Progress in the Utilisation of Organometallic Compounds in the Development of Cancer Drugs.

BACKGROUND: Organometallic compounds are chemical substances containing a carbon-metal bond. From a biological point of view, these compounds are generally considered to be toxic for living organisms. They may exert therapeutic potential, especially as anticancer or antimicrobial drugs. Their structural variability and usually uncharged and mostly lipophilic character are particularly advantageous properties. Platinum derivatives (predominately cisplatin) are the most proven advantageous agents in the medical field. The success of cisplatin has led the scientific community to focus on the synthesis of other organometallic compounds with improved anti-tumour effects and lower cytotoxicity towards healthy tissues. Close attention is focused on compounds bearing atoms of iron, titanium or ruthenium. PURPOSE: Here, we focus on summarising a description of the most important compounds containing iron, titanium or ruthenium atoms in their structure, showing potential application in cancer treatment including the mechanism of action for some of the most commonly studied compounds. The reported structures were used successfully in preclinical studies including animal models and progressed to various stages of human clinical trials. Despite the fail-ure of some of these compounds, there are still several candidates which are expected to progress to the late stages of the clinical trials either alone or as part of combined chemotherapy. Ruthenium-containing substances in particular show high potential for utilisation in cancer treatment due to low cytotoxicity associated with the ability to block neoangiogenesis and metastasis development.

Overview of Current Findings about the Role of Oestrogen Receptor α in Cancer Cell Signalling Pathways

BACKGROUND: Oestrogen receptor α is a key biomarker for breast cancer, and the presence or absence of oestrogen receptor α in breast cancer influences the treatment regimens and patients' prognosis. Oestrogen receptors α are activated after ligand binding, then translocate into the nucleus and activate the transcription of specific genes. This process is called the genomic effect of oestrogen receptor α. Oestrogen receptor α also has nongenomic effects that are exerted mainly in cytoplasm. Due to the important involvement of oestrogen receptor α in cell signalling, these receptors represent a key target for anticancer therapy. PURPOSE: Although oestrogen receptor α was discovered 60 years ago, the corresponding signalling pathways have not yet been fully described due to their complexity. With respect to the considerable extent of oestrogen receptor α signalling, covering all related information is beyond the scope of this review, which is focused mainly on recently discovered aspects of oestrogen receptor α function.

[Molecular Pathology of Colorectal Cancer, Microsatellite Instability - the Detection, the Relationship to the Pathophysiology and Prognosis]. / Molekulární patologie kolorektálního karcinomu, mikrosatelitová nestabilita - zpusob detekce, vztah k patofyziologii a prognóze.

BACKGROUND: Colorectal carcinoma (CRC) is third most common cancer worldwide with very heterogenous character. In most cases, it is caused by sporadic events leading to disruption of epithelial cells of the colon. The minority evolves from germline mutations associated with hereditary cancer syndromes. Mechanisms leading to mutations of oncogenes, tumour suppressors and genes of DNA repair mechanisms include: 1. chromosomal instability, 2. microsatellite instability and 3. CpG island methylator phenotype. Microsatellite instability (MSI) usually arises from a germline mutation of the component of mismatch repair machinery (MMR) or somatic hypermethylation of the MLH1 promoter. The diagnostic approaches include PCR methods and immunohistochemistry for the detection of the loss of MMR part. The aim of our study was to characterise the cohort of ongoing study of gut microbiome in CRC patients considering MSI. MATERIAL AND METHODS: The consecutive study group consisted of 103 patients diagnosed with CRC. The cohort consisted of 45 women (43.7%) and 58 men (56.3%). Patient age at the time of diagnosis was within the range of 31-83 years (median 66 years). The expression of MLH1, MSH2, MSH6 and PMS2 proteins was detected by immunohistochemical method and the positivity was correlated with the stage and the localization of the primary tumour. RESULTS: The MMR status was determined by immunohistochemical method in 43 (41.7%) from the existing total of 103 patients. MSI was detected in 11 (25.6%) cases while 32 (74.4%) were microsatellite stabile. With the respect to cancer clasification the most cases of MSI was detected in stage II (8 cases; 22.2%). In regard to localization of primary tumour, MSI rather correlates to right site CRC, while microsatellite stable tumours do not show any site preferences. CONCLUSION: Considering low number of MMR status determination in study group, statistic evaluation is inaccurate so far. However there is a trend in our cohort in relation to determination of the portion of MSI in CRC population and also in localization of primary tumour according to literature.Key words: colorectal carcinoma - microsatellite instability - Lynch syndrome The work was supported by the project MEYS - NPS I - LO1413 and AZV 16-31966A. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

[MicroRNA Analysis in Epithelial Ovarian Cancer]. / Analýza mikroRNA u epiteliálního karcinomu vajecníku.

