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.

Assessment of SARS-CoV-2 airborne infection transmission risk in public buses.

Public transport environments are thought to play a key role in the spread of SARS-CoV-2 worldwide. Indeed, high crowding indexes (i.e. high numbers of people relative to the vehicle size), inadequate clean air supply, and frequent extended exposure durations make transport environments potential hotspots for transmission of respiratory infections. During the COVID-19 pandemic, generic mitigation measures (e.g. physical distancing) have been applied without also considering the airborne transmission route. This is due to the lack of quantified data about airborne contagion risk in transport environments. In this study, we apply a novel combination of close proximity and room-scale risk assessment approaches for people sharing public transport environments to predict their contagion risk due to SARS-CoV-2 respiratory infection. In particular, the individual infection risk of susceptible subjects and the transmissibility of SARS-CoV-2 (expressed through the reproduction number) are evaluated for two types of buses, differing in terms of exposure time and crowding index: urban and long-distance buses. Infection risk and reproduction number are calculated for different scenarios as a function of the ventilation rates (both measured and estimated according to standards), crowding indexes, and travel times. The results show that for urban buses, the close proximity contribution significantly affects the maximum occupancy to maintain a reproductive number of <1. In particular, full occupancy of the bus would be permitted only for an infected subject breathing, whereas for an infected subject speaking, masking would be required. For long-distance buses, full occupancy of the bus can be maintained only if specific mitigation solutions are simultaneously applied. For example, for an infected person speaking for 1 h, appropriate filtration of the recirculated air and simultaneous use of FFP2 masks would permit full occupancy of the bus for a period of almost 8 h. Otherwise, a high percentage of immunized persons (>80%) would be needed.

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.

Duodenal perforation in a SARS-CoV-2-positive patient with negative PCR results for SARS-CoV-2 in the peritoneal fluid.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has significantly affected health care organizations globally. Many aspects of this disease, as well as the risks for patients treated with multiple drug regimens to control severe COVID-19, are unclear. During emergency surgery for SARS-CoV-2-positive patients, the risk of SARS-CoV-2 exposure and transmission to the surgical staff has yet to be determined. PATIENTS AND METHODS: In this report, we describe a SARS-CoV-2-positive patient with severe respiratory syndrome treated with multiple doses of IL-6 inhibitors who presented with a perforated duodenal ulcer and underwent emergency surgery. During and after surgery, we tested for SARS-CoV-2 at the ulcer site and in the peritoneal fluid. RESULTS: The history of the patient allows for two possible interpretations of the pathogenesis of the duodenal ulcer, which could have been a stress ulcer, or a gastrointestinal ulcer associated to the use of IL-6 inhibitors. We also noticed that the ulcer site and peritoneal fluid repeatedly tested negative for SARS-CoV-2. Therefore, we reviewed the pertinent literature on gastrointestinal bleeding in patients with COVID-19 and on SARS-CoV-2 detection in the peritoneal fluid of surgical patients and discussed possible prevention strategies for bleeding and the actual risk of infection for the surgical staff. CONCLUSIONS: The first implication of this case is that the relation between repeated administration of IL-6 inhibitors and upper gastrointestinal bleeding and perforation must be investigated, and that the threshold for administering prophylactic proton pump inhibitors therapy should be carefully considered for patients with severe COVID-19. The second implication is that further testing should be performed on the peritoneal fluid of COVID-19 patients undergoing emergency surgical procedures to clarify the discordant results for the presence of SARS-CoV-2 in the peritoneal cavity and the possible risk of transmission to the surgical staff.

Review: Danon disease: Review of natural history and recent advances.

Danon disease is a severe multisystem disorder clinically characterized by hypertrophic cardiomyopathy, skeletal myopathy and mental retardation in male patients, and by a milder phenotype (predominantly involving cardiac muscle) in female patients. The disease is inherited as an X-linked dominant trait. The primary deficiency of lysosome-associated membrane protein-2 (LAMP-2) causes disruption of autophagy, leading to an impaired fusion of lysosomes to autophagosomes and biogenesis of lysosomes. We surveyed over 500 Danon disease patients reported in the literature from the first description to the present, in order to summarize the clinical, pathological and molecular data and treatment perspectives. An early molecular diagnosis is of crucial importance for genetic counselling and for therapeutic interventions: in male patients, the prognosis is poor due to rapid progression towards heart failure, and only heart transplantation modifies the disease course.

