Epigenetic changes associated with Bacillus Calmette-Guerin (BCG) treatment in bladder cancer.

Bacillus Calmette-Guérin (BCG) treatment for non-muscle invasive bladder cancer (NMIBC) is an established immunotherapeutic, however, a significant portion of patients do not respond to treatment. Despite extensive research into the therapeutic mechanism of BCG, gaps remain in our understanding. This review specifically focuses on the epigenomic contributions in the immune microenvironment, in the context of BCG treatment for NMIBC. We also summarise the current understanding of NMIBC epigenetic characteristics, and discuss how future targeted strategies for BCG therapy should incorporate epigenomic biomarkers in conjunction with genomic biomarkers.

Chromosomal instability and its effect on cell lines.

BACKGROUND: Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models. RECENT FINDINGS: Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances. CONCLUSION: In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture.

Increased numbers of monocyte-derived dendritic cells during successful tumor immunotherapy with immune-activating agents.

Local treatment with selected TLR ligands or bacteria such as bacillus Calmette-Guérin increases antitumor immune responses and delays tumor growth. It is thought that these treatments may act by activating tumor-associated dendritic cells (DCs), thereby supporting the induction of antitumor immune responses. However, common parameters of successful immune activation have not been identified. We used mouse models to compare treatments with different immune-activating agents for the ability to delay tumor growth, improve priming of tumor-specific T cells, and induce early cytokine production and DC activation. Treatment with polyinosinic-polycytidylic acid or a combination of monosodium urate crystals and Mycobacterium smegmatis was effective at delaying the growth of s.c. B16 melanomas, orthotopic 4T1 mammary carcinomas, and reducing 4T1 lung metastases. In contrast, LPS, monosodium urate crystals, or M. smegmatis alone had no activity. Effective treatments required both NK1.1(+) and CD8(+) cells, and resulted in increased T cell priming and the infiltration of NK cells and CD8(+) T cells in tumors. Unexpectedly, both effective and ineffective treatments increased DC numbers and the expression of costimulatory molecules in the tumor-draining lymph node. However, only effective treatments induced the rapid appearance of a population of monocyte-derived DCs in the draining lymph node, early release of IL-12p70 and IFN-γ, and low IL-10 in the serum. These results suggest that the activation of existing DC subsets is not sufficient for the induction of antitumor immune responses, whereas early induction of Th1 cytokines and monocyte-derived DCs are features of successful activation of antitumor immunity.

Oral vaccination with lipid-formulated BCG induces a long-lived, multifunctional CD4(+) T cell memory immune response.

Oral delivery of BCG in a lipid formulation (Liporale™-BCG) targets delivery of viable bacilli to the mesenteric lymph nodes and confers protection against an aerosol Mycobacterium tuberculosis challenge. The magnitude, quality and duration of the effector and memory immune response induced by Liporale™-BCG vaccination is unknown. Therefore, we compared the effector and memory CD4(+) T cell response in the spleen and lungs of mice vaccinated with Liporale™-BCG to the response induced by subcutaneous BCG vaccination. Liporale™-BCG vaccination induced a long-lived CD4(+) T cell response, evident by the detection of effector CD4(+) T cells in the lungs and a significant increase in the number of Ag85B tetramer-specific CD4(+) T cells in the spleen up to 30 weeks post vaccination. Moreover, following polyclonal stimulation, Liporale™-BCG vaccination, but not s.c. BCG vaccination, induced a significant increase in both the percentage of CD4(+) T cells in the lungs capable of producing IFNγ and the number of multifunctional CD4(+) T cells in the lungs at 30 weeks post vaccination. These results demonstrate that orally delivered Liporale™-BCG vaccine induces a long-lived multifunctional immune response, and could therefore represent a practical and effective means of delivering novel BCG-based TB vaccines.

Induction of T cell responses and recruitment of an inflammatory dendritic cell subset following tumor immunotherapy with Mycobacterium smegmatis.

Mycobacteria and their cell wall components have been used with varying degrees of success to treat tumors, and Mycobacterium bovis BCG remains in use as a standard treatment for superficial bladder cancer. Mycobacterial immunotherapy is very effective in eliciting local immune responses against solid tumors when administered topically; however, its effectiveness in eliciting adaptive immune responses has been variable. Using a subcutaneous mouse thymoma model, we investigated whether immunotherapy with Mycobacterium smegmatis, a fast-growing mycobacterium of low pathogenicity, induces a systemic adaptive immune response. We found that M. smegmatis delivered adjacent to the tumor site elicited a systemic anti-tumor immune response that was primarily mediated by CD8(+) T cells. Of note, we identified a CD11c(+)CD40(int)CD11b(hi)Gr-1(+) inflammatory DC population in the tumor-draining lymph nodes that was found only in mice treated with M. smegmatis. Our data suggest that, rather than rescuing the function of the DC already present in the tumor and/or tumor-draining lymph node, M. smegmatis treatment may promote anti-tumor immune responses by inducing the involvement of a new population of inflammatory cells with intact function.