BACKGROUND: Ovarian carcinoma is a type of cancer with high mortality, which is often diagnosed in late stages indicating the necessity to identify new non-invasive biomarkers for detection this malignancy in early stages of the disease. MicroRNAs (miRNAs) seem to be one of the potential possibilities. MiRNAs are small noncoding RNA molecules, which regulate gene expression and are involved in many cellular processes including carcinogenesis. PATIENTS AND METHODS: Total RNA was isolated from sera of nine ovarian cancer patients treated at Masaryk Memorial Cancer Institute. As a control samples, we used sera from six cancer-free women. Purified RNA was subjected to reverse transcription followed by cDNA preamplification. MiRNA expression was determined using TaqMan MicroRNA Assays. For statistical analysis was used nonparametric Mann-Whitney U test. RESULTS: MiRNAs miR-30a-5p and miR-26b were identified as significantly overexpressed miRNAs in sera from ovarian cancer patients. Other miRNAs showing elevated level were identified as miR-628-5p, 520c-3p, miR-486 and let-7b. On the other hand, miR-596 showed reduced levels in sera from ovarian cancer patients. CONCLUSION: A significantly different level of at least two miRNAs (miR-30a-5p a miR-26b) has been observed in cancer patients in comparison with healthy individuals. These miRNAs would serve as a powerful non-invasive diagnostic tool to detect ovarian epithelial cancer in so called liquid biopsies.Key words: microRNA - biomarkers - ovarian epithelial cancer - qPCR This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

[Cancer Cells as Dynamic System - Molecular and Phenotypic Changes During Tumor Formation, Progression and Dissemination]. / Nádorové bunky jako dynamický systém - molekulární a fenotypové zmeny v prubehu vzniku, progrese a sírení nádoru.

Dynamic, punctual and perfectly coordinated cellular response to internal and external stimuli is a crucial prerequisite for adaptation of mammalian cells to all changes that occur during cellular development under physiological conditions. Hijacking this ability is characteristic for tumor cells that are capable to adapt to unfavorable conditions which contribute to the formation and development of cancer during the process of tumor formation and progression. By changing key mechanisms, malignant cells can avoid cell death and thus allow development and spread of the tumor. The changes at the genetic level are manifested by various phenotypic characteristics, through which tumor cells are able to escape defense mechanisms, to acquire resistance to treatment, to invade and to create secondary tumors. In recent years, one of the most studied properties include changes in energy metabolism, when tumor cells specifically control reprogramming of the main metabolic pathways for their own benefit and to satisfy their increased needs not only for energy, but also for building materials required for increased proliferation. To adapt to extracellular conditions, it is necessary that cells undergo morphological changes, where modifications in the cell shape through reorganization of cytoskeletal filaments allow tumor cells to increase their invasiveness and other aggressive features. Clarifying these changes together with understanding of the switch in the genetic program within cancer cells, which allows them to overcome different stages of differentiation from cancer stem cells to fully differentiated cells, would be an important prerequisite for identification of the cancer cell "weaknesses" and may lead to improved cancer treatment. The ability of tumor cells to alter the rules of their own organism thus represents an important challenge for oncological research.Key words: cellular reprogramming - cancer cell plasticity - cancer metabolism - tumor heterogeneity - cytoskeleton remodeling - metastasis - oncogenesisThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 3. 7. 2016Accepted: 11. 8. 2016.

[Multistep Process of Establishing Carcinoma Metastases]. / Vícestupnový proces vzniku vzdálených metastáz u karcinomu.

