Short-chain fatty acids reverses gut microbiota dysbiosis-promoted progression of glioblastoma by up-regulating M1 polarization in the tumor microenvironment.

Glioblastoma (GBM), known as the most malignant and common primary brain tumor of the central nervous system, has finite therapeutic options and a poor prognosis. Studies have shown that host intestinal microorganisms play a role in the immune regulation of parenteral tumors in a number of different ways, either directly or indirectly. However, the potential impact of gut microbiota on tumor microenvironment, particularly glioma immunological milieu, has not been clarified exactly. In this study, by using an orthotopic GBM model, we found gut microbiota dysbiosis caused by antibiotic cocktail treatment boosted the tumor process in vivo. An obvious change that followed gut microbiota dysbiosis was the enhanced percentage of M2-like macrophages in the TME, in parallel with a decrease in the levels of gut microbial metabolite, short-chain fatty acids (SCFAs) in the blood and tumor tissues. Oral supplementation with SCFAs can increase the proportion of M1-like macrophages in the TME, which improves the outcomes of glioma. In terms of mechanism, SCFAs-activated glycolysis in the tumor-associated macrophages may be responsible for the elevated M1 polarization in the TME. This study will enable us to better comprehend the "gut-brain" axis and be meaningful for the development of TAM-targeting immunotherapeutic strategies for GBM patients.

Neuromedin U regulates the anti-tumor activity of CD8+ T cells and glycolysis of tumor cells in the tumor microenvironment of pancreatic ductal adenocarcinoma in an NMUR1-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a poor prognosis, which is lethal in approximately 90% of cases despite advanced standard therapies. A typical feature of PDAC is the immunosuppressive tumor microenvironment with multiple immunosuppressive factors including neurotransmitters. Recently, neuromedin U (NMU), a highly conserved neuropeptide with many physiological functions, has attracted attention for its roles in tumorigenesis and metastasis in several types of cancers. However, whether NMU affects PDAC progression remains unclear. In this study, using an orthotopic mouse model of PDAC in combination with bioinformatics analysis, we found that NMU was upregulated in tumor tissues from the patients with PDAC and positively correlated with a poor prognosis of the disease. Interestingly, knockout of the Nmu gene in mice enhanced the anti-tumor functions of tumor-infiltrating CD8+ T cells in an NMU receptor 1-dependent manner. Additionally, NMU promoted the glycolytic metabolism of mouse PDAC tumors. The activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH), pivotal enzymes involved in the regulation of lactate production, were markedly reduced in tumor tissues from NMU-knockout mice. In vitro the presence of LDHA inhibitor can reduce the production of lactic acid stimulated by NMU, which can increase the anti-tumor activity of CD8+ T cells. Moreover, treatment of the pancreatic cancer cells with a phosphoinositide 3-kinase (PI3K) inhibitor diminished NMU-induced lactate production and the activities of PK and LDH, suggesting that NMU might regulate glycolysis via the PI3K/AKT pathway.

Gut Lactobacillus contribute to the progression of breast cancer by affecting the anti-tumor activities of immune cells in the TME of tumor-bearing mice.

Studies have proven that gut microbiota dysbiosis may influence the carcinogenesis and outcomes of multiple cancers. However, it is still unclear whether gut microbiota dysbiosis affect the progression of breast cancer, especially triple-negative breast cancer. In the present study, by using gut microbiota dysbiosis murine model established by treatment of mice with streptomycin, we found Lactobacillus and the metabolite-lactic acid are the pivotal factors for 4T1 tumor progression. In fact, streptomycin-treated mice exhibited slower tumor growth, in parallel with less abundance of Lactobacillus in the gut. Supplementation with Lactobacillus resulted in a rapid tumor growth, following a decrease in the expression of mRNAs for anti-tumor-related factors but an increase in the M2 polarization. The elevated percentages of IFN-γ-producing CD4+T cells and CD8+T cells in the tumor microenvironment of streptomycin-treated tumor-bearing mice may be vanished by supplementation of Lactobacillus. It seems likely that lactobacillus-mediated pro-tumor effect is related to the production of lactic acid. A decrease in the levels of lactic acid in the cecal feces and tumor tissues were observed in streptomycin-treated tumor bearing mice. However, supplementation of Lactobacillus can restore streptomycin-reduced concentration of lactic acid in the tumor tissues, suggesting that gut Lactobacillus are the source of lactic acid. Bioinformatics analysis result suggests high concentration of lactic acid in tumor sites may be related to the diminished anti-tumor immunity in the TME. This study reveals a correlation between gut Lactobacillus and tumor progression in a murine 4T1 tumor model, providing experimental evidence for clinical treatment of breast cancer.

