Time-dependent changes in the glycolytic pathway in activated T cells are independent of tumor burden or anti-cancer chemotherapy.

Although activated adoptive T cells therapy (ATC) is an effective approach for cancer treatment, it is not clear how modulation of T cell activation impacts their biochemical signature which significantly impacts the cell function. This study is aimed to investigate the impact of polyclonal activation on the metabolic signature of T cells from tumor-bearing mice under different settings of treatment with chemotherapy. Thirty female Swiss albino mice were divided into 5 groups (n = 6/each), Gp1(PBS), groups Gp2 were inoculated intraperitoneal (i.p) with 1 × 106 cells/mouse Ehrlich ascites carcinoma (EAC), Gp3-Gp5 were treated with cisplatin (20 mg/mice) which were represented as EAC/CIS/1wk Or EAC/CIS/2wk 3 times every other day. Splenocytes were cultured in or presence of concanavalin-A (Con-A) and IL-2 for 24 h or 72 h, then cells were harvested, and processed to determine the enzyme activities of hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and glucose 6 phosphate dehydrogenase(G6PD) enzymes. The results showed that before culture, T cells harvested from EAC/PBS/1wk of mice or inoculated with EAC/CIS/1wk showed higher activity in HK, PFK, LDH, and G6PH as compared to naive T cells. After 24, and 72 h of culture and activation, the enzyme activities in T cells harvested from EAC/CIS/2wk mice or EAC/CIS/3wk mice decreased compared with their control. The late stage of the tumor without chemotherapy gives a low glycolic rate. In late activation, naive and early stages of the tumor with chemotherapy can give high glycolic metabolism. These results show great significance as an application of adoptive T-cell therapy.

COVID-19 Severity Shifts the Cytokine Milieu Toward a Proinflammatory State in Egyptian Patients: A Cross-Sectional Study.

Despite extensive research to decipher the immunological basis of coronavirus disease (COVID-19), limited evidence on immunological correlates of COVID-19 severity from MENA region and Egypt was reported. In a single-center cross-sectional study, we have analyzed 25 cytokines that are related to immunopathologic lung injury, cytokine storm, and coagulopathy in plasma samples from 78 hospitalized Egyptian COVID-19 patients in Tanta University Quarantine Hospital and 21 healthy control volunteers between April 2020 and September 2020. The enrolled patients were divided into 4 categories based on disease severity, namely mild, moderate, severe, and critically ill. Interestingly, interleukin (IL)-1-α, IL-2Rα, IL-6, IL-8, IL-18, tumor necrosis factor-alpha (TNF-α), FGF1, CCL2, and CXC10 levels were significantly altered in severe and/or critically ill patients. Moreover, principal component analysis (PCA) demonstrated that severe and critically ill COVID-19 patients cluster based on specific cytokine signatures that distinguish them from mild and moderate COVID-19 patients. Specifically, levels of IL-2Rα, IL-6, IL-10, IL-18, TNF-α, FGF1, and CXCL10 largely contribute to the observed differences between early and late stages of COVID-19 disease. Our PCA showed that the described immunological markers positively correlate with high D-dimer and C-reactive protein levels and inversely correlate with lymphocyte counts in severe and critically ill patients. These data suggest a disordered immune regulation, particularly in severe and critically ill Egyptian COVID-19 patients, manifested as overactivated innate immune and dysregulated T-helper1 responses. Additionally, our study emphasizes the importance of cytokine profiling to identify potentially predictive immunological signatures of COVID-19 disease severity.

Safety and efficacy of the combination of cryotherapy and photodynamic modalities with imiquimod in patients with actinic keratosis: a systematic review and meta-analysis.

