Entecavir-resistant hepatitis B virus decreases surface antigenicity: A full genome and functional characterization.

BACKGROUND AND AIM: Since polymerase and surface genes overlap in hepatitis B virus (HBV), an antiviral-induced mutation in the polymerase gene may alter the surface antigenicity in patients with chronic hepatitis B (CHB), but this possibility has not been clearly confirmed. This study aimed to determine the drug susceptibility and surface antigenicity of the patient-derived mutants. PATIENTS AND METHODS: Full-length HBV genomes isolated from four entecavir-resistant CHB patients were cloned and sequenced. Around 10 clones of full-length HBV obtained from each patient were analysed and registered in the NCBI GenBank. Representative clones were further characterized by in vitro drug susceptibility and surface antigenicity assays. RESULTS: The rtL180M + rtM204V mutations were common among all the clones analysed. Additionally, the ETV resistance mutations rtT184A/L, rtS202G and rtM250V were found among three patients. Most of the ETV-resistant mutants had amino acid alterations within the known epitopes recognized by T- and B-cells in the HBV surface and core antigens. The in vitro drug susceptibility assay showed that all tested clones were resistant to ETV treatment. However, they were all susceptible to ADV and TDF. More importantly, the rtI169T mutation in the RT domain, led to the sF161L mutation in the overlapping S gene, which decreased in surface antigenicity. CONCLUSIONS: The ETV resistance mutations can affect the antigenicity of the HBsAg proteins due to changes in the overlapping sequence of this surface antigen. Thus, the apparent decline or disappearance of HBsAg needs to be interpreted cautiously in patients with previous or current antiviral resistance mutations.

The Effects of Kangaroo Care in the Neonatal Intensive Care Unit on the Physiological Functions of Preterm Infants, Maternal-Infant Attachment, and Maternal Stress.

UNLABELLED: This study was conducted to identify the effects of kangaroo care on the physiological functions of preterm infants, maternal-infant attachment, and maternal stress. DESIGN AND METHODS: For this study, a quasi-experiment design was used with a nonequivalent control group, and a pre- and post-test. Data were collected from preterm infants with corrected gestational ages of ≥33weeks who were hospitalized between May and October 2011. Twenty infants were assigned to the experimental group and 20 to the control group. As an intervention, kangaroo care was provided in 30-min sessions conducted thrice a week for a total of 10 times. The collected data were analyzed by using the t test, repeated-measures ANOVA, and the ANCOVA test. RESULTS: After kangaroo care, the respiration rate significantly differed between the two groups (F=5.701, p=.020). The experimental group had higher maternal-infant attachment scores (F=25.881, p<.001) and lower maternal stress scores (F=47.320, p<.001) than the control group after the test. In other words, kangaroo care showed significantly positive effects on stabilizing infant physiological functions such as respiration rate, increasing maternal-infant attachment, and reducing maternal stress. CONCLUSION: This study suggests that kangaroo care can be used to promote emotional bonding and support between mothers and their babies, and to stabilize the physiological functions of premature babies. PRACTICE IMPLICATIONS: Kangaroo care may be one of the most effective nursing interventions in the neonatal intensive care unit for the care of preterm infants and their mothers.

Giant axon formation in mice lacking Kell, XK, or Kell and XK: animal models of McLeod neuroacanthocytosis syndrome.

