V. Di Felice

Is chlamydial heat shock protein 60 a risk factor for oncogenesis

Abstract.Heat shock protein 60 (HSP60) plays an important role in the protein folding of prokaryotic and eukaryotic cells. Most of the papers published on chlamydial HSP60 concern its role in immune response during infection. In the last decade, exposure to Chlamydia trachomatis has been consistently associated with the development of cervical and ovarian cancer. Moreover, it has been suggested that chlamydial HSP60 may have an anti-apoptotic effect during persistent infection. We hypothesize that the accumulation of exogenous chlamydial HSP60 in the cytoplasm of actively replicating eukaryotic cells may interfere with the regulation of the apoptotic pathway. The concomitant expression of viral oncoproteins and/or the presence of mutations may lead to the ability to survive apoptotic stimuli, loss of replicative senescence, uncontrolled proliferation and, finally neoplastic transformation.

A comparative analysis of the products of GROEL-1 gene from Chlamydia trachomatis serovar D and the HSP60 var1 transcript from Homo sapiens suggests a possible autoimmune response.

Chlamydia trachomatis serovar D produces large quantities of HSP60‐1 during infections, which accumulate inside the host cell inducing autoimmunity. We compare the aminoacid sequences of the human HSP60 with the bacterial counterpart to better elucidate how CTHSP60 may simulate HSP60 from human origin during infection and may induce an autoimmune response. As a result of the comparison we suggest several possible epitopes of the CTHSP60, which may induce autoimmunity.

Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells.

Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.

Embryonic and foetal Islet-1 positive cells in human hearts are also positive to c-Kit

During embryogenesis, the mammalian heart develops from a primitive heart tube originating from two bilateral primary heart fields located in the lateral plate mesoderm. Cells belongings to the pre-cardiac mesoderm will differentiate into early cardiac progenitors, which express early transcription factors which are also common to the Isl-1 positive cardiac progenitor cells isolated from the developing pharyngeal mesoderm and the foetal and post-natal mice hearts. A second population of cardiac progenitor cells positive to c-Kit has been abundantly isolated from adult hearts. Until now, these two populations have been considered two different sets of progenitor cells present in the heart in different stages of an individual life. In the present study we collected embryonic, foetal and infant hearts, and we tested the hypotheses that c-Kit positive cells, usually isolated from the adult heart, are also present in the intra-uterine life and persist in the adult heart after birth, and that foetal Isl-1 positive cells are also positive to c-Kit. Using immunohistochemistry we studied the temporal distribution of Isl-1 positive and c-Kit/CD105 double positive cells, and by immunofluorescence and confocal analysis we studied the co-localization of c-Kit and Isl-1 positive cells. The results indicated that cardiomyocytes and interstitial cells were positive for c-Kit from the 9th to the 19h gestational week, that cells positive for both c-Kit and CD105 appeared in the interstitium at the 17h gestational week and persisted in the postnatal age, and that the Isl-1 positive cells were a subset of the c-Kit positive population.

Stem cell populations in the heart and the role of Isl1 positive cells.

Cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells, endothelial cells and cardiomyocytes. The embryonic origin of these cells has not yet been demonstrated, but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest. In the last decade, two different populations of cardiac progenitor or stem cells have been identified and isolated, i.e., the Islet1 positive (Isl1+) and c-Kit positive (c-Kit+)/Stem Cell Antigen-1 positive (Sca-1+) cells. Until 2012, these two populations have been considered two separate entities with different roles and a different origin, but new evidence now suggests a connection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells, derived from a common immature primitive cell. To find a common consensus on this concept is very important in furthering the application of stem cells to cardiac tissue engineering.

Acute change of titin at mid-sarcomere remains despite 8 wk of plyometric training