INTRODUCTION: Actinic keratosis (AK) is an intraepithelial tumor that, in most cases, arises in chronically sun-exposed areas. The combination of cryotherapy and photodynamic modalities with imiquimod has been proven to be a potential therapeutic option for AKs. However, there is no comprehensive systematic study that discussed this concept in literature taking into consideration both efficacy and safety. EVIDENCE ACQUISITION: We performed a comprehensive search of the literature for studies assessing the efficacy and toxicity of the combinatorial tripartite regimen, consisting of cryotherapy and photodynamic modalities with imiquimod in AK. Metanalysis was performed using comprehensive meta-analysis version 3.0. EVIDENCE SYNTHESIS: After the screening of 1031 studies, five studies were included. Two trials compared the effect of imiquimod/cryotherapy versus cryotherapy alone or versus cryotherapy/vehicle. Our meta-analysis indicated that imiquimod/cryotherapy effectively induces complete clinical clearance in patients with AKs (OR: 6.26; 95%CI: 1.56-24.1; P=0.01). Moreover, another two studies, which were not meta-analyzed, indicated a substantial clinical clearance in the number of AK lesions in the imiquimod plus photodynamic therapy arm as compared to 5% imiquimod or PDT alone. No serious systemic adverse events were reported in all the treatment arms. CONCLUSIONS: Combined PDT or cryotherapy with imiquimod is more effective in the complete recovery of AK than treatment with imiquimod alone.

IL-12 conditioning of peripheral blood mononuclear cells from breast cancer patients promotes the zoledronate-induced expansion of γδ T cells in vitro and enhances their cytotoxic activity and cytokine production.

BACKGROUND: In a series of our preclinical studies, we have reported that conditioning of α/ß CD8+ T cells in vitro with interleukin-12 (IL-12) during their expansion improves their homing phenotype and anti-tumor cytolytic function upon their adoptive transfer in vivo. Vγ9+Vδ2+ T cells can also be expanded in vitro with amino bisphosphonates such as zoledronate (ZOL) for the purpose of adoptive therapy. AIM: We aimed in this study to use IL-12 to enhance the expansion and cytotoxic functions of ZOL-expanded Vγ9+Vδ2+T cells. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were separated from healthy donors and stage II breast cancer patients. PBMCs (1 × 106 cells/mL) were cultured and treated with ZOL/IL2, ZOL/IL2/IL12, or IL2/IL12. Cultured cells were harvested on days 7 and 14 of culture and their numbers, phenotype, and cytolytic activity were assessed. The levels of pro- and inflammatory cytokines/chemokines in the plasma and supernatants of the cultured cells were analyzed by Luminex. RESULTS: In healthy subjects, the addition of IL-12 to ZOL/IL2-stimulated PBMCs increased the expansion and the cytotoxic activity of Vγ9+Vδ2+ T cells on days 7 and 14 of culture. The latter was measured by the expression level of the cytolytic molecules granzyme B (GZB) and perforin (PER). Of note, αß CD8 + T cells were also activated under the same condition but with a lesser extent addition of IL-12 to ZOL/IL2-stimulated PBMCs from cancer patients also induced similar effects but were lower than in control subjects. Interestingly, ZOL/IL2/IL12-treated PBMCs showed higher levels of cytokines/chemokines, in particular, CCL, CCL4, GM-CSF, IL-1rα; IL-12, IL-13, TNF, and IFNγ measured on days 7 and 14. CONCLUSION: The addition of IL12 at the start of the expansion protocol can enhance the activity of γδ T cells which might be mediated in part by the activation of αß T cells.

Beneficial Modulatory Effects of Treatment With Bone Marrow Lysate on Hematopoietic Stem Cells and Myeloid Cells in Tumor-Bearing Mice.

Introduction: Leukopenia is one of the major side effects of myelosuppressive chemotherapy such as cyclophosphamide (CTX). We and others have used CTX either alone or in combination with G-CSF for the mobilization of hematopoietic stem cells (HSCs). This mobilization can induce expansion of myeloid cells with immunosuppressive phenotype. In this pilot study, we aimed to test whether bone marrow lysate (BML)/CTX, a rich source of growth factors, can lower the expansion of myeloid cells with immunosuppressive phenotypes in tumor-bearing mice without interfering with the anti-tumor effects of CTX or with the mobilization of HSCs. Methods: Female CD1 mice were treated on day 0 with an i.p. injection of Ehrlich ascites carcinoma (EAC). On day 7, the mice were i.p. injected with CTX followed by s.c. injection of G-CSF for 5 consecutive days, single s.c. injection of BML/PBS or BML/CTX or single i.v. injection of BMC/PBS or BMC/CTX. Results: Treatment of EAC-bearing mice with BML/PBS or BML/CTX did not interfere with the anti-tumor effect of CTX. EAC increased the numbers of immature polymorphonuclear cells (iPMN; neutrophils) in both blood and spleen. Treatment of EAC-bearing mice with CTX further increased the numbers of these cells, which were decreased upon treatment with BML/CTX. Treatment with BML/PBS or BML/CTX increased the numbers of stem cells (C.Kit+Sca-1+) in BM; the effect of BML/CTX was higher, but with no significant effect on the numbers of HSCs. Future studies are needed to analyze the molecular components in BM lysate and to determine the underlying mechanisms.