BACKGROUND: Dissemination of cancer cells from the primary tumor and establishment of therapy-resistant distant metastases is the most common cause of human cancer deaths. The primary tumor consists of a heterogeneous population of cancer cells that have to overcome activity of the immune system, insufficient delivery of nutrients and oxygen, chemotherapy, radiotherapy etc. that lead to the selection of resistant and plastic cancer cells. Another selection pressure during metastatic spread gives rise to resistant subpopulations of cells, capable of surviving and proliferating in the hostile microenvironment of distant tissues. AIM: In this article, individual steps of the metastatic cascade are described as well as the mechanisms and signaling pathways that cancer cells use to deal with them. Metastatic process is generally inefficient and only very few cells released from the primary tumor develop into metastases. This success is enabled by pro-metastatic mutations, accumulated due to the selection pressure and also by cooperation of non-transformed cells that secrete supporting factors. CONCLUSION: Recent advances in research provide deeper insights into the complex processes that lead to formation and dissemination of cancer cells. Deciphering the key points of metastatic cascade and principles of its regulation will perhaps lead to development of efficient therapeutics targeting metastatic cells.Key words: metastasis - carcinoma - vascular endothelial growth factor A - epithelial-mesenchymal transitioThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 19. 5. 2016.

[Circulating Tumor DNA in Blood and Its Utilization as a Potential Biomarker for Cancer]. / Cirkulující nádorová DNA v krvi a její vyuzití jako potenciálního bio-markeru nádorových onemocnení.

Pursuing sensitive methods for detection and monitoring of oncologic diseases, that would limit the stress for patients, represents a longstanding challenge in cancer diagnostics. As an ideal noninvasive bio-markers may be considered  bio-logical molecules that can be detected in blood and that provide most relevant picture about the state and development of disease. In fact, all types of cancer cells carry somatic mutations that enable the cells to escape from regulation and to grow and progress. These mutations are only present in the DNA of tumor cells and thus are hallmarks of cancer cells. Genotyping of tumor tissues becomes a common technique in clinical oncology, but it has its limits. Tissue biopsy only yields information about a very small area of tumor at the time of extraction and in some cases it is difficult or impossible to obtain the tissue sample. Furthermore, it is an invasive method that can stress patients. Analysis of circulating tumor DNA from blood--the so-called liquid biopsy--represents one possible solution. Dying tumor cells release fragments of their DNA into the blood stream. From blood, they can be isolated and subjected to analysis using new, sensitive and precise methods that detect genomic changes. These changes are evolving over time because cancer disease is characterized by evolution and ability to select new mutations that bring growth advantages or resistance to treatment. Our inability to capture the heterogeneity during tumor development is one of the major reasons responsible for failure of cancer treatment. Recent technological progress in detection and characterization of circulating DNA could enable tumor evolution monitoring in real time and become a guideline for an accurate and prompt treatment choice.

Electrochemical analysis of a novel ferrocene derivative as a potential antitumor drug.

Ferrocenes represent an interesting group of drugs with potential antitumor properties. Moreover, their electronic properties make them suitable for electrochemical studies. We determined an uptake of a novel ferrocene derivative in low µM concentrations by selected cancer cell lines and showed its localization predominantly in cytoplasm, using glassy carbon electrodes.

[Development of PCR methods and their applications in oncological research and practice]. / Vývoj metod zalozených na PCR a jejich aplikace v onkologickém výzkumu a praxi.

Since its discovery, PCR has become a conventional method of molecular biology research laboratories and an indispensable tool in diagnostic medicine. Multiple variants of the PCR technique were developed, which enable the analysis of different bio-logical materials at different amounts and reaction conditions. This article briefly summarizes the PCR approaches and points out their applications in oncological research and practice.

Second primary cancers -  causes, incidence and the future.

Thanks to continually improving screening programs, diagnostic, and treatment methods, the survival rate in newly diagnosed cancer patients is increasing. With this improvement in survival, attention is now being focused on potential longterm complications such as multiple primary tumors, which represent a leading cause of late nonrelapse mortality. The number of patients who survive cancer dia-gnosis is growing by 2% each year. Multiple primary neoplasms have become the third most common finding in oncology since 1890s, when they were first described. This review aims to summarize recent information regarding the multiple primary neoplasms, elucidate the definition, etiology, association with the primary cancer treatment, genetic and environmental dispositions and finally, it recapitulates new approaches to identification of the risk factors for multiple cancers.

