Effect of vitamin K2 on osteoblast apoptosis: vitamin K2 inhibits apoptotic cell death of human osteoblasts induced by Fas, proteasome inhibitor, etoposide, and staurosporine

Vitamin K2 is used for the treatment of osteoporosis, but the precise mode of action is still not clear. We investigated the effects of vitamin K2 on apoptosis of human osteoblasts. Human osteoblastic cell line MG63 cells and human primary osteoblast-like cells obtained from bone fragments in corrective surgery were used as human osteoblasts. Cells were cultured with or without various concentrations of vitamin K2 and tumor necrosis factor-alpha (TNF-alpha). We then determined the proliferative response, expression of Fas and Bcl-2-related proteins, and Fas-mediated apoptosis of these cells induced by anti-Fas immunoglobulin M (IgM). In addition, the effect of vitamin K2 in osteoblast apoptosis induced by Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or staurosporine was also examined. Human osteoblasts did not show spontaneous apoptosis in culture, even in the presence of vitamin K2 or TNF-alpha. Furthermore, proliferation of the cells was not influenced by vitamin K2 or TNF-alpha. Fas was functionally expressed on human osteoblasts, and the treatment with TNF-alpha significantly enhanced both Fas expression and Fas-mediated apoptosis of osteoblasts. The addition of vitamin K2 to the culture resulted in a dose-dependent inhibition of functional Fas expression on osteoblasts, in the presence or absence of TNF-alpha. Treatment of human osteoblasts with vitamin K2 clearly suppressed Bax expression of the cells, although the expression of Bcl-2 was not influenced by vitamin K2. Fas ligand (FasL) cDNA transformants were cytotoxic against osteoblasts, and the cytotoxicity was increased when osteoblasts were treated with TNF-alpha. The addition of vitamin K2 to osteoblasts significantly decreased the cytotoxic effects of FasL cDNA transformants. Furthermore, apoptosis of human osteoblasts induced by LLL-CHO, etoposide, or staurosporine was also clearly suppressed in vitamin K2-treated osteoblasts. Our results suggest that vitamin K2 inhibits apoptotic cell death of osteoblasts and maintains the number of osteoblasts. These actions may explain the therapeutic efficacy of vitamin K2 in osteoporosis.     

Impacts of Humanized Mouse Models on the Investigation of HIV-1 Infection: Illuminating the Roles of Viral Accessory Proteins in Vivo

Human immunodeficiency virus type 1 (HIV-1) encodes four accessory genes: vif, vpu, vpr, and nef. Recent investigations using in vitro cell culture systems have shed light on the roles of these HIV-1 accessory proteins, Vif, Vpr, Vpu, and Nef, in counteracting, modulating, and evading various cellular factors that are responsible for anti-HIV-1 intrinsic immunity. However, since humans are the exclusive target for HIV-1 infection, conventional animal models are incapable of mimicking the dynamics of HIV-1 infection in vivo. Moreover, the effects of HIV-1 accessory proteins on viral infection in vivo remain unclear. To elucidate the roles of HIV-1 accessory proteins in the dynamics of viral infection in vivo, humanized mouse models, in which the mice are xenotransplanted with human hematopoietic stem cells, has been utilized. This review describes the current knowledge of the roles of HIV-1 accessory proteins in viral infection, replication, and pathogenicity in vivo, which are revealed by the studies using humanized mouse models.     