Dissecting memory T cell responses to TB: concerns using adoptive transfer into immunodeficient mice.

Several studies have used adoptive transfer of purified T cell subsets into immunodeficient mice to determine the subset of T cells responsible for mediating protection against Mycobacterium tuberculosis. These studies suggested that CD62L(hi) memory CD4(+) T cells from BCG-vaccinated mice are key for protection against tuberculosis. Importantly, we observed that transfer of naïve CD4(+) T cells into Rag1-/- recipients protected against a mycobacterial challenge as well as transfer of BCG-experienced CD4(+) T cells. We found that transfer of total CD4(+) T cells from naïve mice or enriched CD62L(hi)CD4(+) T cells from BCG-vaccinated mice into Rag1-/- recipients induced severe colitis by 3 weeks post cell transfer, whereas transfer of CD62L(lo)CD4(+) T cells from BCG-vaccinated mice did not. Naïve and CD62L(hi)CD4(+) T cells proliferated extensively upon transfer and developed an activated effector phenotype in the lung, even in the absence of infectious challenge. The induction of colitis and systemic cytokine response induced by the transfer and subsequent activation of CD4(+) T cells from naïve mice or CD62L(hi)CD4(+) T cells from BCG-vaccinated mice, into immunodeficient recipients, may heighten their ability to protect against mycobacterial challenge. This raises doubts about the validity of this model to study CD4(+) T cell-mediated protection against tuberculosis.

A key role for lung-resident memory lymphocytes in protective immune responses after BCG vaccination.

The immune mechanisms that orchestrate protection against tuberculosis as a result of BCG vaccination are not fully understood. We used the immunomodulatory properties of fingolimod (FTY720) treatment to test whether the lung-resident memory T lymphocytes generated by BCG vaccination were sufficient to maintain immunity against challenge infection with mycobacteria (BCG). Mice were given daily fingolimod treatment, starting either immediately before s.c. BCG vaccination or during subsequent BCG i.n. challenge, to prevent LN effector and memory lymphocytes from entering the periphery either during priming or challenge, respectively. Treatment with fingolimod during vaccination reduced vaccine-mediated protection against subsequent infection. By contrast, BCG-vaccinated mice were protected when fingolimod was given during the infectious challenge, suggesting that memory lymphocytes that migrate to the lung following vaccination are sufficient for protection. Notably, the antigen-reactive IFN-gamma or multicytokine-producing CD4(+) T cells present in the lung when fingolimod was given during BCG challenge did not correlate with protection; however, expression of MHC class II on macrophages isolated from the lungs post BCG challenge was increased in the protected mice. We conclude that protection conferred by BCG vaccination is dependent on memory lymphocytes retained in the lung, although IFN-gamma production by this population is not correlated with vaccine-mediated protection.

Geographical differences in the proportion of human group A rotavirus strains within New Zealand during one epidemic season.

The prospect of future rotavirus vaccine programs means it is important to understand rotavirus strain diversity within New Zealand, especially if this was to influence vaccine effectiveness. The G-genotype of 359 group A rotavirus strains isolated from 416 stool samples collected from June 2005 to May 2006 (inclusive) from children less than 5 years of age in multiple centers throughout New Zealand was determined. G1 was the dominant circulating strain (55.8%) followed by G4 (21.4%), G3 (3.4%), G9 (3.4%) G2 (1.0%), and mixed infection (1.0%). Two less common strains, G6 and G8, were identified for the first time in New Zealand. P genotypes were determined for a random 10% of samples containing the common G-type strains, and all samples with an unusual G-type. All samples able to be tested contained P[8] bearing strains, except for G1P[4], G2P[4], and G8P[14] strains. Importantly, significant differences in strain frequency were found between samples collected from the North and South Islands of New Zealand. G1 was the most commonly identified strain in the North Island (81.9%); whereas G4 predominated in the South Island (39.6%). Of note, no significant differences in the relative frequency of rotavirus strains were observed between samples collected from children treated in hospital compared to samples collected from children seen by their primary healthcare provider in the community. Regional strain variation highlights the importance of ensuring multi-center surveillance to help monitor program effectiveness when rotavirus vaccines are introduced into New Zealand's national childhood immunization schedule.