McLeod neuroacanthocytosis syndrome (MLS) is a rare X-linked multisystem disease caused by XK gene mutations and characterized by hematological and neurological abnormalities. XK, a putative membrane transporter, is expressed ubiquitously and is covalently linked to Kell, an endothelin-3-converting enzyme (ECE-3). Absence of XK results in reduction of Kell at sites where both proteins are coexpressed. To elucidate the functional roles of XK, Kell, and the XK-Kell complex associated with pathogenesis in MLS, we studied the pathology of the spinal cord, anterior roots, sciatic nerve, and skeletal muscle from knockout mouse models, using Kel(-/-), Xk(-/-), Kel(-/-)Xk(-/-), and wild-type mice aged 6 to 18 months. A striking finding was that giant axons were frequently associated with paranodal demyelination. The pathology suggests probable anterograde progression from the spinal cord to the sciatic nerve. The neuropathological abnormalities were found in all three genotypes, but were more marked in the double-knockout Kel(-/-)Xk(-/-) mice than in either Kel(-/-) or Xk(-/-) mice. Skeletal muscles from Xk(-/-) and Kel(-/-)Xk(-/-) mice showed mild abnormalities, but those from Kel(-/-) mice were similar to the wild type. The more marked neuropathological abnormalities in Kel(-/-)Xk(-/-) mice suggest a possible functional association between XK and Kell in nonerythroid tissues.

Vascular inflammation with eosinophils after the use of n-butyl cyanoacrylate liquid embolic system.

OBJECTIVE AND IMPORTANCE: Currently, n-butyl cyanoacrylate (n-BCA) is one of the most widely used liquid embolic agents in the treatment of intracranial arteriovenous malformations (AVMs). The cases of three patients are reported who underwent endovascular embolization with n-BCA, followed by resection in two and post-embolization hemorrhage with emergent evacuation in one, with histologic demonstration of an eosinophilic vasculitis found in resected AVM specimens. This is probably the first report of this tissue reaction, which may have theoretically serious clinical implications. CLINICAL PRESENTATION: In this series, three patients (2 women, 1 man) presented with intracranial AVMs (Spetzler-Martin I-III) with the lesions located in the frontal lobe in two of the patients and in the parietal lobe in one. All patients presented with headache, and one also had new-onset seizures. INTERVENTION: All patients underwent embolization with n-BCA before a planned, staged surgical resection of the embolized AVMs. One patient had four embolizations over a 5-month period, one had three embolizations over 3 months complicated by hemorrhage after embolization requiring emergent evacuation of the hematoma, and the third patient had a single embolization. In all three patients, surgical and autopsy specimens showed an inflammatory response within the embolized vasculature with a prominent eosinophilic infiltrate. CONCLUSION: The eosinophilic vasculitis seen in the pathology specimens may represent a previously undocumented hypersensitivity reaction following exposure to n-BCA, with the potential for adverse sequelae, including increased risk of hemorrhage as was seen in one of our patients.

Neurosarcoidosis presenting as an anterior horn syndrome.

We report a 43 year old man who developed progressive weakness of all extremities with fasciculation over four months. Neurological examination was consistent with an anterior horn syndrome. CSF examination showed elevated opening pressure and a lymphocytic pleocytosis. The diagnosis of sarcoidosis was confirmed by muscle and lacrimal gland biopsies. He was treated with the combination of corticosteroids and intravenous immunoglobulin with almost complete resolution of his symptoms a few weeks after discharge. We hypothesize that meningeal granulomatous inflammation compressed the exiting anterior roots which resulted in motor dysfunction with preservation of peripheral sensory fibers.

Identification and characterization of clevudine-resistant mutants of hepatitis B virus isolated from chronic hepatitis B patients.

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV) infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to in vitro drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common. In vitro phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.

Immunoglobulins and complement in postmortem multiple sclerosis tissue.