Update on polyglucosan storage diseases.

An abnormal structural form of glycogen (with less branching points or amylopectin-like polysaccharide) called polyglucosan (PG) may accumulate in various tissues such as striated and smooth muscles, brain, nerve, liver and skin, and cause a group of nine different genetic disorders manifesting with a variety of clinical phenotypes that affect mainly the nervous system (Lafora disease, adult PG body disease), the heart (glycogen storage disease type XV, hypertrophic cardiomyopathy type 6, PG body myopathy type 1) and the skeletal muscle (glycogen storage disease type IV, glycogen storage disease type VII, PG body myopathy type 2), depending on the organs which are mostly affected by the PG aggregates. The pathological feature of PG storage in tissues is a hallmark of these disorders. Whole-genome sequencing has allowed to obtain a diagnosis in a large number of patients with a previously unrecognized disorder. We describe the clinical, pathological and molecular features of these genetic disorders, for many of which the pathological mechanisms underlying the corresponding mutant gene have been investigated and, at least in part, understood.

Subpopulations of dermal skin fibroblasts secrete distinct extracellular matrix: implications for using skin substitutes in the clinic.

BACKGROUND: While several commercial dermoepidermal scaffolds can promote wound healing of the skin, the achievement of complete skin regeneration still represents a major challenge. OBJECTIVES: To perform biological characterization of self-assembled extracellular matrices (ECMs) from three different subpopulations of fibroblasts found in human skin: papillary fibroblasts (Pfi), reticular fibroblasts (Rfi) and dermal papilla fibroblasts (DPfi). METHODS: Fibroblast subpopulations were cultured with ascorbic acid to promote cell-assembled matrix production for 10 days. Subsequently, cells were removed and the remaining matrices characterized. Additionally, in another experiment, keratinocytes were seeded on the top of cell-depleted ECMs to generate epidermal-only skin constructs. RESULTS: We found that the ECM self-assembled by Pfi exhibited randomly oriented fibres associated with the highest interfibrillar space, reflecting ECM characteristics that are physiologically present within the papillary dermis. Mass spectrometry followed by validation with immunofluorescence analysis showed that thrombospondin 1 is preferentially expressed within the DPfi-derived matrix. Moreover, we observed that epidermal constructs grown on DPfi or Pfi matrices exhibited normal basement membrane formation, whereas Rfi matrices were unable to support membrane formation. CONCLUSIONS: We argue that inspiration can be taken from these different ECMs, to improve the design of therapeutic biomaterials in skin engineering applications.

Juvenile dermatomyositis: A report of three cases.

Juvenile dermatomyositis (JDM), an autoimmune idiopathic myositis, is characterized by rash and proximal muscle weakness. Immunohistopathology typically shows perivascular inflammatory infiltrate with predominance of CD4+ T lymphocytes, perifascicular atrophy, and upregulation of major histocompatibility complex class I. JDM has been attributed to a humoral-driven muscle microangiopathy probably implicating the type I interferon pathway. Tubulo-reticular inclusions present in endothelial cell of muscle are biomarkers of interferon exposure, and so may be an indirect data of this myopathy especially in the absence of rash and inflammatory infiltrate. We report on three patients in which electron microscopy solves the differential diagnosis among infantile myositis showing peculiar inclusions.

Building blocks of the GIPU, Italian Group of Ultrastructural Pathology.

