Cancer progression: a single cell perspective.

Tumor tissues are constituted by a dynamic diversity of malignant and non-malignant cells, which shape a puzzling biological ecosystem affecting cancer biology and response to treatments. Over the course of the tumoral disease, cancer cells acquire genotypic and phenotypic changes, allowing them to improve cellular fitness and overcome environmental and treatment constraints. This progression is depicted by an evolutionary process in which single cells expand as a result of an interaction between single-cell changes and the local microenvironment. Recent technological developments have made it possible to depict the development of cancer at the single-cell level, offering a novel method for understanding the biology of this complex disease. Here, we review those complex interactions from the perspective of single cells and introduce the concept of omics for single-cell studies. This review emphasizes the evolutionary dynamics that control cancer progression and the capacity of single cells to escape the local environment and colonize distant sites. We are assisting a rapid progression of studies carried out at the single-cell level, and we survey relevant single-cell technologies looking at multi-omics studies. These forefront approaches will address the combined contribution of both genetic and non-genetic factors to cancer progression and will pave the path for precision medicine in cancer.

Porcine coronary arteries: immunohistochemical profile of TNF-alpha, IL-1beta, TGF-beta1 and ICAM-1.

BACKGROUND: In our study we used immunohistochemical technique to demonstrate the presence of the cytokines tumour necrosis factor alpha (TNF-α), interleukin 1beta (IL-1ß), transforming growth factor beta1 (TGF-ß1) and intercellular adhesion molecule-1 (ICAM-1) in porcine coronaries even in physiological conditions. MATERIALS AND METHODS: Inflammatory cytokines are polypeptide mediators which act as a communication signal between immune system cells and other types of cellsin different organs and tissues, both in human and pig coronary circulation. RESULTS: Our results show that pro-inflammatory cytokines TNF-α, IL-1ß, TGF-ß1 and ICAM-1 are also present in the medium tunica of the coronary arteries under physiological conditions. These results may be compared with those found in coronary atherosclerosis, where the increase in TNF-α has a dramatic effect on the function of the left ventricle, and the high value of IL-1 correlates directly with the extent of myocardial necrosis. In our study we observe the damage and activation of endothelial cells; this induces endothelial dysfunction by accumulation and oxidation of low density lipoproteins (LDL). The formation of oxidized LDL could play a central role in the amplification of the inflammatory response causing an increased expression of pro-inflammatory cytokines which promotes leukocyte recruitment in the intimal layer. These leukocytes, after the adhesion to the endothelium, penetrate the intimate tunic. CONCLUSIONS: Therefore inflammatory processes promote the onset and evolution of atheroma and the development of thrombotic complications.

The role of congenital malformations of the thoracic outlet in the development of the syndrome.

BACKGROUND: Thoracic outlet syndrome (TOS) represents a clinical condition caused by compression of the neurovascular structures that cross the thoracic outlet. TOS can be classified in: 1) neurogenic TOS (NTOS), 2) venous TOS (VTOS), 3) arterial TOS (ATOS). Many different causes can determine the syndrome: congenital malformations, traumas, and functional impairments. MATERIALS AND METHODS: This manuscript reviews how the congenital malformations play an important role in adult age; however, TOS also affects patients of all ages. RESULTS: Radiological imaging like X-ray (radiography), magnetic resonance and computed tomography can provide useful information to assess TOS causes and decide a potential surgery. 79% of the patients included in the first two stages of nerve, artery, vein (NAV) staging experienced excellent results with kinesiotherapy; whereas patients included in the third and fourth stage of NAV staging were subject to surgery. CONCLUSIONS: The treatment of acute forms of TOS involves thrombolysis and anticoagulant therapy; surgery is appropriate for true NTOS, vascular TOS and in some cases when conservative treatment fails.

Keratoconus: the possible involvement of inflammatory cytokines in its pathogenesis. An experimental study and review of the literature.

