Mycoplasma DnaK expression increases cancer development in vivo upon DNA damage.

Well-controlled repair mechanisms are involved in the maintenance of genomic stability, and their failure can precipitate DNA abnormalities and elevate tumor risk. In addition, the tumor microenvironment, enriched with factors inducing oxidative stress and affecting cell cycle checkpoints, intensifies DNA damage when repair pathways falter. Recent research has unveiled associations between certain bacteria, including Mycoplasmas, and various cancers, and the causative mechanism(s) are under active investigation. We previously showed that Mycoplasma fermentans DnaK, an HSP70 family chaperone protein, hampers the activity of proteins like PARP1 and p53, crucial for genomic integrity. Moreover, our analysis of its interactome in human cancer cell lines revealed DnaK's engagement with several components of DNA-repair machinery. Finally, in vivo experiments performed in our laboratory using a DnaK knock-in mouse model generated by our group demonstrated that DnaK exposure led to increased DNA copy number variants, indicative of genomic instability. We present here evidence that expression of DnaK is linked to increased i) incidence of tumors in vivo upon exposure to urethane, a DNA damaging agent; ii) spontaneous DNA damage ex vivo; and iii) expression of proinflammatory cytokines ex vivo, variations in reactive oxygen species levels, and increased ß-galactosidase activity across tissues. Moreover, DnaK was associated with increased centromeric instability. Overall, these findings highlight the significance of Mycoplasma DnaK in the etiology of cancer and other genetic disorders providing a promising target for prevention, diagnostics, and therapeutics.

Mycoplasma DnaK increases DNA copy number variants in vivo.

The human microbiota affects critical cellular functions, although the responsible mechanism(s) is still poorly understood. In this regard, we previously showed that Mycoplasma fermentans DnaK, an HSP70 chaperone protein, hampers the activity of important cellular proteins responsible for DNA integrity. Here, we describe a novel DnaK knock-in mouse model generated in our laboratory to study the effect of M. fermentans DnaK expression in vivo. By using an array-based comparative genomic hybridization assay, we demonstrate that exposure to DnaK was associated with a higher number of DNA copy number variants (CNVs) indicative of unbalanced chromosomal alterations, together with reduced fertility and a high rate of fetal abnormalities. Consistent with their implication in genetic disorders, one of these CNVs caused a homozygous Grid2 deletion, resulting in an aberrant ataxic phenotype that recapitulates the extensive biallelic deletion in the Grid2 gene classified in humans as autosomal recessive spinocerebellar ataxia 18. Our data highlight a connection between components of the human urogenital tract microbiota, namely Mycoplasmas, and genetic abnormalities in the form of DNA CNVs, with obvious relevant medical, diagnostic, and therapeutic implications.

Combined cART including Tenofovir Disoproxil, Emtricitabine, and Dolutegravir has potent therapeutic effects in HIV-1 infected humanized mice.

HIV-1 reservoirs persist in the presence of combined antiretroviral therapy (cART). However, cART has transformed HIV-1 infection into a chronic disease marked by control of HIV-1 viral load and mortality reduction. Major challenges remain, including viral resistance upon termination of cART and persistence and identification of tissue distribution of HIV-1 reservoirs. Thus, appropriate animal models that best mimic HIV-1 pathogenesis are important, and the current study complements our previously published validation of the CD34+ hematopoietic humanized mouse model for this purpose. Here we analyze viral suppression using the recently developed combination of antiretrovirals that include Tenofovir Disoproxil (TDF), Emtricitabine (FTC), and Dolutegravir (DTG), a choice based on recent clinical outcomes showing its improved antiretroviral potency, CD4+ T cell preservation, tolerability, and prevention of viral drug resistance compared to that of previous regimens. We used quantitative Airyscan-based super resolution confocal microscopy of selected mouse tissues. Our data allowed us to identify specific solid tissue reservoirs of human T cells expressing the HIV-1 core protein p24. In particular, lymph node, brain, spleen, and liver were visualized as reservoirs for residual infected cells. Marked reduction of viral replication was evident. Considering that detection and visualization of cryptic sites of HIV-1 infection in tissues are clearly crucial steps towards HIV-1 eradication, appropriate animal models with pseudo-human immune systems are needed. In fact, current studies with humans and non-human primates have limited sample availability at multiple stages of infection and cannot easily analyze the effects of differently administered combined antiretroviral treatments on multiple tissues. That is easier to manage when working with humanized mouse models, although we realize the limitations due to low human cell recovery and thus the number of cells available for thorough and comprehensive analyses. Nonetheless, our data further confirm that the CD34+ humanized mouse model is a potentially useful pre-clinical model to study and improve current anti-HIV-1 therapies.

Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Nitrosative Stress Is Associated with Dopaminergic Dysfunction in the HIV-1 Transgenic Rat.

