Myeloperoxidase expression in human colonic mucosa is related to systemic oxidative balance in healthy subjects

Objectives: To improve understanding of the preclinical stage of colonic inflammation by exploring the existence of a link between early inflammatory changes in the colonic mucosa and the systemic redox balance. Methods: Clinical characteristics, a fasting blood draw, and mucosal biopsies from the right, left, and sigmoid-rectum colonic tracts collected from 28 healthy individuals (14/14 males/females) who underwent colonoscopy. Myeloperoxidase (MPO) positive cells infiltrating colonic mucosa specimens were assessed by immunohistochemistry, and patients divided into high or low MPO expressing cells/optical field groups (MPOhighor MPOlow, respectively).The systemic oxidative balance has been studied through derived-Reactive Oxygen Metabolites (d-ROMs), Biological Antioxidant Potential (BAP), and Lipoperoxide-cholesterol Oxidizing (LP-CHOLOX) tests on serum. Results: MPOhighpatients demonstrated an increased systemic oxidative stress compared to MPOlowindividuals (P = 0.035), especially when MPO is referred to the left-sided colonic mucosa (P = 0.007). MPOlowsubjects in the sigmoid-rectum showed a significant higher antioxidant capacity in the serum (P < 0.02). Sex-specific differences in MPO expression (male and female: 4.6 \ub1 3.2 and 2.6 \ub1 1.5 MPO-positive cells/optical field, respectively, P = 0.044), and a decreasing gradient in MPO expression moving from the cecum to the rectum (ascendant, descendant, and sigmoid-rectum: 3.7 \ub1 2.8, 3.1 \ub1 1.7, and 1.4 \ub1 0.5, respectively, P = 0.012) were also found and discussed. Discussion: The study is the first demonstrating a connection between systemic redox balance and MPO expression in the colonic mucosa, according to the colonic tract and patient gender. Further research evaluating the MPO expression in the human colon and its relationship with pathological conditions could benefit from these results

Effect of PTH (1-34) on trabecular bone of rat vertebral body in induced-biochemical osteoporosis by calcium- deprived diet

Rats fed calcium-deprived diet were used as experimental model for studying bone modelling alterations during biochemical osteoporosis and recovery of bone loss. Such model is suitable to evaluate the possible effects exerted by PTH(1-34) in preventing as well as in recovering metabolic osteoporosis. Three-month-old Sprague Dawley male rats were divided in different groups: some fed normal diet or calcium-deprived diet with/without 40µg/Kg/day PTH(1-34), provided by Eli Lilly-USA, for 4 weeks and some with restoration of normal diet with/without PTH (1-34) for further 4 weeks. To evaluate the occurrence of osteogenesis during the first 4 weeks of the experimental period, rats received three labels of bone deposition at 1st, 20th and 27th day (and then were sacrificed); during the successive 4 weeks (in which those rats previously fed with calcium-deprived diet had restoration of normal diet), animals received three labels of bone deposition at 1st, 7th and 14th day. Histomorphometrical analyses were performed on cortical and trabecular bone taken from the central level of the 5th lumbar vertebral body, transversely sectioned. The results showed that differences among the groups were observed mainly in trabecular bone with respect to cortical one, thus underlining the different role of the two types of bone architecture in mineral and skeletal homeostasis. Concerning trabecular bone, the observations showed that administration of PTH (1-34) during calcium-deprived diet and/or during the restoration of normal diet induces higher deposition of trabecular bone with respect to that recorded in rats that never received PTH(1-34), neither during calcium-deprived diet nor during restoration of normal diet. Since increments of trabecular bone are detectable only after the period of diet restoration (but not before), the authors suggest that a chronic administration of PTH (1-34) is necessary to achieve appreciable results on bone mass recovery

Effects of PTH(1-34) during fracture healing after experimental bone drilling in rat femur: novel aspects

The study concerns the role of PTH(1-34) during bone lesion repair. 3-month-old male Sprague-Dawley rats, in which trans-cortical holes were drilled at femur middiaphysis, were divided in groups with/without Teriparatide administration (40g/ Kg/day), and sacrificed at different times (10, 28, 45 days). In 2002 (1) we demonstrated the occurrence of two successive bone forming processes during both skeletal organogenesis and bone repair, i.e. static (SO) and dynamic (DO) osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. In brief, the primary function of SO is to provide a rigid scaffold, containing osteocytes (i.e. mechano-sensors), to DO-osteoblastic laminae; therefore, in DO mechanical factors can play a crucial role in transduction of mechanical stresses into biological signals. In the present work, histomorphometric analysis showed that, already after 10 days from drilling, notwithstanding the holes are temporarily filled by the same amount of newly-formed trabecular bone (produced by SO) independently from the treatment, the number of movable osteoblast laminae (typical of DO), covering the trabecular surface, is statistically higher in animals submitted to PTH(1-34) administration than in the control ones; this suggests that the mere effect of Teriparatide is to anticipate the occurrence of dynamic osteogenesis involved in the production of good quality bone more suitable to loading. These findings are also supported by the higher values of microhardness as well as the more ordered-fibered texture (observed by polarized light) in treated animals with respect to control ones that strongly indicates the qualitative (instead of quantitative) effect of PTH (1-34) in improving bone healing. The present investigation could be of crucial importance in further translational clinical research in humans to define the best therapeutic strategies in recovering skeletal lesions, particularly in terms of time of administration of PTH(1-34)