Accelerating the secondary immune response by inactivating CD4(+)CD25(+) T regulatory cells prior to BCG vaccination does not enhance protection against tuberculosis.

CD4(+)CD25(+) natural T regulatory cells (Tregs) have been shown to suppress protective immune responses in several different vaccination models. Since the effect of Tregs on vaccination against tuberculosis (Tb) was unknown, we used a murine model to investigate whether natural Tregs suppress the development of protective immunity following Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. Using a monoclonal antibody against CD25, natural Tregs were inactivated prior to vaccination with BCG. The primary immune response was evaluated after BCG vaccination and the secondary immune response was assessed after an intranasal BCG challenge 42 days after vaccination. Inactivation of natural Tregs prior to vaccination led to an increased immune response 14 days after vaccination, increased numbers of antigen-responsive lymphocytes immediately prior to secondary challenge and the earlier appearance of IFN-gamma-producing CD4(+) and CD8(+) lymphocytes in the draining lymph nodes and lungs after challenge. Despite this, protection from virulent Mycobacterium tuberculosis or M. bovis aerosol challenge was unaffected by natural Treg inactivation prior to BCG vaccination. This suggests that increasing the primary and accelerating the secondary immune responses by inactivating natural Tregs at the time of vaccination, does not affect the development of protective immunity to Tb.

Distinct patterns of evolution between respiratory syncytial virus subgroups A and B from New Zealand isolates collected over thirty-seven years.

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract infections in infants and children worldwide. In New Zealand, infants with RSV disease are hospitalized at a higher rate than other industrialized countries, without a proportionate increase in known risk factors. The molecular epidemiology of RSV in New Zealand has never been described. Therefore, we analyzed viral attachment glycoprotein (G) gene sequences from 106 RSV subgroup A isolates collected in New Zealand between 1967 and 2003, and 38 subgroup B viruses collected between 1984 and 2004. Subgroup A and B sequences were aligned separately, and compared to sequences of viruses isolated from other countries during a similar period. Genotyping and clustering analyses showed RSV in New Zealand is similar and temporally related to viruses found in other countries. By quantifying temporal clustering, we found subgroup B viruses clustered more strongly than subgroup A viruses. RSV B sequences displayed more variability in stop codon usage and predicted protein length, and had a higher degree of predicted O-glycosylation site changes than RSV A. The mutation rate calculated for the RSV B G gene was significantly higher than for RSV A. Together, these data reveal that RSV subgroups exhibit different patterns of evolution, with subgroup B viruses evolving faster than A.

Inactivation of CD4+ CD25+ regulatory T cells during early mycobacterial infection increases cytokine production but does not affect pathogen load.

Mycobacterium tuberculosis uses numerous mechanisms to avoid elimination by the infected host. In this study, we investigated the possibility whether, similar to other pathogens, M. tuberculosis exploits natural CD4+ CD25+ T-regulatory cells (Treg) to suppress the effector function of responding host lymphocytes, thus enhancing its survival. During a Mycobacterium bovis bacille calmette guerin (BCG) pulmonary infection, we observed a 2.8-fold increase in forkhead box P3 (Foxp3+) CD25+ Treg in the lung. To inactivate the Treg in vivo, an mAb was given against CD25 (PC61) 3 days before a pulmonary infection with BCG or M. tuberculosis. Following PC61 treatment, we observed significantly decreased CD25 expression on CD4+ T lymphocytes for at least 23 days in the blood, spleen and lung when compared with the control mice. To determine whether Treg inactivation affected the protective antimycobacterial immune response, we measured cytokine production by flow cytometry. We observed small, but significant increases in the percentages of both IFN-gamma-producing and IL-2-producing CD4+ cells from the spleen and the IL-2-producing CD4+ cells from the lungs of PC61-treated BCG-infected mice compared with the infected control mice. Despite this, there was neither a difference between the lung bacterial burdens of PC61-treated mice and control mice, measured until day 44 postinfection, nor was there an effect on infection-induced lung pathology. Together, these data imply that the absence of natural Treg early after infection results in a small increase in cytokine production, but this does not alter the course of either M. tuberculosis or BCG infections. This contrasts with the important role that natural Treg play in the pathogenesis of many other intracellular infectious organisms.