The Italian Group of Ultrastructural Pathology, GIPU, is a scientific organization committed to promote the art and science of Electron Microscopy (EM) in the pathology field in Italy, sharing its professional work with a public audience. The history of the GIPU goes back to 1990s when a founder group set up the Italian Group of Ultrastructural Diagnostic (GIDU) in Milan. The central focus of annual meetings was on EM, transmission and scanning one, about interesting cases in which it was instrumental in diagnosis. In the 1990s, ultrastructure was still the gold standard for cell/tissue morphology, biology, biochemistry, diagnostic pathology, and played an important role in tailored medicine. So, especially transmission EM, could play a critical role in the diagnosis of various diseases as in human as in animals. Best topics of the annual scientific meetings of the group were kidney, muscle, heart, and liver pathology, infertility, neuropathology, respiratory diseases, skin diseases, storage diseases, tumor pathology, infectious diseases, parasitology, veterinary pathology and more. Nowadays, EM is a method whose importance for diagnosis and pathology is well established: it is still essential in several pathologies, helpful in others, and welcome implemented in eclectic research pathology. Omission of EM likely makes the studies suboptimal and wasteful. So, from 2007 the name of the group has been changed to the Italian Group of Ultrastructural Pathology (GIPU) to favor broader applications of EM also to pathology research field. During last decades, GIDU/GIPU has interconnected with international (Society for Ultrastructural Pathology) and european (European Society of Pathology and Joint Meeting with the European Electron Microscopy Working Group) scientific society, according its statute. By 1991, GIPU has had 40 members: membership in this Group is still open and welcome to all pathologists, PhD, electron microscopy technologists, pathology trainees, and researchers interested in pathology and electron microscopy.

Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PURPOSE: Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). METHODS: Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. RESULTS: Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. CONCLUSIONS: Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

Use of the Wasserman equation in optimization of the duration of the power ramp in a cardiopulmonary exercise test: a study of Brazilian men

This study aimed to analyze the agreement between measurements of unloaded oxygen uptake and peak oxygen uptake based on equations proposed by Wasserman and on real measurements directly obtained with the ergospirometry system. We performed an incremental cardiopulmonary exercise test (CPET), which was applied to two groups of sedentary male subjects: one apparently healthy group (HG, n=12) and the other had stable coronary artery disease (n=16). The mean age in the HG was 47±4 years and that in the coronary artery disease group (CG) was 57±8 years. Both groups performed CPET on a cycle ergometer with a ramp-type protocol at an intensity that was calculated according to the Wasserman equation. In the HG, there was no significant difference between measurements predicted by the formula and real measurements obtained in CPET in the unloaded condition. However, at peak effort, a significant difference was observed between oxygen uptake (V˙O2)peak(predicted)and V˙O2peak(real)(nonparametric Wilcoxon test). In the CG, there was a significant difference of 116.26 mL/min between the predicted values by the formula and the real values obtained in the unloaded condition. A significant difference in peak effort was found, where V˙O2peak(real)was 40% lower than V˙O2peak(predicted)(nonparametric Wilcoxon test). There was no agreement between the real and predicted measurements as analyzed by Lin’s coefficient or the Bland and Altman model. The Wasserman formula does not appear to be appropriate for prediction of functional capacity of volunteers. Therefore, this formula cannot precisely predict the increase in power in incremental CPET on a cycle ergometer.

The endocrine disruptor cadmium alters human osteoblast-like Saos-2 cells homeostasis in vitro by alteration of Wnt/ß-catenin pathway and activation of caspases.

PURPOSE: The pollutant Cadmium (Cd) is widespread in the environment and causes alterations of human health by acting as an endocrine disruptor. Bone tissue seems to be a crucial target of Cd contamination. Indeed, we have previously demonstrated that this endocrine disruptor induces osteoblast apoptosis and necrosis. Thus, aim of this study was to further evaluate the effect of Cd on osteoblasts homeostasis, investigating potential modification of the Wnt/ß-catenin intracellular pathway, the intracellular process involved in programmed cellular death and the cytoskeletal alterations. MATERIAL AND METHODS: To this purpose, human osteoblastic Saos-2 cells, a human osteosarcoma osteoblast-like cell line, were cultured and treated with Cd. RESULTS: Osteoblastic cells were treated for 6 h with 10µM Cd, which induced nuclear translocation of ß-catenin and increased expression of Wnt/ß-catenin target genes. Longer exposure to the same Cd concentration induced osteoblastic cell apoptosis. To better characterize the intracellular events involved in these Cd-induced alterations, we evaluated the effect of Cd exposure on actin filaments and proteins associated to cytoskeletal actin, characterized by the presence of LIM domains. Long (15, 24 h) exposure of osteoblasts to Cd reduced LIM proteins expression and induced actin filaments destruction and a significant caspase-3 activation after 24 h. In addition, to prove that Cd induces osteoblastic cells apoptosis after long exposure, we performed TUNEL assay which demonstrated increase of cell apoptosis after 24 h. CONCLUSION: In conclusion, our study shows that osteoblasts exposed to Cd for short intervals of time demonstrated an increase in cell proliferation through a Wnt/ß-catenin dependent mechanism, likely as a compensatory mechanism in response to cell injury. Longer exposure to the same Cd concentration induced cells apoptosis through cytoskeleton disruption-mediated mechanisms and caspase activation.