Reduced fitness of the mosquito Culex pipiens (Diptera: Culicidae) after feeding on a blood meal with hepatitis C virus.

Hepatitis C virus (HCV) is a blood-borne virus. Given that mosquitoes can take blood meals from HCV patients, we aimed to test whether HCV in the blood meal can induce alterations in the biology of Culex pipiens. To address this aim, Cx. pipiens females were fed HCV-negative blood from healthy individuals or HCV-positive fresh blood samples harvested from viremic HCV patients. Replication of HCV in mosquitoes was confirmed by negative strand-specific RT-PCR and sequencing of RNA extracted from the mosquito bodies 7 days post-feeding. In addition, several parameters that determine the fitness of the mosquitoes were measured. Virus acquisition was associated with alterations in the architecture of the gut microvilli and the immune response, indicated by an increase in phenol oxidase activity. Interestingly, the mosquitoes that were fed the HCV-positive blood meal showed shorter median longevity (8 days) and laid fewer eggs than the control mosquitoes. Furthermore, the offspring of females fed the HCV-positive blood meal demonstrated a lower emergence rate than the controls. In sum, the results indicate that feeding on HCV by Cx. pipiens decreases fitness, which may, in turn, affect its potential as a vector.

Immunosuppressive role of Benzo[a]pyrene in induction of lung cancer in mice.

AIM: Benzo[a]pyrene [BP] is one of the major carcinogenic precursors of cigarette smoke that primary affects the lung at its first proximity. The goal of the current research was to elucidate new mechanisms underlying the tumorigenic impact of oral BP in the lung of mice, with focus on immunosuppressive effects and cancer stemming properties. METHODS: Female albino mice (n = 44) were divided into 2 groups: normal control and BP group. BP was administered orally to mice (50 mg/kg body weight), twice a week for four weeks in succession. At the end of experiment (22 weeks), gene expression were measured for transforming growth factor-ß (TGF-ß), cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death ligand 1(PD-L1), forkhead box protein P3 (FOXP3) and interleukin 12 (IL-12) and CD83+, CD8+ and CD166+ cell percentage were measured in lung tissue. RESULTS: The results indicated the tumorigenic role of BP in the lung which was evidenced by histopathological examination. BP group also showed immunosuppressive role which evidenced by increased expression of lung TGF-ß, CTLA-4, PD-L1, FOXP3 genes and decreased expression of lung IL-12 gene compared with normal control group. BP group also showed decreased CD83+ cells, CD8+ cells and increased number of CD166+ cells. CONCLUSION: Our findings indicated that BP has immunosuppressive role in lung cancer besides increasing the percentage of cancer stem like cells.

Ramadan intermittent fasting and immunity: An important topic in the era of COVID-19.

WITH the growing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome (SARS)-related coronavirus (SARS-CoV-2) infection, a parallel growing interest arose concerning potential preventive and adjunct therapies, dietary and lifestyle modifications, and remedies that may boost the immunity against SARS-CoV-2 infection. Furthermore, as Ramadan intermittent religious fasting that is practiced by about one and a half billion Muslims throughout the globe is coincide this year with COVID-19 pandemic, a growing debate rose concerning the expected impact of fasting during Ramadan month and the associated dietary and lifestyle behaviors on the body's immunity against the pandemic infection. Published literature was searched to find out how intermittent fasting (IF) and its model of Ramadan affect the various aspects related to the body's immunity against microbial infections. IF was found to impact immunity by changing different related elements, including oxidative stress and inflammation, metabolism, body weight, and body composition. Dietary and lifestyle modifications during Ramadan month and their impact on immunity, such as water intake and hydration status, sleep duration and timing, caloric intake and mealtime, and social and spirtual activities, were addressed. Further research is warranted to figure out how IF during ramadan affects immunity against SARS-CoV-2 infection.