OBJECTIVE: To identify evidence of a discrete, specific immune response in multiple sclerosis (MS) by analyzing the distribution of immunoglobulins and complement in tissue derived from cases of MS, and from control inflammatory white matter diseases known to express viral and autoantigens in the brain and spinal cord. METHODS: Autopsy tissue from 25 MS patients and 24 patients with other neurological diseases was examined immunohistochemically for immunoglobulins and activated complement (C3d and C9neo). RESULTS: In tissue remote from focal lesions in MS and other neurological diseases, IgG was detected in many normal structures but not in myelin or ramified microglia. Disrupted myelin in areas of active myelin breakdown and in phagocytes stained positively for C3d and C9neo, and equivocally for IgG in MS and all other neurological diseases examined, including ischemic infarcts. Disease-specific deposits of IgG or complement were detected in virus-infected cells in progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, and cytomegalovirus encephalitis; in glial-limiting membranes in neuromyelitis optica; and in senile plaques in Alzheimer's dementia. Specific to MS were unusual microglial nodules containing short, linear deposits of activated complement (C3d) on partly demyelinated axons located in normal-appearing periplaque white matter. INTERPRETATION: IgG and complement immunostaining of disrupted myelin in MS lesions, frequently cited as an indication of pathogenic anti-myelin antibodies, is a nonspecific feature that cannot be interpreted as evidence of a distinct pathogenesis or serve to define particular variants of the disease. The unusual microglial nodules described in this study may constitute a specific biomarker with pathogenetic significance in MS.

Idiopathic hypertrophic pachymeningitis mimicking lymphoplasmacyte-rich meningioma.

A 28-year-old woman with a 6-year history of optic neuropathy and 8 years of hearing loss had enhancing dural lesions around the brain stem and in both internal auditory canals on MRI. Histopathology from cranial procedures performed in 1990 and 1993 was originally interpreted as inflammatory meningioma, now known as lymphoplasmacyte-rich meningioma (LRM). Because the clinical course was more consistent with a relapsing process, the original surgical specimens were restudied with additional immunocytochemical stains. The review led to a pathologic diagnosis of idiopathic hypertrophic pachymeningitis (IHP). IHP and LRM can be confused on both imaging and histopathologic grounds.

Ionizing radiation-induced micronucleus formation is mediated by reactive oxygen species that are produced in a manner dependent on mitochondria, Nox1, and JNK.

Ionizing radiation (IR) is known to induce genotoxic damage to DNA, chromosomes, and the nucleus. However, the damage that IR causes to the nucleus has received much less attention. Given that reactive oxygen species (ROS) are involved in IR-induced DNA breaks and chromosomal aberrations, this study examined the role of ROS in IR-induced damage to the nucleus. Human Jurkat T cells were irradiated with gamma-rays at a dose of 2.5 Gy, which resulted in a dramatic increase in both the cellular ROS levels and the number of micronuclei. This latter event was attenuated when the IR-induced ROS were eliminated through the exogenous application of an antioxidant enzyme catalase. The ability of IR to induce the accumulation of ROS and micronucleus formation was also reduced either when the cells were irradiated in the presence of rotenone, a mitochondrial respiratory chain inhibitor, or when the cellular Nox1 levels were reduced by RNA interference. These results suggest that IR stimulates both the mitochondria and Nox1 to produce ROS, and that these ROS are involved in the IR-induced formation of micronuclei. IR also activated c-Jun N-terminal kinase (JNK), which was reversed by catalase, rotenone, or Nox1 RNA interference. SP600125, a JNK-specific inhibitor, suppressed the IR-induced accumulation of ROS. This inhibitor consistently attenuated the IR-induced formation of micronuclei. Therefore, ROS and JNK appear to act in a positive mutual manner in IR-induced signaling processes. Overall, IR appears to induce the formation of micronuclei by inducing ROS through mitochondria, Nox1, and JNK.

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts.

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.

Rolipram, a phosphodiesterase 4 inhibitor, stimulates inducible cAMP early repressor expression in osteoblasts.

Phosphodiesterase (PDE) 4 inhibitors have been shown to induce the cAMP-mediated signaling pathway by inhibiting cAMP hydrolysis. This study investigated the effect of a PDE4 inhibitor on the expression of the inducible cAMP early repressor (ICER), which is an endogenous inhibitor of CRE- mediated transcription, in osteoblastic cells. RT-PCR analysis revealed that rolipram, a PDE4 inhibitor, stimulates the ICER mRNA in a dose dependent manner. The induction of ICER mRNA expression by rolipram was suppressed by the inhibitors of protein kinase A (PKA) and p38 MAPK, suggesting the involvement of PKA and p38 MAPK activation in ICER expression by rolipram. It was previously shown that rolipram induced the expression of TNF-related activation-induced cytokine (TRANCE, also known as RANKL, ODF, or OPGL) in osteoblasts. This paper provides evidences that a transcriptional repressor like ICER might modulate TRANCE mRNA expression by rolipram in osteoblasts.

