Abstract: We previously developed a hybrid small molecule SNIPER (Specific and Nongenetic IAP-dependent Protein ERaser) against transforming acidic coiled-coil-3 (TACC3), SNIPER(TACC3), that induces proteasomal degradation of TACC3 protein. In this study, we found that SNIPER(TACC3) induces cytoplasmic vacuolization derived from endoplasmic reticulum (ER) and paraptosis-like cell death selectively in cancer cells. Mechanistic analysis suggests that accumulation of ubiquitylated protein aggregates that requires X-linked inhibitor of apoptosis protein (XIAP) induces ER stress, which results in ER-stress responses involving X-box binding protein-1 (XBP-1) and ER-derived vacuolization in cancer cells. Importantly, inhibition of proteasome enhanced the SNIPER(TACC3)-induced vacuolization, and the combination treatment of SNIPER(TACC3) and bortezomib exhibited a synergistic anticancer activity in several cancer cell lines. The induction of paraptosis-like cell death in cancer cells by SNIPER(TACC3) could be applied to treat cancer cells resistant to undergo apoptosis by overexpression of XIAP.

Abstract: Methuosis is a form of non-apoptotic cell death involving massive vacuolization of macropinosome-derived endocytic compartments, followed by a decline in metabolic activity and loss of membrane integrity. To explore the induction of methuosis as a potential therapeutic strategy for killing cancer cells, we have developed small molecules (indole-based chalcones) that trigger this form of cell death in glioblastoma and other cancer cell lines. Here, we report that in addition to causing fusion and expansion of macropinosome compartments, the lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), disrupts vesicular trafficking at the lysosomal nexus, manifested by impaired degradation of EGF and LDL receptors, defective processing of procathepsins, and accumulation of autophagosomes. In contrast, secretion of the ectodomain derived from a prototypical type-I membrane glycoprotein, β-amyloid precursor protein, is increased rather than diminished. A closely related MOMIPP analog, which causes substantial vacuolization without reducing cell viability, also impedes cathepsin processing and autophagic flux, but has more modest effects on receptor degradation. A third analog, which causes neither vacuolization nor loss of viability, has no effect on endolysosomal trafficking. The results suggest that differential cytotoxicity of structurally similar indole-based chalcones is related, at least in part, to the severity of their effects on endolysosomal trafficking pathways.

Abstract: Astrocytes have been implicated in epileptogenesis and seizure-induced brain injury. Pathological studies reveal a variety of structural abnormalities in astrocytes, such as vacuolization and astrogliosis. While in vivo imaging methods have demonstrated rapid changes in astrocytes under a variety of physiological and pathological conditions, the acute effects of seizures on astrocyte morphology in vivo and corresponding mechanisms of seizure-induced astrocytic injury have not been documented. In this study, we utilized in vivo two-photon imaging to directly monitor the acute structural effects of kainate-induced seizures on cortical astrocytes. Kainate seizures cause an immediate, but transient, vacuolization of astrocytes, followed over several days by astrogliosis. These effects are prevented by pre- or post-treatment with rapamycin, indicating the mTOR pathway is involved in mediating seizure-induced astrocyte injury. These finding have clinical implications for mechanisms of seizure-induced astrocyte injury and potential therapeutic applications with mTOR inhibitors.

Abstract: Background/aim To study the effect of kisspeptin, a gonadotropin release stimulator, on the testicular tissue of the rat. Materials and methods Four groups were formed as follows: control, Kiss-10 501397645907nmol administration for 1 day, Kiss-10 administration for 13 days, and one last group kept for 7 days following Kiss-10 applied for 13 days. Testicular tissues were stained with hematoxylin-eosin, periodic acid Schiff, Masson trichrome staining, terminal deoxynucleotidyl transferased UTP nick-end labeling, and Ki-67 immune staining. Serum testosterone levels were determined. Results Serum testosterone level increased following acute application, while it was reduced by chronic treatment. Spermatogenic cells as stained by Ki-67 and TUNEL increased in the treated groups compared to the controls. Following a 7-day rest after treatment, a decrease in testosterone levels and Ki-67-stained cell numbers and an increase in TUNEL-stained cells were observed. Leydig cells showed increased vacuolization in the Kiss-1 group. Leydig cell vacuolization continued in the Kiss (13) group and was reduced in the Kiss (13 + 7) group. Conclusion Kiss-10 increased spermatogenic cell proliferation, while testosterone level and proliferation decreased and apoptosis increased during the waiting period.

