Phenol-Soluble Modulin α3 Stimulates Autophagy in HaCaT Keratinocytes.

BACKGROUND: Phenol-soluble modulins (PSMs) are pore-forming toxins (PFTs) produced by staphylococci. PSMs exert diverse cellular effects, including lytic, pro-apoptotic, pro-inflammatory and antimicrobial actions. Since the effects of PSMs on autophagy have not yet been reported, we evaluated the autophagic activity in HaCaT keratinocytes treated with recombinant PSMα3. METHODS: The autophagic flux and levels of autophagic marker proteins were determined using Western blot analysis. Subcellular localization of LC3B and Beclin-1 was investigated using an indirect immunofluorescence assay. The ultrastructural features of control and PSMα3-treated cells were evaluated via transmission electron microscopy. Cytoplasmic acidification was measured via acridine orange staining. Phosphorylation levels of protein kinases, implicated in autophagy regulation, were studied using a phospho-kinase array and Western blot analysis. RESULTS: PSMα3 facilitated the intracellular redistribution of LC3B, increased the average number of autophagosomes per cell, promoted the development of acidic vesicular organelles, elevated the levels of LC3B-II, stimulated autophagic flux and triggered a significant decrease in the net autophagic turnover rate. PSMα3 induced the accumulation of autophagosomes/autolysosomes, amphisomes and multilamellar bodies at the 0.5, 6 and 24 h time points, respectively. The phospho-Akt1/2/3 (T308 and S473), and phospho-mTOR (S2448) levels were decreased, whereas the phospho-Erk1/2 (T202/Y204 and T185/Y187) level was increased in PSMα3-treated cells. CONCLUSIONS: In HaCaT keratinocytes, PSMα3 stimulates autophagy. The increased autophagic activity elicited by sub-lytic PSM concentrations might be an integral part of the cellular defense mechanisms protecting skin homeostasis.

Herpes Simplex Virus Infection Alters the Immunological Properties of Adipose-Tissue-Derived Mesenchymal-Stem Cells.

The proper functioning of mesenchymal stem cells (MSCs) is of paramount importance for the homeostasis of the body. Inflammation and infection can alter the function of MSCs, which can also affect the regenerative potential and immunological status of tissues. It is not known whether human herpes simplex viruses 1 and 2 (HSV1 and HSV2), well-known human pathogens that can cause lifelong infections, can induce changes in MSCs. In non-healing ulcers, HSV infection is known to affect deeper tissue layers. In addition, HSV infection can recur after initially successful cell therapies. Our aim was to study the response of adipose-derived MSCs (ADMSCs) to HSV infection in vitro. After confirming the phenotype and differentiation capacity of the isolated cells, we infected the cells in vitro with HSV1-KOS, HSV1-532 and HSV2 virus strains. Twenty-four hours after infection, we examined the gene expression of the cells via RNA-seq and RT-PCR; detected secreted cytokines via protein array; and determined autophagy via Western blot, transmission electron microscopy (TEM) and fluorescence microscopy. Infection with different HSV strains resulted in different gene-expression patterns. In addition to the activation of pathways characteristic of viral infections, distinct non-immunological pathways (autophagy, tissue regeneration and differentiation) were also activated according to analyses with QIAGEN Ingenuity Pathway Analysis, Kyoto Encyclopedia of Genes and Genome and Genome Ontology Enrichment. Viral infections increased autophagy, as confirmed via TEM image analysis, and also increased levels of the microtubule-associated protein light chain 3 (LC3B) II protein. We identified significantly altered accumulation for 16 cytokines involved in tissue regeneration and inflammation. Our studies demonstrated that HSV infection can alter the viability and immunological status of ADMSCs, which may have implications for ADMSC-based cell therapies. Alterations in autophagy can affect numerous processes in MSCs, including the inhibition of tissue regeneration as well as pathological differentiation.

Transcriptome dataset of six human pathogen RNA viruses generated by nanopore sequencing.