Oral administration of Clostridium butyricum rescues streptomycin-exacerbated respiratory syncytial virus-induced lung inflammation in mice.

Changes in the intestinal microbiota indirectly impact the health of mucosa distal to the intestine, particularly the respiratory tract. However, the effects of intestinal microbiota dysbiosis on the regulation of respiratory syncytial virus (RSV) infection are not clear. In this study, we examined the effects of altering the intestinal microbiota on the pulmonary immune response against RSV infection. BALB/c mice were treated with streptomycin before infection with RSV to study the altered immune response. The ingestion of streptomycin led to a marked alteration in the intestinal microbiota with a reduced abundance of Lactobacillus and Clostridium genera, followed by greatly aggravated pulmonary inflammation in response to RSV infection. This aggravated inflammation was associated with a dysregulated immune response against RSV infection, characterized by the increased expression of IFN-γ and IL-17 and increased pulmonary M1-like macrophage polarization, and decreased expression of IL-5. Supplementation of Clostridium butyricum (CB) prevented aggravated inflammation and the dysregulated immune response characterized by greater M2 polarization of pulmonary macrophages and decreased release of IFN-γ and IL-17 as well as increased IL-5 levels. Furthermore, in vitro and in vivo experiments identified that butyrate, the main metabolite produced by CB, promoted M2 polarization of macrophages in RSV-infected mice exposed to streptomycin. Together, these results demonstrate the mechanism by which intestinal microbiota modulate the pulmonary immune response to RSV infection.

Resveratrol attenuates TNBC lung metastasis by down-regulating PD-1 expression on pulmonary T cells and converting macrophages to M1 phenotype in a murine tumor model.

Triple-negative breast cancer (TNBC) is an invasive breast cancer with the characteristics of easy to develop distant metastasis. Immune escape is one of the main reasons for TNBC growth and metastasis. Enhancement of T cell-mediated anti-tumor activity may benefit to inhibit tumor metastasis and improve the efficacy of cancer therapy. As a natural bioactive substance, resveratrol shows potential capability to prevent or suppress the development of a variety of cancers through direct or indirect effects, including immunoregulatory effect. However, whether resveratrol might affect lung metastasis of TNBC, and whether the effect of resveratrol might be associated with resveratrol-regulated immune responses in tumor microenvironment is still unknown. In this study, by using an experimental metastatic mouse 4 T1 tumor model, we identified that resveratrol may suppress TNBC lung metastasis by elevating local anti-tumor immunity. Indeed, an increase in the cytotoxic activity of CD8+T cells as well as the levels of type 1 cytokine IFN-γ and IL-2 in the lungs of resveratrol-treated tumor bearing mice were observed. The enhanced CD8+T cell activity and Th1 immune responses by resveratrol administration might be related to the down-regulated PD-1 expression on pulmonary CD8+T cells and CD4+T cells. Resveratrol may also convert macrophages to M1 phenotype in the lungs of tumor bearing mice. However, it seems likely resveratrol has no effect on pulmonary myeloid-derived suppressor cell activation. Our results provide an evidence that resveratrol might be a promising candidate agent for adjuvant therapy in the process of TNBC metastasis.

Group 2 innate lymphoid cells promote TNBC lung metastasis via the IL-13-MDSC axis in a murine tumor model.