Abstract: Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4 days, using 24 h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes—alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase—was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8 days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations.

Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Neuronal vacuolization and glial activation are pathologic hallmarks in the superoxide dismutase 1 (SOD1) mouse model of ALS. Previously, we found the neuropeptide calcitonin gene-related peptide (CGRP) associated with vacuolization and astrogliosis in the spinal cord of these mice. We now show that CGRP abundance positively correlated with the severity of astrogliosis, but not vacuolization, in several motor and non-motor areas throughout the brain. SOD1 mice harboring a genetic depletion of the βCGRP isoform showed reduced CGRP immunoreactivity associated with vacuolization, while motor functions, body weight, survival, and astrogliosis were not altered. When CGRP signaling was completely disrupted through genetic depletion of the CGRP receptor component, receptor activity-modifying protein 1 (RAMP1), hind limb muscle denervation, and loss of muscle performance were accelerated, while body weight and survival were not affected. Dampened neuroinflammation, i.e., reduced levels of astrogliosis in the brain stem already in the pre-symptomatic disease stage, and reduced microgliosis and lymphocyte infiltrations during the late disease phase were additional neuropathology features in these mice. On the molecular level, mRNA expression levels of brain-derived neurotrophic factor (BDNF) and those of the anti-inflammatory cytokine interleukin 6 (IL-6) were elevated, while those of several pro-inflammatory cytokines found reduced in the brain stem of RAMP1-deficient SOD1 mice at disease end stage. Our results thus identify an important, possibly dual role of CGRP in ALS pathogenesis.

Abstract: Activation of macrophages may be one of the possible approaches in modulating inflammation. We previously reported that Bougainvillea xbuttiana extract showed an immunomodulatory activity. Here we compare the activation of macrophages exposed to B. xbuttiana extract and compare it with the other treatments such as LPS, IL-4, and IL-10. The cytotoxic effect of extract on peritoneal macrophages was determined by the technique of violet crystal staining. To verify the activation of macrophages we used the tests of vacuolization, hydrogen peroxide production, and percentages of cellular expansion and phagocytosis. The levels of interleukins secreted by macrophages treated with the extract, LPS, and cytokines were determined by the biological assay for the determination of TNF levels and by ELISA for all other interleukins. NO levels were evaluated by colorimetric reactions using Griess reagent. Our results showed that B. xbuttiana extract induced (a) low cytotoxicity percentages, (b) increased vacuolization, hydrogen peroxide production and cell expansion and phagocytosis percentages, and (c) decreased production of TNF-α, IFN-γ, IL-1β, and IL-6 and potentiated production of IL-4, IL-10 and TGF-β. These results suggest that B. xbuttiana extract was able to activate the murine macrophages in a manner similar to those macrophages exposed to IL-4 and IL-10.

Abstract: Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.

Abstract: The triterpenoid, bauerenol, from Suregada angustifolia (Baill. ex Muell.-Arg.) Airy Shaw (Euphorbiaceae) was screened for anti-cancer property using hepatocellular carcinoma cell line, HepG2. Bauerenol exhibited growth inhibitory and apoptosis inducing potential against HepG2 cancer cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxic assay revealed that bauerenol treatment significantly reduced the growth of HepG2 cells in a time- and dose-dependent manner with 50% growth inhibitory concentration doses of 45 and 25 µg/mL at 24 and 48 h treatments, respectively. Bauerenol-induced cell death reflected apoptotic morphological features, that is, cell membrane blebbing, vacuolization, chromatin condensation, and nuclear fragmentation. In addition, bauerenol treatment diminished the mitochondrial membrane potential, by inducing the efflux of cytochrome c, downregulating the levels of anti-apoptotic Bcl-2 as well as upregulating the levels of pro-apoptotic Bax, and inducing caspase act...