Long-read sequencing (LRS) approaches shed new light on the complexity of viral (Kakuk et al., 2021 [1]; Boldogkoi et al., 2019 [2]; Depledge et a., 2019 [3]), bacterial (Yan et al., 2018 [4]) and eukaryotic (Tilgner et al., 2014 [5]) transcriptomes. Emerging RNA viruses are zoonotic (Woolhouse et al., 2016 [6]) and create public health problems, e.g. influenza pandemic caused by H1N1 virus in (Fraser et al., 2009 [7]), as well as the current SARS-CoV-2 pandemic (Kim et al., 2020 [8]). In this study, we carried out nanopore sequencing for generating transcriptomic data valuable for structural and kinetic profiling of six important human pathogen RNA viruses, the H1N1 subtype of Influenza A virus (IVA), the Zika virus (ZIKV), the West Nile virus (WNV), the Crimean-Congo hemorrhagic fever virus (CCHFV), the Coxsackievirus [group B serotype 5 (CVB5)] and the Vesicular stomatitis Indiana virus (VSIV), and the response of host cells upon viral infection. The raw sequencing data were filtered during basecalling and only high quality reads (Qscore ≥ 7) were mapped to the appropriate viral and host genomes. Length distribution of sequencing reads were assessed and statistics of data were plotted by the ReadStat.4 python script. The datasets can be used to profile the transcriptomic landscape of RNA viruses, provide information for novel gene annotations, can serve as resource for studying the virus-host interactions, and for the analysis of RNA base modifications. These datasets can be used to compare the different sequencing techniques, library preparation approaches, bioinformatics pipelines, and to analyze the RNA profiles of viruses with small RNA genomes.

IL-36α and Lipopolysaccharide Cooperatively Induce Autophagy by Triggering Pro-Autophagic Biased Signaling.

Autophagy is an intracellular catabolic process that controls infections both directly and indirectly via its multifaceted effects on the innate and adaptive immune responses. It has been reported that LPS stimulates this cellular process, whereas the effect of IL-36α on autophagy remains largely unknown. We therefore investigated how IL-36α modulates the endogenous and LPS-induced autophagy in THP-1 cells. The levels of LC3B-II and autophagic flux were determined by Western blotting. The intracellular localization of LC3B was measured by immunofluorescence assay. The activation levels of signaling pathways implicated in autophagy regulation were evaluated by using a phosphokinase array. Our results showed that combined IL-36α and LPS treatment cooperatively increased the levels of LC3B-II and Beclin-1, stimulated the autophagic flux, facilitated intracellular redistribution of LC3B, and increased the average number of autophagosomes per cell. The IL36α/LPS combined treatment increased phosphorylation of STAT5a/b, had minimal effect on the Akt/PRAS40/mTOR pathway, and reduced the levels of phospho-Yes, phospho-FAK, and phospho-WNK1. Thus, this cytokine/PAMP combination triggers pro-autophagic biased signaling by several mechanisms and thus cooperatively stimulates the autophagic cascade. An increased autophagic activity of innate immune cells simultaneously exposed to IL-36α and LPS may play an important role in the pathogenesis of Gram-negative bacterial infections.

Nanopore Assay Reveals Cell-Type-Dependent Gene Expression of Vesicular Stomatitis Indiana Virus and Differential Host Cell Response.

Vesicular stomatitis Indiana virus (VSIV) of genus Vesiculovirus, species IndianaVesiculovirus (formerly as Vesicular stomatitis virus, VSV) causes a disease in livestock that is very similar to the foot and mouth disease, thereby an outbreak may lead to significant economic loss. Long-read sequencing (LRS) -based approaches already reveal a hidden complexity of the transcriptomes in several viruses. This technique has been utilized for the sequencing of the VSIV genome, but our study is the first for the application of this technique for the profiling of the VSIV transcriptome. Since LRS is able to sequence full-length RNA molecules, it thereby provides more accurate annotation of the transcriptomes than the traditional short-read sequencing methods. The objectives of this study were to assemble the complete transcriptome of using nanopore sequencing, to ascertain cell-type specificity and dynamics of viral gene expression, and to evaluate host gene expression changes induced by the viral infection. We carried out a time-course analysis of VSIV gene expression in human glioblastoma and primate fibroblast cell lines using a nanopore-based LRS approach and applied both amplified and direct cDNA sequencing (as well as cap-selection) for a fraction of samples. Our investigations revealed that, although the VSIV genome is simple, it generates a relatively complex transcriptomic architecture. In this study, we also demonstrated that VSIV transcripts vary in structure and exhibit differential gene expression patterns in the two examined cell types.

