CROCC-mutated rhabdoid colorectal carcinoma showing in intercellular spaces lamellipodia and cellular projections revealed by electron microscopy.

BACKGROUND: Rhabdoid colorectal carcinoma (RC) is a rare lesion localized to the proximal colon of patients with a mean age at diagnosis of around 70 years. This tumor shows an aggressive behavior with an overall survival period shorter than 12 months. The diagnostic hallmark is the presence of rhabdoid cells. Alterations in chromatin remodeling (SMARCB1) and in the centrosome structure (CROCC) are reported in RC usually BRAFmut and MSI-H. RKO intestinal neoplastic cells culture (BRAFmut, SMARCB1wt, MSI-H) with CROCC knockdown exhibit rhabdoid features and develop prominent projections from the edge of the cell. METHODS: Here, we investigated two cases of CROCCmutSMARCB1wt RC by scanning and transmission electron microscopy (SEM, TEM). RESULTS: TEM confirmed the diagnostic presence of intermediate cytoplasmic filaments and nucleolar margination. SEM showed cellular protrusions (lamellipodia) in the intercellular spaces not evident at light microscopy. CONCLUSIONS: These protrusions CROCC-related might represent the pathogenetic mechanism underlying the rhabdoid aggressive behavior, independently of tumor staging. To our knowledge, the SEM technique was applied in the study of this neoplasm for the first time.

ALDH2 Activity Reduces Mitochondrial Oxygen Reserve Capacity in Endothelial Cells and Induces Senescence Properties.

Endothelial cells (ECs) are dynamic cells that turn from growth into senescence, the latter being associated with cellular dysfunction, altered metabolism, and age-related cardiovascular diseases. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme metabolizing acetaldehyde and other toxic aldehydes, such as 4-hydroxynonenal (4-HNE). In conditions in which lipid peroxidation products and reactive oxygen species (ROS) are accumulated, ECs become dysfunctional and significantly contribute to the progression of vascular-dependent diseases. The aim of the present study has been to investigate whether inhibition of ALDH2 alters endothelial functions together with the impairment of bioenergetic functions, accelerating the acquisition of a senescent phenotype. HUVECs transfected with siRNA targeting ALDH2 or treated with daidzin, an ALDH2 inhibitor, were used in this study. We observed an alteration in cell morphology associated with endothelial dysfunctions. Loss of ALDH2 reduced cell proliferation and migration and increased paracellular permeability. To assess bioenergetic function in intact ECs, extracellular flux analysis was carried out to establish oxygen consumption rates (OCR). We observed a decrease in mitochondrial respiration and reserve capacity that coincided with SA-ß-Gal accumulation and an increase in p21 and p53 expression in siALDH2 or daidzin-treated HUVECs. Treatment with N-acetyl-L-cysteine (NAC) reduced endothelial dysfunctions mediated by siALDH2, indicating that oxidative stress downstream to siALDH2 plays an instrumental role. Our results highlight that ALDH2 impairment accelerates the acquisition of a premature senescent phenotype, a change likely to be associated with the observed reduction of mitochondrial respiration and reserve capacity.

Chromium-induced diffuse dermatitis with lymph node involvement resulting from Langerhans cell histiocytosis after metal-on-metal hip resurfacing.

Total hip arthroplasty (THA) is a highly effective surgical treatment for severe joint involvement. However, due to the release of metal ions in the blood, the patients who undergo hip replacement with metal-on-metal (MOM) bearings may develop signs of allergic skin disease. We report a case of a 60-year-old man who had received MOM hip resurfacing 5 years earlier for osteoarthritis. He presented with a 3-year history of diffuse dermatitis that did not respond to antihistamines and corticosteroids and also had elevated serum levels of chromium and cobalt. A patch test revealed chromium-sulfate hypersensitivity. A skin biopsy showed nonspecific perivascular lymphocytic infiltrate associated with histiocytes. A biopsy of an inguinal lymph node demonstrated large aggregates of Langerhans cells, suggesting type IV delayed-type hypersensitivity. The prosthesis was replaced using ceramic-on-ceramic bearings and the dermatitis resolved after 3 months. The lymph nodes decreased in volume and the serum chromium levels normalized within 24 months of revision surgery. The high levels of serum ions associated with the metal debris from MOM-THAs may induce sensitization and type IV hypersensitivity reactions. Replacing the prosthesis using alternative coupling surfaces is the only approach that has the capacity to resolve these symptoms. Physicians who are not familiar with this issue may misdiagnose systemic symptoms and provide inadequate treatment.