Abstract: Nonmotor neuron-related pathology is a feature of amyotrophic lateral sclerosis (ALS), both in patients and in animal models. There is emerging evidence that sensory systems (olfaction and vision) are affected in humans. Here, we asked whether such sensory neuropathology is recapitulated in the superoxide dismutase 1 (SOD1G93A) mouse model of ALS. Neuronal vacuolization in olfaction and vision pathways was assessed in tissue sections from presymptomatic and symptomatic disease stages, and compared to wild type. In both, the olfactory bulb and retina, vacuolization started around postnatal day 60, and vacuole sizes increased until disease end-stage. Notably, vacuolization was largely restricted to the external plexiform layer of the olfactory bulb and to the inner plexiform layer of the retina. In both layers, hSOD1-immunoreactive vacuoles localized to dendrites of excitatory neurons. Downstream olfaction and vision pathway fiber tracts and relay stations did not display obvious vacuolization. Finally, on a morphological level, there was no evidence for an activation of astrocytes and microglia in the 2 affected areas. Thus, we identified a new pathology hallmark in SOD1G93A ALS mice: a glutamatergic sensory neuron dendropathy restricted to olfactory bulb mitral cells and retinal ganglionic cells.

Abstract: Repeated oral gavages silver nanoparticle colloidal solution ingestion procedures for AgNps neural toxicity have been investigated in Swiss albino mouse fetuses’ brain at the ultra-structural (TEM) level by transmission electron microscopy and Nikon bright field microscopy. Transmission electron microscopy probe identified neural toxicity in the brain of mice fetuses in the form of mitochondrial autophagy, dysmorphology & dysfunction of cellular organelles vacuolization of neuronal cell, blood brain barrier inflammation, astrocytic swelling, Gliosis and a well visualized differentiation to the reaction products. Multiple reaction product localization was visible in the form of smaller size silver nano particles (1 to 20 nm range) percolation and perimembranous multiple patchy condensations focuses appeared from inside and outside of neuronal cell membrane. Histologically cell and tissue dysmorphology was observed in the form of unipolar brush cell and lugaro cells degeneration with dysmorphology of corticomedullary layers of cerebrum, cerebellum and hippocampus. In twice, lower dose (0.5 and 1 mg/kg b.w./ day) and higher dose (10 and 15 mg/kg b.w./day) small size silver nano particles were found adhered both side of cell membrane, mitochondria and endocytotic vesicle. Neural histochemistry of fetal cortex by Golgi staining procedure showed reduction number of dendrite arborisation and unipolar neuron with basket cells degeneration and dysmorphology. But the intensity of such was found almost low or equal to control in lower dose and high in higher dose and concentration. Small size silver nano particles exhibits more neurotoxic effect comparatively to other metal nanoparticles because of easy penetration and disintegrity to BBB.

Abstract: Downregulation of apoptotic signal pathway and activation of protective autophagy mainly contribute to the chemoresistance of tumor cells. Therefore, exploring efficient chemotherapeutic agents or isolating novel natural products that can trigger nonapoptotic and nonautophagic cell death such as lysosome-associated death is emergently required. We have recently extracted a saponin, gypenoside L (Gyp-L), from Gynostemma pentaphyllum and showed that Gyp-L was able to induce nonapoptotic cell death of esophageal cancer cells associated with lysosome swelling. However, contributions of vacuolization and lysosome to cell death remain unclear. Herein, we reveal a critical role for NADPH oxidase NOX2-mediated vacuolization and transcription factor EB (TFEB) activation in lysosome-associated cell death. We found that Gyp-L initially induced the abnormal enlarged and alkalized vacuoles, which were derived from lipid rafts dependent endocytosis. Besides, NOX2 was activated to promote vacuolization and mTORC1-indepe...

