The potency of allospecific Tregs cells appears to correlate with T cell receptor functional avidity.

CD4(+) CD25(+) regulatory T cells (T(reg) cells) are an attractive adoptive cell therapy in mediating transplantation tolerance. T-cell receptor (TcR) activation is critical for T(reg) function, suggesting that the TcR avidity of T(reg) cells used in therapy may affect the therapeutic outcome. To address this, we compared the regulatory capacity of T(reg) lines expressing TcRs derived from two TcR transgenic mice shown to have the same specificity but different functional avidities. T(reg) lines generated from CD4(+)CD25(+) T cells from C57BL/6 mice were transduced with one of either of these TcRs. The antigen specificity of the transduced T(reg) lines was confirmed in vitro. T(reg) lines expressing the TcR with higher functional avidity showed stronger suppressive capacity in a linked suppression model in vitro. Furthermore, the same T(reg) lines demonstrated a stronger proliferation in vivo following antigen exposure. Pretreatment of recipient BL/6 mice with these T(reg) cells, together with anti-CD8 antibody and Rapamycin therapies, prolonged survival of BALB/c skins, as compared with mice that received T(reg) lines with lower TcR avidity. Taken together, these data suggest that the TcR functional avidity may be important for T(reg) function. It highlights the fact that strategies to select T(reg) with higher functional avidity might be beneficial for immunotherapy in transplantation.

Risk factors associated with low birth weight infants in the Malaysian population.

This study aimed to identify the risk factors which were significantly associated with low birth weight (LBW, <2500 g) infants among the Malaysian population. This was a case-control study carried out at the Tuanku Jaafar Hospital, Seremban, Malaysia over a five-month period. Cases were all infants born with birth weight less than 2500 g. Control infant were selected with the help a random sampling table from among infants with birth weight of > or =2500 g born on the same day in the hospital. Of 3341 livebirths delivered in the hospital, 422 (12.6%) were LBW infants. Logistic regression analysis showed that, after controlling for various potential confounders, the only significant risk factors associated with infants of LBW were gestational age (adjusted odds ratio (OR)=0.6, 95% C.I.: 0.5, 0.6; < 0.0001), maternal pre-pregnancy weight (adjusted OR = 0.97, 95% C.I.: 0.95, 0.99; p < 0.0001), nulliparity (adjusted OR = 3.4, 95% C.I.: 2.2, 5.1; p < 0.0001), previous history of LBW infants (adjusted OR = 2.3, 95% C.I.: 1.4, 3.8; p=0.001) and PIH during current pregnancy (adjusted OR=3.3, 95% C.I.: 1.6, 6.6; p = 0.001). A number of potentially preventable or treatable risk factors were identified to be associated with LBW infants in Malaysia.

Expression of the Fe65 adapter protein in adult and developing mouse brain.

Fe65 is a multimodular adaptor protein expressed mainly in the nervous system. Fe65 binds to the Alzheimer's disease amyloid precursor protein (APP) and the interaction is mediated via a phosphotyrosine binding domain in Fe65 and the carboxy-terminal cytoplasmic domain of APP. Fe65 modulates trafficking and processing of APP, including production of the beta-amyloid peptide that is believed to be central to the pathogenesis of Alzheimer's disease. Fe65 also facilitates translocation of a carboxy-terminal fragment of APP to the nucleus and is required for APP-mediated transcription events. In addition, Fe65 functions in regulation of the actin cytoskeleton and cell movement. Here we report the distribution profile of Fe65 immunoreactivity in adult mouse brain. Fe65 expression was found to be widespread in neurones in adult brain. The areas of highest expression included regions of the hippocampus in which the earliest abnormalities of Alzheimer's disease are detectable. Fe65 was also highly expressed in the cerebellum, thalamus and selected brain stem nuclei. Fe65 was evident in a sub-set of astrocytes within the stratum oriens and radiatum in the hippocampus. Expression of Fe65 was found to be developmentally regulated with levels reducing after embryonic day 15 and increasing again progressively from post-partum day 10 up to adulthood, a developmental pattern that partially parallels that of APP. These data indicate a widespread distribution of Fe65 in neurones throughout mouse brain and also suggest that Fe65 may have functions independent of APP and any potential role in the pathogenesis of Alzheimer's disease.