Chemotherapeutic induction of mitochondrial oxidative stress activates GSK-3α/ß and Bax, leading to permeability transition pore opening and tumor cell death.

Survival of tumor cells is favored by mitochondrial changes that make death induction more difficult in a variety of stress conditions, such as exposure to chemotherapeutics. These changes are not fully characterized in tumor mitochondria, and include unbalance of the redox equilibrium, inhibition of permeability transition pore (PTP) opening through kinase signaling pathways and modulation of members of the Bcl-2 protein family. Here we show that a novel chemotherapeutic, the Gold(III)-dithiocarbamato complex AUL12, induces oxidative stress and tumor cell death both favoring PTP opening and activating the pro-apoptotic protein Bax of the Bcl-2 family. AUL12 inhibits the respiratory complex I and causes a rapid burst of mitochondrial superoxide levels, leading to activation of the mitochondrial fraction of GSK-3α/ß and to the ensuing phosphorylation of the mitochondrial chaperone cyclophilin D, which in turn facilitates PTP opening. In addition, following AUL12 treatment, Bax interacts with active GSK-3α/ß and translocates onto mitochondria, where it contributes to PTP induction and tumor cell death. These findings provide evidence that targeting the redox equilibrium maintained by mitochondria in tumor cells allows to hit crucial mechanisms that shield neoplasms from the toxicity of many anti-tumor strategies, and identify AUL12 as a promising chemotherapeutic compound.

A large outbreak of Opisthorchis felineus in Italy suggests that opisthorchiasis develops as a febrile eosinophilic syndrome with cholestasis rather than a hepatitis-like syndrome.

We describe the greatest Italian human acute opisthorchiasis outbreak acquired from eating raw tenches. Out of 52 people with suspected opisthorchiasis, 45 resulted in being infected. The most frequent symptoms and laboratory findings were fever, abdominal pain and eosinophilia. Seven tri-phasic computed tomography (CT) scans were done, showing multiple hypodense nodules with hyper-enhancement in the arterial phase. All patients took one day of praziquantel 25 mg/kg TID without failures. Reported symptoms suggested a febrile eosinophilic syndrome with cholestasis rather than a hepatitis-like syndrome. It seems common to find hepatic imaging alterations during acute opisthorchiasis: CT scan could be the most suitable imaging examination. Even if stool test remains the diagnostic gold standard, we found earlier positivity with the serum antibody test. Without previous freezing, the consumption of raw freshwater fish should be avoided.

The trochanteric fat pad.

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis.

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.

Calcium and cell death: the mitochondrial connection.

Physiological stimuli causing an increase of cytosolic free Ca2+ [Ca2+], or the release of Ca2+ from the endoplasmic reticulum invariably induce mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free [Ca2+] ([Ca2+]m). The [Ca2+]m rise occurs despite the low affinity of the mitochondrial Ca2+ uptake systems measured in vitro and the often limited amplitude of the cytoplasmic [Ca2+]c increases. The [Ca2+]m increase is typically in the 0.2-3 microM range, which allows the activation of Ca2(+)-regulated enzymes of the Krebs cycle; and it rapidly returns to the resting level if the [Ca2+], rise recedes due to activation of mitochondrial efflux mechanisms and matrix Ca2+ buffering. Mitochondria thus accumulate Ca2+ and efficiently control the spatial and temporal shape of cellular Ca2+ signals, yet this situation exposes them to the hazards of Ca2+ overload. Indeed, mitochondrial Ca2+, which is so important for metabolic regulation, can become a death factor by inducing opening of the permeability transition pore (PTP), a high conductance inner membrane channel. Persistent PTP opening is followed by depolarization with Ca2+ release, cessation of oxidative phosphorylation, matrix swelling with inner'membrane remodeling and eventually outer membrane rupture with release of cytochrome c and other apoptogenic proteins. Understanding the mechanisms through which the Ca2+ signal can be shifted from a physiological signal into a pathological effector is an unresolved problem of modern pathophysiology that holds great promise for disease treatment.