Emerging roles for the pro-oncogenic anterior gradient-2 in cancer development.

Clinical studies have defined the core 'genetic blueprint' of a cancer cell, but this information does not necessarily predict the cancer phenotype. Signalling hubs that mediate such phenotype have been identified largely using OMICS platforms that measure dynamic molecular changes within the cancer cell landscape. The pro-oncogenic protein anterior gradient 2 (AGR2) is a case in point; AGR2 has been shown using a range of expression platforms to be involved in asthma, inflammatory bowel disease, cell transformation, cancer drug resistance and metastatic growth. AGR2 protein is also highly overexpressed in a diverse range of human cancers and can be secreted and detected in extracellular fluids, thus representing a compelling pro-oncogenic signalling intermediate in human cancer. AGR2 belongs to the protein disulphide isomerase family with all the key features of an endoplasmic reticulum-resident protein-this gives clues into how it might function as an oncoprotein through the regulation of protein folding, maturation and secretion that can drive metastatic cell growth. In this review, we will describe the known aspects of AGR2 molecular biology, including gene structure and regulation, emerging protein interaction networks and how its subcellular localization mediates its biological functions. We will finally review the cases of AGR2 expression in human cancers, the pathophysiological consequences of AGR2 overexpression, its potential role as a tumour biomarker that predicts the response to therapy and how the AGR2 pathway might form the basis for drug discovery programmes aimed at targeting protein folding/maturation pathways that mediate secretion and metastasis.

C-terminal phosphorylation of Hsp70 and Hsp90 regulates alternate binding to co-chaperones CHIP and HOP to determine cellular protein folding/degradation balances.

Heat shock proteins Hsp90 and Hsp70 facilitate protein folding but can also direct proteins for ubiquitin-mediated degradation. The mechanisms regulating these opposite activities involve Hsp binding to co-chaperones including CHIP and HOP at their C-termini. We demonstrated that the extreme C-termini of Hsp70 and Hsp90 contain phosphorylation sites targeted by kinases including CK1, CK2 and GSK3-ß in vitro. The phosphorylation of Hsp90 and Hsp70 prevents binding to CHIP and thus enhances binding to HOP. Highly proliferative cells contain phosphorylated chaperones in complex with HOP and phospho-mimetic and non-phosphorylable Hsp mutant proteins show that phosphorylation is directly associated with increased proliferation rate. We also demonstrate that primary human cancers contain high levels of phosphorylated chaperones and show increased levels of HOP protein and mRNA. These data identify C-terminal phosphorylation of Hsp70 and Hsp90 as a switch for regulating co-chaperone binding and indicate that cancer cells possess an elevated protein folding environment by the concerted action of co-chaperone expression and chaperone modifications. In addition to identifying the pathway responsible for regulating chaperone-mediated protein folding/degradation balances in normal cells, the data provide novel mechanisms to account for the aberrant chaperone activities observed in human cancer cells and have implications for the application of anti-chaperone therapies in cancer treatment.

Cancer as a metabolic disease and diabetes as a cancer risk?

The prevailing aerobic glycolysis (so called Warburg effect) in cancer cells is according to current understanding the consequence of reprogramming of cellular metabolism during the process of malignant transformation. Metabolic regulation is inseparable component of cell proliferation machinery and has a tight link with activities of oncogenes and suppressor genes. The purpose of metabolic reprogramming of cancer (but also normal intensively proliferating cells) is to incorporate greater fraction of glucose metabolites into newly synthesised macromolecules. Apart from that, aerobic glycolysis confers several other selective advantages to cancer cells. Epidemiological data indicate that type 2 diabetes mellitus is associated with increased incidence of several types of cancer and that cancer mortality can be influenced by certain types of anti-diabetic treatment, however future research is needed to explain whether this relationship might be causal. Deeper knowledge about metabolic properties of rapidly proliferating cells can be exploited for further improvement of anti-cancer, immunosuppressive or anti-inflammatory therapies.

The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers.