Abstract: The gill mitochondria-rich cells of the juvenile Amazonian fish Colossoma macropomum were analyzed using light and scanning and transmission electron microscopy after 96 h exposure to 0.04 and 0.2 mM nitrite. Although the number of mitochondria-rich cells decreased significantly in the lamellar epithelium, no decrease was found in the interlamellar region of the gill filament. Nitrite exposure caused significant reduction on the apical surface area of individual mitochondria-rich cells (p < 0.05), with a resulting reduction of the fractional area of these cells in both the lamellar and filament epithelium. Swelling of endoplasmic reticulum cisternae, nuclear envelope and mitochondria were the main changes found in the mitochondria-rich cells. Cristae lysis and matrix vacuolization characterized the mitochondrial changes. The overall ultrastructural changes indicated cellular functional disruption caused by exposure to nitrite. The changes observed in the gill indicate that the cellular structures involved in the process of energy production become severely damaged by exposure to nitrite indicating irreversible damage conducting to cell death.

Abstract: A significant part of morbidity in elderly male patients involves the pelvic organs and their autonomic neural regulation. The aim of the current study was to report the histopathological changes in the peri-prostatic ganglia in elderly males. The sympathetic ganglia from 36 prostatectomy specimens, 26 due to carcinoma of the prostate and 10 prostates from total cystectomies for transitional cell carcinoma, were examined. The age range was 54-88 years. A total of 5,075 ganglion cells were counted in all the specimens. Pathological changes were identified in 1,696 neuron cells as follows: Neuronophagia in 746 neuron cells, neuron cell vacuolization (330 cells), satellite cells vacuolization (423 cells), cell pyknosis (148 cells) and nageotte nodules (49 cells). A number of these changes increased with age. All the changes were more marked in the peri-prostatic ganglion cells of patients with prostatic adenocarcinoma compared with those with benign prostate hyperplasia, which may be due to local environmental changes associated with the presence of malignancy.

Abstract: Most studies on the effects of glucocorticosteroid therapy in rhinitis relate to their inhibitory effect on activation and the number of inflowing cells that are involved in the development and maintenance of inflammation. It is also very important to determine the range of effect of budesonide on residing cells (epithelial cells). The purpose of this study was to evaluate the effect of local budesonide therapy on the cytological image of the nasal mucosa, with attention paid to columnar cells in patients with rhinitis. The in vivo results obtained were analyzed in correlation with changes in normal CHO-K1 cells exposed to budesonide at concentrations falling within the pharmacological dose range. Fifty patients diagnosed with rhinitis with suspected allergic background without nasal polyps were included in clinical trials. The control group were 10 healthy people without clinical signs of rhinitis. Only in patients with homogeneous cytological picture, exfoliative cytology was performed before treatment and after 4 weeks of therapy with budesonide used in aerosol form. Papanicolaou and Pappenheim - stained smears were evaluated qualitatively and quantitatively for changes in nasal mucosal cells. The nasal mucosal image of the patients before treatment clearly indicated the pathological state confirmed by the presence of numerous neutrophils, eosinophils, abundant bacterial flora and goblet or epithelial cells prevalence. In contrast, in smears of patients post-treatment budesonide observed a clear improvement in their nasal mucosa by reducing inflammation. There was a significant increase in the number of columnar cells and the appearance of very numerous epithelial cells with increased cytoplasmic vacuolization and visible leucophagocytosis. In vitro studies were performed on normal CHO-K1 cells that were treated with budesonide at concentrations of 0.5 μM - 45 μM. After 48 hours of incubation with the test agent, the samples were prepared for optical microscopy using the H&E method and transmission electron microscopy. Comparison of cells exposed to budesonide with control cells (without addition of test agent) revealed vacuolization changes with autophagy. Apoptotic changes have also been demonstrated, which occured to a lesser extent than vacuolization. The changes observed after budesonide treatment in the cytological picture of patients with allergic rhinitis indicate the therapeutic effect of this drug. On the other hand, the changes observed in the cytoplasm of epithelial cells, such as autophagy (clearly promoted in CHO-K1 cells) and leucophagocytosis, may indicate an additional mechanism of action for budesonide.

