitis incidence together with benign prostate hyperplasia, prostate malignancies, and biochemical recurrence in high-risk prostate cancer as a model for human study.

The aim of this study was to evaluate the prevalence of acute and chronic inflammation, benign prostatic hyperplasia (BPH), and cancer of the prostate glands in the canine as a human model in prostate disorders. The study was carried out on 12 cases of different male dogs of terrier (50%), German shepherd (25%) breeds, and Greden (25%), and the age of the dogs ranged from 6 to 13 years (average age 7.8 ± 3.6). The bodyweight ranged from 3.6 to 7.9 kg. Signalment, clinical signs, and diagnostic tools such as ultrasonography, urinary cytology, and histopathology are presented. Dysuria was the most common clinical sign in this study and occurred in 10/12 canine (83.3%) included. Other clinical signs included lameness (5/12 canine, 41.6%) and constipation (3/12 canine, 25%). The range of duration of clinical signs was 5 days to 7 months. Moreover, in the present study, the urinary biochemical markers of different prostate lesions include blood, protein, and glucose and were detected in 11/12 cases (91.6%), 5/12 cases (41.6%), and 2/12 cases (16.6%), respectively. Taken together, sonographic data were classified into four groups based on histological diagnosis. In 7/12 cases (58.4%), the prostate appeared to have BPH lesions, and the remaining lesions included inflammation (3/12 cases, 25%), abscess (1 case, 8.3%), and adenocarcinoma (1 case, 8/3%) on ultrasound. In all cases, prostate tissue had an irregular echotexture. None of the dogs had sonographic evidence of sublumbar lymph node enlargement. Histopathologically, we looked at the prevalence of inflammation (33.3% chronic and 8.3% acute) and BPH (58.4%) in dogs of different ages and breeds, and also, we observed chronic inflammation in >20% of dogs, which was about 25% in 3 cases of the 12 cases referred. More chronic inflammation was associated with more BPH. The majority of the asymptomatic inflammation that is detected in the prostate is classified as chronic inflammation (i.e., as evidenced by the presence of monocytic and/or lymphoplasmacytic inflammatory cell infiltrates); however, acute inflammation is also observed to a lesser degree. Acute inflammation, as is typically evidenced by the infiltration of neutrophils, is classically an indicator of an infectious process. Finally, the patients included seven castrated, four castrated together with antibiotic therapy, and one castrated together with chemotherapy intact male dogs, which were treated with the mentioned cases. In conclusion, chronic prostatic inflammation could be a central mechanism in BPH progression, but the pathological features of tissue inflammation were different between BPH and prostate cancer (PCa). Nevertheless, the histological examination of prostate biopsies remains the only way to diagnose prostatic disorders.

Clinico-histopathologic and outcome features of cutaneous infundibular keratinizing acanthoma: a case report and literature review.

The infundibular keratinizing acanthoma (IKA) is a rare epithelial benign keratin-containing neoplasm of hair follicles. The purpose of this study was to evaluate the defining histopathologic architecture of IKA. A typical IKA consisted of a keratin-filled crypt in the dermis and subcutis that opened to the skin surface. Most of this tumor occurred on the back, neck, head, and the shoulders. Microscopically, the dermal nodules were focally contiguous in both the dermis and subcutis. Furthermore, most histological lesions are consistent with a simple or multiloculated cyst filled with keratin and lined by a wall of stratified squamous epithelium; keratin appears as a concentric lamellar mass, with a keratotic pearly aspect. Histological examination of the cutaneous lesions revealed that the growths were comprised of IKA. IKA of man and dog were compared, and it was concluded that although they are similar in many respects, they are not identical entities. To the best of our knowledge, this is the first report on the prevalence of IKAs among the population of owned dogs in Iran.

Immunoregulatory Effect of Calcitriol on Experimental Autoimmune Encephalomyelitis (EAE) Mice.