Transcriptomic screens in breast cancer cell lines have identified a protein named anterior gradient-2 (AGR2) as a potentially novel oncogene overexpressed in estrogen receptor (ER) positive tumours. As targeting the ER is responsible for major improvements in cure rates and prevention of breast cancers, we have evaluated the pro-oncogenic function of AGR2 in anti-hormone therapeutic responses. We show that AGR2 expression promotes cancer cell survival in clonogenic assays and increases cell proliferation and viability in a range of cancer cell lines. Chromatin immunoprecipitation and reporter assays indicate that AGR2 is transcriptionally activated by estrogen through ERalpha. However, we also found that AGR2 expression is elevated rather than inhibited in response to tamoxifen, thus identifying a novel mechanism to account for an agonistic effect of the drug on a specific pro-oncogenic pathway. Consistent with these data, clinical analysis indicates that AGR2 expression is related to treatment failure in ERalpha-positive breast cancers treated with tamoxifen. In contrast, AGR2 is one of the most highly suppressed genes in cancers of responding patients treated with the anti-hormonal drug letrozole. These data indicate that the AGR2 pathway represents a novel pro-oncogenic pathway for evaluation as anti-cancer drug developments, especially therapies that by-pass the agonist effects of tamoxifen.

MicroRNA-181 family predicts response to concomitant chemoradiotherapy with temozolomide in glioblastoma patients.

MicroRNAs are endogenously expressed regulatory noncoding RNAs. Previous studies showed altered expression levels of several microRNAs in glioblastomas. In this study, we examined the expression levels of selected microRNAs in 22 primary glioblastomas and six specimens of adult brain tissue by real-time PCR method. In addition, we examined methylation status of MGMT promoter by methylation-specific real-time PCR, as this has been shown to be a predictive marker in glioblastomas. MGMT methylation status was not correlated with response to concomitant chemoradiotherapy with temozolomide (RT/TMZ). MiR-221 (p=0.016), miR-222 (p=0.038), miR-181b (p=0.036), miR-181c (p=0.043) and miR-128a (p=0.001) were significantly down-regulated in glioblastomas. The most significant change was observed for up-regulation in miR-21 expression in glioblastomas (p<0.001). MiR-181b and miR-181c were significantly down-regulated in patients who responded to RT/TMZ (p=0.016; p=0.047, respectively) in comparison to patients with progredient disease. Our data indicate for the first time that expression levels of miR-181b and miR-181c could serve as a predictive marker of response to RT/TMZ therapy in glioblastoma patients.

Significant overexpression of Hsp110 gene during colorectal cancer progression.

Colorectal cancer (CRC) is one of the most frequent malignant diseases in the world. Metastatic spread of the cancer to the lymph nodes is a crucial factor for progression and therapeutic management of the disease. We analysed gene expression profiles of CRC patiens by low-density cancer-focused oligonucleotide microarrays to identify new predictive markers of the extent of the disease and for better understanding of CRC progression. Relative expression levels of 440 genes known to be involved in cancer biology were obtained by low-density oligonucleotide microarrays from 20 tumor samples. Statistical analysis of gene expression data identified 3 genes (HSP110, HYOU1 and TCTP) significantly up-regulated in primary tumors of patients who developed lymph node metastasis. We have shown, for the first time, that up-regulation HSP110 and HYOU1 expression is associated with lymph node involvement in CRC. We validated the differences in HSP110 expression in an independent group of 30 patients of all clinical stages by real-time PCR. We identified significant up-regulation of HSP110 expression in colorectal tumors compared to adjacent non-tumoral tissue (p<0.0003). We observed significant differences of HSP110 gene expression between metastatic and localized disease (p=0.031) and negative trend of HSP110 gene expression and overall survival of CRC patients. We suggest that HSP110 gene is a promising molecular predictor in CRC.

[The significance of methylation in HPV16 genome to cervix cancerogenesis]. / Význam metylace v genomu lidského papillomaviru 16 u karcinomu delozního hrdla.