Molecular cloning and expression analysis of human glycogen synthase kinase-3 alpha promoter.

Human glycogen synthase kinase-3 alpha (GSK-3 alpha) is a serine/threonine kinase that phosphorylates a variety of cytoplasmic and nuclear proteins. It also phosphorylates components of the neuronal cytoskeleton including tau and neurofilament heavy chain. Hyperphosphorylated tau is found in neurofibrillary tangles, a hallmark of Alzheimer's disease and aberrant phosphorylation of neurofilament heavy chain is observed in motor neuron disease. Alterations in GSK-3 alpha activity may therefore contribute to the disease process in these disorders. As a first step to understand the transcriptional regulation of GSK-3 alpha, a 2-kb (p-1751/+243) DNA fragment upstream of the GSK-3 alpha initiation codon was obtained from a YAC clone and characterised. Using primer extension assays, a putative transcriptional start site was located to a G nucleotide 244 bp upstream of the ATG codon. Several transcription factor-binding sites were identified on the promoter region, but no TATA-like element was located close to the start site. Deletion mutants of the 2-kb DNA fragment were generated and fused to a promoterless chloramphenicol acetyltransferase (CAT) gene. Transfection study in a neuroblastoma cell line revealed the 1-kb (p-719/+243) fragment carried strong promoter activity, while the 2-kb construct that contains an Alu-like sequence was only 50% active.

Fe65 and X11beta co-localize with and compete for binding to the amyloid precursor protein.

The Fe65s and X11s are two families of adaptor proteins that bind to the Alzheimer's disease amyloid precursor protein (APP). Although both the X11s and Fe65s bind to similar regions of APP, they have opposing effects on Abeta production and hence may represent novel therapeutic targets. However, there is no evidence that the Fe65s and X11s are present within the same cell type or cell compartment and are thus capable of competing for binding to APP. Here we show that in neurones and transfected cells, APP, Fe65 and X11beta show overlapping subcellular distributions. Furthermore, we demonstrate that Fe65 and X11beta compete for binding to APP.

Predictive value of surveillance skin and hub cultures in central venous catheters sepsis.

In a prospective study of septic complications of central venous catheters used for total parenteral nutrition, daily surveillance catheter hub cultures and twice weekly skin cultures at the catheter entry site were evaluated for their predictive value for catheter sepsis, i.e. bacteraemia with an identical species as that recovered from the catheter tip, or catheters which grew greater than or equal to 15 cfus by a semiquantitative method and/or greater than or equal to 10(3) cfus by a quantitative method. Of 142 catheters studied, 29 were identified to have catheter sepsis. For these the sensitivity of the surveillance hub culture was 34.5% and the sensitivity of the skin culture was 37.9%. When either the hub or the skin culture result was considered as an indication of catheter sepsis, the sensitivity increased to 79.3%. The positive and negative predictive value of the combined result was 44.2% and 93.3% respectively. This study suggests that simultaneous hub and skin cultures are required for a satisfactory surveillance.

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3ß (GSK3ß). KLC2 phosphorylation by GSK3ß induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3ß on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-ß (TGFß) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFß-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

Theory for protein mutability and biogenesis.

Using an elementary physical model for protein folding, of self-avoiding short copolymer chains on two-dimensional square lattices, we address two questions regarding the evolution and origins of globular proteins. (i) How will protein native structures and stabilities be affected by single-and double-site mutations? (ii) What is the probability that a randomly chosen sequence of amino acids will be compact and globular under folding conditions? For a large number of different sequences, we search the conformational space exhaustively to find unequivocally the "native" conformation(s), of global minimum free energy, for each sequence. We find that replacing nonpolar residues in the core by polar residues is generally destabilizing, that surface sites are less sensitive than core sites, that some mutations increase the degeneracy of native states, and that overall it is most probable that a mutation will be neutral, having no effect on the native structure. These results support a "Continuity Principle," that small changes in sequence seldom have large effects on structure or stability of the native state. The simulations also show that (i) the number of "convergent" sequences (different sequences coding for the same native structure) is extremely large and (ii) most sequences become quite dense under folding conditions. This implies that the probability of formation of a globular protein from a random sequence of amino acids by prebiotic or mutational methods is significantly greater than zero.

Evaluation of central venous catheter sepsis by differential quantitative blood culture.

The accuracy of differential quantitative blood culture in the diagnosis of central venous catheter sepsis was evaluated in 24 parenterally-fed patients in whom catheter sepsis was suspected. The pour-plate quantitative culture technique was performed immediately before removal of the catheter on blood drawn through the central venous catheter and a peripheral vein. If bacterial colonies in the catheter blood specimen were sevenfold more frequent than identical bacterial colonies in the peripheral blood specimen, the test was considered positive and indicative of catheter sepsis. Catheter-tip culture identified 9 of the 24 patients as positive for catheter sepsis. A positive differential quantitative blood culture result was found for seven of the nine infected catheters. Sensitivity of this test was 77.8%, specificity was 100%, and overall accuracy was 91.7%. It is concluded that differential quantitative blood culture is a reliable method for the exclusion of catheter sepsis.

In vitro and in vivo study of fosfomycin in methicillin-resistant Staphylococcus aureus septicaemia.

Five hundred strains of methicillin-resistant Staphylococcus aureus were tested against various anti-staphylococcal agents. Vancomycin, fusidic acid and fosfomycin were found to be the most effective. Only 1 strain out of 500 was resistant to fosfomycin. Three patients with methicillin-resistant Staphylococcus aureus septicaemia were successfully treated by fosfomycin. We conclude that fosfomycin could be the drug of choice for methicillin-resistant Staphylococcus aureus infection.

Molecular cloning and characterization of the human glycogen synthase kinase-3beta promoter.

Glycogen synthase kinase 3beta (GSK-3beta) is a proline-directed kinase that forms part of the wingless signaling pathway. Recent studies have shown that GSK-3beta phosphorylates the microtubule-associated protein tau in vitro and in cell culture. Tau is the principal component of the paired helical filaments (PHFs) found in the brains of patients with Alzheimer disease, and PHF-tau is hyperphosphorylated. GSK-3beta is therefore one of the candidate kinases for phosphorylating tau in Alzheimer disease. GSK-3beta activity is negatively regulated by phosphorylation on serine 9 and positively regulated by phosphorylation on tyrosine 216. However, since overexpression of GSK-3beta by transfection leads to increased activity in the absence of any stimuli, GSK-3beta activity may also be regulated at the transcriptional level. Indeed, increased GSK-3beta protein levels are found in Alzheimer disease brains, and GSK-3beta is found associated with PHFs in Alzheimer disease. To understand how GSK-3beta activity may be regulated at the transcriptional level, we have isolated the human GSK-3beta promoter. The GSK-3beta promoter does not contain a conventional TATA box although several other transcription factor binding sites were identified. A putative transcription start site was mapped by 5' RACE. Transfection of various GSK-3beta promoter CAT reporter genes into both COS-7 cells and SHSY5Y neuronal cells revealed that the GSK-3beta promoter is more active in neuronal cells. Such transfection studies involving promoter deletion mutants revealed that a negative transcriptional response element may be present at position -1421 to -1363 and an activator sequence at position -427 to -384. CP2 binding sites were also present within the promoter. CP2 has recently been shown to interact with the Alzheimer disease amyloid precursor protein binding protein Fe65. The significance of these results with respect to Alzheimer disease pathogenesis are discussed.

Expression analysis of glycogen synthase kinase-3 in human tissues.

Human glycogen synthase kinase-3 (GSK-3) is a multisubstrate, proline-directed kinase that phosphorylates tau, beta-amyloid and neurofilaments. In this study, the expression levels of the two GSK-3 isoforms, alpha and beta, RNA and proteins in different human tissues were examined. Northern analysis demonstrated that GSK-3alpha is encoded by a 2.6-kb mRNA and GSK-3beta by 8.3- and 2.8-kb mRNAs. The two GSK-3beta mRNA species were variably expressed in different tissues. Northern and quantitative polymerase chain reaction demonstrated that both GSK-3alpha and GSK-3beta mRNA were prominently expressed in testis, thymus, prostate and ovary but were low in adult lung and kidney. Western blot analysis showed that the 51-kDa GSK-3alpha protein was highly expressed in lung, ovary, kidney and testis, whereas the 46-kDa GSK-3beta protein was highly expressed in lung, kidney and brain. The differential expression of GSK-3alpha and GSK-3beta mRNA and proteins and the lack of relationship between transcription and translation in some tissues indicate that GSK-3alpha and GSK-3beta are subject to different means of regulation.

X11 alpha and x11 beta interact with presenilin-1 via their PDZ domains.

X11 alpha and X11 beta are two neuronal adaptor proteins that interact with the Alzheimer's disease amyloid precursor protein (APP). X11 alpha and X11 beta stabilise APP and inhibit production of proteolytic APP fragments including the A beta peptide that is deposited in the brains of Alzheimer's disease patients. The mechanisms by which X11 alpha and X11 beta modulate APP processing are not clear but one possibility is that they influence the activity of the secretases that cleave APP to give rise to A beta. Presenilin-1 is required for gamma-secretase activity and here we demonstrate that both X11 alpha and X11 beta interact with presenilin-1. X11/presenilin-1 binding is via two X11 PDZ domains and sequences within the carboxy-terminus of presenilin-1. We also demonstrate that both X11 alpha and X11 beta mediate the formation of complexes between APP and presenilin-1. These results suggest that the X11 regulation of APP processing is controlled, at least in part, via their interactions with APP and presenilin-1.

Once daily administration of netilmicin compared with thrice daily, both in combination with metronidazole, in gangrenous and perforated appendicitis.

The safety and efficacy of a single daily dose of netilmicin plus metronidazole after appendicectomy for gangrenous and perforated appendicitis was compared with the traditional thrice daily dosage. Twenty patients were enrolled in each group. The antibiotics were given intramuscularly for seven days after operation. Eradication of infection was observed in all patients and the postoperative wound sepsis was the same for each group. A significantly higher peak serum netilmicin level was achieved in the group receiving a single daily dose but nephrotoxicity was not observed. We concluded that the single daily dose of netilmicin was well tolerated and was as efficacious in this small series as the thrice daily regimen. The single-dose regimen has the advantage of simplicity and potentially increased bactericidal activity.

Intracranial venous thrombosis and pulmonary embolism with antiphospholipid syndrome in systemic lupus erythematosus.

The presence of antiphospholipid antibodies is associated with arterial and venous thrombosis. A 14-year-old girl, with systemic lupus erythematosus (SLE), developed headache and cough and was found to have intracranial venous sinus thrombosis with secondary pulmonary embolism associated with antiphospholipid syndrome. Clinical and radiological improvement occurred with anticoagulation therapy. Because SLE is commonly associated with antiphospholipid antibodies, thromboembolic events should be considered in the differential diagnosis of both cough and headache in children with SLE.

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity.

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.

The bacteriology and septic complication of patients with appendicitis.

A detailed bacteriologic study was done on 161 patients operated for appendicitis. Aerobic and anaerobic cultures were taken from the blood, the appendicular lumen, mucosa, serosa, fossa, and from the wound after closure of the peritoneum. There is no correlation between the degree of appendicitis and the incidence of positive blood culture. The infection spread through the appendicular wall as the disease progressed. Aerobic infection was common in early appendicitis but a mixed aerobic and anaerobic infection was predominant in late cases. Late appendicitis, a positive wound culture at the end of the operation, the duration of symptoms of over 36 hours before operation and the age of the patient over 50 years were all associated with an increased incidence of septic complication. From the antibiotic sensitivity on the bacteria isolated, the most effective agent against anaerobes was metronidazole. Effective agents against the aerobes were aminoglycosides and cephalosporins. The best single agent against both anaerobes and aerobes was moxalactum.