Combined nanopore and single-molecule real-time sequencing survey of human betaherpesvirus 5 transcriptome.

Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.

COVID-19-associated diarrhea.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged as a highly virulent respiratory pathogen that is known as the causative agent of coronavirus disease 2019 (COVID-19). Diarrhea is a common early symptom in a significant proportion of patients with SARS-CoV-2 infection. SARS-CoV-2 can infect and replicate in esophageal cells and enterocytes, leading to direct damage to the intestinal epithelium. The infection decreases the level of angiotensin-converting enzyme 2 receptors, thereby altering the composition of the gut microbiota. SARS-CoV-2 elicits a cytokine storm, which contributes to gastrointestinal inflammation. The direct cytopathic effects of SARS-CoV-2, gut dysbiosis, and aberrant immune response result in increased intestinal permeability, which may exacerbate existing symptoms and worsen the prognosis. By exploring the elements of pathogenesis, several therapeutic options have emerged for the treatment of COVID-19 patients, such as biologics and biotherapeutic agents. However, the presence of SARS-CoV-2 in the feces may facilitate the spread of COVID-19 through fecal-oral transmission and contaminate the environment. Thus gastrointestinal SARS-CoV-2 infection has important epidemiological significance. The development of new therapeutic and preventive options is necessary to treat and restrict the spread of this severe and widespread infection more effectively. Therefore, we summarize the key elements involved in the pathogenesis and the epidemiology of COVID-19-associated diarrhea.

Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus.

BACKGROUND: Varicella zoster virus (VZV) is a human pathogenic alphaherpesvirus harboring a relatively large DNA molecule. The VZV transcriptome has already been analyzed by microarray and short-read sequencing analyses. However, both approaches have substantial limitations when used for structural characterization of transcript isoforms, even if supplemented with primer extension or other techniques. Among others, they are inefficient in distinguishing between embedded RNA molecules, transcript isoforms, including splice and length variants, as well as between alternative polycistronic transcripts. It has been demonstrated in several studies that long-read sequencing is able to circumvent these problems. RESULTS: In this work, we report the analysis of the VZV lytic transcriptome using the Oxford Nanopore Technologies sequencing platform. These investigations have led to the identification of 114 novel transcripts, including mRNAs, non-coding RNAs, polycistronic RNAs and complex transcripts, as well as 10 novel spliced transcripts and 25 novel transcription start site isoforms and transcription end site isoforms. A novel class of transcripts, the nroRNAs are described in this study. These transcripts are encoded by the genomic region located in close vicinity to the viral replication origin. We also show that the ORF63 exhibits a complex structural variation encompassing the splice sites of VZV latency transcripts. Additionally, we have detected RNA editing in a novel non-coding RNA molecule. CONCLUSIONS: Our investigations disclosed a composite transcriptomic architecture of VZV, including the discovery of novel RNA molecules and transcript isoforms, as well as a complex meshwork of transcriptional read-throughs and overlaps. The results represent a substantial advance in the annotation of the VZV transcriptome and in understanding the molecular biology of the herpesviruses in general.

Propionibacterium acnes Induces Autophagy in Keratinocytes: Involvement of Multiple Mechanisms.