Treatment options for autoimmune bullous dermatoses other than systemic steroids: A systematic review and network meta-analysis.

Autoimmune blistering diseases can eventually cause life-threatening complications if left untreated. Although there is no cure for these bullous diseases; their therapy is based on suppressing the immune system to cease the de novo formation of the generated antibodies. The current study aimed to assess the safety and efficacy of using standing alone alternative therapies beyond systemic steroids for management of autoimmune bullous diseases. We searched six literature databases for both randomized and quasi-randomized clinical trials that assessed the efficacy of drugs other than systemic steroids in autoimmune bullous diseases. Outcomes were calculated as odds ratios with 95% confidence-interval. We used the R software to perform conventional and network meta-analyses with a frequentist approach. The network ranking order for 629 bullous pemphigoid patients, from the best to the worst was, clobetasol propionate cream (40 mg; (P-score = .87), clobetasol propionate cream (10-30 mg; P-score = .77), nicotinamide plus tetracycline (P-score = .56), steroids (P-score = .29) and doxycycline (P-score = .01). Limitations of this study are the small sample of the included studies except for blister trial and lack of randomization in most trials. To conclude, Combined doxycycline and nicotinamides are safer and more effective option for extensive bullous pemphigoid patients than the usual use of systemic steroids. For limited disease, topical corticosteroid (40 mg/d) use provides a safer and better response modality than the other proposed treatments.

Anti-neoplastic and immunomodulatory potency of co-treatment based on bovine lactoferrin and/or muramyl dipeptide in tumor-bearing mice.

The current study investigates anti-neoplastic and immunomodulatory activities of co-treatment based on bovine lactoferrin (bLF) and/or muramyl dipeptide (MDP) with or without cisplatin (Cis) in tumor-bearing mice. In the present study, bLF (100 mg/kg; orally) and MDP (0.5 mg/kg; subcutaneously) was administered alone or together. MDP or bLF was co-treated with Cis (1 mg/kg; intraperitoneally) in mice-bearing Ehrlich solid carcinoma. Tumor size, tumor mass proliferation, apoptosis using immunohistochemistry, the alteration in spleen cell proliferation, phenotype using flow cytometry and white blood cells total and differential counts were detected. Treatment with Cis or (bLF and MDP) significantly reduced tumor size, upregulated the pro-apoptotic p53 expression and downregulated the anti-apoptotic Bcl-2 and proliferative marker PCNA expression compared to non-treated tumor-bearing animals. Moreover, co-treatment of MDP and Cis significantly potentiated the reduction of the tumor size, downregulated the Bcl-2 and PCNA expression and upregulated the p53 expression compared to Cis-treated animals. While bLF and Cis co-treatment positively controlled PCNA and p53 expression compared to tumor-bearing animals, it significantly potentiated the reduction of the tumor size and downregulated the Bcl-2 expression compared to Cis-treated animals. Co-treatment of (bLF and MDP), (bLF and Cis) or (MDP and Cis) increased the spleen cell proliferation and altered the immunological profile of the CD3+CD4+, CD3+CD8+, CD3+CD4+CD69+, CD3+CD8+CD69+ and CD11b+Ly6G+ cells to achieve better immune response against tumor. In conclusion, co-treatments based on bLF and/or MDP are promising therapies against cancer, through their potency to control proliferation, enhance apoptosis and improve the immune status against tumor cells.

Correlations between MHLC scores and Indicators of Immune Response in Egyptian Women with Breast Cancer.