Induced Biochemical osteoporosis: Effects of 1-month calcium–deprived diet on rat bone remodelling with/without contemporary administration of PTH(1-34)

It is known that rats fed calcium-deprived diet develop osteoporosis due to en-hanced bone resorption secondary to parathyroid overactivity resulting from nutritional hypocalcemia. Therefore, rats provide a good experimental animal model for studying bone remodelling alterations during biochemical osteoporosis. This preliminary study is performed in 3 month-old Sprague Dawley male rats, divided into 4 groups (5 rats each): 1) base line, 2) normal diet for 4 weeks, 3) calcium-deprived diet for 4 weeks; 4) calcium-deprived diet for 4 weeks plus contemporary administration of PTH(1-34) 40µg/kg/day. Three labels of osteogenesis were performed at 1st , 20th and 27th day of experimental period in order to evaluate bone formation during animal treatment. His-tomorphometrical analyses were performed on cortical bone of femoral diaphyses, as well as on trabecular bone of distal femoral metaphyses, both transversely sectioned. The preliminary results showed that at femur mid-diaphyseal level the diet induced a reduction of cortical bone area (even if not significant) with enlargement of the medul-lary canal due to endosteal resorption, while periosteal neo-deposition is similar in all groups and particularly abundant in those periosteal regions mainly devoted in answering the mechanical demands. PTH(1-34) treatment seems to reduce endosteal resorption only in those surfaces where periosteal mechanical loading are less consistent. Conversely, PTH(1-34) treatment doesn't seem to affect osteoblast activity. Moreover, in distal femoral metaphyses, diet induced osteoclast activity, with a decrease in the amount of trabecular bone volume, confirming that this architecture is mainly devoted in answering the metabolic demands. The novelty of the proposed model Is the contemporary administration of PTH(1-34) together with calcium deprived diet to evaluate induced-biochemical osteoporosis. This model seems a good starting point for successive studies in order to study bone alterations during unbalanced calcium metabolism frequently occurring in aging and to define time and manner of bone mass recovery

Up-regulation of the chemo-attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments

To study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures from each patient of the fractured and control groups were subjected to immunofluorescence staining and quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken from the fractured and control groups were processed for Western blot experiments and densitometric analysis. The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemoattractive receptor ChemR23, compared with control cultures. On the basis of these observations, the authors hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of proinflammatory factors, could enhance the deterioration of cartilage tissue with consequent development of post-traumatic arthritis following intra-articular bone fracture

Proposed roles of the immune response regulator-ThPOK in human colorectal cancer progression

Solid tumours are commonly infiltrated by several immune cells [1-3]. In cancer, immune cells play conflicting roles with both the potentials to eliminate or to promote malignancy. In contrast to infiltration of cells responsible for chronic inflammation, the presence of high numbers of lymphocytes, especially T cells, has been reported to be important as indicator of good prognosis in many types of cancer [4-7]. The thorough knowledge of both manners and pathways with which tumors are able to evade immune-mediated attack, once established, is therefore of crucial importance. The strategies to escape anti-tumor immune responses include the limited priming or differentiation of antitumor T cells and the role of tumor microenvironment in order to prevent infiltration or activation of effector phase functions. We proposed to evaluate the role of Th inducing POZ-Kruppel Factor (ThPOK), a transcriptional regulator of T cell fate, in tumour-induced immune system plasticity during colorectal carcinogenesis. Data were collected on the amounts of CD4+, CD8+ and CD56+ as well as on ThPOK+ cells infiltrated in normal colorectal mucosa (NM), in dysplastic aberrant crypt foci (microadenomas, MA, the earliest detectable lesions in colorectal carcinogenesis) and in colorectal carcinomas (CRC); moreover, the colocalization of ThPOK with the above-mentioned markers of immune cells was evaluated using confocal microscopy. Interestingly, ThPOK showed a prominent increase since MA. A strong colocalization of ThPOK with CD4 both in NM and in MA was observed, weaker in carcinomas. Surprisingly, there was a peak in the colocalization levels of ThPOK with CD8 in MA, which was evident, although to a lesser extent, also in carcinomas. In conclusion, according to the data of the present study, ThPOK may be considered a central regulator of the earliest events in the immune system during colorectal cancer development. The novelty of the present study is the proposed role of ThPOK in influencing the immune response against cancer cells. References [1] Dunn et al. (2004) The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21: 137-148. [2] Knaapen et al. (2006) Neutrophils and respiratory tract DNA damage and mutagenesis: a review. Muta-genesis 21: 225-236. [3] Coussens and Werb (2002) Inflammation and cancer. Nature 420: 860-867. [4] Watt and House (1978) Colonic carcinoma: a quantitative assessment of lymphocyte infiltration at the periphery of colonic tumors related to prognosis. Cancer 41: 279-282. [5] Galon et al. (2006) Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313: 1960-1964. [6] Pagès et al. (2009) In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J Clin Oncol 27: 5944-5951. [7] Mlecnik et al. (2010) Biomolecular network reconstruction identifies T-cell homing factors associated with survival in colorectal. Gastroenterology 138: 1429-1434