Propagation of prions causing synucleinopathies in cultured cells

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)–YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson’s disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.James Parkinson first described a progressive deterioration of the nervous system in 1817 and called it “shaking palsy” (1). Almost one century later, Friederich Heinrich Lewy described the neuropathological hallmark now known as Lewy bodies (LBs) (2). Progress toward discerning the etiology of Parkinson’s disease (PD) was achieved 85 years later when the first of several studies identified mutations in or multiplications of the gene encoding α-synuclein, SNCA, in inherited cases of PD (3–5). These studies were corroborated by immunostaining for α-synuclein in brain sections from PD patients (6) and subsequently from dementia with Lewy bodies (DLB) cases (7, 8), which found that LBs are surrounded by a halo of α-synuclein polymers.Along with point mutations in SNCA (3), and duplication and triplication of the gene (4, 5) as causes of inherited PD, meta-analysis of genome-wide association studies (9) have identified common variations in SNCA as a risk factor for sporadic PD cases. Combined, these data strongly support an etiological role for α-synuclein in the pathogenesis of both the inherited and sporadic forms of PD.In 1998, brain sections from cases classified as multiple system atrophy (MSA) were analyzed for α-synuclein. Although no LBs were found, abundant immunostaining in the cytoplasm of glial cells was identified (8, 10, 11). A decade earlier, these large immunopositive deposits of α-synuclein were called glial cytoplasmic inclusions (GCIs) based on silver staining (12); they are primarily found in oligodendrocytes but have been occasionally observed in astrocytes and neurons. Limited ultrastructural studies performed on GCIs suggest that they are collections of poorly organized bundles of α-synuclein fibrils (8).In addition to the accumulation of α-synuclein into LBs in PD and GCIs in MSA, depigmentation of the substantia nigra pars compacta is a hallmark of both PD and the majority of MSA cases (13). This loss of dopaminergic neurons results in diminished input to the basal ganglia that is reflected in the motor deficits exhibited by patients. In the 1990s, fetal tissue transplants into the substantia nigra of PD patients were performed in an attempt to counteract the effects of dopamine loss. Strikingly, upon autopsy of patients that survived at least 10 years posttransplant, LBs were found in the grafted fetal tissue. Because these grafts were no more than 16 years old, the findings argued for host-to-graft transmission of LBs (14, 15). The results of these transplant studies offered evidence supporting the hypothesis that PD is a prion disease, characterized by a misfolded protein that self-propagates and gives rise to progressive neurodegeneration (16, 17). Additional support for this hypothesis came from studies on the spread of α-synuclein deposits from the substantia nigra to other regions of the CNS in PD patients (18).Even more convincing support for α-synuclein prions came from animal studies demonstrating the transmissibility of an experimental synucleinopathy. The first report used transgenic (Tg) mice expressing human α-synuclein containing the A53T mutation found in familial PD; the mice were designated TgM83 (19). Homozygous mice (TgM83^+/+) were found to develop spontaneous motor deficits along with increased amounts of insoluble phosphorylated α-synuclein throughout the brain between 8–16 months of age. Ten years later, Mougenot et al. (20) intracerebrally inoculated brain homogenates from sick TgM83^+/+ mice into ∼2-months-old TgM83^+/+ mice and found a substantial reduction in the survival time with incubation periods of ∼130 days. Similar observations were reported from two other groups using either homozygous TgM83^+/+ (21) or hemizygous TgM83^+/− (22) mice.Although our initial attempts to transmit PD to TgM83^+/− mice failed (23), the transmission of MSA to the same mouse line was the first demonstration of α-synuclein prions in human brain (22). The TgM83^+/− mice, which differ from their homozygous counterparts by not developing spontaneous disease, exhibited progressive CNS dysfunction ∼120 days following intrathalamic inoculation of brain homogenates from two MSA patients. Inoculation of brain fractions enriched for LBs from PD patients into wild-type (WT) mice and macaque monkeys induced aberrant α-synuclein deposits, but neither species developed neurological disease (24). In a similar approach, inoculation of WT mice with the insoluble protein fraction isolated from DLB patients also induced phosphorylated α-synuclein pathology after 15 months, but it failed to induce neurological disease characteristic of DLB (25).Because α-synuclein prions from MSA patients were transmissible to TgM83^+/− mice, we asked whether a more rapid cell-based bioassay could be developed to characterize the MSA prions. With the cell bioassay for progressive supranuclear palsy (PSP) in mind (26, 27), we began by constructing WT and mutant α-synuclein cDNAs fused to yellow fluorescent protein (YFP) (28–30) and expressed these in human embryonic kidney (HEK) cells. By testing the cells with full-length recombinant mutant human α-syn140*A53T fibrils, we induced aggregate formation in HEK cells expressing WT and mutant human SNCA transgenes. To expand these findings beyond synthetic prions and to examine natural prions, we report here that phosphotungstic acid (PTA) (31) can be used to selectively precipitate α-synuclein from MSA patients. Screening PTA-precipitated brain homogenate with our cellular bioassay, we detected MSA prions in all six of the cases examined. By measuring the distribution of prions in the substantia nigra, basal ganglia, cerebellum, and temporal gyrus, we found evidence to suggest that at least three different strains of α-synuclein prions may give rise to MSA. We also found that after enrichment by PTA precipitation, ∼6 million α-synuclein molecules comprised an infectious unit of MSA prions in cell culture. Importantly, we transmitted neurodegenerative disease to TgM83^+/− mice using PTA-precipitated brain homogenate from an MSA patient, confirming that the aggregate isolation methods used successfully purify prions from patient samples.     