BACKGROUND: Recent studies indicate the immune dysfunction in cancer patients in comparison with healthy individuals. The quality and quantity of this dysfunction are not equal in all patients even with similar cancer type. AIM: This study aims to correlate health locus of control (HLC) beliefs with CD4+ helper T (Thelper) cells, T regulatory (Treg) cells, NK cells, IL-1ß and TNF-a in breast cancer patients. PATIENTS AND METHODS: The study included 30 early diagnostic breast cancer patients who responded to Form C of the MHLC questionnaire that assessed internal (IHLC), chance (CHLC), doctor (DHLC) and other person's (OHLC) control of the patient's health status. Peripheral blood samples were collected to analyze the numbers and phenotype of Thelper cells, Treg cells and NK cells by flow cytometry and to measure gene expression of IL-1ß and TNF-a with real time PCR. RESULTS: A significant positive correlation was found between IHLC with Thelper cells and NK cells. However, a significant inverse correlation was found between DHLC with NK, Thelper and Treg cells. CONCLUSION: There is strong probability that the quality of immunity in cancer patients is related to their MHLC beliefs. Further research is recommended for studying whether MHLC beliefs of patients with other types of cancer can improve their immune responses and how beliefs control immune system.

The Toll-Like Receptor 3 Agonist Polyriboinosinic Polyribocytidylic Acid Increases the Numbers of NK Cells with Distinct Phenotype in the Liver of B6 Mice.

One of the activating factors of the cells of the innate immune system is the agonists of toll-like receptors (TLRs). Our earlier publications detailed how poly(I:C), a TLR3 agonist, elevates the NK cell population and the associated antigen-specific CD8+ T cell responses. This study involved a single treatment of the B6 mice with poly(I:C) intraperitoneally. To perform a detailed phenotypic analysis, mononuclear cells were prepared from each of the liver, peripheral blood, and spleen. These cells were then examined for their NK cell population by flow cytometric analysis following cell staining with indicated antibodies. The findings of the study showed that the NK cell population of the liver with an NK1.1highCD11bhighCD11chigh B220+Ly6G- phenotype was elevated following the treatment with poly(I:C). In the absence of CD11b molecule (CR3-/- mice), poly(I:C) can still increase the remained numbers of NK cells with NK1.1+CD11b- and NK1.1+Ly6G- phenotypes in the liver while their numbers in the blood decrease. After the treatment with anti-AGM1 Ab, which induced depletion of NK1.1+CD11b+ cells and partial depletion of CD3+NK1.1+ and NK1.1+CD11b- cell populations, poly(I:C) normalized the partial decreases in the numbers of NK cells concomitant with increased numbers of NK1.1-CD11b+ cell population in both liver and blood. Regarding mice with a TLR3-/- phenotype, their injection with poly(I:C) resulted in the partial elevation in the NK cell population as compared to wild-type B6 mice. To summarise, the TLR3 agonist poly(I:C) results in the elevation of a subset of liver NK cells expressing the two myeloid markers CD11c and CD11b. The effect of poly(I:C) on NK cells is partially dependent on TLR3 and independent of the presence of CD11b.

A new strategy for enhancing antitumor immune response using dendritic cells loaded with chemo-resistant cancer stem-like cells in experimental mice model.

BACKGROUND AND AIM: Cancer stem cells (CSCs) are rare cell population present in the tumor bulk that are thought to be the reason for treatment failure following chemotherapy in terms of their intrinsic chemo-resistance. Our study aimed to develop an effective therapeutic strategy to target chemo-resistant cancer stem - like cells population in solid Ehrlich carcinoma (SEC) mice model using dendritic cells (DCs) loaded with enriched tumor cells lysate bearing CSC-like phenotype as a vaccine. MATERIALS AND METHODS: Ehrlich carcinoma cell line was exposed to different concentrations of cisplatin, doxorubicin, or paclitaxel. Drug treatment that resulted in drug surviving cells with the highest expression of CSCs markers (CD44+/CD24-) was selected to obtain enriched cell cultures with resistant CSCs population. Dendritic cells were isolated from mice bone marrow, pulsed with enriched CSC lysate, analyzed and identified (CD11c, CD83 and CD86). SEC-bearing mice were treated with loaded or unloaded DCs either as single treatment or in combination with repeated low doses of cisplatin. IFN- γ serum level and p53gene expression in tumor tissues were determined by ELISA and real-time PCR, respectively. RESULTS AND CONCLUSION: The results revealed that vaccination with CSC loaded DCs significantly reduced tumor size, prolonged survival rate, increased IFN-γ serum levels, and upregulated p53gene expression in SEC bearing mice. These findings were more evident and significant in the group co-treated with CSC-DC and cisplatin rather than other treated groups. This study opens the field for combining CSC-targeted immunotherapy with repeated low doses chemotherapy as an effective strategy to improve anticancer immune responses.