Role of osteocyte apoptosis in peculiar ossicles of the hearing sense organ: preliminary observations on hearing loss and osteoporosis

Starting point of the present study is the osteocyte role in bone remodelling that allows bone adaptation to mechanical load [1-3]. Bone remodelling has been investigated in relation to the occurrence of apoptosis [4] to understand if and how the process of programmed cell death interferes with bone turnover. In 1998, in a study on human middle ear, Marotti et al. [5] demonstrated that: 1) over 40% of osteo-cytes are dead within the 2nd year of age (but the authors were not able to demonstrate if osteocyte death occurred by degeneration or apoptosis); 2) bone remodelling occurs only occasionally. Recently [6], we showed that: 1) osteocytes of human auditory ossicles die by apoptosis; 2) also osteocytes located inside scleral ossicles of lower vertebrate eye (reptiles and birds) phylogenetically so far from human auditory ossicles are widely affected by apoptosis (about 60%); 3) in scleral ossicles bone turnover never occur. It is to be noted that both auditory ossicles of human ear and scleral ossicles of vertebrate eye are peculiar bony segments continuously submitted to stereotyped stresses and strains, with specialized func-tions: the first are involved in sound wave transmission and the latter protect the eyeball against deformation during the movement and have a role in visual accomodation, providing attachment for the ciliary muscles. In both cases, bone remodelling might severely impair, by resorption, the mechanical resistance of these extremely small specialized bony segments. Thus, we suggested that in auditory and scleral ossicles, submitted to stereotyped loading for all life, bone mechanical adaptation is not needed and osteocyte programmed death could represent the mechanism to avoid bone remodelling and to make stable, when necessary, bone structure and mechanical resistance. More recently, to confirm this hypothesis, clinical data were collected from a cohort of patients aged 55-85 years affected by hearing loss. The main target of the present study is to exclude any correlation between hearing loss and osteoporosis. During osteoporosis, unbalanced bone turnover causes the bone depletion in skeletal segments; such condition, in the peculiar ossicles of human middle ear, should imply hearing impairment. Our preliminary observations indicate, instead, that osteoporotic patients do not show higher percentage of hearing loss with respect to non osteoporotic ones. This evidence is ascribable to osteocyte apoptosis of auditory ossicles that avoid bone remodelling, thus assuring the integrity of such bony segments also in osteoporotic conditions. References [1] Turner (1991) Omeostatic control of bone structure: an application of feed-bach theory. Bone 12: 203-217. [2] Turner and Forwood (1995) What role does the osteocyte network play in bone adaptation? Bone 16: 283-285. [3] Marotti (1996) The structure of bone tissue and the cellular control oftheir deposition. IJAE 101(4): 26-79. [4] Noble et al. (1997) Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 20: 273-282. [5] Marotti et al. (1998) Morphometric investigation on osteocytes in human auditory ossicles. Ann Anat 180: 449-453. [6] Palumbo et al. (2012) Osteocyte apoptosis in human auditory ossicles and scleral ossicles of lower ver-tebrates: a mere coincidence or linked processes? Calcif. Tissue Int. 90: 211-218

Chondrocyte expression of apoptotic and pro-inflammatory factors in the development of post- traumatic arthritis in humans