Histological changes of the liver in experimental graft-versus-host disease across minor histocompatibility barriers. VIII. Role of eosinophil infiltration

Abstract: Although eosinophil infiltrate has been recognized in hepatic graft-versus-host disease, its significance in relation to hepatic graft-versus-host disease lesions is unknown. In the present study, we analyzed hepatic eosinophil infiltration in relation to bile duct damage in experimental mouse graft-versus-host disease across minor histocompatibility barriers up to 14 months after transplantation. Portal eosinophil infiltration was found from 1 week after transplantation throughout the entire 14-month observation period. It was most striking during the early chronic stage of hepatic graft-versus-host disease between 2 to 7 months, with a peak at 5 months after transplantation. Microscopic and electron microscopic study revealed eosinophils infiltrated around the bile duct as well as in the bile duct epithelial layer. They were commonly found together with lymphocytes but were also occasionally found singly around the bile duct and in the bile duct epithelial layer. Bile duct epithelial cells in contact with and in the vicinity of eosinophils showed a variety of degenerative changes, occasionally associated with the presence of extracellular eosinophil granules. Bile duct epithelial cells with eosinophil infiltration just beneath the basement membrane frequently showed further characteristic severe degenerative changes with shedding or dropping-off into the lumen, which features were quite similar to those seen in the bronchial epithelium in asthma patients. These results indicate that not only lymphocytes but also eosinophils may be involved in the production of the bile duct injury in hepatic graft-versus-host disease, especially in its early chronic stage.     

Value of shear wave velocity measurements for the risk assessment of hepatocellular carcinoma development in patients with nonalcoholic fatty liver disease

Purpose

To evaluate the value of measuring shear wave velocity evoked by acoustic radiation force impulse (VTTQ) for the risk assessment of hepatocellular carcinoma (HCC) development in patients with nonalcoholic fatty liver disease (NAFLD).

Methods

VTTQ was measured three times in each of the four liver segments in 163 NAFLD patients, including 14 HCC cases; the results were statistically evaluated.

Results

The VTTQ was 3.04 ± 0.17 m/s (median ± median absolute deviation) and 1.27 ± 0.25 m/s in patients with and without HCC, respectively, and was significantly higher in HCC cases (p < 0.001). When the patients were classified as F0–F4 based on VTTQ cutoff values, VTTQ was significantly higher in the left lobe than in the right lobe for F0 (p < 0.0001) and for F1 and F2 combined (p < 0.0001), but not significantly higher for F3 and F4 combined (p = 0.070). The robust coefficient of variation was significantly higher in the left than in the right (p = 0.018) and significantly increased as VTTQ increased (p = 0.0002). Multivariate analysis showed that total bilirubin concentration {p = 0.014, 38.9 (2.08–727) [odds ratio (95 % confidence interval)]} and VTTQ [p = 0.006, 113 (3.91–3245)] were the only independent explanatory factors for HCC presence among the seven variables identified by univariate analysis. The area under the receiver-operating characteristic curve in the differentiation of HCC from non-HCC was 0.943 for VTTQ and was comparable to that for other noninvasive markers such as Fib-4 (0.964) or higher than that in BARD (0.838).

Conclusions

These results suggest that fibrosis occurs heterogeneously throughout the liver and that VTTQ measurements are useful in HCC risk evaluation in a NAFLD cohort.     

Interactions between epithelial and mesenchymal cells in intrahepatic peribiliary glands in normal and hepatolithiatic livers

The anatomy and pathology of the intrahepatic peribiliary glands were evaluated. In this study, we ultrastructuraly examined the peribiliary glands of normal and hepatolithiatic livers using common and serial ultrathin section observations. It is well known that these glands proliferate markedly in hepatolithiasis. These glands were composed of several acini surrounded by thickened and multilayered basement membranes, and there were mesenchymal cells (the majority were fibroblasts) in the periacinar fibrous connective tissue. Some cytoplasmic processes of acinar epithelial cells and mesenchymal cells in the periacinar connective tissue were in close contact with each other within the thickened and multilayered basement membranes. Such cell-to-cell interaction was most frequent in cases of hepatolithiasis, in which peribiliary glands proliferated markedly. In hepatolithiatic livers, some unmyelinated nerve fibers or axonal button profiles were in close contact with periacinar mesenchymal cells and also with cytoplasmic processes of glandular epithelial cells. Such contacts were rare in normal livers. These findings suggest that such epithelial and mesenchymal cell interactions and innervations play a part in the normal regulation of peribiliary glands and also in the proliferation of peribiliary galnds in hepatolithiasis.     