Abstract: To determine whether basal lipid vacuolization characteristic of ketoacidosis could be induced with short-term hypertriglyceridemia, adult Sprague Dawley rat kidneys were perfused in an isolated perfused kidney model with, and without, 11.3 mM (10 g/L) of triglycerides in Krebs-Henseleit buffer, for 1 and 2 h (n = 5/group). Additional treatments included perfusion with triglycerides with 20 mM of β-hydroxybutyrate and 2 mM of acetoacetate (n = 5) and perfusion with triglycerides with 70 mM of glucose (n = 1). Basal vacuolization was produced in all groups, but differed in morphology to that reported in postmortem studies. There was no further increase in vacuolization after 2 h of perfusion compared to 1 h (p = 0.24), and the addition of ketones did not alter the morphology or extent of vacuolization. This study using an ex vivo model has confirmed that isolated hypertriglyceridemia is sufficient to cause basal lipid vacuolization in renal tubular epithelial cells, but with different morphology to vacuoles observed in lethal ketoacidosis at autopsy.

Abstract: Nephrotoxicity is one of the life-threatening side effects of cisplatin when used in the treatment of a wide variety of both pediatric and adult malignancies. Accumulating evidence suggests that cisplatin treatment induces oxidative stress and reduces antioxidant status leading to apoptosis of renal cells. The purpose of this study was aimed to explore the effect of butein, a potent antioxidant, against cisplatin induced nephrotoxicity in mice. Single intraperitoneal administration of dose of cisplatin (20 mg/kg) caused marked renal damage, characterized by a significant increase in serum creatinine and blood urea nitrogen (BUN). Cisplatin also increased relative weight of kidney with higher kidney lipid peroxidation levels, and lowered kidney superoxide dismutase (SOD) and catalase (CAT) activities. Histological examinations revealed renal tubular degeneration, extensive epithelial vacuolization and a large number of infiltrated inflammatory cells in cisplatin alone treated mice. The results showed that treatment with butein attenuated cisplatin induced kidney injury and significantly inhibited serum creatinine and BUN levels and restored antioxidant enzyme activities in kidney tissue. Histological studies exhibited that butein treatment reduced tubular degeneration, and epithelial vacuolization with reduce infiltrated inflammatory cells in kidney. The present study demonstrates that butein can effectively suppress cisplatin induced nephrotoxicity by inhibiting oxidative stress.

Abstract: Tissue trauma induces migration and activation of neutrophils through specific mediators. Vacuolated neutrophils in peripheral blood smear of septic patients correlated with mortality. However, scarce data exist with respect to findings in hemorrhagic shock (HS) trauma patients. The aim of this work was to evaluate the number and size of cytoplasmic and nuclear vacuoles in polymorphonuclear neutrophil (PMN) obtained from a peripheral blood smear stained with the May-Grunwald-Giemsa method in trauma patients with hemorrhagic shock. Seven sequential blood samples were taken from 20 patients with severe hemorrhagic shock and 20 patients who sustained mild thoracic trauma (control group). The first sample was obtained shortly after admission to the hospital followed by new samples taken at 6, 12, 18, 24, 48 and 72 h. Blood smears from both groups were processed to assess vacuolization and vacuole morphology in one hundred PMNs at each time point. The number and the area of vacuoles in the nucleus and the cytoplasm were determined using the program Image-Pro Express version 4.0 for Windows (Media Cybernetics, Bethesda, MD, USA). The number and the area of vacuoles in the cytoplasm and nucleus were significantly different (p <0.05) between shock and control groups. Moreover, serum lactate and heart rate correlated directly with the number (r=0.634) and the area (r=0.624) of cytoplasmic vacuoles as shown by multivariate analysis (p<0.05). Severe hemorrhagic shock induces greater vacuolization of PMNs as compared to mild trauma. PMN vacuolization has direct correlation with serum lactate, a known marker of severe shock. Key words: Hemorrhagic shock, trauma, lactate, inflammatory response, blood smear, neutrophils, vacuolization, apoptosis.