OBJECTIVE: To summarize the significance of methylation in HPV16 genome to cervix cancerogenesis. DESIGN: Review. SETTING: Department of Oncological and Experimental Pathology, Masaryk Memorial Cancer Institute, Brno. SUBJECT AND METHOD: Human papillomaviruses, especially HPV16, are the most frequent causative agents of cervical intraepithelial neoplasia and cervical carcinoma. Their ability to initiate transformation of infected epithelial cells fully depends up production of viral early phase proteins E6 and E7. Affected keratinocytes activate defensive mechanisms based on inhibition of viral DNA transcription by changes in chromatin structure like DNA methylation or histon deacetylation and therefore prevent transcriptional factors from binding to target promoters and from the production of viral oncoproteins. CONCLUSION: Research into epigenetic mechanisms of gene silencing clearly showed their important roles in etiology of cancer. Recent findings confirm the significance of methylation of HPV16 oncogenes leading to block of neoplastic transformation, and simultaneously they indicate new therapeutic possibilities linked with reactivation of methylated tumor supressors.

[Involvement of microRNAs in cancer biology and possibilities of their application to diagnostic and predictive oncology]. / Zapojení mikroRNA do patogeneze nádorových onemocnení a moznosti jejich vyuzití v diagnostické a prediktivní onkologii.

MicroRNAs (miRNAs) are large class of non-coding RNAs that post-transcriptionally regulate gene expression. Their ability of translational repression applied for example on oncogenes or tumor-suppressor genes indicates involvement of miRNAs in multi-step carcinogenesis. Evidences of miRNAs linkage to biological processes like apoptosis, proliferation, differentiation and cell survival are rapidly accumulating. Approximately 50% of miRNAs are located at fragile sites of chromosomes or regions known to be amplified or deleted in human cancer. That is why, non-coding miRNAs seem to be another level of genetic information which regulation is altered or lost during neoplastic growth. Expression profiles of miRNAs are successfully used for molecular classification, more exact diagnosis and prognosis of human cancers and reached analogical analytical characteristics like studies based on DNA micro-arrays technology and profiling of coding transcripts. In this review we attempt to introduce basic knowledge of miRNAs biogenesis and biological functions and in particular summarise reports focused on miRNAs in oncology research area.

Chaperone-dependent stabilization and degradation of p53 mutants.

p53 missense mutant proteins commonly show increased stability compared to wild-type p53, which is thought to depend largely on the inability of mutant p53 to induce the ubiquitin ligase MDM2. However, recent work using mouse models has shown that the accumulation of mutant p53 occurs only in tumour cells, indicating that stabilization requires additional factors. To clarify the stabilization of p53 mutants in tumours, we analysed factors that affect their folding and degradation. Although all missense mutants that we studied are more stable than wild-type p53, the levels correlate with individual structural characteristics, which may be reflected in different gain-of-function properties. In the absence of Hsp90 activity, the less stable unfolded p53 mutants preferentially associate in a complex with Hsp70 and CHIP (carboxy terminus of Hsp70-interacting protein), and we show that CHIP is responsible for ubiquitination and degradation of these mutants. The demonstration of a complex interplay between Hsp90, Hsp70 and CHIP that regulate the stability of different p53 mutant proteins improves our understanding of the pro-tumorigenic effects of increased Hsp90 activity during multi-stage carcinogenesis. Understanding the roles of Hsp90, Hsp70 and CHIP in cancers may also provide an important avenue through which to target p53 to enhance treatment of human cancers.

Francisella tularensis strain LVS resides in MHC II-positive autophagic vacuoles in macrophages.

The Francisella tularensis strain LVS phagosome disintegrates during the first few hours after bacterial entry and microbes are released to the cytosol. Within 12 h both rapid multiplication of microbes and a steep increase of apoptosis of infected macrophages occur. We searched for signals involved in the death of macrophages and detected molecules associated with the autophagy machinery cathepsin D, PTEN, p53 and LC3, whose levels or modification were influenced by ongoing in vitro tularemic infection. The sequestration of cytoplasmic F. tularensis LVS into autophagosomes was confirmed by co-localization of the LVS strain containing vacuoles with LC3 (an autophagosomal marker). We also demonstrated the presence of MHC II antigens in these autophagosomes, indicating that they might act as a source of endogenous tularemic antigens for presentation to CD4+ T lymphocytes.