OBJECTIVE: Keratoconus (KC) is generally described as a non-inflammatory disease, characterized by thinning in the central region of the cornea with consequent tissue degradation producing impaired visual acuity. MATERIALS AND METHODS: In our experimental study, we analyzed the presence and implications of several inflammatory cytokines in the corneal tissues of patients suffering from keratoconus by immunohistochemical analysis. RESULTS: The analysis showed increased levels of inflammatory factors in the pathological tissues compared to controls, confirming that KC cannot be considered an entirely non-inflammatory pathology and that its etiopathogenesis includes several chronic inflammatory events. CONCLUSIONS: In the light of these results, the classification of KC as an inflammatory pathology or as a pathology related to inflammation might be useful in directing future research aimed at developing effective anti-inflammatory therapies to pharmacologically target the inflammatory mediators which contribute to the development and progression of the disease.

Melatonin role in skeletal muscle disorders.

OBJECTIVE: This review discusses the impact of the neuro-hormone melatonin on skeletal muscle disorders based on recent literature data with the aim to clarify the utility of the melatonin therapy in patients affected by muscle diseases. MATERIALS AND METHODS: It has been pointed out the possible role of melatonin as a food supplement to cure muscular disorders characterized by muscle wasting. Oxidative damage has been proposed as one of the major contributors of the skeletal muscle decline occurring both in physiological and pathological conditions. It is known that excessive oxidant levels lead to mitochondrial damage, and in turn, contribute to apoptotic signaling activation and autophagic impairment. This condition is common in a variety of skeletal muscle disorders. RESULTS: The scientific evidence enhances the antioxidant effect of melatonin, that has been demonstrated by several studies both in vitro and in vivo. This effect counteracts mitochondrial impairments and reduces oxidative stress and autophagic alterations in muscle fibers. Its beneficial role in restoring muscle decline, takes place mainly in atrophic conditions correlated to muscle aging. CONCLUSIONS: The findings of the research suggest that melatonin may be considered as a valid dietary supplement, useful to prevent muscle wasting, in particular, in sarcopenia-associated diseases.

Arthrogenic human synovial cysts: immunohistochemical profile of interleukin-1beta, interleukin-6, tumour necrosis factor-alpha.

BACKGROUND: Synovial cysts are currently classified as degenerative lesions affecting the joint capsule or adjacent structures. MATERIALS AND METHODS: In our study we describe the results obtained in an immunohistochemical study comprising 18 patients with synovial cysts, performed to evaluate the pathophysiological role of some inflammatory cytokines such as: interleukin (IL)-1ß, IL-6 and tumour necrosis factor-alpha (TNF-α). RESULTS: Results showed an over-expression of TNF-α, IL-1ß and IL-6 which appears to be involved in the onset and progression of the disease. At the present time it is not possible to affirm that these molecules play a direct role also due to the absence of further and more specific investigations. The authors therefore hypothesize that inhibition of inflammation may have a significant role in the pathogenesis and regression of synovial cysts. CONCLUSIONS: Hence, these inflammatory cytokines may be considered potential therapeutic targets. The development of synthetic inhibitors of these inflammatory factors could lead to a reduction in the intensity of inflammation, thus inhibiting the onset and development of the disease.

The role of inflammation in diabetic retinopathy: a review.

OBJECTIVE: Diabetic retinopathy and diabetes represent serious health conditions, being considered among the main causes of blindness. In recent years, anti-VEGF therapies have been of great help in the treatment of retinal pathology and, until now, they represent the primary choice therapy for diabetic retinopathy. Nevertheless, many patients do not experience significant benefits of vision after an anti-VEGF monotherapy. For this reason, several researchers recently focused their attention on the mechanisms that play a central role in the development and progression of diabetic retinopathy. RESULTS: Available scientific evidence confirms that diabetic retinopathy requires other molecules capable of modifying the mechanisms that, together with angiogenesis, contribute to the development of the condition, such as vascular and neuroinflammation. CONCLUSIONS: This review summarizes the current knowledge of the pathological changes that occur in diabetic retinopathy and that might contribute to identify possible new strategies for the treatment of this condition.