Group 2 innate lymphoid cells (ILC2s) are reportedly associated with the progression of many tumors. However, the role of ILC2s in triple-negative breast cancer (TNBC) lung metastasis remains unclear. In this study, we found that ILC2s may be a key element in the process of TNBC lung metastasis since the adoptive transfer of pulmonary ILC2s increased the numbers of metastatic lung nodules and reduced the survival of tumor-bearing mice. ILC2-promoted 4 T1 lung metastasis appears to be related to ILC2-derived IL-13. An expansion of IL-13-producing ILC2s and an elevated expression of IL-13 mRNA in pulmonary ILC2s were determined in tumor-bearing mice, in parallel with an increase in the levels of local IL-13 by ILC2 transfer. The neutralization of IL-13 reduced the increased pulmonary metastatic nodules and improved the decreased survival rate caused by ILC2-adoptive transfer. Interestingly, adoptive transfer of ILC2s elevated IL-13Ra1 expression in myeloid-derived suppressor cells (MDSCs). Treatment of ILC2-transferred tumor-bearing mice with anti-IL-13 antibodies significantly diminished the number of pulmonary MDSCs and inhibited MDSC activation. Moreover, when pulmonary MDSCs were cocultured with ILC2s in the presence of an anti-IL-13 mAb, the number and activation of MDSCs were reduced. Depletion of MDSCs may promote the proliferation of CD4+ T cells and CD8+ T cells, but reduce the expansion of regulatory T cells (Tregs) in the lungs of ILC2-transferred tumor-bearing mice. Our results suggest that pulmonary ILC2s may promote TNBC lung metastasis via the ILC2-derived IL-13-activated MDSC pathway.

Role of gut microbiome in the outcome of cancer immunotherapy.

Nearly 3 × 1013 types of bacteria colonize the human intestine. These colonized bacteria help in maintaining intestinal homeostasis by establishing a complex relationship with the intestinal epithelium and lymphoid tissue. Alteration in the composition of the intestinal microbiota is associated with susceptibility to various pathological conditions, such as autoimmune disorders, diabetes, inflammation and cancer. Of late, several researchers have focused on examining the effects of gut microbiota on the outcome of various cancer treatment protocols. Side effects and complications of traditional chemotherapy and allogeneic hematopoietic cell transplantation are associated with intestinal dysbiosis. Gut microbiota affects the efficacy of immune checkpoint inhibitor-based immunotherapy. The gut is inhabited by diverse resident bacteria, of which, few enhance, while others inhibit the host response to immunotherapy. This review focuses on the correlation between intestinal microbiota and the outcome of tumor immunotherapy. Additionally, the molecular mechanisms underlying the effects of gut microbiota on the efficacy of cancer immunotherapy have been reviewed. Further studies are needed for the identification of distinct gut microbiota and their efficacy in tumor immunotherapy as certain types of intestinal bacteria could function as novel adjuvant drugs to enhance the effectiveness of antitumor therapy in humans.

Critical role of OX40/OX40L in ILC2-mediated activation of CD4+T cells during respiratory syncytial virus infection in mice.

CD4+T cells are crucial cellular source of type 2 cytokines and responsible for RSV-induced asthma-like symptoms and asthma exacerbations. However, the mechanism for regulating the activation of CD4+T cells during RSV infection is not clear completely. We show in this study that infection with RSV may induce an expansion and activation of CD4+T cells in the lungs of BALB/c mice. RSV-induced CD4+T cell expansion and activation seems to depend upon the pulmonary group 2 innate lymphoid cells (ILC2s), since adoptive transfer of lung ILC2s can enhance not only the numbers of CD4+T cells but also the cytokine production by CD4+T cells. Interestingly, blockade of the contact between ILC2s and CD4+T cells, may significantly diminish the CD4+T cell expansion and cytokine production, suggesting that membrane molecules may be involved in ILC2-regulated CD4+T cell activation. In fact, infection with RSV resulted in an increase in the numbers of OX40+CD4+T cells as well as OX40L+ILC2s in the lungs of mice. Moreover, the mRNA expressions of OX40 and OX40L as well as the levels of OX40 and OX40L proteins in the lung CD4+T cells and ILC2s were elevated respectively. When co-culture of CD4+T cells with ILC2s in the presence of anti-OX40L antibody, the cytokine productions by CD4+T cells were reduced markedly, suggesting that lung ILC2s may regulate RSV-induced CD4+T cell expansion and activation perhaps via OX40/OX40L interaction.