Propionibacterium acnes is a dominant member of the cutaneous microbiota. Herein, we evaluate the effects of different P. acnes strains and propionic acid on autophagy in keratinocytes. Our results showed that P. acnes strain 889 altered the architecture of the mitochondrial network; elevated the levels of microtubule-associated protein 1 light chain 3B-II, Beclin-1, and phospho-5'-adenosine-monophosphate-activated protein kinase α; stimulated autophagic flux; facilitated intracellular redistribution of microtubule-associated protein 1 light chain 3B; increased average number of autophagosomes per cell; and enhanced development of acidic vesicular organelles in the HPV-KER cell line. Propionic acid increased the level of phospho-5'-adenosine-monophosphate-activated protein kinase α, enhanced lipidation of microtubule-associated protein 1 light chain 3B, stimulated autophagic flux, and facilitated translocation of microtubule-associated protein 1 light chain 3B into autophagosomes in HPV-KER cells. P. acnes strains 889 and 6609 and heat-killed strain 889 also stimulated autophagosome formation in primary keratinocytes to varying degrees. These results indicate that cell wall components and secreted propionic acid metabolite of P. acnes evoke mitochondrial damage successively, thereby triggering 5'-adenosine-monophosphate-activated protein kinase-associated activation of autophagy, which in turn facilitates the removal of dysfunctional mitochondria and promotes survival of keratinocytes. Thus, we suggest that low-level colonization of hair follicles with noninvasive P. acnes strains, by triggering a local increase in autophagic activity, might exert a profound effect on several physiological processes responsible for the maintenance of skin tissue homeostasis.

Long-Read Sequencing of Human Cytomegalovirus Transcriptome Reveals RNA Isoforms Carrying Distinct Coding Potentials.

The human cytomegalovirus (HCMV) is a ubiquitous, human pathogenic herpesvirus. The complete viral genome is transcriptionally active during infection; however, a large part of its transcriptome has yet to be annotated. In this work, we applied the amplified isoform sequencing technique from Pacific Biosciences to characterize the lytic transcriptome of HCMV strain Towne varS. We developed a pipeline for transcript annotation using long-read sequencing data. We identified 248 transcriptional start sites, 116 transcriptional termination sites and 80 splicing events. Using this information, we have annotated 291 previously undescribed or only partially annotated transcript isoforms, including eight novel antisense transcripts and their isoforms, as well as a novel transcript (RS2) in the short repeat region, partially antisense to RS1. Similarly to other organisms, we discovered a high transcriptional diversity in HCMV, with many transcripts only slightly differing from one another. Comparing our transcriptome profiling results to an earlier ribosome footprint analysis, we have concluded that the majority of the transcripts contain multiple translationally active ORFs, and also that most isoforms contain unique combinations of ORFs. Based on these results, we propose that one important function of this transcriptional diversity may be to provide a regulatory mechanism at the level of translation.

Well begun is half done: Rubella virus perturbs autophagy signaling, thereby facilitating the construction of viral replication compartments.

The rubella virus is the causative agent of postnatal German measles and the congenital rubella syndrome. The majority of the rubella virus replication complexes originate from the endomembrane system. The rubella virus perturbs the signaling pathways regulating the formation of autophagic membranes in the infected cells, including the Ras/Raf/MEK/ERK and PI3K/Akt pathways. It is widely accepted that these pathways inhibit autophagy. In contrast, the class III PI3K enzymes are essential for autophagy initiation. By manipulating the Ras/Raf/MEK/ERK, class I PI3K/Akt and class III PI3K axes of signal transduction, the rubella virus may differentially regulate the autophagic cascade, with consequent stimulation of the initiation and strong suppression of the later phases. Dysregulation of autophagy by this virus can have a significant impact on the construction of replication compartments by regulating membrane trafficking. We hypothesize that the rubella virus perturbs the autophagic process in order to prevent the degradation of the virus progeny, and to ensure its replication by hijacking omegasomes for the construction of the replication complexes. The virus is therefore able to utilize an antiviral mechanism to its own advantage. Therapeutic modalities targeting the autophagic process may help to ameliorate the serious consequences of the congenital rubella syndrome.

IL-17A and IL-17F induce autophagy in RAW 264.7 macrophages.