Neuroprotective role of phosphoserine in primary open-angle glaucoma patients.

OBJECTIVE: To evaluate the neuroprotective role of phosphoserine (P-Ser) in primary open-angle glaucoma (POAG) patients and to compare its therapeutic effectiveness to placebo treatment. PATIENTS AND METHODS: Fifty-one patients (24 males and 27 females) between 35 and 61 years (average 46 years ± 3.8 SD) affected by POAG were enrolled in this study. Patients were divided in two groups: group A included 28 subjects that received an oral P-Ser treatment for 12 months; and group B included 23 subjects that received an oral placebo treatment for 12 months. Complete ophthalmological examination, standard automated perimetric examination, analysis of ON fibers via scanning laser polarimetry and glaucoma staging was performed in all patients at enrolment and 1, 3, 6, and 12 months after. Statistical analysis was performed using STATA 14.0 (Collage Station, TX, USA). RESULTS: Mean deviation (MD) and pattern standard deviation (PSD) analysis by means of 30-2 full threshold of the visual fields (VFs), retinal nerve fiber layer (RNFL) thickness by means of GDx, and IOP were considered to evaluate P-Ser therapy effectiveness in both groups. A statistically significant improvement (p<0.05) in VF, RNFL thickness and IOP compared to pre-treatment was found in patients in group A. CONCLUSIONS: Our study shows a significant improvement in several variables in patients with glaucoma treated with P-Ser compared to placebo and suggests a potential neuroprotective effect of P-Ser in treating glaucoma patients in association with the traditional hypotonic topical therapy.

Drugs inducing hearing loss, tinnitus, dizziness and vertigo: an updated guide.

OBJECTIVE: The awareness of audio-vestibular side effects of drugs, such as hearing loss, tinnitus, dizziness and vertigo, has widely increased in the recent years. The present guide represents an update of the previous documents published by the authors in 2005 and 2011 on drug-induced ototoxicity and vestibulotoxicity. MATERIALS AND METHODS: The authors performed a comprehensive analysis of audio-vestibular side effects of commercially available drugs based on the British National Formulary, a pharmaceutical reference book that contains a wide range of useful information and advice on prescription and pharmacology. RESULTS: Commercially available drugs and their active principles have been classified based on their audio-vestibular side effects, as reported by the pharmaceutical companies and/or health agencies. Drugs have been categorized based on the field of application, the therapeutic indication and the pharmacological properties. CONCLUSIONS: General practitioners, otolaryngology, neurology and audiology specialists should be aware of possible audio-vestibular side effects of drugs, such as hearing loss, tinnitus, dizziness and vertigo. The present guide represents a practical tool to rapidly identify potential audio-vestibular side effects of drugs as reported by the pharmaceutical companies and/or health agencies.

Brain gliomas and growth factors: immunohistochemical, immunofluorescence, flow cytometry and RT-PCR profile in pediatric age.

Gliomas represent over 50% of tumors occurring in children. Evidence suggests that glioma stem cells (GSCs), maintained by the transforming growth factor-beta (TGF-ß1) pathway, and vascularization substantially contribute to tumor aggressiveness. The identification of important angiogenic factors such as vascular endothelial growth factor (VEGF) may represent a crucial step in the therapeutic approach against tumor growth and metastatic diffusion. The aim of this study was to identify the expression of TGF-ß1, VEGF and VEGF-receptors in brain gliomas. Specimens of 16 gliomas and 4 controls from children aged 0.2-14 years were used in the study. Immunohistochemical analysis and gene expression study from specimens was performed. Flow cytometry analysis on GSCs was performed to ascertain the expression of VEGF and VEGF-R2 in the tumor stem cell compartment. Newly diagnosed gliomas mainly showed moderate to strong VEGF immunostaining and increased expression of pro-inflammatory molecules in glioma cells. The proportion of TGF-ß1 positive endothelial cells was markedly lower in normal brain vessels compared to tumor vessels. These findings demonstrate that the glioma mass is constituted by a phenotypically immature anoxic central area with a proliferating hypoxic layer; the peripheral area is characterized by cell types with a higher degree of differentiation expressing pro-angiogenic factors. Our data have proven that GSCs play a central role in promoting glioma neovascularization. These findings are useful to understand glioma vascularization, have relevant implications in the therapeutic options and may favor new insights into stem cells biology and suggest therapeutic opportunities for the anti-vascular treatment strategy.

Anatomical variations of the median nerve and of the vascular-nervous structures at the wrist.

Three cases of anatomical variation of the median nerve at the wrist found during our surgical activity led us to take the opportunity to expose anatomical variations by reviewing already published reviews. Consequently, on the basis of anatomical studies, clinical reports and imaging, as a result of careful examination of the published literature, it has been observed that the interventions in such anatomical area must take into account these variations. In particular, the most performed procedure is the lysis of the transverse carpal ligament (TCL), which is not free from complications. In our opinion it is therefore necessary, in order to avoid the complications of the nervous, vascular and tendinous sections, to use some specific technical procedures.

Anteriorly placed tumors to the conus: removal by interradicular window.

Tumors anteriorly situated to the medullary conus are rarely encountered and represent a true surgical challenge. We examined the literature on this topic, concluding that there are no previous reports on alternative surgical techniques different to the traditional one. We report two cases of intradural extramedullary tumor operated on by a technique performed through a window opened between the spinal roots, which allows an easy, effective and useful resection. We describe a new operative technique which ensures a complete removal of these tumors and discuss clinical implications in the light of the available literature on this topic.

D’Andrea’s disease (angiomegaly): a currently well-defined nosological entitys.

In 1997 D’Andrea et al. described a new nosological entity the characteristics of which consisted of lengthening, dilation and tortuosity of blood vessels, arteries or veins, less prominent, but also less circumscribed than an aneurysm. This condition does not necessarily imply specific aneurysm formation although aneurysms at multiple sites are a frequent observation. The term used by authors for angiomegaly of the venous system was venomegaly and the analogous condition of the arterial system was termed arteriomegaly. Although tortuosity and dilation of arteries and veins have been widely reported, suggesting a systemic disorder which affects the structural integrity of all vessels, most papers dealing with this intriguing condition did not describe any alterations in the components of vessel walls. In the present paper, the authors describe a well-defined condition, D’Andrea’s Disease (or DD, in this article), analyzing its salient morphological and clinical features and clarifying this pathological condition as a distinct and now well-defined nosological entity.

Age-related ultrastructural and monoamine oxidase changes in the rat optic nerve.

The aim of this paper is to study the morphology and the distribution of the monoamine oxidase enzymatic system in the optic nerve of 4 month-old Wistar (young) and 28 month-old Wistar (old) rats. The optic nerve was harvested from 20 young and old rats. The segment of optic nerve was divided longitudinally into two pieces, each 0.1 mm in length. The first piece was used for transmission electron microscopy. The second piece was stained with histochemical reaction for monoamine oxidase. The agerelated changes in the optic nerve of rats include micro-anatomical details, ultrastructure and monoamine oxidase histochemical staining. A strong decrease of the thin nerve fibers and a swelling of the thick ones can be observed in optic nerve fibers of old rats. Increased monoamine oxidase histochemical staining of the optic nerve of aged rats is well demonstrated. The increase of meningeal shealth and the decrease of thin nerve fibers of the optic nerve in old rats are well documented. Morphological, ultrastructural and histochemical changes observed in optic nerve fibers of the old rats show a close relation with aging.