Respiratory syncytial virus prevents the subsequent development of ovalbumin-induced allergic responses by inhibiting ILC2 via the IL-33/ST2 pathway.

AIM: How respiratory syncytial virus (RSV) influences the development of ovalbumin (OVA)-induced asthma remains elusive. As potent T helper (Th)2 cytokine producers, group 2 innate lymphoid cells (ILC2s) are known to serve important functions in the pathogenesis of allergic inflammation. However, how RSV infection affects innate immunity, especially with regard to the function of ILC2s in OVA-induced allergic airway inflammation, is largely unknown. MATERIALS & METHODS: RSV was used to infect adult BALB/c mice intranasally prior to sensitization and subsequent challenge with OVA. ILC2 frequencies and Th2 cytokine production by ILC2s were assessed by flow cytometry. Cytokine levels were detected both by real-time PCR and ELISA. RESULTS: Previous infection with RSV attenuated airway inflammation and decreased Th2 cytokine production in mice sensitized and challenged with OVA. Furthermore, previous infection with RSV inhibited the influx of ILC2s into the lung, and constrained their Th2 cytokine production. Adoptive transfer of ILC2s increased asthma-associated airway inflammation in mice previously infected with RSV. These results indicate that previous infection with RSV prevents OVA-induced asthma development via inhibition of ILC2s. Previous infection with RSV attenuated IL-33 production in lung tissue and reduced relative ST2L expression in lung ILC2s, meaning that previous infection with RSV may alter ILC2 function via the IL-33/ST2 signaling pathway. CONCLUSION: These results demonstrate that previous infection with RSV attenuates OVA-induced airway inflammation by inhibiting the recruitment and Th2 cytokine production of ILC2s via the IL-33/ST2 pathway.

Natural helper cells mediate respiratory syncytial virus-induced airway inflammation by producing type 2 cytokines in an IL-33-dependent manner.

AIM: Type 2 cytokine production during respiratory virus infection is considered to be linked with asthma exacerbation. As potent Th2 cytokine producers, natural helper (NH) cells play a key role in influenza virus-induced airway hyper-responsiveness. However, it is unclear whether NH cells contribute to respiratory syncytial virus (RSV)-induced airway inflammation, and how the cytokine profile in NH cells is changed during RSV infection. METHODS: BALB/c mice were infected intranasally with RSV. The number of NH cells in lungs was detected by flow cytometry. The expression of cytokine mRNAs was performed by real-time RT-PCR. Cytokines levels were determined by ELISA. RESULTS: Following intranasal infection with RSV, BALB/c mice showed an increase in the expression of mRNAs for Th2-like cytokines in NH cells. Furthermore, adoptive transfer of pulmonary NH cells resulted in a massive infiltration of mononuclear cells, in particular eosinophils and neutrophils in lungs, in parallel with an augmented production of Th2-associated cytokines, such as IL-4, IL-5 and IL-10 in bronchoalveolar lavage fluids, providing convincing evidence that NH cells contribute to RSV-induced lung pathogenesis by producing type 2 cytokines. It should be noted that blocking IL-33 with antibody can diminish the absolute number of pulmonary NH cells and the relative expression of mRNAs for type 2 cytokines in pulmonary NH cells, suggesting that IL-33 is necessary for activating Th2-type NH cells. CONCLUSION: These results reveal that pulmonary NH cells might participate in RSV-induced airway inflammation by producing large quality of type 2 cytokines in an IL-33-dependent manner.

Respiratory macrophages and dendritic cells mediate respiratory syncytial virus-induced IL-33 production in TLR3- or TLR7-dependent manner.