Previous studies noted an imbalance in T helper (Th) 17 and regulatory T cells (Tregs) in experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis animal model. calcitriol, vitamin D's active form, was found to ameliorate EAE symptoms by favoring Tregss over Th17 cells, suggesting immunomodulatory effects. This study aimed to assess calcitriol's impact on EAE manifestations and cytokine profile in mice. In this study, we recruited twenty-eight C57BL/6 mice and divided them into 4 groups: healthy controls, EAE, EAE with calcitriol treatment, and healthy mice with calcitriol treatment. CD4+ T cells were isolated from splenocytes using magnetic-activated cell sorting. Real-time polymerase chain reaction was employed to quantify the genes associated with Th9 cells (i.e., SPI1 encoding PU.1 and IL9 encoding interleukin [IL]-9). Moreover, the levels of IL-17 and transforming growth factor beta (TGF-ß) were evaluated through enzyme-linked immunosorbent assay in the supernatant of CD4+ T cell culture stimulated by anti-CD3 and anti-CD28 antibodies for 72 hours. In the supernatant of CD4+ T cell cultures, the levels of interleukin-17 (IL-17) were significantly increased, while the levels of transforming growth factor beta (TGF-ß) were decreased in the EAE Group compared to the healthy control group. Calcitriol treatment reversed these changes and attenuated EAE symptoms, as confirmed in hematoxylin and eosin, and luxol fast blue stains. Notably, calcitriol increased IL9 gene expression in both EAE and healthy mice.  This study provides further evidence of the anti-inflammatory effects of calcitriol and its role in attenuating EAE.

Mouse Model of Irritant Contact Dermatitis.

Background: Irritant contact dermatitis is a common inflammatory skin disease characterized by skin barrier dysfunction, eczematous dermatitis, and chronic itching. This disease severely affects the quality of life. Considering that the current treatment approaches are not ideal, more extensive research is needed to develop new treatments. Mainly, a mouse model is needed to investigate the effectiveness of new drugs to treat this disease. Objectives: This study was conducted to create a mouse model of irritant contact dermatitis. Methods: In the current study, we used BALB/c female mice to prepare a mouse model of irritant contact dermatitis. To induce irritant contact dermatitis, we used a dinitrochlorobenzene mixture with acetone/olive oil as an irritant. After 10 days of application, the mouse skin tissue was isolated and examined in terms of histopathology. Results: The introduced protocol created an irritant contact dermatitis model clinically and histopathologically. Conclusions: In the present study, we introduced a new protocol using a mixture of dinitrochlorobenzene and acetone/olive oil to create an irritant contact dermatitis model. Mouse models have been extensively used to discover the complex mechanisms of irritant contact dermatitis and provide a preclinical platform before conducting clinical interventional research on humans to evaluate a new therapeutic approach. However, one should always look for models that cause the least pain and suffering in the animal and simultaneously are simple and reliable for the desired studies. Thus, our protocol is a new approach that can be effective and painless in creating a model of irritant contact dermatitis.

Dendritic cell immunotherapy with miR-155 enriched tumor-derived exosome suppressed cancer growth and induced antitumor immune responses in murine model of colorectal cancer induced by CT26 cell line.

Nowadays, various strategies are considered to prime Dendritic cells (DCs) with tumor antigens. The tumor cell-derived exosomes are recognized as one of the most efficient strategies for achieving this purpose. In this regard, MicroRNA 155 (miR-155) is employed as one of the most prominent miRNAs, which play substantial roles in DCs maturation and IL-12 production. This study investigates the tumor growth suppression and antitumor effects of DCs primed with miR-155-enriched exosome on the BALB/c murine model of colorectal cancer induced by CT-26 cell lines. Therefore, a holistic framework is proposed for the analysis procedure. In the first stage, miRNA-155 was electroporated into texosomes. In the second stage, bonemarrow-derived DCs were treated with miRNA-155 enriched texosomes. Then, antitumor properties of manipulated DC have been evaluated in the BALB/c mice model of colorectal cancer. After DC immunotherapy, several features have been assessed for each animal, including survival, body weight, tumor volume/size, histopathology, and serum cytokine levels. Also, flow cytometric evaluation has been performed for the spleen and the tumor tissue T-cell subsets. The findings demonstrated that the primed DCs could significantly increase IL-12p70 and IFN-γ in serum and accelerate the differentiation, proliferation, and cytotoxicity effects on the Th and CTL cells. Also, the treatment also increased the infiltration of Th and CTL cells into the tumor microenvironment while decreasing Tregs. This situation causes tumor growth control, and survival improvement. Therefore, DC immunotherapywith miR-155-enriched texosomes can be employed as a the desired approach for inducing antitumor immune responses, controlling tumor growth, and improving survival in mice with colorectal cancer. However, it is essential to perform more investigations to confirm the clinical application of this approach in humans and other types of tumors.