The purpose of this study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise (PlyEx) both before and after PlyEx training, to understand if titin is affected differently after PlyEx training. Healthy untrained individuals (N = 11) completed the 1stPlyEx (10 × 10 squat-jumps, 1-min rest). Thereafter, six subjects completed 8 wk of PlyEx, while five controls abstained from any jumping activity. Seven days after the last training session, all subjects completed the 2ndPlyEx. Blood samples were collected before and 6 h and 1, 2, 3, and 4 days after each acute bout of PlyEx, and muscle biopsies 4 days before and 3 days after each acute bout of PlyEx. The 1stPlyEx induced an increase in circulating myoglobin concentration. Muscle sample analysis revealed Z-disk streaming, a stretch or a fragmentation of titin (immunogold), and increased calpain-3 autolysis. After training, 2ndPlyEx did not induce Z-disk streaming or calpain-3 activation. The previously observed post-1stPlyEx positional change of the titin COOH terminus was still present pre-2ndPlyEx, in all trained and all control subjects. Only two controls presented with Z-disk streaming after 2ndPlyEx, while calpain-3 activation was absent in all controls. Eccentric explosive exercise induced a stretch or fragmentation of titin, which presented as a positional change of the COOH terminus. Calpain-3 activation does not occur when titin is already stretched before explosive jumping. Enzymatic digestion results in titin fragmentation, but since an increase in calpain-3 autolysis was visible only after the 1stPlyEx acute bout, fragmentation cannot explain the prolonged positional change.

Atrial natriuretic peptide and CD34 overexpression in human idiopathic dilated cardiomyopathies.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause characterized by ventricular chamber enlargement with impaired contractile function. In familial forms of IDCM, mutations of genes coding for cytoskeletal proteins related to force transmission, such as dystrophin, cardiac actin, desmin, and δ‐sarcoglycan, have been identified. Here, we report the data of a retrospective investigation carried out to evaluate the expression of atrial natriuretic peptide (ANP), CD34, troponin T and nestin in the myocardium of patients affected with IDCM. Formalin‐fixed and paraffin‐embedded consecutive tissue sections from the ventricular wall of 10 human normal hearts (NH) following forensic autopsy and 22 IDCM (living explanted hearts) were studied using primary monoclonal antibodies against ANP, CD34, troponin T and nestin by immunohistochemistry. Myocardial fibers were counted independently by three pathologists. Statistics included analysis of variance, log‐rank test for Kaplan‐Meier analysis, and kappa assessment for intra‐ and inter‐observer variability. ANP and CD34 were significantly overexpressed in IDCM compared to NH (p<0.05). Conversely, troponin T and nestin expression levels did not show significant variation. Inter‐observer kappa statistics showed a value of 0.87 and intra‐observer kappa statistics a value of 0.98. Evaluation of the marker distribution in the myocardium of patients with IDCM CD34 expression curve was similar to that of troponin T (p<0.0001), although two groups could be identified. Patients with a difference of more than 20 myocardial fibers in expression of CD34 and troponin T had a somewhat less favorable survival although the difference was not significant. The analysis of cells positive for troponin T resulted in a similar number of cardiac fibers between NH and IDCM. This is in agreement with cardiac enlargement present in IDCM, which is due to ventricular dilatation rather than increased number of myocytes. Moreover, the expression of nestin, a marker of activation of myocardial precursors, did not change either, and this may confirm that there are no hyperplastic phenomena in the IDCM pathogenesis. The increase in ANP‐positive cells in IDCM could be a consequence of neurohormonal activation due to a decline in the impaired myocyte contractility. Furthermore, since it was already shown that ANP could be important in the control of vascular remodeling, we postulated that the increase in CD34‐positive cells might be functionally correlated with the increase in ANP production. Differential expression of CD34 and troponin T might be used in future studies to evaluate their prognostic value.

Presence of Oxytocin, Vasopressin and Atrial Natriuretic Peptide and Their Modification in Rat Hypothalamic Paraventricular Nucleus During Resistance Training

Many studies have demonstrated the physiological effects of oxytocin (OT), atrial natriuretic peptide (ANP) and vasopressin (VP) in the homoeostasis of body fluids during physical exercise. However, a little information is available about the related immunohistochemical changes in hypothalamic magnocellular neurosecretory system during and after the training. The aim of the present work was to study the immunohistochemical changes in OT, ANP and VP levels in the hypothalamic paraventricular nucleus during and after resistance exercise protocol. Three groups of Wistar rats were trained by a rung ladder protocol for 15, 30 and 45 days, respectively; a fourth group was left to rest for 15 days after the training. Finally, four sedentary groups were used as controls. The results show that resistance training induces a significant reduction in the percentage of OT‐positive neurons, compared with sedentary controls. In contrast, this protocol did not induce any change in VP levels, and ANP levels did not change significantly. However, VP increased after the resting period of 15 days. Our work shows that neurons of the paraventricular nucleus are involved in body fluid homoeostasis during and after resistance exercise. The functional significance of these changes in OT and VP levels, during and after the protocol, needs to be further investigated.