Abstract: An isolated perfused kidney model was used to evaluate the effect of hyperglycemia on renal tubular epithelial cell morphology Ten Sprague-Dawley rat kidneys were perfused with Krebs-Henseleit buffer containing 70 mmol/L of glucose (five for 1 h and five for 2 h) Two control groups consisted of 10 kidneys perfused with Krebs-Henseleit buffer without hyperglycemia (five for 1 h and five for 2 h), and 10 nonperfused contralateral kidneys placed in the same environment for the same duration The hyperglycemia group had significantly increased renal tubular vacuolization (p < 0001) compared to both control groups at 1 and 2 h The isolated perfused kidney model recapitulates the renal tubular vacuolization phenotype found in hyperglycemia and may be a potential tool for the investigation into causal factors in renal histology The full pattern of the Armanni-Ebstein phenomenon was not, however, reproduced, suggesting that this change requires more time or involves more complex factors

Abstract: Kidney cells damage and subsequent renal adverse effects with oxaliplatin are less reported phenomena, whereas cisplatin (CDDP, first generation platinum compound) has therapeutic limitations due to renal toxicity. This experimental study reports oxaliplatin (third generation platinum compound) induced direct damage in rat kidney tissues and alterations in renal biochemical profile. Oxaliplatin was administered in albino wistar rats with 5-FU (5 Fluorouracil) to mimic as model of FOLFOX, the mainstay chemotherapeutic regimen in colorectal cancer (CRC). This study reports changes in renal biochemical profile (serum creatinine and urea) in rats treated in different treatment groups with cisplatin, oxaliplatin, 5-FU, cisplatin+5-FU and oxaliplatin+5-FU which are compared with group of rats treated with normal saline (control group). Subjective renal toxicity in tissues was compared among rats treated with oxaliplatin alone and cisplatin, with and without 5-FU by light microscopy. Cast formation, medial hypertrophy of the vessel wall, vacuolization and necrosis was seen in kidney tissues of oxaliplatin treated rat. Changes in serum creatinine well-above diagnostic risk levels were noted. Apparent tubular degenerative sequence associated with vacuolization and cast formation was observed in 5-FU treated rats. Kidney damage ensued after treatment with 5-FU and oxaliplatin are slightly comparable to massive tubular damages, hemorrhage, casts and vacuolization along with multiple foci of alterations induced by cisplatin.