Learning Self-care After Left Ventricular Assist Device Implantation

The number of heart failure (HF) patients living with a left ventricular assist device (LVAD) as destination therapy is increasing. Successful long-term LVAD support includes a high degree of self-care by the patient and their caregiver, and also requires long-term support from a multidisciplinary team. All three components of self-care deserve special attention once an HF patient receives an LVAD, including activities regarding self-care maintenance (activities related both to the device and lifestyle), self-care monitoring (e.g., monitoring for complications or distress), and self-care management (e.g., handling alarms or coping with living with the device). For patients to perform optimal self-care once they are discharged, they need optimal education that focuses on knowledge and skills through a collaborative, adult learning approach.     

Tongue Pressure During Swallowing in Adults with Down Syndrome and Its Relationship with Palatal Morphology

In individuals with Down syndrome, hypotonicity of the tongue and an underdeveloped maxilla may lead to poor oral motor coordination, which adversely affects the oral phase of swallowing. This study aimed to evaluate the characteristics of pressure produced by the tongue against the hard palate during swallowing in individuals with Down syndrome. In addition, the relationship between tongue pressure and palatal morphology was examined. We studied nine adults with Down syndrome and ten healthy adults as controls. Tongue pressure while swallowing 5 mL water was recorded by a sensor sheet system with five measuring points attached to the hard palate. Palatal length, depth, width, curvature, and slope were measured by three-dimensional digital maxillary imaging. The order of onset of tongue pressure on the median line of the hard palate was the same in all participants, except for three with Down syndrome. The duration and maximal magnitude of tongue pressure on the median line in nine participants with Down syndrome were significantly shorter and lower than those of controls. In participants with Down syndrome, significant positive correlations were observed between the duration of tongue pressure at the mid-median part of the hard palate and palatal depth and width, and between the duration and maximal magnitude of tongue pressure at the posterior-median part and palatal length. These findings suggest that impaired tongue activity, poor tongue control, and constrained tongue motion due to a short and narrow palate contribute to swallowing difficulty in individuals with Down syndrome.     

Intravascular ultrasound morphology of culprit lesions and clinical demographics in patients with acute coronary syndrome in relation to low-density lipoprotein cholesterol levels at onset

Despite current standards of care aimed at achieving targets for low-density lipoprotein cholesterol (LDL-C), many patients remain at high residual risk of cardiovascular events. We sought to assess the LDL-C-dependent differences in culprit intravascular ultrasound (IVUS) morphologies and clinical characteristics in patients with acute coronary syndrome (ACS). Eighty-six consecutive ACS patients whose culprit lesions imaged by preintervention IVUS were divided into two groups based on the fasting LDL-C level on admission: a low-LDL-C group (LDL-C <2.6 mmol/l, n = 45) and a high-LDL-C group (LDL-C ≥2.6 mmol/l, n = 41). Patients with stable angina with LDL-C <2.6 mmol/l (n = 30) were also enrolled as an age- and gender-matched control. The low-LDL-C ACS group was significantly older (72 ± 12 vs 64 ± 14 years, P = 0.007) and more diabetic (47 % vs 15 %, P = 0.001). Importantly, IVUS morphologies were comparable between low- and high-LDL-C ACS groups (all P not significant), whereas culprit plaque was more hypoechoic and less calcified in the low-LDL-C ACS group than in the low-LDL-C stable angina group. Furthermore, compared with the low-LDL-C ACS nondiabetic group, the low-LDL-C ACS diabetic group was more obese, more triglyceride rich (1.3 ± 0.6 vs 0.9 ± 0.4 mmol/l, P = 0.003), and more endothelially injured, but no different for the culprit IVUS morphologies. In multivariate analysis, diabetes was independently associated with a low LDL-C level on admission in patients with ACS. There was no relationship between the LDL-C level at onset and culprit-plaque IVUS morphologies in ACS patients, although culprit plaque in the low-LDL-C ACS group was more vulnerable than in the low-LDL-C stable angina group. In patients with low-LDL-C levels, diabetes with atherogenic dyslipidemia might be the key residual risk.