Cyclophosphamide eradicates murine immunogenic tumor coding for a non-self-antigen and induces antitumor immunity.

Although the majority of cancers respond to chemotherapy, most cancer types relapse, at least in part, due to the poor immunogenicity of most tumor. We have reported before that treatment of tumor bearing mice with a combination of the anti-cancer chemotherapy cyclophosphamide (CTX) and immunotherapy can result in complete tumor regression using T-cell receptor (TCR) transgenic CD8+ T cells specific to antigens. This study aimed to determine whether chemotherapy can cure immunogenic tumor which expresses non-self-tumor antigen and result in antitumor immunity. Either EL4 cell line, a poorly immunogenic thymoma, or EG7, a clone of EL4 cells transfected with ovalbumin (OVA), as a non-self-antigen were inoculated subcutaneously into wild type or splenectomized C57BL/6 mice and then treated once with intraperitoneal (i.p.) injection of 4 mg CTX/mouse. In certain experiments, the mice were rechallenged with the same tumor type 1-2 months after the primary challenge. Treatment of EL4 bearing mice with CTX induced transient antitumor effect followed by tumor progression. Interestingly, however, treatment of EG7-bearing mice with CTX resulted in regression of early and advanced tumors. EG7 tumor-free mice rejected the second and the third challenges with EG7 cells, but not with challenge EL4 cells. These antitumor effects did not require spleen, since splenectomized mice showed similar antitumor effects of CTX on EG7 cells. Taken together, these data indicate that expression of non-self-antigen by poorly immunogenic tumor might be a reliable means to increase its immunogenicity and its response to chemotherapy.

Loading of doxorubicin and thymoquinone with F2 gel nanofibers improves the antitumor activity and ameliorates doxorubicin-associated nephrotoxicity.

AIMS: This study aimed to elucidate the benefits of nanoformulation of doxorubicin (DOX) and thymoquinone (TQ) loaded with nanofibers of poly-N-acetyl glucosamine (pGlcNAc), which is known as F2 gel, over their conventional free forms. Moreover, evaluate the role of TQ in improving chemotherapeutic effect and ameliorating nephrotoxicity of DOX. MAIN METHODS: The drugs were loaded into F2 gel followed by measurement of physicochemical characterization. Next, MCF-7 and HEPG2 cells were treated with the prepared formulations and assessed for apoptosis alongside with cellular proliferation. Furthermore, we experimentally induced Heps liver carcinoma in mice and at the end of the treatment, mice were sacrificed and serum samples were used to assess nephrotoxicity markers; blood urea nitrogen (BUN) and creatinine. Additionally, renal tissue was used for determination of oxidative markers and antioxidant enzymes; whereas, tumor tissue was utilized to measure nuclear factor kappa B (NF-κB) and caspase 3. KEY FINDINGS: Nanoformulation showed dramatic increase in apoptosis, caspase 3, and antioxidant enzymes; in contrast to, dramatic fall in cell viability, tumor volume, oxidative and nephrotoxicity markers, and NF-κB compared to corresponding free therapies. Combined therapy was superior in conserving the measured parameters compared to other treated groups. SIGNIFICANCE: F2 gel loaded with DOX and TQ revealed enhanced antitumor activity with minimal toxicity. Moreover, using TQ as an adjuvant with DOX could augment its cytotoxicity and ameliorate nephrotoxicity.

A novel potential effective strategy for enhancing the antitumor immune response in breast cancer patients using a viable cancer cell-dendritic cell-based vaccine.