Respiratory syncytial virus (RSV) infection can increase the production of IL-33 in lungs of mice. However, little is known about cellular source of IL-33, particularly the types of IL-33-producing cells in innate immune cells during RSV infection. In this study, by using BALB/c mice that were infected intranasally with RSV, it became clear that RSV infection can enhance not only the number of IL-33(+)-alveolar macrophages (AMs) and dendritic cells (DCs), but also the expression of IL-33 mRNA in these cells, suggesting that innate immune cells participate in the production of IL-33. Indeed, in vitro experiments by using murine cell lines found that RSV infection results in more expression of IL-33 mRNA in AMs and DCs, further confirming that these cell types may be an important source of IL-33 during RSV infection. It should be noted that the expression of mRNA for TLR3 and TLR7 was up-regulated in pulmonary AMs during RSV infection. Blockade of TLRs by TLR3 or TLR7 antagonist significantly reduces the levels of IL-33 mRNA in AMs and DCs, suggesting that RSV-induced IL-33 production might be TLRs-dependent manner. Although the expression of TLRs mRNA in pulmonary interstitial macrophages (IMs) was enhanced after RSV infection, stimulation with agonists or inactivated RSV cannot alter the expression of IL-33 mRNA in IMs, suggesting that pulmonary IMs may not be a source of IL-33 during RSV infection. Thus, these results demonstrate that during RSV infection, respiratory macrophages and dendritic cells mediate the production of IL-33 in a TLR-dependent manner.

Infection with respiratory syncytial virus influences FasL-mediated apoptosis of pulmonary γδ T cells in a murine model of allergen sensitization.

BACKGROUND: It has been reported that adoptive transfer of γδ T cells increases the cellular infiltration, especially eosinophils, in the lungs of allergic mice, suggesting that γδ T cells may play a proinflammatory role in allergic airway inflammation. Respiratory syncytial virus (RSV) infection can decrease the number of Th2-type γδ T cells. However, the underlying mechanisms remain unknown. METHODS: BALB/c mice were inoculated intranasally with RSV before or after sensitization to OVA. The amounts of Th1/Th2 cytokines as well as the levels of specific antibodies were determined by ELISA. The apoptotic death of pulmonary γδ T cells was analyzed by flow cytometry. RESULTS: Adoptive transfer of γδ T cells increased the production of Th2 cytokines in the lungs and allergy-related antibodies in the serum, further confirming that γδ T cells act as pro-inflammatory cells or a promoter for the development of allergic asthma. RSV infection before sensitization to OVA enhanced apoptotic death of pulmonary γδ T cells. The percentage and absolute number of FasL-expressing γδ T cells in the lungs of allergic mice were elicited significantly by prior RSV infection. Blocking FasL with monoclonal antibody diminished apoptotic death of γδ T cells, suggesting that FasL is important for RSV-induced apoptosis of pulmonary γδ T cells. CONCLUSIONS: This work provides evidence that RSV infection suppresses the subsequent development of OVA-induced allergic responses partly by enhancing FasL-mediated apoptosis of pulmonary γδ T cells.

Respiratory syncytial virus protects against the subsequent development of ovalbumin-induced allergic responses by inhibiting Th2-type γδ T cells.

Respiratory syncytial virus (RSV) infection has been hypothesized to be a risk factor for the development of allergy and asthma, but epidemiologic studies in humans still remain inconclusive. The association between RSV infection and allergic diseases may be dependent on an atopic background and previous history of RSV infection. It has been reported that RSV infection before sensitization to an allergen decreased the production of Th2-like cytokines in the lung and the levels of allergen-specific Th2-type antibodies in the serum. However, the underlying mechanisms are largely unknown. In the present study, the role of pulmonary γδ T cells in RSV-affected, allergen-induced airway inflammation was investigated. BALB/c mice were sensitized to or challenged with ovalbumin (OVA) and infected with RSV either before or after the sensitization period. It became clear that sensitization and challenge of mice with OVA induced a large influx of γδ T cells to the lungs. However, prior RSV infection inhibited the infiltration of γδ T cells as well as activated γδ T cells, characterized by expression of CD40L or CD69 molecular in the cell surface. Moreover, prior RSV infection elevated the type 1 cytokine gene expression but suppressed type 2 cytokine expression in the lung γδ T cells. Adoptive transfer of γδ T cells from OVA-sensitized and challenged mice increased airway inflammation, suggesting that γδ T cells may play a proinflammatory role in allergic responses. These results described here support the idea of an unknown γδ T cell-dependent mechanism in the regulation of RSV-affected, allergen-induced allergic airway responses.