The development of post-traumatic arthritis following intra-articular fracture remains an important unsolved clinical problem. The possibility that extensive chondrocyte apoptosis occurs following intra-articular fracture, thus contributing to the development of post-traumatic arthritis, has received increasing attention [1]. It has been demonstrated the existence of a direct correlation between the rate of apoptosis and the severity of osteoarthritis [2]. Pharmacologic inhibitors of enzymes involved in apoptosis have been explored as potential therapeutic agents [3]. In the present study we aimed to deepen the characterization of apoptotic mediators, expressed by chondrocytes, involved in human post-traumatic arthritis following intra-articular fracture and the possible implication of pro-inflammatory receptors in arthritis. The expression of a panel of pro/anti apoptotic factors (Caspase-3, PARP-1, BCL2) and inflammation-related receptors (ChemR23) were analysed in chondrocytes from patients undergoing surgery for intra-articular calcaneal fractures. The factors were investigated by immunofluorescence coupled with confocal analysis and western blotting, followed by densitometric evaluation of chondrocyte cultures harvested from patients with intra-articular fractures compared with control ones. The results clearly demonstrated that a statistically significant difference exists in the expression of pro/anti apoptotic factors and ChemR23 between fractured and control patients. In conclusion our data suggest that increased chondrocyte death, occurring after cartilage injury together with inflammatory process, could play a pivotal role in the onset of arthritic disease. References [1]. Hembree W.C. et al. (2007) Viability and apoptosis of human chondrocytes in osteochon-dral fragments following joint trauma. J Bone Joint Surg Br 89(10): 1388-95. [2] Kim H.A. et al. (2000) Apoptotic chondrocyte death in human osteoarthritis. J Rheumatol 27: 455\u2013462. [3] D'Lima D. et al. (2006) Caspase inhibitors reduce severity of cartilage lesions in experi-mental osteoarthritis. Arthritis Rheum 54(6): 1814-1821

Preliminary observations on scleral ossicles in performing functionalized 3D vascularized scaffolds for “critical-size” bone defect healing

The problem of “critical-size” bone defects occurs when a severe lesion is difficult to be self-recovered. Many strategies of regenerative medicine were used in the last decade, with translational approaches, to mimic both structure and function of the native bone tissue, making use of synthetic materials, nanotechnologies, bio/synthetic constructs or some of their combination. The main obstacle to engineering strategies is mostly due to the lack of a proper vascularization of the construct used. In this feasibility study, our attention is directed towards the main tissue engineering items: scaffolds, cells and conditioning factors. We propose the use of scleral ossicles of lower vertebrates (1), as natural scaffolds which will be functionalized to allow the best adhesion of endothelial cells along a geometrically controlled pattern on the bony surface of the construct; successively, on the functionalized scaffold, osteogenic cell lines will be cultured. In the preliminary phases of the study, the ossicles were scratched to remove soft tissue residues, variously flattened with different methods to reach a regular morphology on both sides, and finally autoclaved to eliminate cellular remnants and to annul antigenic properties. Ossicles were observed under SEM and subjected to micro-assay, to establish the best scaffold preparation and to characterize morphological properties more suitable for engineering phases. Functionalization will be made by immobilizing on the engineered ossicles specific growth factors for endothelial cells; later, mouse primary lung endothelial cells (ECs) and immortalized osteogenic cells (IDG-SW3) will be used. As expected results ECs should adhere to the ossicle surface and organize to form lumenized microvascular-like structures; later, supported by the vascular-like network, the osteogenic lineage should produce bone matrix on the construct. The production of newly-formed bone around vascular-like buds will be verify. 3D tissue constructs generated in vitro will be used in successive in vivo study for the healing of “critical-size” bone defects experimentally induced in mice

Matrix metalloproteinases 15 and 19 are stromal regulators of colorectal cancer development from the early stages

Matrix metalloproteinases (MMPs) have been well characterized for their ability to degrade extracellular matrix proteins and, thus, they have been studied to elucidate their involvement in both tumor development and progression. In the present study, attention was focused on MMP-15 and MMP-19, two less known members of the MMP family. The expression profile of MMP-15 and -19 was assayed in samples of normal colorectal mucosa, microadenomas and cancer using confocal analysis, western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both qRT-PCR and western blotting showed that MMP-15 and MMP-19 appeared to be upregulated during colorectal tumorigenesis, with different expression patterns: MMP-15 expression level increases from normal mucosa to microadenomas, with a reduced level in cancer with respect to microadenomas; the semiquantitative immunofluorescence analysis showed a stromal localization of this protein in the early phases of neoplastic transformation. Increasing amount of MMP-19 mRNA and protein levels were observed in the progression of colonic lesions; MMP-19 staining increased in the normal mucosa-microadenoma-carcinoma sequence. Such different expression patterns, are probably due to the different roles played in colorectal tumorigenesis by these two molecules. Conflicting data on the role of these proteins in tumor progression have been reported, thus, an improved understandingof the biological roles of MMPs, in particular the lesser known members such as MMP-15 and 19, in colorectal cancer may lead to a re-evaluation of the use of MMP inhibitors and suggests the need of integrated translational studies on MMP expression patterns