Dendritic cells (DCs) have been used in a number of clinical trials for cancer immunotherapy; however, they have achieved limited success in solid tumors. Consequently the aim of the present study was to identify a novel potential immunotherapeutic target for breast cancer patients through in vitro optimization of a viable DC-based vaccine. Immature DCs were primed by viable MCF-7 breast cancer cells and the activity and maturation of DCs were assessed through measuring CD83, CD86 and major histocompatibility complex (MHC)-II expression, in addition to different T cell subpopulations, namely CD4+ T cells, CD8+ T cells, and CD4+CD25+ forkhead box protein 3 (Foxp3)+ regulatory T cells (Tregs), by flow cytometric analysis. Foxp3 level was also measured by enzyme-linked immunosorbent assay (ELISA) in addition to reverse-transcription quantitative polymerase chain reaction. The levels of interleukin-12 (IL-12) and interferon-γ (IFN-γ) were determined by ELISA. Finally, the cytotoxicity of cytotoxic T lymphocytes (CTLs) was evaluated through measuring lactate dehydrogenase (LDH) release by ELISA. The results demonstrated that CD83+, CD86+ and MHC-II+ DCs were significantly elevated (P<0.001) following priming with breast cancer cells. In addition, there was increased activation of CD4+ and CD8+ T-cells, with a significant decrease of CD4+CD25+Foxp3+ Tregs (P<0.001). Furthermore, a significant downregulation of FOXP3 gene expression (P<0.001) was identified, and a significant decrease in the level of its protein following activation (P<0.001) was demonstrated by ELISA. Additionally, significant increases in the secretion of IL-12 and IFN-γ (P=0.001) were observed. LDH release was significantly increased (P<0.001), indicating a marked cytotoxicity of CTLs against cancer cells. Therefore viable breast cancer cell-DC-based vaccines could expose an innovative avenue for a novel breast cancer immunotherapy.

Fibroblast growth factor 2 decreases bleomycin-induced pulmonary fibrosis and inhibits fibroblast collagen production and myofibroblast differentiation.

Fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of pulmonary fibrosis. Mice lacking FGF2 have increased mortality and impaired epithelial recovery after bleomycin exposure, supporting a protective or reparative function following lung injury. To determine whether FGF2 overexpression reduces bleomycin-induced injury, we developed an inducible genetic system to express FGF2 in type II pneumocytes. Double-transgenic (DTG) mice with doxycycline-inducible overexpression of human FGF2 (SPC-rtTA;TRE-hFGF2) or single-transgenic controls were administered intratracheal bleomycin and fed doxycycline chow, starting at either day 0 or day 7. In addition, wild-type mice received intratracheal or intravenous recombinant FGF2, starting at the time of bleomycin treatment. Compared to controls, doxycycline-induced DTG mice had decreased pulmonary fibrosis 21 days after bleomycin, as assessed by gene expression and histology. This beneficial effect was seen when FGF2 overexpression was induced at day 0 or day 7 after bleomycin. FGF2 overexpression did not alter epithelial gene expression, bronchoalveolar lavage cellularity or total protein. In vitro studies using primary mouse and human lung fibroblasts showed that FGF2 strongly inhibited baseline and TGFß1-induced expression of alpha smooth muscle actin (αSMA), collagen, and connective tissue growth factor. While FGF2 did not suppress phosphorylation of Smad2 or Smad-dependent gene expression, FGF2 inhibited TGFß1-induced stress fiber formation and serum response factor-dependent gene expression. FGF2 inhibition of stress fiber formation and αSMA requires FGF receptor 1 (FGFR1) and downstream MEK/ERK, but not AKT signaling. In summary, overexpression of FGF2 protects against bleomycin-induced pulmonary fibrosis in vivo and reverses TGFß1-induced collagen and αSMA expression and stress fiber formation in lung fibroblasts in vitro, without affecting either inflammation or epithelial gene expression. Our results suggest that in the lung, FGF2 is antifibrotic in part through decreased collagen expression and fibroblast to myofibroblast differentiation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Enhanced Lymphodepletion Is Insufficient to Replace Exogenous IL2 or IL15 Therapy in Augmenting the Efficacy of Adoptively Transferred Effector CD8+ T Cells.

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15.Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067-74. ©2018 AACR.