A prophylactic effect of an oligodeoxynucleotide containing a cytidine-guanosine motif against Japanese cedar pollen-induced T-helper type 2 allergic response.

BACKGROUND: Over 10% of entire population in Japan suffer from allergic diseases induced by Japanese cedar pollen (JCP) every spring. In terms of preventive medicine, it has become a matter of urgency to establish successful prophylactic and therapeutic strategies for controlling the disorders. The effect of an oligodeoxynucleotide containing a cytidine-guanosine motif (CpG ODN) on the regulation of immune responses induced by JCP was investigated in this study. METHODS: BALB/c mice were inoculated with CpG ODN intraperitoneally before intranasal sensitization to JCP. Cellular infiltration in the lung of BALB/c mice after treatment with CpG ODN or JCP was performed by hematoxylin and eosin (H&E) staining. Antibody titers and cytokines levels were determined by ELISA. RESULTS: Intranasal inoculation of BALB/c mice with JCP induced a T-helper type 2 (Th2-type) dominant immune response, as characterized by the production of interleukin (IL)-4 and IL-5 in the lung and of JCP-specific IgE antibody in serum. Prior intraperitoneal administration of CpG ODN to mice suppressed the subsequent JCP-induced antibody production and infiltration of inflammatory cells in the lung. The inhibitory mechanism of CpG ODN seemed to be attributable to a CpG ODN-induced Th1-type dominant environment, which down-regulated Th2-type response subsequently induced by JCP allergen sensitization. Furthermore, administration with CpG ODN decreased the production of JCP-induced IL-17, which has been found to play a pivotal role in several inflammatory diseases including allergic asthma. The decreased production of IL-17, together with reduced secretion of IL-4 and IL-5, may contribute to diminish the inflammation in the lung of JCP-sensitized mice. CONCLUSION: This work provides evidence that the CpG ODN has a prophylactic effect on the JCP-induced Th2-type allergic responses by establishing or restoring a Th1-type shift of immune environments.

Identification of the risk for liver fibrosis on CHB patients using an artificial neural network based on routine and serum markers.

BACKGROUND: Liver fibrosis progression is commonly found in patients with CHB. Liver biopsy is a gold standard for identifying the extent of liver fibrosis, but has many draw-backs. It is essential to construct a noninvasive model to predict the levels of risk for liver fibrosis. It would provide very useful information to help reduce the number of liver biopsies of CHB patients. METHODS: 339 chronic hepatitis B patients with HBsAg-positive were investigated retrospectively, and divided at random into 2 subsets with twice as many patients in the training set as in the validation set; 116 additional patients were consequently enrolled in the study as the testing set. A three-layer artificial neural network was developed using a Bayesian learning algorithm. Sensitivity and ROC analysis were performed to explain the importance of input variables and the performance of the neural network. RESULTS: There were 329 patients without significant fibrosis and 126 with significant fibrosis in the study. All markers except gender, HB, ALP and TP were found to be statistically significant factors associated with significant fibrosis. The sensitivity analysis showed that the most important factors in the predictive model were age, AST, platelet, and GGT, and the influence on the output variable among coal miners were 22.3-24.6%. The AUROC in 3 sets was 0.883, 0.884, and 0.920. In the testing set, for a decision threshold of 0.33, sensitivity and negative predictive values were 100% and all CHB patients with significant fibrosis would be identified. CONCLUSIONS: The artificial neural network model based on routine and serum markers would predict the risk for liver fibrosis with a high accuracy. 47.4% of CHB patients at a decision threshold of 0.33 would be free of liver biopsy and wouldn't be missed.

Evaluation of a virus derived from MDCK cells infected persistently with influenza A virus as a potential live-attenuated vaccine candidate in the mouse model.