Advances in antiretroviral therapy have resulted in significantly decreased HIV-related mortality. HIV-associated neurocognitive disorders, however, continue to be a major problem in infected patients. The neuropathology underlying HIV-associated neurocognitive disorders has not been well characterized, and evidence suggests different contributing mechanisms. One potential mechanism is the induction of oxidative stress. Using the HIV-1 transgenic (Tg) rat model of HIV, we found increased striatal NADPH oxidase-4 and neuronal nitric oxide synthase expression in the adult (7- to 9-month-old) Tg rat compared with control rats but not in the young (1-month-old) Tg rats. This was accompanied by increased 3-nitrotyrosine (3-NT) immunostaining in the adult Tg rats, which worsened significantly in the old Tg rats (18 to 20 months old). There was, however, no concurrent induction of the antioxidant systems because there was no change in the expression of the nuclear factor-erythroid 2-related factor 2 and its downstream targets (thioredoxin and glutathione antioxidant systems). Colocalization of 3-NT staining with neurofilament proteins and evidence of decreased tyrosine hydroxylase and dopamine transporter expression in the old rats support dopaminergic involvement. We conclude that the HIV-1 Tg rat brain shows evidence of nitrosative stress without appropriate oxidation-reduction adaptation, whereas 3-NT modification of striatal neurofilament proteins likely points to the ensuing dopaminergic neuronal loss and dysfunction in the aging HIV-1 Tg rat.

Altered expression pattern of Nrf2/HO-1 axis during accelerated-senescence in HIV-1 transgenic rat.

Chronic oxidative stress plays a central role in the pathogenesis of many diseases, including HIV-1 associated disorders. Concomitantly with the decline of endogenous antioxidant systems, it was reported that HIV-1-related proteins increase the production of radical species in cells and tissues that are not directly infected by the virus. In the context of HIV-1 infection, the role of Nrf2, a key transcription factor that contributes to the maintenance of cellular redox homeostasis, remains largely uncharacterized. One of the major stress-responsive player regulated by Nrf2 is the antioxidant enzyme HO-1. The Nrf2/HO-1 axis constitutes a crucial cell survival mechanism to counteract oxidative stress and inflammation. The present study aims to investigate the age-related patterns of Nrf2 and HO-1 in different brain regions and tissues of HIV-1 transgenic rat. Since HIV-1 induces an accelerated aging and the redox imbalance may actively promote senescence, we also evaluated the senescence phenotype-switching by quantifying levels of ß-galactosidase activity. Our results showed changes in gene expression, with different trends depending on the brain regions and tissues examined. However, compared to age-matched controls, we observed in HIV-1 transgenic rats a significant reduction in the protein levels of Nrf2 and HO-1, suggesting a weakening in the protection exerted by Nrf2/HO-1 system. Moreover, we show that senescence occurs more rapidly in HIV-1 transgenic rats than in control animals. To our knowledge this is the first in vivo report showing the involvement of Nrf2/HO-1 pathway in a rat model of HIV-1.

Identification of degenerated synaptic boutons in the dorsal claustrum of the cat after electrolytic lesions of the intralaminar thalamic nuclei.

The aim of this study is to investigate whether the dorsal claustrum receives afferent input from the intralaminar thalamic nuclei - centromedian nucleus, central lateral nucleus and paracentral nucleus. The intralaminar thalamic nuclei of eight cats were electrolytically lesioned. We obtained samples from the dorsal claustrum for electron microscopic analysis from the second to the seventh post-procedural day. Two types of degenerated synaptic boutons were observed: electron-dense which formed the majority of boutons, and electron-lucent comprising the remaining samples. Between the second and seventh post-procedural day, we observed a steady increase in the number of electron-dense boutons which were diffusely distributed throughout the dorsal claustrum. Electron-dense degenerated boutons formed asymmetrical contacts with dendritic spines as well as with small and medium-sized dendrites. In contrast, electron-lucent degenerated boutons were observed in earlier post-procedural periods and formed symmetrical axodendritic contacts.

The HIV-1 Transgenic Rat: Relevance for HIV Noninfectious Comorbidity Research.

HIV noninfectious comorbidities (NICMs) are a current healthcare challenge. The situation is further complicated as there are very few effective models that can be used for NICM research. Previous research has supported the use of the HIV-1 transgenic rat (HIV-1TGR) as a model for the study of HIV/AIDS. However, additional studies are needed to confirm whether this model has features that would support NICM research. A demonstration of the utility of the HIV-1TGR model would be to show that the HIV-1TGR has cellular receptors able to bind HIV proteins, as this would be relevant for the study of cell-specific tissue pathology. In fact, an increased frequency of HIV receptors on a specific cell type may increase tissue vulnerability since binding to HIV proteins would eventually result in cell dysfunction and death. Evidence suggests that observations of selective cellular vulnerability in this model are consistent with some specific tissue vulnerabilities seen in NICMs. We identified CXCR4-expressing cells in the brain, while specific markers for neuronal degeneration demonstrated that the same neural types were dying. We also confirm the presence of gp120 and Tat by immunocytochemistry in the spleen, as previously reported. However, we observed very rare positive cells in the brain. This underscores the point that gp120, which has been reported as detected in the sera and CSF, is a likely source to which these CXCR4-positive cells are exposed. This alternative appears more probable than the local production of gp120. Further studies may indicate some level of local production, but that will not eliminate the role of receptor-mediated pathology. The binding of gp120 to the CXCR4 receptor on neurons and other neural cell types in the HIV-1TGR can thus explain the phenomena of selective cell death. Selective cellular vulnerability may be a contributing factor to the development of NICMs. Our data indicate that the HIV-1TGR can be an effective model for the studies of HIV NICMs because of the difference in the regional expression of CXCR4 in rat tissues, thus leading to specific organ pathology. This also suggests that the model can be used in the development of therapeutic options.