Oral chitin treatment improved demyelination in murine autoimmune encephalomyelitis model by inhibition of inflammatory responses.

This study aimed to determine whether chitin microparticles (CMP), glucosamine-based polymers, have an anti-inflammatory response in a murine model of autoimmune encephalomyelitis. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunization with myelin antigens emulsified in complete Freund adjuvant. A standard clinical and histological method (Luxol Fast Blue staining) was used to validate the model and document the impact of CMP treatment. ELISA was used to determine the production of spleen cell cytokines and serum levels of anti-chitin antibodies. Flowcytometry was used to determine the percentage of regulatory lymphocytes. The relative expression of the breast regression protein 39 (BRP-39) gene was examined through real time-PCR amplification. Clinical signs were significantly improved in mice given CMP compared with untreated mice. Histological analysis of the spinal cord revealed that treatment significantly reduced demyelination. The levels of interferon-γ, interleukin-17, and tumor necrosis factor-α were also reduced; conversely, no significant change was detected in interleukin-10 level and regulatory T cell count. The CMP-fed mice showed lower BRP-39 expression compared with the control group. It was ultimately determined that CMP modulates immune responses which could indirectly alter the pathology of an injured central nervous system. The data suggests that CMP may be used as an effective and cheap oral therapeutic agent for multiple sclerosis.

Immunomodulatory Effects of Calcitriol through DNA Methylation Alteration of FOXP3 in the CD4+ T Cells of Mice.

Vitamin D plays a variety of physiological functions, such as regulating mineral homeostasis. More recently, it has emerged as an immunomodulator player, affecting several types of immune cells, such as regulatory T (Treg) cells. It has been reported that vitamin D exerts some mediatory effects through an epigenetic mechanism. In this study, the impacts of calcitriol, the active form of vitamin D, on the methylation of the conserved non-coding sequence 2 (CNS2) region of the forkhead box P3 (Foxp3) gene promoter, were evaluated. Fourteen C57BL/6 mice were recruited in this study and divided into two intervention and control groups. The CD4+ T cells were isolated from mice splenocytes. The expression of Foxp3, IL-10, and transforming growth factor-beta (TGF-ß1) genes were relatively quantified by real-time PCR technique, and the DNA methylation percentage of every CpG site in the CNS2 region was measured individually by bisulfite-sequencing PCR. Vitamin D Intervention significantly (p<0.05) could increase the expression of Foxp3, IL-10, and TGF-ß1 gene in the CD4+ T cells of mice comparing with the control group. Meanwhile, methylation of the CNS2 region of Foxp3 promoter was significantly decreased in three of ten CpG sites in the vitamin D group compared to the control group. The results of this study showed that vitamin D can engage the methylation process to induce Foxp3 gene expression and probably Treg cytokines profile. Further researches are needed to discover the precise epigenetic mechanisms by which vitamin D modulates the immune system.

Chitin Micro Particles Regulate Splenocytes Immune Response in Experimental Autoimmune Encephalomyelitis.