A temperature-sensitive mutant virus unable to replicate at 38 degrees C was recovered from passage 189 (IVpi-189) of Madin-Darby canine kidney cells infected persistently with influenza A. Immunofluorescent staining of the IVpi-189 virus-infected cells revealed disrupted transport of the matrix (M) 1 protein into the nucleus at non-permissive temperatures, resulting in retention of the nucleoprotein (NP) in the nucleus. Upon comparison with the parental influenza A E61-24-P15 strain used to establish persistent infection, amino acid exchanges were found in the M1 protein of IVpi-189 virus; arginine to glutamine at position 72 and threonine to alanine at position 139. When mice were inoculated intranasally with IVpi-189 virus, virus growth in the lungs was restrained and terminated rapidly. Prior intranasal inoculation with only a small dose of IVpi-189 virus induced humoral and cellular immune responses and protected mice against subsequent virulent virus challenge. These results indicate that IVpi-189 virus, an avirulent temperature-sensitive mutant, is a promising candidate for use as a live-attenuated vaccine.

Fetal calf serum inhibits virus genome expression in Madin-Darby canine kidney cells persistently infected with influenza A virus.

A cell line of Madin-Darby canine kidney (MDCK) cells persistently infected with human influenza A virus has been established and designated as MDCK-IVpi cells. Production of progeny virus in MDCK-IVpi cells was suppressed when the cells were incubated in the presence of 10% fetal calf serum (FCS). FCS impaired virus mRNA synthesis in MDCK-IVpi cells, which resulted in a scarcity of virus proteins for virion formation. However, MDCK-IVpi cells well supported the growth of superinfecting heterologous influenza viruses, even in the presence of FCS. A certain fetuin-like substance in FCS might be responsible for the observed inhibition of virus replication.

Influenza A virus derived from persistently virus-infected cells shows attenuated cytotoxicity in cultured cells but virulent pathogenicity in mice.

The IVpi-43 strain of influenza A virus, a progeny virus derived from persistently virus-infected Madin-Darby canine kidney (MDCK) cells, showed a more attenuated nature in cytopathology in cultured cells than the parental wild-type influenza virus (IVwt) that was used for establishment of the virus carrier culture. Upon infection of MDCK cells, growth of the IVpi-43 virus was restrained with an impaired synthesis of virus structural proteins in the cells. Apoptosis induced by IVpi-43 virus was confined at a low level. The IVpi-43 virus was able to easily cause persistent infection in fresh MDCK cells. In contrast to the in vitro phenotype, the IVpi-43 virus proved highly virulent in mice, with massive and broadly disseminated virus multiplication in the lungs. It was suggested that impaired activity of the neuraminidase molecule of the IVpi-43 virus was responsible for the delayed and faint appearance of apoptosis in the IVpi-43 virus-infected respiratory cells, which made it possible for the virus to replicate for a longer period and to spread to a broader area of the lungs and that abundant numbers of the virus-infected lung cells were killed within a short period by the subsequently established virus-specific immune responses, leading to unrecoverable serious pneumonia.

An immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif protects senescence-accelerated mice from lethal influenza virus by augmenting the T helper type 1 response.

The SAM-P1 strain of senescence-accelerated model mice shows an impaired T helper type 1 (Th1) immune response upon infection with influenza virus, which results in high susceptibility to the virus. Treatment of spleen cells from SAM-P1 mice with an immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif (CpG ODN) in vitro increased the ratio of the titre of IFN-gamma to that of IL-4. Administration of CpG ODN to SAM-P1 mice generated satisfactory virus-specific cytotoxic T-lymphocyte responses and natural killer cell activation and the virus-specific immunoglobulin (Ig) isotype switched from IgG1 to IgG2a. Virus growth in the lungs of CpG ODN-treated SAM-P1 mice was cleared quickly and mice survived the lethal influenza virus infection. It could be inferred that a possible mechanism of CpG ODN for normalization of senescence-associated dysregulation of the Th1/Th2 balance involves the upregulated expression of CD154 and CD40 molecules on immune-competent cells. These results suggest that CpG ODN could contribute to the development of a protective strategy against infectious diseases, especially among immunocompromised elderly persons, by stimulating Th1 immune responses.