Characterization of neuropathology in the HIV-1 transgenic rat at different ages.

The transgenic HIV-1 rat (Tg) is a commonly used neuroHIV model with documented neurologic/behavioral deficits. Using immunofluorescent staining of the Tg brain, we found astrocytic dysfunction/damage, as well as dopaminergic neuronal loss/dysfunction, both of which worsening significantly in the striatum with age. We saw mild microglial activation in young Tg brains, but this decreased with age. There were no differences in neurogenesis potential suggesting a neurodegenerative rather than a neurodevelopmental process. Gp120 CSF levels exceeded serum gp120 levels in some animals, suggesting local viral protein production in the brain. Further probing of the pathophysiology underlying astrocytic injury in this model is warranted.

Establishment of an ex vivo model of monocytes-derived macrophages differentiated from peripheral blood mononuclear cells (PBMCs) from HIV-1 transgenic rats.

It has been developed an HIV type 1 transgenic rat model (HIV-1 Tg Rat) which contains a gag-pol-deleted HIV-1 provirus regulated by the viral LTR promoter. Although it harbors a non infectious provirus, efficient viral expression occurs in different tissues and disease manifestations as well as immune-response alterations and pathologies similar to humans can be observed. Regulation of HIV-1 expression is influenced by various cellular factors and it is well known that macrophages are one of the major reservoir of HIV-1 infection and a vehicle for virus spread to other tissues. Purpose of our work was to establish an antigen presenting cells (monocyte-derived macrophages, MDM) ex vivo model from these HIV-1 transgenic rats. This model can be used to study function of HIV-infected MDM and their behavior like HIV-1 reservoir. Ultimately, these studies may be helpful in defining approaches to control HIV-1 spread.

Topographical distribution and morphology of NADPH-diaphorase-stained neurons in the human claustrum.

We studied the topographical distribution and morphological characteristics of NADPH-diaphorase-positive neurons and fibers in the human claustrum. These neurons were seen to be heterogeneously distributed throughout the claustrum. Taking into account the size and shape of stained perikarya as well as dendritic and axonal characteristics, Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd)-positive neurons were categorized by diameter into three types: large, medium and small. Large neurons ranged from 25 to 35 µm in diameter and typically displayed elliptical or multipolar cell bodies. Medium neurons ranged from 20 to 25 µm in diameter and displayed multipolar, bipolar and irregular cell bodies. Small neurons ranged from 14 to 20 µm in diameter and most often displayed oval or elliptical cell bodies. Based on dendritic characteristics, these neurons were divided into spiny and aspiny subtypes. Our findings reveal two populations of NADPHd-positive neurons in the human claustrum-one comprised of large and medium cells consistent with a projection neuron phenotype, the other represented by small cells resembling the interneuron phenotype as defined by previous Golgi impregnation studies.

A novel HIV-1 transgenic rat model of childhood HIV-1-associated nephropathy.

BACKGROUND: A characteristic finding of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is the presence of heavy proteinuria, focal or global glomerulosclerosis, and microcystic tubular dilatation leading to renal enlargement, and rapid progression to end-stage renal disease (ESRD). METHODS: We have recently developed the first HIV-1 transgenic rat model that carry a noninfectious HIV-1 DNA construct lacking 3.1 kb of sequence overlapping the gag and pol sequences, and develop many of the clinical lesions seen in HIV-infected patients, including HIVAN. To gain further insight into the pathogenesis of childhood HIVAN, we followed the clinical and renal pathologic outcome of 165 HIV-1 transgenic (HIV-Tg) rats and their respective control littermates for a period of 18 months. RESULTS: HIV-1 Tg rats progressively developed proteinuria and renal histologic lesions similar to those seen in children with HIVAN, leading to chronic renal failure. By in situ hybridization, HIV-1 genes were detected in glomerular and tubular epithelial cells and infiltrating mononuclear cells, which also expressed the HIV-1 envelop protein gp120. The development of HIVAN was associated with the accumulation of basic fibroblast growth factor (bFGF) in the kidney. CONCLUSION: These data support the notion that HIV-1 plays a direct role in the pathogenesis of HIVAN, by affecting the function and growth of renal epithelial cells, inducing the recruitment of mononuclear cells, and accumulating bFGF in the kidney, even in the absence of viral replication. These rats may provide an excellent model system to study the pathogenesis of childhood HIVAN.