Contrasting studies are reported on the induction of IL-10 and IFN-γ via chitin microparticles (CMPs) during immune stimulation. Our previous studies have shown marked protection among CMP treated Leishmania-infected mice via regulated IL-10/IFN-γ response, at the present study, once more, examined the inconsistent responses regarding the immunologic response of CMPS. To verify whether CMPs could indeed up-regulate IL-10/IFN-γ axis, isolated spleen cells from the myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (EAE) mice were cultured in the presence of MOG peptide and/or CMPs. The effects of CMPs on IL-10, IFN-γ and IL-17 production were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Moreover, GATA binding protein 3 (Gata3), T-box transcription factor TBX21 (Tbx21), and RAR-related orphan receptor gamma (RORγT) expressions (real-time PCR) were investigated. MOG alone stimulated the production of IFN-γ (p≤0.004) but not, IL-10 (p≤0.140). MOG/chitin stimulation resulted in a significant increase in IFN-γ and IL-10 levels, respectively; (p≤0.004 and p≤0.003) rather than MOG. Additionally, the expression of Tbx21 (p≤0.001), but not Gata3 (p≤0.08), was increased in the MOG/chitin-treated spleen cells. All in all, CMP supports Gata3 independent IL-10 production and promotes Tbx21 dependent IFN-γ induction. These results, alongside our previous data, indicate that CMPs has particular adjuvant effects.

Sublingual Immunotherapy with Sal k1 Expressing Lactococcus lactis Down-regulates Th2 Immune Responses in Balb/c Mice.

Sublingual immunotherapy (SLIT) has been introduced as a noninvasive and safer approach for allergen-specific immunotherapies. In this study we investigated the efficacy of oral immunotherapy with recombinant Salsola kali 1 protein (Sal k 1) on Th1/Th2 balance in a mouse model of allergy. Female Balb/c mice were intraperitoneally sensitized with rSal k1, followed by a respiratory challenge with 1% (w/v) rSal k1. The sensitized mice were subjected to SLIT using rSal K1 expressing Lactobacillus lactis strain for three weeks. Each week the experimental group underwent SLIT protocol twice. Finally, serum levels of specific immunoglobulins including IgE, IgG1 and IgG2a, as well as secretion of different cytokines from splenocytes including IL-2, IL-4, IL-10, IFNγ and TGFß into culture media were measured by ELISA. Following immunotherapy, the levels of specific IgE and IgG1 in mice sera as well as IL-4 level in supernatant of splenocytes were significantly lower than allergic controls. While serum IgG2a, IgG2a/IgG1 ratio as well as concentration of IL-2, IL-10, IFNγ, and TGFß were higher in the SLIT group compared to the controls. The histopathological examination of intestinal tissues revealed no sign of inflammatory response following SLIT. This study revealed that Th2 immune responses are reduced in allergic mice after feeding them with allergen expressing probiotic bacteria as a SLIT approach. Since the safety of this procedure was previously approved, thus, it seems that a similar protocol using human based probiotics could be applied for Salsola kali sensitive patients.

Anti-inflammatory and anti-tumor effects of α-l-guluronic acid (G2013) on cancer-related inflammation in a murine breast cancer model.

Cancer-related inflammation (CRI) is associated with the malignant progression of several cancer types. Targeting these pathways is a novel promising strategy for cancer prevention and treatment. In this present study, we evaluated the efficacy of ?-l-guluronic acid (ALG), a potent anti-inflammatory agent on breast cancer-related inflammation both in vitro and in vivo conditions. Our results indicated that ALG can effectively inhibit the CRI and tumor-promoting mediators (COX-2, MMP2, MMP9, VEGF and proinflammatory cytokines) without direct toxic effects on the cells. Moreover, it was found that, ALG can effectively inhibit the tumor cell adhesion to extracellular matrix, seeding in implantation tissue, reduce accumulation of immunosuppressive and inflammatory cells in tumor-bearing mice. These findings were associated with decreased tumor growth, metastasis, angiogenesis and prolonged mice survival. In conclusion, our data provide a cellular and molecular justification for the use of nonsteroidal anti-inflammatory drugs (NSAIDs) in treating cancer and imply the potential anti-tumor activity of ALG therapy via inhibition of CRI. These findings could lead to the establishment of novel NSAID-based cancer therapy in the near future and open a new horizon for cancer treatment.

Targeting of crosstalk between tumor and tumor microenvironment by ß-D mannuronic acid (M2000) in murine breast cancer model.