Use of the Wasserman equation in optimization of the duration of the power ramp in a cardiopulmonary exercise test: a study of Brazilian men.

This study aimed to analyze the agreement between measurements of unloaded oxygen uptake and peak oxygen uptake based on equations proposed by Wasserman and on real measurements directly obtained with the ergospirometry system. We performed an incremental cardiopulmonary exercise test (CPET), which was applied to two groups of sedentary male subjects: one apparently healthy group (HG, n=12) and the other had stable coronary artery disease (n=16). The mean age in the HG was 47±4 years and that in the coronary artery disease group (CG) was 57±8 years. Both groups performed CPET on a cycle ergometer with a ramp-type protocol at an intensity that was calculated according to the Wasserman equation. In the HG, there was no significant difference between measurements predicted by the formula and real measurements obtained in CPET in the unloaded condition. However, at peak effort, a significant difference was observed between oxygen uptake (V˙O2)peak(predicted)and V˙O2peak(real)(nonparametric Wilcoxon test). In the CG, there was a significant difference of 116.26 mL/min between the predicted values by the formula and the real values obtained in the unloaded condition. A significant difference in peak effort was found, where V˙O2peak(real)was 40% lower than V˙O2peak(predicted)(nonparametric Wilcoxon test). There was no agreement between the real and predicted measurements as analyzed by Lin's coefficient or the Bland and Altman model. The Wasserman formula does not appear to be appropriate for prediction of functional capacity of volunteers. Therefore, this formula cannot precisely predict the increase in power in incremental CPET on a cycle ergometer.

Does a minimum number of 16 retrieved nodes affect survival in curatively resected gastric cancer?

BACKGROUND: According to the TNM classification, the analysis of 16 or more lymph nodes is required for the appropriate staging of gastric cancer. The aim of this study was to evaluate whether this number of resected lymph nodes also affects survival. METHODS: This was a multicenter retrospective study based on an analysis of 992 patients with gastric adenocarcinoma who underwent curative resection between January 1980 and December 2009. Patients were classified according to the number of resected lymph nodes (<16 and ≥16 lymph nodes), the anatomical extent of lymph node dissection (D2 vs. D1), and the staging criteria of the seventh edition of the UICC/AJCC TNM staging system. Survival estimates were determined by univariate and multivariate analyses. RESULTS: Based on the univariate and multivariate analyses, the resection of 16 or more lymph nodes was associated with significantly better survival [p = 0.002; hazard ratio (HR) (95% confidence interval [CI]): 0.519 (0.345-0.780)]. Patients with a lymph node count <16 had a significantly worse survival rate than patients with a lymph node count ≥16 in the pN0 (p = 0.001), pN1 (p = 0.007) and pN2 (p = 0.001) stages. In the majority of cases, ≥16 lymph nodes were retrieved when D2 dissection was performed. CONCLUSIONS: In gastric cancer the retrieval of less than 16 lymph nodes may cause inaccurate staging and/or inadequate treatment, thus affecting survival rates. These patients should be considered a high-risk group for stage migration and worse survival compared with those who have a retrieval of more than 16 lymph nodes.

Lean mass in obese adult subjects correlates with higher levels of vitamin D, insulin sensitivity and lower inflammation.