High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening.

To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 microM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33,342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity ( approximately 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12+/-22). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30x the maximal efficacious concentration or 100 microM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS.

Apoptosis to necrosis switching downstream of apoptosome formation requires inhibition of both glycolysis and oxidative phosphorylation in a BCL-X(L)- and PKB/AKT-independent fashion.

Human T-lymphoma Jurkat cells treated with several intrinsic death stimuli readily undergo a stepwise apoptotic program. Treatment with 1,9-dideoxyforskolin (ddFSK), an inactive analogue of the adenylate cyclase activator forskolin, induces necrotic cell death and switches to necrosis the response to the apoptosis inducers in Jurkat and in other cell models. Yet, in the presence of ddFSK, mitochondrial changes are enhanced and apoptosome formation takes place. We show that ddFSK does not inhibit the catabolic steps of apoptosis, but rather elicits a profound ATP depletion that in turn tunes the mode of cell demise towards necrosis. Treatment with ddFSK impairs both glycolysis and oxidative phosphorylation in a Bcl-X(L)- and PKB/Akt-independent fashion, and inhibition of both processes is needed to affect apoptosis progression. Apoptosis is not blocked per se by ATP depletion, as engagement of the Fas receptor directly activates caspases, thus bypassing ddFSK inhibition.

The selection between apoptosis and necrosis is differentially regulated in hydrogen peroxide-treated and glutathione-depleted human promonocytic cells.

Treatment with 0.2 mM hydrogen peroxide (H(2)O(2)) or with 0.5 mM cisplatin caused caspase-9 and caspase-3 activation and death by apoptosis in U-937 human promonocytic cells. However, treatment with 2 mM H(2)O(2), or incubation with the glutathione suppressor DL-buthionine-(S,R)-sulfoximine (BSO) prior to treatment with cisplatin, suppressed caspase activation and changed the mode of death to necrosis. Treatment with 2 mM H(2)O(2) caused a great decrease in the intracellular ATP level, which was partially prevented by 3-aminobenzamide (3-ABA). Correspondingly, 3-ABA restored the activation of caspases and the execution of apoptosis. By contrast, BSO plus cisplatin did not decrease the ATP levels, and the generation of necrosis by this treatment was not affected by 3-ABA. On the other hand, while all apoptosis-inducing treatments and treatment with 2 mM H(2)O(2) caused Bax translocation from the cytosol to mitochondria as well as cytochrome c release from mitochondria to the cytosol, treatment with BSO plus cisplatin did not. Treatment with cisplatin alone caused Bid cleavage, while BSO plus cisplatin as well as 0.2 and 2 mM H(2)O(2) did not. Bcl-2 overexpression reduced the generation of necrosis by H(2)O(2), but not by BSO plus cisplatin. These results indicate the existence of different apoptosis/necrosis regulatory mechanisms in promonocytic cells subjected to different forms of oxidative stress.

Correlation MRI/ultrastructure in cerebral ischemic lesions: application to the interpretation of cortical layered areas.

The origin and fate of cortical ischemic lesions, showing a stratified appearance at in vivo MRI-examination, was studied on rats in which a focal brain ischemia was induced by occlusion of the middle cerebral artery. One week after ischemia induction, six rats were selected in which three layers of different intensity were visible in the lesioned cortex. Two animals were sacrificed and studied by histology and electron microscopy. The external hyperintense layer was composed of pial and lesioned nervous tissue, the intermediate of degenerating nervous tissue in which an accumulation of macrophages was found, the deepest of edematous nerve tissue without a marked accumulation of macrophages. The remaining rats underwent further MRI examinations showing that, in the lesioned areas, cerebral blood volume was 14-69% lower than the contralateral healthy cortex. At histological and ultrastructural examination, a large part of the lesion was occupied by enlarged pial tissue and marginal glia. A dilatation of the ventricular cavity and cystic structures were also visible. In three animals an increase of the transverse diameter of the caudo-putamen ipsilateral to the lesion was found. The study suggests that the layered appearance is mainly due to an accumulation of macrophages in the intermediate layer and that several processes contribute to the occlusion of the space created by the removal of the necrotic tissue in stratified ischemic lesions (i.e. expansion of the pial tissue, thickening of the marginal glia; expansion of the caudo-putamen, enlargement of the ventricular cavity and development of cystic structures).