Autophagy is an important cellular catabolic process for the lysosomal degradation of cytoplasmic organelles, proteins and microorganisms. The autophagic process is intertwined with the immune response: autophagy regulates both innate and adaptive immunity, conversely, cytokines produced during the course of the immune response modulate various functions of the autophagic cascade. The IL-17 family member cytokines play a pivotal role in immune protection against extra- and intracellular bacterial pathogens. Since the effects of IL-17A and IL-17F on autophagy have not yet been reported, we have evaluated the autophagic activity in the RAW 264.7 cell line treated with either IL-17A or IL-17F. Both IL-17A and IL-17F proved to promote microtubule-associated protein 1 light chain 3 B-II (LC3B-II) accumulation, enhance the autophagic flux, facilitate the intracellular redistribution of LC3B, increase both the average number and the size of autophagosomes per cell, and foster the formation of acidic vesicular organelles. IL-17F was considerably more efficient than IL-17A in promoting the autophagic process. Further experiments to determine the potential effect of IL-17-induced autophagy on the antibacterial activity of RAW macrophages revealed that IL-17F significantly decreased the intracellular counts of Mycobacterium terrae, while the colony-forming unit values remained comparable in the IL-17A-treated cells and the control cultures. In conclusion, these results demonstrate that IL-17A and IL-17F are capable of inducing autophagy in macrophages, and thereby contribute to the elimination of Mycobacterium terrae. These data may bear on the pathogenesis of infections caused by Mycobacterium terrae, as IL-17 plays a pivotal role in the immune response to mycobacteria. IL-17-mediated activation of autophagy may also be implicated in various infections and other pathological conditions.

Herpes simplex virus types 1 and 2 modulate autophagy in SIRC corneal cells.

Autophagy and apoptosis function as important early cellular defense mechanisms in infections and other diseases. The outcome of an infection is determined by a complex interplay between the pathogenic microorganism and these intracellular pathways. To better understand the cytopathogenicity of Herpes simplex virus types 1 and 2 (HSV-1 and - 2), we studied the effect of these viruses on the autophagic and apoptotic processes in the SIRC corneal cell line. Infection with the KOS strain of HSV-1 and a wild-type strain of HSV-2 enhanced autophagosome formation, triggered cytoplasmic acidification, increased LC3B lipidation and elevated the ratio of apoptotic cells. The autophagy inhibitor bafilomycin A1 triggered a significant increase in the apoptotic responses of HSV-1 and HSV-2-infected cells. Thus, both HSV types affect autophagy and apoptosis in a coordinated fashion, and autophagy plays cytoprotective role in HSV-infected cells via antagonizing apoptosis. Together these data implicate autophagy in the pathogenic mechanism of herpetic keratitis.

Rubella virus perturbs autophagy.

Autophagy is a cellular catabolic process implicated in numerous physiological processes and pathological conditions, including infections. Viruses have evolved different strategies to modulate the autophagic process. Since the effects of rubella virus (RV) on autophagy have not yet been reported, we evaluated the autophagic activity in the Statens Seruminstitut Rabbit Cornea cell line infected with the To336 strain of RV. Our results showed that RV lowered the levels of microtubule-associated protein 1 light chain 3 B-II (LC3B-II) and the autophagy-related gene 12-autophagy-related gene 5 conjugate, inhibited the autophagic flux, suppressed the intracellular redistribution of LC3B, decreased both the average number and the size of autophagosomes per cell and impeded the formation of acidic vesicular organelles. Induction of autophagy by using rapamycin decreased both the viral yields and the apoptotic rates of infected cultures. Besides its cytoprotective effects, autophagy furnishes an important antiviral mechanism, inhibition of which may reorchestrate intracellular environment so as to better serve the unique requirements of RV replication. Together, our observations suggest that RV utilizes a totally different strategy to cope with autophagy than that evolved by other positive-stranded RNA viruses, and there is considerable heterogeneity among the members of the Togaviridae family in terms of their effects on the cellular autophagic cascade.

Inducible expression of human ß-defensin 2 by Chlamydophila pneumoniae in brain capillary endothelial cells.

Defensins are an important family of natural antimicrobial peptides. Chlamydophila pneumoniae, a common cause of acute respiratory infection, has a tendency to cause persistent inflammatory diseases such as atherosclerosis, which may lead to cardiovascular disease or stroke. As endothelial cells are related to the physiopathology of stroke, the effects of in vitro C. pneumoniae infection on the expression of human ß-defensin 2 (HBD-2) in brain capillary endothelial cells (BB19) was investigated. A time-dependent increase in HBD-2 mRNA was observed by means of real-time reverse transcription PCR (RT-PCR) in BB19 cells following C. pneumoniae infection, with a maximum increase at 24 h. A gradual induction of HBD-2 protein in the C. pneumoniae-infected endothelial cells was detected by immunoblotting. Immunofluorescence revealed the staining of HBD-2 in the cytoplasm of endothelial cells following C. pneumoniae infection. The secretion of HBD-2 (confirmed by ELISA) was significantly elevated 24 h after C. pneumoniae infection. These novel results indicate that HBD-2 is expressed and produced in the human brain capillary endothelial cells upon infection with C. pneumoniae, and provide evidence that HBD-2 plays a role in the early immune responses to C. pneumoniae and probably in the immunopathogenesis of atherosclerosis.

Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells.

BACKGROUND: The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC). METHODS: SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins. RESULTS: Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of DeltaNp63alpha. The expressions of the Bax-beta and TAp63gamma isoforms were considerably increased, whereas the level of DeltaNp63alpha was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63gamma. CONCLUSION: These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival DeltaN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.

The herpes simplex virus-induced demise of keratinocytes is associated with a dysregulated pattern of p63 expression.

p63 plays a pivotal role in the development and maintenance of stratified epithelial tissues. In an effort to gain insight into the pathogenic mechanisms of skin infections caused by HSV-1 and HSV-2, we determined the patterns of p63 expression in primary keratinocytes and in the HaCaT cell line. The levels of DeltaNp63alpha and a 50kDa p73 isoform were decreased, Bax-alpha remained unaffected, while the expressions of the Bax-beta, TAp63gamma and a 44.5kDa p73 isoform were highly increased in both HSV-1-infected HaCaT cells and primary keratinocytes. In contrast, in response to HSV-2 infection the levels of DeltaNp63alpha, a 50kDa p73 isoform and a 44.5kDa p73 protein were decreased, Bax-alpha and TAp63gamma remained unaffected, while the expression of Bax-beta was slightly increased. The knockdown of TAp63 expression enhanced the viability of HSV-1-infected cells. Thus, HSV-1 and HSV-2 modulate the patterns of p63 and Bax expression in a serotype-specific manner. The dysregulated pattern of p63 expression observed in HSV-infected keratinocytes may comprise part of a mechanism by which these viruses perturb the functions of keratinocytes and lead to their demise.

Vesicular stomatitis virus infection triggers apoptosis associated with decreased DeltaNp63alpha and increased Bax levels in the immortalized HaCaT keratinocyte cell line.

In view of the powerful inherent oncolytic activity exhibited by the vesicular stomatitis virus (VSV) in several tumor types, we set out to investigate the susceptibility of the immortalized HaCaT keratinocyte cell line to VSV, and analyzed the role of apoptosis in the VSV-mediated induction of cell death. Indirect immunofluorescence assays, Western blot analyses and plaque titrations demonstrated that the HaCaT cell line was permissive to VSV replication. The results of ELISA for detection of the enrichment of nucleosomes in the cytoplasm of apoptotic cells revealed that VSV infection elicits the apoptotic death of HaCaT cells. Mock-infected HaCaT cells displayed the endogenous expression of DeltaNp63alpha, p53 mutated on UV hot spots (p53(mt)), Bcl-2 and p21 Bax. The levels of DeltaNp63alpha and p53(mt) were decreased, Bcl-2 remained unaffected, while the expressions of p21Bax and p18 Bax were increased in VSV-infected HaCaT cells. Together, these data demonstrate that VSV replicates efficiently and triggers apoptosis in the immortalized HaCaT keratinocyte cell line. The VSV-mediated alterations in the expressions of DeltaNp63alpha and Bax may be implicated in the apoptotic responses of infected cells and may also sensitize to other apoptotic stimuli. These findings may stimulate further studies with the goal of developing VSV-based virotherapy into an effective modality for the treatment of epithelial-derived malignant tumors of the skin.

The role of histamine in the intracellular survival of Mycobacterium bovis BCG.

The course and outcome of infection with mycobacteria are determined by a complex interplay between the immune system of the host and the survival mechanisms developed by the bacilli. Histamine plays an important role in various processes, including cell division, metabolism, and apoptosis, and it modulates innate and adaptive immune responses. In the present study we investigated the intracellular survival of Mycobacterium bovis BCG in murine bone-marrow macrophages isolated from wild-type (WT) and histidine-decarboxylase knock-out [HDC (-/-)] mice. Mycobacterial titers were significantly higher in the HDC (-/-) macrophages as compared with the WT cells. M. bovis BCG growth in WT macrophages could be enhanced by pyrilamine and cimetidine. Exogenously added histamine decreased the intracellular counts of M. bovis BCG in HDC (-/-) macrophages. Infection of activated macrophages with M. bovis BCG elicited apoptosis, but there was no significant difference between the WT and the HDC (-/-) cells. These bacilli induced comparable levels of tumor necrosis factor-alpha production in the WT and the HDC (-/-) macrophages. M. bovis BCG stimulated interleukin-18 (IL-18) production in the macrophages from WT mice, but not in the HDC (-/-) cells. Exogenously added IL-18 decreased the titers of intracellular mycobacteria in HDC (-/-) cells. In conclusion, these data implicate histamine in the intracellular survival of M. bovis BCG. The cellular control mechanisms restricting the growth of M. bovis BCG are complex and involve H1 and H2 receptor-mediated events. Histamine might be an important mediator of M. bovis BCG-induced IL-18 production, which in turn contributes to immune protection.

Vesicular stomatitis virus induces apoptosis in the Wong-Kilbourne derivative of the Chang conjunctival cell line.

BACKGROUND: Virotherapy represents a novel therapeutic modality for the treatment of malignant diseases. Vesicular stomatitis virus (VSV) has been shown to exert antitumor effect in several tumor types. Since the potential oncolytic activity of VSV has not yet been evaluated in epithelial tumors of the conjunctiva, we set out to investigate the susceptibility of the immortalized Wong-Kilbourne derivative of the Chang conjunctival cell line (WK) to VSV and analyze the role of apoptosis in VSV-mediated induction of cell death. METHODS: WK cells were infected with VSV at various multiplicities and maintained for different periods of time. VSV-infected cells were analyzed by inverted microscopy for the development of cytopathic effects (CPE). Virus replication was measured by indirect immunofluorescence assay, Western blot analysis and plaque titration. The apoptotic response of the infected cells was quantitated by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of Bcl-2 and Bax proteins. RESULTS: The WK cell line was highly permissive to VSV replication and was highly susceptible for the CPE of this virus. VSV infection elicited the apoptotic death of WK cells. Mock-infected cells exhibited endogenous expression of Bcl-2 and p21 Bax proteins. VSV infection caused a significant decrease in the expression level of Bcl-2. Moreover, in parallel with a slight decrease in the level of p21 Bax, p18 Bax protein accumulated in VSV-infected WK cells. CONCLUSIONS: VSV is a powerful inducer of apoptosis in immortalized WK cells. The VSV-mediated alterations in the expressions of Bcl-2 and Bax proteins may play important roles in the apoptotic responses of infected cells and may also sensitize to other apoptotic stimuli. This virus may possess oncolytic activity in epithelial tumors of the conjunctiva.