AIM: Several chronic metabolic alterations are present in obese subjects. While it is well known about the detrimental effect of abdominal adipose tissue on chronic metabolic clinical condition, less is known on the role of lean mass in obese subjects. Thus, the aim of our study was to evaluate the potential correlation of muscle mass, metabolic condition and inflammation status in obese individuals. METHODS: The study included 426 obese subjects (86 men and 340 female; mean age 44.8 ± 14 years; BMI: 34.9 ± 6.1 kg/m(2)). Exclusion criteria were chronic medical conditions or use of medications affecting bone metabolism, alterations of hormonal and nutritional status, vitamin D supplementation, recent weight loss and prior bariatric surgery. Patients underwent measurements of bone mineral density (lumbar and hip) and body composition (lean mass, total and trunk fat mass) by dual X-ray absorptiometry and were evaluated for hormonal and metabolic profile and inflammatory markers. RESULTS: Higher lean body mass (LM%) was inversely correlated with homeostasis model assessment of insulin resistance (p < 0.0091; r(2) 0.03938) and associated with lower fibrinogen levels (p < 0.0001; r(2) 0.1263). Interestingly, in obese subjects, LM% was associated with higher levels of vitamin D (p < 0.0001, r(2) 0.1140), osteocalcin (p < 0.0001, r(2) 0.2401) and insulin-like growth factor-1 (IGF-1) (p < 0.0002, r(2) 0.1367). CONCLUSION: Our results show for the first time that in obese patients, higher amounts of lean mass are directly linked to a lower inflammatory profile and to better insulin sensitivity, but also to the presence of higher level of vitamin D and IGF-1. Moreover, these data suggest that higher levels of lean mass in obese people correlate with a better metabolic profile and, thus, strongly suggest the need to develop programs to facilitate an increase in physical activity in obese people.

The environmental pollutant cadmium induces homeostasis alteration in muscle cells in vitro.

BACKGROUND: Cadmium (Cd) is a heavy metal widely distributed throughout the environment as a result of contamination from a variety of sources. It exerts toxic effects in many tissues but scarce data are present as yet on potential effects on skeletal muscle tissue. AIM: To evaluate the potential alteration induced by Cd in skeletal muscle cells. MATERIALS AND METHODS: C2C12 skeletal muscle cells were treated with Cd at different times of cellular differentiation and gene expression was evaluated. RESULTS: Exposure to Cd decreased significantly p21 mRNA expression and strongly up-regulated cyclin D1 mRNA expression in committed cells and in differentiated myotubes. Moreover, myogenin, fast MyHC-IIb and slow MyHC-I mRNAs expression were also significantly decreased both in committed cells and in myotubes. Moreover, Cd exposure induced a strong increase of Pax3, Pax7 and Myf5 mRNAs expression and stimulated an up-regulation of IL6 and TNF-α proinflammatory cytokines. CONCLUSION: These data lead to hypothesize that environmental Cd exposure might trigger an injury-like event in muscle tissue, possibly by an estrogen receptor-mediated mechanism.

MMP activity in the hybrid layer detected with in situ zymography.

Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application.

Type 1 diabetes mellitus and hyperadrenocorticism in a ferret.

Diabetes mellitus (DM) was diagnosed in a 6-year-old neutered male ferret with polyuria/polydipsia, symmetrical alopecia, and weight loss. Laboratory tests revealed severe hyperglycemia, glucosuria, and increased steroid hormone profile. Abdominal ultrasound revealed a bilateral enlargement of the adrenal glands. Significant clinical improvement was achieved with insulin- and leuprolide acetate-based therapy. After 2 months of therapy, the ferret showed a severe ketoacidosis, and the owner decided to euthanize the animal. Histological findings revealed carcinoma of the left adrenal cortex and cortical hyperplasia of the right adrenal gland. Moderate, chronic, and active pancreatitis with a marked decrease in the number of beta-cells was also present. This is the first reported case of type 1 DM associated with hyperadrenocorticism and chronic pancreatitis in a ferret.

Erucylphosphohomocholine, the first intravenously applicable alkylphosphocholine, is cytotoxic to acute myelogenous leukemia cells through JNK- and PP2A-dependent mechanisms.

Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6-12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34(+) leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34(+) CD38(Low/Neg) CD123(+)) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix.

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule.Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.