A 915-MHz antenna for microwave thermal ablation treatment: physical design, computer modeling and experimental measurement.

A 915-MHz antenna design that produces specific absorption rate distributions with preferential power deposition in tissues surrounding and including the distal end of the catheter antenna is described. The design features minimal reflected microwave current from the antenna flowing up the transmission line. This cap-choke antenna consists of an annular cap and a coaxial choke which matches the antenna to the coaxial transmission line. The design minimizes heating of the coaxial cable and its performance is not affected by the depth of insertion of the antenna into tissue. The paper provides a comparison of results obtained from computer modeling and experimental measurements made in tissue equivalent phantom materials. There is excellent agreement between numerical modeling and experimental measurement. The cap-choke, matched-dipole type antenna is suitable for intracavitary microwave thermal ablation therapy.

A mitochondrial perspective on cell death.

The role of mitochondria as crucial participants in cell death programs is well established, yet the mechanisms responsible for the release of mitochondrial activators and the role of BCL2 family proteins in this process remain controversial. Here, we point out the limitations of current approaches used to monitor the physiological responses of mitochondria during cell death, the implications arising from modern views of mitochondrial structure, and briefly assess two proposed mechanisms for the release of mitochondrial proteins during apoptosis.

Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling.

We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent depolarization or respiratory inhibition, suggesting a specific effect on the PT pore. When added to intact MH1C1 cells, arachidonic acid but not palmitic acid caused a mitochondrial PT in situ that was accompanied by cytochrome c release and rapidly followed by cell death. All these effects of arachidonic acid could be prevented by cyclosporin A but not by the phospholipase A(2) inhibitor aristolochic acid. In contrast, tumor necrosis factor alpha caused phospholipid hydrolysis, induction of the PT, cytochrome c release, and cell death that could be inhibited by both cyclosporin A and aristolochic acid. These findings suggest that arachidonic acid produced by cytosolic phospholipase A(2) may be a mediator of tumor necrosis factor alpha cytotoxicity in situ through induction of the mitochondrial PT.

The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ.

We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells. Treatment with arachidonic acid or induces PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co(2+) quenching technique (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725-734). Yet depolarization, as assessed from the changes of mitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence, is rapid and extensive with arachidonic acid and slow and partial with. Cyclosporin A-inhibitable release of cytochrome c and cell death correlate with the changes of TMRM fluorescence but not with those of calcein fluorescence. Since pore opening must be accompanied by depolarization, we conclude that short PTP openings are detected only by trapped calcein and may have little impact on cell viability, while changes of TMRM distribution require longer PTP openings, which cause release of cytochrome c and may result in cell death. Modulation of the open time appears to be the key element in determining the outcome of stimuli that converge on the PTP.

Alpha-gustducin-immunoreactive solitary chemosensory cells in the developing chemoreceptorial epithelium of the rat vallate papilla.

The presence of solitary chemosensory cells was studied in rat vallate papillae during the first week of post-natal life by alpha-gustducin immunocytochemistry. In 1- to 3-day-old rats, isolated alpha-gustducin-immunoreactive cells were found within the epithelium of the vallate papilla. These cells, mainly located in the basal layer, were scattered among keratocytes and wrapped in alpha-gustducin-negative epithelial cells in a glia-like fashion. The alpha-gustducin-immunoreactive cells were usually round and some of them gave rise to short, large processes directed towards the lumen of the oral cavity or the basal lamina. Rarely, some cells showed an evident bipolar shape. Small taste buds containing either alpha-gustducin-immunoreactive or alpha-gustducin-negative cells appeared in the vallate papillae of 4-day-old rats in which isolated, bipolar-shaped alpha-gustducin-immunoreactive cells were also found. After the first week of post-natal life, the taste buds appeared basically similar to those of adult animals. In conclusion, the present study demonstrates that the presence of epithelial cells with characteristics of solitary chemosensory cells precedes the development of the taste buds.

Mitochondria and cell death. Mechanistic aspects and methodological issues.

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ.