Giovanni Peri

Interleukin 2 and interleukin 15 differentially predispose natural killer cells to apoptosis mediated by endothelial and tumour cells

Human natural killer (NK) cells constitutively express the β‐ and γ‐chains of the interleukin 2 (IL‐2)/IL‐15 receptor, and both IL‐2 and IL‐15 are able to activate NK cell proliferation and cytotoxicity. When IL‐2‐primed human NK cells are exposed to sensitive targets (i.e. K562) they undergo apoptosis mediated by the β2‐integrin CD18. Here, we demonstrate that: (i) endothelial cells, similar to K562 tumour target cells, induce apoptosis of IL‐2‐primed NK cells; (ii) endothelial‐ and K562 cell‐induced apoptosis is significantly lower in IL‐15 than in IL‐2‐stimulated NK cells; (iii) a critical role in the apoptosis of IL‐2‐primed NK cells is played by the α‐chain of the IL‐2 receptor. Our data show for the first time that IL‐2‐activated NK cells can die by apoptosis upon contact with the vascular endothelium, which is a necessary step for their extravasation, with a direct pathophysiological relevance on the strategy of adoptive immunotherapy of cancer. On the other hand, IL‐15, although generating a similar level of activation of NK cells, largely prevents their apoptotic fate. Therefore, IL‐15 produced early in the immune response, when T cells are not yet activated, generates lymphokine‐activated killer cells that are efficient killers relatively protected from apoptosis. Once activated, T cells produce IL‐2 that overcomes the effect of IL‐15 on NK cells, paving the way for their death by apoptosis.

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.

Involvement of Caspase-3 and GD3 Ganglioside in Ceramide-induced Apoptosis in Farber Disease

Farbers disease (FD) is a rare genetic disorder caused by ceramidase deficiency, which results in ceramide accumulation in lung, liver, colon, skeletal muscle, cartilage, and bone. Although this disease has been symptomatically characterized, little is known about its molecular pathogenetic process. Because recent studies reported that ceramide accumulation induces GD3 ganglioside formation and apoptosis, we investigated, in tissue obtained via colonoscopy from seriously involved patients, the possible involvement of ceramide in FD colonocyte destruction. Histochemical and TUNEL analyses of paraffin-embedded sections revealed that 45 ± 4.3% of FD colonocytes showed morphological signs of apoptosis compared with the 8 ± 2.3% of constitutive epithelial cell death. Importantly, immunohistochemical study for pro-apoptotic factors showed that GD3 accumulation co-localized with active caspase-3 and cleaved K18 in FD colon tissue. These findings provide evidence for a role of the apoptotic ceramide pathway in the pathogenesis of FD.

Hsp10: anatomic distribution, functions, and involvement in human disease

There is growing evidence that molecular chaperones/heat shock proteins are involved in the pathogenesis of a number of human diseases, known as chaperonopathies. A better molecular understanding of the pathogenetic mechanisms is essential for addressing new strategies in diagnostics, therapeutics and clinical management of chaperonopathies, including those in which Hsp10 is involved. This chaperonin has been studied for a long time as a member of the mitochondrial protein-folding machine. However, although in normal cells Hsp10 is mainly localized in the mitochondrial matrix, it has also been found during and after stress in other subcellular compartments, such as cytosol, vesicles and secretory granules, alone or in combination with other proteins. In these extramitochondrial locales, Hsp10 plays an active role in cell signalling. For example, cancer cells often show altered levels of Hsp10, compared to normal cells. Hsp10 may also be found in the extracellular space and in the bloodstream, with a possible immunomodulatory activity. This minireview focuses on some studies to date on the involvement of Hsp10 in human disease pathogenesis.

Patient-specific instrumentation for total knee arthroplasty: a literature review

Abstract During the past decade, total knee arthroplasty (TKA) has been markedly increased. Recently, patient-specific custom cutting guides have been commercially introduced in order to achieve an accurate component alignment during TKA. In fact, these cutting blocks are specific to a patient’s knee anatomy and should help the surgeons to perform bone cuts, reducing the complexity of conventional alignment and sizing tools. Nevertheless, there are critical arguments against patient-specific cutting guides for routine use, such as poor evidence and higher costs. Additionally, there are still no mild and long-term results available that describe the clinical outcomes following patient-specific instrumentation of TKR, cost-effectiveness and lower revision rates. Aim of the current manuscript was to describe the recent improvements of the surgical technique and instrumentation of TKA, reviewing the recent literature concerning the PSI technology.

Presence and interaction in tissues of atrial natriuretic peptide, oxytocin and vasopressin: new insights

Atrial natriuretic peptide, oxytocin and vasopressin are three well known and widely studied molecules since many years. They have been fully characterised from a genetic and biomolecular point of view and a number of receptor-dependent functions have been recognised for them. Nevertheless, in the last years our group has conducted morphologic studies, using an immunohistochemical approach complemented by molecular biology techniques, and could show non-canonical localization and co-localization of these peptides in normal and pathologic tissues, that permitted us to postulate that they may be involved in a wider range of functions than usually assumed and not yet fully understood. In this minireview we summarise some of the main results that open new scenarios in the comprehension of the biologic activities of these peptides and allow to postulate a role for them as diagnostic tools.

Anatomical variations of the carotid arteries: kinking, coiling, and tortuosity. Anatomical and functional considerations

1 Department of Human Anatomy, University of Medicine and Pharmacy “Carol Davila”, Bucharest 2 Department of Human Anatomy, “Ovidius” University of Constanta 3 Department of Radiology and Medical Imaging, Fundeni Hospital, Bucharest 4 Department of Vascular Surgery, ARNAS Civico Di Cristina Benfratelli Hospital, Palermo, Italy 5 Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Universita degli Studi di Palermo, Italy 6 Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, Universita degli Studi di Palermo, Italy

Anatomy of the Cervical Spine

The vertebral column, or spine, consisting of a coordinated series of 33–34 vertebrae separated from each other by intervertebral disks, is divided in five segments or sections: cervical, dorsal, lumbar, sacral and coccygeal [1–7].

Role of Adrenomedullin in LSP-mediated lung injury

Acute Respiratory Distress Syndrome is a life-threatening disease characterized by diffuse lung injury that leads to respiratory failure and death. Among various endogenous protective peptides, adrenomedullin (AM) has been demonstrated to play a major role. The aim of our study was to assess the significance of AM in the complex pathophysiological cascade underlying lipopolysaccharide (LPS) mediated inflammatory response. In the first set of our experiments we showed that LPS induced a significant increase in the activation of the mitogen-activated protein kinase (MAPK) transduction signals in epithelial respiratory cells. In particular, our results showed a time and dose dependent activation of ERK and JNK pathways. No significant changes were observed for p38MAPK phosphorylation. Luminex analysis further confirmed the significant increase of IL-6 release along with a significant increase of MCP-1 , VEGF and IL-8. Pre-treatment of cells with AM (0.5 and 1 ng/ml) showed that AM was able to prevent JNK and ERK phosphorylation. Such effect turned into a significant reduction of IL-6 and TNF- gene transcription and induction of heme oxygenase-1 (Hsp32). Taken all together our data suggest that AM may play a major role in reducing LPS mediated inflammatory response by reducing the activation of MAPK signal transduction pathway.

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM).