Impact of control measures including decolonization and hand hygiene for orthopaedic surgical site infection caused by MRSA at a Japanese tertiary-care hospital.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) is the most common pathogen in orthopaedic surgical site infections (SSIs). However, few studies have investigated the transmission process of orthopaedic MRSA SSI. AIM: To investigate the transmission process of orthopaedic MRSA SSI using epidemiological and molecular analyses and to determine a method to prevent MRSA SSI in nosocomial orthopaedic surgery. METHODS: Active MRSA surveillance, preoperative decolonization and contact precautions for MRSA-positive cases was performed at our institution. Changes in epidemic strains were evaluated and the possibility of transmission from patients in an orthopaedic ward of a Japanese tertiary-care hospital was assessed by genotyping stored MRSA strains. In addition, data on the prevalence of MRSA SSI, MRSA colonization, and use of an alcohol antiseptic agent (mL/patient-days) during 2005-2022 were retrospectively assessed. FINDINGS: SCCmec type II strain in the SSI group decreased over time, associated with fewer outbreaks. Even during a period of high infection rates, no cases of transmission-induced SSI from nasal MRSA carriers were identified. The infection rate correlated negatively with the use of an alcohol antiseptic agent (r = -0.82; P < 0.0001). Two cases among five nasal carriers developed MRSA SSI caused by strains different from those related to nasal colonization. CONCLUSION: The infection control measures for transmission from the hospital reservoirs including strict adherence to hand hygiene and decolonization of carriers is likely to be important for the prevention of orthopaedic MRSA SSI. However, the need for contact precautions for decolonized nasal carriers might be low.

Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes.

In primary cultured adult rat hepatocytes stimulated by epidermal growth factor and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concentrated in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was observed 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggest that primary cultured hepatocytes are suitable for examining the relation between subcellular localization of metallothionein and cell growth.

Expression of IFN-gamma in cerebrovascular endothelial cells from aged mice.

Recently, it has become clear that interferon-gamma (IFN-gamma) plays a role in the central nervous system (CNS) as well as in the immune system. However, the reason for the alteration in IFN-gamma production in the brain with aging remains unknown. In this study, we investigated the expression of IFN-gamma in the brain in terms of both mRNA and protein and compared the expression in young adult brain with that in aged mice. The cerebrum and cerebellum were collected from young adult (8-10 weeks old) and aged (24-26 months old) BALB/c mice, and the expressions of IFN-gamma and IFN-gamma receptor-1 (IFNGR-1) mRNA were examined by RT-PCR. Expression of IFN-gamma mRNA was detected in the brains from aged mice but not in those from young adult mice. However, IFNGR-1 mRNA was expressed in the brains from both young adult and aged mice. Moreover, IFN-gamma levels in the cerebrum and cerebellum from aged mice were detectable by ELISA, but IFN-gamma was undetectable in these tissues from young adult mice. To identify the cellular source of IFN-gamma in the brain of aged mice, immunostaining using antimouse IFN-gamma monoclonal antibody (mAb) was done. Immunoreactivity of IFN-gamma appeared to be located in cerebrovascular endothelial cells, including the choroid plexus of the cerebellum from aged mice. Expression of IFN-gamma and IFNGR-1 was also identified in isolated microvessels from brains. These results suggest that IFN-gamma plays a role in age-associated changes.

Sympathetic activation and contribution of genetic factors in hypertension with neurovascular compression of the rostral ventrolateral medulla.

OBJECTIVE: The rostral ventrolateral medulla is an important center for the regulation of sympathetic and cardiovascular activities. Reportedly, neurovascular compression of the rostral ventrolateral medulla may be causally related to essential hypertension. We aimed to determine the mechanism behind elevated blood pressure in hypertensive patients with compression of the rostral ventrolateral medulla and to investigate whether genetic factors contribute to the etiology of hypertension with compression. DESIGN AND METHODS: The study included 56 patients with essential hypertension and 25 normotensive individuals. With the use of magnetic resonance imaging, the essential hypertension group was subdivided into hypertension with compression and without compression groups. We compared plasma levels of hormones that raise blood pressure and family histories of hypertension between the two hypertension groups and the normotension group. RESULTS: Plasma norepinephrine levels, but not plasma renin activity, aldosterone, epinephrine, or vasopressin levels, were significantly higher in the hypertension with compression group (389+/-53 pg/ml) than in the hypertension without compression group (217+/-38, P<0.05) or in the normotension group (225+/-30, P<0.05). The percentage of individuals who had two hypertensive parents was significantly higher in the hypertension with compression group (39.4%) than in the hypertension without compression group (13.0%, P<0.05) or in the normotension group (8.0%, P<0.01). CONCLUSIONS: These results indicate that neurovascular compression of the rostral ventrolateral medulla might be, at least in part, causally related to essential hypertension by increasing sympathetic nerve activity. They also suggest that genetic factors might contribute to the etiology of hypertension with neurovascular compression.

Nerve growth factor-induced neuronal differentiation is accompanied by differential induction and localization of the amyloid precursor protein (APP) in PC12 cells and variant PC12S cells.

PC12 cells and the morphological variant PC12S cells in culture were examined by immunochemical methods for the presence of the amyloid precursor protein (APP), before and after treatment with the nerve growth factor (NGF). In untreated PC12, untreated PC12S and in NGF-treated PC12 cells, APP was localized in the cytoplasm, whereas in NGF-treated PC12S cells, APP was localized at growth cones, processes and cytoplasm. In PC12 cells, three major forms of APP (695 and 751/770) were detected by Western blot. After NGF treatment, only the level of APP 695 was increased. Immunoprecipitation studies in PC12 cells revealed six protein species, corresponding to immature and mature forms of each of the three APP 695, 751 and 770 proteins. Addition of NGF increased the synthesis of the immature and mature forms of APP695. In PC12S cells, only the higher molecular weight forms of APP (751/770) were detected by both Western blot and immunoprecipitation. Addition of NGF had no effect on their levels. In both cell types, the level of the secreted form of APP showed a significant transient increase after NGF treatment. These results suggest that NGF can differentially regulate the molecular forms of APP and the localization of APP within the cell.

Human long-chain acyl-CoA synthetase: structure and chromosomal location.

A complementary DNA clone encoding the entire human long-chain acyl-CoA synthetase was isolated and the total 698-amino acid sequence was deduced. The amino acid sequence of human long-chain acyl-CoA synthetase shows 84.9% identity to that of rat long-chain acyl-CoA synthetase. The nucleotide sequences of the protein coding regions between human and rat long-chain acyl-CoA synthetase mRNAs are highly conserved (85.6%), whereas those of the 3' untranslated regions are less conserved (72%). The location of the human long-chain acyl-CoA synthetase gene was identified on chromosome 4 by spot hybridization of flow-sorted chromosomes. Computer-assisted homology search revealed a significant similarity of the enzyme with the enzymes of the luciferase family. Based on this similarity, the structure of human long-chain acyl-CoA synthetase can be divided into five domains: the N-terminus, two domains similar to those in enzymes of the luciferase family, a long gap region between the similar domains and the C-terminus.

Muramyl dipeptide injected into crushed sciatic nerve, activates macrophages and promotes recovery of walking locomotion in rats.

We injected an immunoadjuvant, muramyl dipeptide (MDP), intrafascicularly into crushed rat sciatic nerve so as to test whether activation of macrophages promotes regeneration of peripheral nerve from crush injury and improves walking locomotion in rats. Immunohistochemical staining of Schwann cells and macrophages with anti-S100 and ED-1 monoclonal antibodies revealed that macrophages are more abundant and phagocytic in nerve injected with MDP than in control. Axonal elongation in damaged nerve and locomotion recovery in rats were evaluated with pinch test and measurement of sciatic nerve functional index (SFI), respectively. Pinch test showed a 15.5% increase in length (n = 6, p < 0.05) of elongating axons for MDP-injected group 5 days after the crush injury comparing to the control group. The value of SFI obtained from the rats injected with MDP showed a 18.3 % increase (n = 4-6, p < 0.01) comparing to the control 3 weeks after the crush injury. Activation of macrophages in the nerve injected with MDP was monitored by detecting gene expression of marker molecules for macrophages such as apolipoprotein E (ApoE), interleukin-1beta (IL-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) using reverse transcription-PCR (RT-PCR) technique 2 days and 1 week after the injection. Levels of transcripts of IL-1beta and MIP-1alpha were up-regulated in the nerves injected with MDP 1 week after MDP injection. These results suggest that an intrafascicular injection of MDP activates macrophages infiltrating into damaged nerve and that the macrophages support elongation of regenerating axon, resulting in functional recovery of sciatic nerve from injury in rats.

Purification of glial fibrillary acidic protein (GFAP) from normal bovine brain.

Glial fibrillary acidic protein (GFAP) was purified from normal bovine brain by a modification of the procedure used to isolate vimentin in order to avoid contamination by other cytoskeletal components: vimentin, neurofilament triplet proteins, tubulin and actin. GFAP is thought to be separated from vimentin in the DE cellulose column chromatography step. The three other major proteins were also separable through ion exchange and gel filtration column chromatographies. A purified 49 kDa polypeptide was estimated to be GFAP from peptide mapping and subsequent immunoblotting analysis. We obtained 4.4 mg GFAP/1 g bovine brain white matter in less than 3 days. The polyclonal antibody raised against purified GFAP was able to detect 49 kDa GFAP by immunoblotting analysis. This isolation method is simpler and more rapid than previous methods.

Development and application of a modified monoclonal hybridoma technique for isolating monoclonal antibodies to human brain regions.

We developed a modified monoclonal hybridoma technique that combines two conventional methods: a conventional immunosuppression method with cyclophosphamide treatment and an in vitro immunization method. This technique is advantageous over conventional methodologies because it requires a shorter period for immunization of mice and a smaller quantity of antigen, and gives rise to antibody-secreting hybridomas with higher efficiency. One monoclonal hybridoma line, designated as BG5, was established by this technique after activation of lymphocytes with muramyl dipeptide and with the immunogen obtained from human entorhinal cortex. Western blot analysis showed a relatively high expression of BG5 antigen in human entorhinal cortex. Our results suggest that this modified hybridoma technique may rapidly facilitate the acquisition of brain region-specific antibodies. We call this technique 'suppression immunization followed by in vitro stimulation procedure' (SOFISTIC).

Development and application of a modified monoclonal hybridoma technique for isolating monoclonal antibodies to human brain regions.

We developed a modified monoclonal hybridoma technique that combines two conventional methods: a conventional immunosuppression method with cyclophosphamide treatment and an in vitro immunization method. This technique is advantageous over conventional methodologies because it requires a shorter period for immunization of mice and a smaller quantity of antigen, and gives rise to antibody-secreting hybridomas with higher efficiency. One monoclonal hybridoma line, designated as BG5, was established by this technique after activation of lymphocytes with muramyl dipeptide and with the immunogen obtained from human entorhinal cortex. Western blot analysis showed a relatively high expression of BG5 antigen in human entorhinal cortex. Our results suggest that this hybridoma technique may rapidly facilitate the acquisition of brain region-specific antibodies. We call this technique 'suppression immunization followed by in vitro stimulation procedure' (SOFISTIC).

Isolation of a monoclonal antibody reactive to brain microsome-associated antigen, lap-1, and its preferential localization in limbic system of rat brain.

The distribution and subcellular localization of AH9 antigen, recognized by a monoclonal antibody AH9, were examined in rat brain. Highest expression was observed in the lamina lucidum of the dentate gyrus of the rat hippocampus. Synaptic subfields of other limbic areas also expressed AH9 antigen at a substantial level. The molecular size of the AH9 antigen is 15 kDa and it was found in the microsomal fraction of brain but not of heart or kidney. These results indicate that AH9 antigen is a novel synaptosomal protein that is relatively specific to the limbic system, at least in the rat brain. We designated AH9 antigen as a limbic system associated protein-1, lap-1.

Gene expression of ND4, a subunit of complex I of oxidative phosphorylation in mitochondria, is decreased in temporal cortex of brains of Alzheimer's disease patients.

Gene expression of mitochondrial DNA-encoded ND4 in brains of Alzheimer's disease (AD) patients and age-matched controls was measured using Northern blot. The level of ND4 message in temporal cortex of control subjects was higher than in motor cortex, whereas the level of ND4 gene expression in temporal cortex of AD brains was decreased compared with that in temporal cortex of controls. A control probe showed no difference in expression between the two areas of AD and control brains. These and previous data suggest that neurons vulnerable to AD express higher levels of enzymes of oxidative phosphorylation than do spared neurons, and that this difference may promote selective neuronal vulnerability of AD.

beta-Amyloid polypeptide increases calcium-uptake in PC12 cells: a possible mechanism for its cellular toxicity in Alzheimer's disease.

Calcium-uptake into PC12 cells was measured by incubation with 45Ca after the cells were exposed for 24 h to beta-amyloid peptide(1-40) at concentrations between 0 and 46 microM. The rate of influx of 45Ca into PC12 cells was constant for the first 10 min. For 46 microM beta-amyloid peptide(1-40), the rate of influx was about 1,300 ions/s/microns 2 and the number of cells decreased significantly. There was no significant decrease in cell number when cells were exposed to beta-amyloid in calcium-free medium. These results indicate that beta-amyloid increases calcium uptake into PC12 cells, and suggest that the increased uptake is responsible for the toxicity of beta-amyloid in PC12 cells.

beta-Amyloid increases choline conductance of PC12 cells: possible mechanism of toxicity in Alzheimer's disease.

When beta-amyloid-(1-40) is added to PC12 cells, there is an increase in choline conductance that is proportional to the beta-amyloid concentration. If a similar effect occurs in cholinergic brain cells of Alzheimer's disease patients, the intracellular choline concentration would be reduced, leading to a decrease in the production of acetylcholine. This could explain the reduced level of acetylcholine that has been found in post-mortem brain tissue of Alzheimer's disease patients.

A monoclonal antibody, raised against rat embryo hippocampus, identifies a nuclear protein enriched in hippocampal and other neurons of the adult rat brain.

We report characteristics of a monoclonal antibody (MAb) produced by immunizing mice against rat embryo hippocampus, and its cellular distribution in the brains of adult and embryonic rats. This antibody, designated as MAb 4A4, brightly stained granular and pyramidal neurons of the adult rat hippocampus, as well as some cortical neurons. Also, MAb 4A4 labeled granular and Purkinje cells of the cerebellum with less intensity. While glial cells were labeled relatively faintly. At embryonic day 18 in the rat, most brain neurons, primitive neuroepithelium, connective tissues and glia were labeled with this antibody, indicating that the expression of 4A4 antigen is regulated developmentally. The 4A4 antigen appeared to be localized to the nucleus of cells except in choroid plexus in which the focal membrane staining was observed. The nuclear localization of 4A4 antigen was further confirmed by the staining of cultured cell lines with MAb 4A4. Western blot analysis demonstrated a single band of the 4A4 antigen from cultured cells, with an apparent molecular weight of 220 kDa. Both the molecular weight and the distribution of the 4A4 antigen in the embryonic and adult rat brain and cultured cells suggest that this antigen is a novel nonhistone nuclear type, which is preferentially expressed in neurons of the rodent brains and is under developmental regulation.

Beta-amyloid induced increase in choline flux across PC12 cell membranes.

Beta-amyloid peptide is the main constituent of senile plaques and is implicated in the pathogenesis of Alzheimer's disease. It has been shown to be both neurotoxic and neurotrophic in vivo, and its effects have been suggested to be mediated in part by alterations in membrane transport. In the present study, we investigated the effect of beta-amyloid (1-40) on choline transport in cultured PC12 cells. We found that exposure to 46 or 92 microM beta-amyloid (1-40) increased [14C]choline flux in PC12 cells in a concentration-dependent manner, whereas exposure to reverse sequence beta-amyloid (40-1) had no effect. If there is a similar effect in vivo, the increased beta-amyloid dependent permeability to choline could lead to depletion of cellular choline stores and could contribute to the selective vulnerability of cholinergic neurons in Alzheimer's disease.

Mutated in colorectal cancer, a putative tumor suppressor for serrated colorectal cancer, selectively represses beta-catenin-dependent transcription.

Mutated in colorectal cancer (MCC) was originally identified as a candidate gene for familial adenomatous polyposis (FAP) but further study identified adenomatous polyposis coli (APC) as responsible for FAP and the physiologic/pathologic roles of MCC remained poorly understood. Recently, MCC promoter methylation was discovered as a frequent early event in a distinct subset of precursor lesions and colorectal cancer (CRC) associated with the serrated CRC pathway. Here we provide the first evidence of the biological significance of MCC loss in CRC and the molecular pathways involved. We show MCC expression is dramatically decreased in many CRC cell lines and the distinct subset of sporadic CRC characterized by the CpG island methylator phenotype and BRAF(V600E) mutation due to promoter methylation as reported previously. Importantly, we find MCC interacts with beta-catenin and that reexpression of MCC in CRC cells specifically inhibits Wnt signaling, beta-catenin/T-cell factor/lymphoid-enhancer factor-dependent transcription and cellular proliferation even in the presence of oncogenic mutant APC. We also show that MCC is localized in the nucleus and identify two functional nuclear localization signals. Taken together, MCC is a nuclear, beta-catenin-interacting protein that can act as a potential tumor suppressor in the serrated CRC pathway by inhibiting Wnt/beta-catenin signal transduction.