Phosphodiesterase inhibitors stimulate osteoclast formation via TRANCE/RANKL expression in osteoblasts: possible involvement of ERK and p38 MAPK pathways.

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. In the present study, 3-isobutyl-1-methylxanthine (IBMX) and pentoxifylline, PDE inhibitors, induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. These inhibitors induced the expression of the osteoclast differentiation factor, TNF-related activation induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL), in calvarial osteoblasts. IBMX induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in osteoblasts. Induction of TRANCE expression by IBMX was partially suppressed by the inhibitors of protein kinase A (PKA), ERK, and p38 MAPK, suggesting that activation of ERK and p38 MAPK, as well as PKA, is involved in TRANCE expression by IBMX. Osteoblasts expressed PDE4, a PDE subtype, and rolipram, a selective inhibitor of PDE4, induced TRANCE expression. These results suggest that PDE4 is a key regulator of TRANCE expression in osteoblasts, which in turn controls osteoclast formation.

Serum withdrawal kills U937 cells by inducing a positive mutual interaction between reactive oxygen species and phosphoinositide 3-kinase.

Reactive oxygen species (ROS) can be generated following cell stimulation and function as intracellular signaling molecules. To determine signaling components involved in ROS induction, human U937 blood cells grown in 10% serum were exposed to serum-free media. It was previously reported that serum withdrawal (SW) killed cells by elevating cellular ROS levels. This study showed that SW activates phosphoinositide 3-kinase (PI3K). PI3K activation was evident after the ROS levels began increasing, and an antioxidant blockade of this increase resulted in PI3K activation suppression. Interestingly, the inhibition of PI3K activity/activation using either its specific inhibitor or dominant-negative mutant attenuated the subsequent additional increase in the ROS levels. These results suggest that SW-induced ROS activate PI3K, which in turn promotes the process leading to ROS accumulation. The present study also revealed that both ROS and PI3K support SW-induced cell death by activating stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Overall, it appears that SW triggers a positive mutual interaction between ROS and PI3K, which amplifies signals required for the induction of an SAPK-dependent death pathway.

Isolation and characterization of a strain of Bacillus thuringiensis subsp. morrisoni PG-14 encoding delta-endotoxin Cry1Ac.

Bacillus thuringiensis 656-3, isolated from a soil sample collected at mushroom houses, showed high toxicity to mushroom flies, Lycoriella mali and Coboldia fuscipes. B. thuringiensis 656-3 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis subsp. morrisoni (H8a8b). The plasmid and protein profiles of B. thuringiensis 656-3 were similar to those of its reference strain, subsp. morrisoni PG-14. However, PCR analysis using cry gene primers showed that B. thuringiensis 656-3, unlike its reference strain, had cry4A, cry4B, cry10A, cry11A, and cry1Ac genes, suggesting that B. thuringiensis 656-3 was a unique strain with respect to gene type. In addition, B. thuringiensis 656-3 showed a high level of toxicity against mushroom flies, L. mali and C. fuscipes.

Rolipram, a phosphodiesterase 4 inhibitor, stimulates osteoclast formation by inducing TRANCE expression in mouse calvarial cells.

Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. This activity was not observed in the absence of calvarial osteoblasts, suggesting that calvarial osteoblasts are likely target cells of rolipram. Osteoclast formation by rolipram was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for the osteoclast differentiation factor, TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL). Northern blot analysis revealed the effect of rolipram to be associated with the increased expression of TRANCE mRNA in mouse calvarial osteoblasts. Collectively, these data indicate that PDE4 inhibitor up-regulates the TRANCE mRNA expression in osteoblasts, which in turn controls osteoclast formation.