Metastasis is the main cause of death in breast cancer patients. Inflammatory processes following crosstalk between tumor cells and tumor microenvironment play an important role in progression and metastasis of cancer. Hence, targeting of these interactions may represent a novel promising strategy for breast cancer therapy. So, we investigated the effects of ß-D mannuronic acid (BDM), a new antiinflammatory agent, on 4T1 breast cancer cell line both in vitro and in vivo. Proliferation assays revealed low-cytotoxic effect of BDM on 4T1 cells. However, BDM reduced activity of MMP-2, MMP-9 and significantly decreased the adhesion of 4T1 cells to extracellular matrix (ECM) in a dose-dependent manner. The in vivo results demonstrated that BDM strongly inhibits tumor growth and increases lifespan as compared with control mice. The decrease in tumor mass was associated with decreased metastasis, recruitment, and frequency of inflammatory cells in tumor tissue. Our preclinical findings demonstrated that BDM therapy not only prevents formation of chronic inflammatory response but also inhibits crosstalk between tumor cells and their microenvironment, which is associated with reduction of tumor growth and metastasis arrest. Our data imply the use of BDM therapy in future clinical trials to open a new horizon for breast cancer therapy.

Placenta-specific1 (PLAC1) is a potential target for antibody-drug conjugate-based prostate cancer immunotherapy.

Our recent findings strongly support the idea of PLAC1 being as a potential immunotherapeutic target in prostate cancer (PCa). Here, we have generated and evaluated an anti-placenta-specific1 (PLAC1)-based antibody drug conjugate (ADC) for targeted immunotherapy of PCa. Prostate cancer cells express considerable levels of PLAC1. The Anti-PLAC1 clone, 2H12C12, showed high reactivity with recombinant PLAC1 and selectivity recognized PLAC1 in prostate cancer cells but not in LS180 cells, the negative control. PLAC1 binding induced rapid internalization of the antibody within a few minutes which reached to about 50% after 15 min and almost completed within an hour. After SN38 conjugation to antibody, a drug-antibody ratio (DAR) of about 5.5 was achieved without apparent negative effect on antibody affinity to cell surface antigen. The ADC retained intrinsic antibody activity and showed enhanced and selective cytotoxicity with an IC50 of 62 nM which was about 15-fold lower compared to free drug. Anti-PLAC1-ADC induced apoptosis in human primary prostate cancer cells and prostate cell lines. No apparent cytotoxic effect was observed in in vivo animal safety experiments. Our newly developed anti-PLAC1-based ADCs might pave the way for a reliable, efficient, and novel immunotherapeutic modality for patients with PCa.

An experimental model of colitis induced by dextran sulfate sodium from acute progresses to chronicity in C57BL/6: correlation between conditions of mice and the environment.

AIM: To induce acute colitis progresses to chronicity in C57BL/6 mice by dextran sulfate sodium. BACKGROUND: Murine models are essential tools to understand IBD pathogenesis. Among different types of chemically induced colitis models, the dextran sulfate sodium (DSS)-induced colitis model is the most common model of IBD, due to its simplicity. PATIENTS AND METHODS: Male C57BL/6 mice 6-8 weeks old, were collected and matched by age with controls. C57BL/6 mice treated with 2 cycles of 3.5% DSS for 4 days and 4 days of pure water between each cycle. After that, mice were sacrificed and the entire colon was removed. Small sections of the colon were fixed in formaldehyde, embedded in paraffin and sectioned with a microtome. Sections were stained with hematoxylin eosin to analyses the degree of inflammation. RESULTS: After the first cycle oral administration of DSS, mice with severe and visible rectal bleeding and diarrhea entered into the acute phase. After day 4-5, bleeding and diarrhea were improved and mice entered into the chronic phase with peak levels of weight loss. Macroscopically, the inflammation was predominantly located in the distal colon. Microscopically, examination of the distal colon sections showed a decrease number of goblet cells, loss of crypts, signs of surface epithelial regeneration and moderate to severe infiltration of inflammatory cells in the mucosa. CONCLUSION: In order to achieve an experimental colitis model, our protocol is recommended for future therapies in IBD experimental modeling.