Occlusion of retinal capillaries caused by glial cell proliferation in chronic ocular inflammation.

The inner blood-retinal barrier is a gliovascular unit in which glial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. During chronic ocular inflammation, microvascular complications can give rise to vascular proliferative lesions, which compromise visual acuity. This pathologic remodelling caused by proliferating Müller cells determines occlusion of retinal capillaries. The aim of the present study was to identify qualitative and quantitative alterations in the retinal capillaries in patients with post-traumatic chronic ocular inflammation or post-thrombotic vascular glaucoma. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in retinal inflammation. Our electron microscopy findings demonstrated that during chronic ocular inflammation, thickening of the basement membrane, loss of pericytes and endothelial cells and proliferation of Müller cells occur with irreversible occlusion of retinal capillaries. Angiogenesis takes place as part of a regenerative reaction that results in fibrosis. We believe that VEGF and pro-inflammatory cytokines may be potential therapeutic targets in the treatment of this disease although further studies are required to confirm these findings.

Growth factors, their receptor expression and markers for proliferation of endothelial and neoplastic cells in human osteosarcoma.

Osteosarcoma is the most common primary malignant tumour of the bone. Although new therapies continue to be reported, osteosarcoma-related morbidity and mortality remain high. Modern medicine has greatly increased knowledge of the physiopathology of this neoplasm. Novel targets for drug development may be identified through an understanding of the normal molecular processes that are deeply modified in pathological conditions. The aim of the present study is to investigate, by immunohistochemistry, the localisation of different growth factors and of the proliferative marker Ki-67 in order to determine whether these factors are involved in the transformation of osteogenic cells and in the development of human osteosarcoma. We observed a general positivity for NGF - TrKA - NT3 - TrKC - VEGF in the cytoplasm of neoplastic cells and a strong expression for NT4 in the nuclear compartment. TGF-beta was strongly expressed in the extracellular matrix and vascular endothelium. BDNF and TrKB showed a strong immunolabeling in the extracellular matrix. Ki-67/MIB-1 was moderately expressed in the nucleus of neoplastic cells. We believe that these growth factors may be considered potential therapeutic targets in the treatment of osteosarcoma, although proof of this hypothesis requires further investigation.

Cholinergic innervation of human mesenteric lymphatic vessels.

BACKGROUND: The cholinergic neurotransmission within the human mesenteric lymphatic vessels has been poorly studied. Therefore, our aim is to analyse the cholinergic nerve fibres of lymphatic vessels using the traditional enzymatic techniques of staining, plus the biochemical modifications of acetylcholinesterase (AChE) activity. MATERIALS AND METHODS: Specimens obtained from human mesenteric lymphatic vessels were subjected to the following experimental procedures: 1) drawing, cutting and staining of tissues; 2) staining of total nerve fibres; 3) enzymatic staining of cholinergic nerve fibres; 4) homogenisation of tissues; 5) biochemical amount of proteins; 6) biochemical amount of AChE activity; 6) quantitative analysis of images; 7) statistical analysis of data. RESULTS: The mesenteric lymphatic vessels show many AChE positive nerve fibres around their wall with an almost plexiform distribution. The incubation time was performed at 1 h (partial activity) and 6 h (total activity). Moreover, biochemical dosage of the same enzymatic activity confirms the results obtained with morphological methods. CONCLUSIONS: The homogenates of the studied tissues contain strong AChE activity. In our study, the lymphatic vessels appeared to contain few cholinergic nerve fibres. Therefore, it is expected that perivascular nerve stimulation stimulates cholinergic nerves innervating the mesenteric arteries to release the neurotransmitter AChE, which activates muscarinic or nicotinic receptors to modulate adrenergic neurotransmission. These results strongly suggest, that perivascular cholinergic nerves have little or no effect on the adrenergic nerve function in mesenteric arteries. The cholinergic nerves innervating mesenteric arteries do not mediate direct vascular responses.