Abstract: Sir, It is with great interest that we read the article “Septic ketoacidosis—a potentially lethal entity with renal tubular epithelial vacuolization” by Zhou and Byard, recently published in the Journal of Forensic Sciences (1). In this article, Zhou and Byard reported the results of a retrospective, 5-year review pertaining to forensic autopsy cases of sepsis and/or cases that had attributed death to severe bacterial infection. The authors identified four situations (with no history of diabetes mellitus, chronic alcohol abuse, or prolonged fasting) characterized by increased vitreous beta-hydroxybutyrate levels (ranging from 6.26 to 13.32 mmol/L) and normal or decreased vitreous glucose levels (ranging from <0.6 to 1.4 mmol/L, thus excluding the existence of hyperglycemia at the time of death), along with the presence of macroscopic/microscopic findings suggesting bacterial infections (bilateral pneumonia in case number 1, acute bilateral confluent pneumonia and morphological findings consistent with hypothermia in case number 2, acute bronchopneumonia and morphological findings consistent with hypothermia in case number 3, and meningitis and endocarditis in case number 4). The authors concluded that the postmortem identification of significantly increased beta-hydroxybutyrate levels at biochemistry in certain situations could reasonably evoke the diagnosis of “septic ketoacidosis”. This might occur in cases characterized by the morphological findings of bacterial infections and the absence of documented histories of diabetes mellitus, chronic alcohol abuse, or prolonged fasting. Septic ketoacidosis defines a glucose metabolism impairment that has been described during sepsis, potentially responsible for significant ketoacidosis, and which is most likely caused by the relative lack of insulin and impaired metabolic function that characterizes the host response to infection (2). The report by Zhou and Byard is extremely interesting and deals with a topic that we have recently examined in our facility. Furthermore, they emphasized that morphological findings consistent with the hypothesis of antemortem cold exposure were observed in two of four individuals in their case series, thereby suggesting that hypothermia may have enhanced or aggravated increased ketone body production promoted by the septic situation (1,2). Compared to these researchers, we found significantly lower beta-hydroxybutyrate concentrations in our case series. The range in peripheral blood was from 770 to 1350 lmol/L, whereas vitreous beta-hydroxybutyrate levels ranged from 6.26 to 13.32 mmol/L in the case series presented by Zhou and Byard. We also found blood acetone concentrations ranging from 2.1 to 4.9 mg/dL (corresponding to 316–843 lmol/L) and vitreous glucose levels ranging from 0.4 to 4.2 mmol/L. The latter excluded the existence of hyperglycemia at the time of death (the vitreous glucose levels found in our case series are quite comparable to those found by Zhou and Byard in their case series). Furthermore, compared to Zhou and Byard, we did not notice macroscopic and/or microscopic findings consistent with the hypothesis of antemortem cold exposure in any of the cases of our series. We therefore concluded that the measured blood beta-hydroxybutyrate and acetone levels were not likely to have played a contributing role in the death process, although they did suggest increased ketone production as the consequence of probable metabolic impairment (of septic origin) in the absence of documented histories of diabetes mellitus, ethanol addiction, or prolonged fasting (2). Irrespective of these slight differences in measured beta-hydroxybutyrate concentrations, it is scientifically reassuring when two independent teams of researchers encounter very common conclusions and when distinct forensic facilities independently replicate significant biochemical findings. Moreover, we applaud the authors for dealing with a very specific, interesting topic, albeit not well known among forensic pathologists, and emphasizing that bacterial infections and sepsis-related fatalities might be characterized by higher ketone levels in the absence of any additional biochemical results consistent with ketoacidosis of another origin. As far as our research is concerned, we stress that increased ketone concentrations in sepsis-related deaths should always be interpreted carefully and in combination with data obtained from all postmortem investigations to ensure that the origin and magnitude of the biochemical impairment are correctly understood and not overestimated.

Abstract: Titanium Dioxide Nanoparticles (TiO2-NPs) applications are widely used in the daily life and their potential toxicity to the living organism is necessary to be insured. The nanomaterial may or may not exhibit the same toxic potential as the original material. Therefore, this study used TiO2-NPs and their original bulk to ensure their safety on the histology, immunohistochemistry and ultrastructure of the liver of male albino mice (Mus musculus). For this purpose, 25 and 50 mg/kg b.wt.; 55 μm size; anatase TiO2-NPs compared with 50 mg/kg b.wt.; 106 μm size; anatase TiO2 micro-sized (bulk form) were daily injected intraperitoneally into the mice for ten successive days. The results showed numerous alterations in the liver of anatase TiO2-NPs treated animals in a dose-dependent manner that were more than these shown in anatase TiO2-bulk material. However, histopathological disruption of the normal cellular architecture of liver, vacuolization and congestion of blood capillary following higher doses of TiO2-NPs exposure were revealed. In addition, quantitative analysis of both Bcl-2 and PCNA immunostaining density data showed significant increase as compared with the control indicating activation of apoptosis and proliferation in liver cells. Moreover, ultrastructural observation displayed dramatic potential alteration in nucleus, mitochondria, rER, numerous lysosomes, bile canaliculi and a Kupffer cell was detected. Besides, obvious agglomerations of TiO2-NPs were taken up by hepatocytes cytoplasm and its organelles, nucleus and kupffer cells. These results show that TiO2-NPs induced potential toxicity in mice liver following both doses used that varied severely when compared with TiO2-bulk form. Therefore, it could be concluded that both tested doses of nano-anatase TiO2 induced potential liver toxicity than the dose of bulk anatase TiO2.