Brain metastases in Asian HER2-positive breast cancer patients: anti-HER2 treatments and their impact on survival.

BACKGROUND: In Asia, large-scale studies on anti-HER2 treatment in HER2-positive breast cancer patients with brain metastases are limited. We studied the treatment patterns of these patients in Asia to evaluate the impact of anti-HER2 treatment on the time to occurrence of brain metastases (TTBM) and survival after brain metastasis (BM). METHODS: A retrospective study of HER2-positive breast cancer patients diagnosed with BM between January 2006 and December 2008 in six Asian countries was conducted. Demographics, tumour characteristics, treatment details, and events dates were collected from medical records. RESULTS: Data from 280 patients were analysed. Before BM, 63% received anti-HER2 treatment. These patients had significantly longer TTBM than those without anti-HER2 treatment (median 33 vs 19 months; P<0.002). After BM, 93% received radiotherapy, 57% received chemotherapy, and 41% received anti-HER2 treatment (trastuzumab and/or lapatinib). Use of both anti-HER2 agents, primarily sequentially, after BM demonstrated the longest survival after BM and was associated with a significant survival benefit over no anti-HER2 treatment (median 26 vs 6 months; hazard ratio 0.37; 95% CI 0.19-0.72). CONCLUSION: Anti-HER2 treatment before BM was associated with longer TTBM. Anti-HER2 treatment after BM was associated with a survival benefit, especially when both trastuzumab and lapatinib were utilised.

Ultrasound-derived fetal size nomogram for a sub-Saharan African population: a longitudinal study.

OBJECTIVES: To create a fetal size nomogram for use in sub-Saharan Africa and compare the derived centiles with reference intervals from developed countries. METHODS: Fetal biometric measurements were obtained at entry to antenatal care (11-22 weeks' gestation) and thereafter at 4-week intervals from pregnant women enrolled in a longitudinal ultrasound study in Kinshasa, Democratic Republic of Congo. The study population comprised 144 singleton gestations with ultrasound-derived gestational age within 14 days of the menstrual estimate. A total of 755 monthly ultrasound scans were included with a mean +/- SD of 5 +/- 1 (range, 2-8) scans per woman. Estimated fetal weight (EFW) was calculated at each ultrasound examination using the Hadlock algorithm. A general mixed-effects linear regression model that incorporated random effects for both the intercept and slope was fitted to log-transformed EFW to account for both mean growth and within-fetus variability in growth. Reference centiles (5(th), 10(th), 50(th), 90(th) and 95(th) centiles) were derived from this model. RESULTS: Nomograms derived from developed populations consistently overestimated the 50(th) centile EFW value for Congolese fetuses by roughly 5-12%. Differences observed in the 10(th) and 90(th) centiles were inconsistent between nomograms, but generally followed a pattern of overestimation that decreased with advancing gestational age. CONCLUSIONS: In low-resource settings, endemic malaria and maternal nutritional factors, including low prepregnancy weight and pregnancy weight gain, probably lead to lower fetal weight and utilization of nomograms derived from developed populations is not appropriate. This customized nomogram could provide more applicable reference intervals for diagnosis of intrauterine growth restriction in sub-Saharan African populations.

Impact of maternal malaria and under-nutrition on intrauterine growth restriction: a prospective ultrasound study in Democratic Republic of Congo.

Maternal malaria and under-nutrition are established risk factors for small-for-gestational-age (SGA) births; however, whether malaria is associated with intrauterine growth restriction (IUGR) is unknown. We investigated IUGR risk among 177 HIV-negative pregnant women enrolled in a longitudinal ultrasound study conducted in Democratic Republic of Congo from May 2005 to May 2006. Malaria infection, maternal anthropometrics, and ultrasound estimated fetal weight were measured monthly. All positive malaria cases were treated and intermittent presumptive therapy (IPTp) provided. Log-binomial regression models for IUGR were fitted using generalized estimating equations to account for statistical clustering of repeat IUGR measurements. Twenty-nine percent of fetuses experienced an episode of IUGR with the majority occurring in the third trimester. The risk of IUGR associated with malaria was greatest after three or more cumulative infections (RR 3.3, 95% CI 1.3-8.2) and was two- to eight-fold higher among women with evidence of under-nutrition. Receiving antimalarial treatment in the previous month (for IPTp or treatment) was significantly protective against IUGR (RR 0.5, 95% CI 0.3-0.7). The interaction observed between malaria and under-nutrition suggests that antenatal programmes in malaria endemic areas should incorporate nutritional screening and supplementation in addition to IPTp.

Quantitative characterizations of a nanopatterned bonded wafer: force determination for nanoimprint lithography stamp removal.

The nanoimprint lithography process consists of two mechanical steps: molding and stamp removal. While many publications dealing with anti-sticking layer properties or the understanding of polymer flow during imprinting have recently been published, only a few studies have been carried out to deeply characterize the demolding step. Regarding the small amount of theoretical work dedicated to this issue, in this paper both experimental and first theoretical approaches are proposed to characterize the demolding process in a peeling scheme. Full 200 mm stamp and imprinted wafers were used to identify the experimental limitation of such a full wafer peeling demolding scheme. A rectangular stamp and substrate samples with or without nanoscale features combined with an augmented beam theory are proposed to extract quantitative data for the required demolding force as well as the friction stress along the feature sidewall. Therefore both adhesion and friction forces were characterized on single stamp structures.

Stamp design effect on 100 nm feature size for 8 inch NanoImprint lithography.

Sub-100 nm resolution on a 200 mm silicon stamp has been hot embossed into commercial Sumitomo NEB 22 resist. A single pattern, exposed with electron beam lithography, has been considered to define the stamp and thus make it possible to point out the impact of stamp design on the printing. These results may be considered as a first attempt to define rules to solve the proximity printing effects (PPEs). Moreover, a large range of initial resist thickness, from 56 to 506 nm, has been spin coated to assess the effect of polymer flow properties for the stamp cavity filling and the printed defects. A detailed analysis of the printed resist in dense hole patterns showed that the application volume conservation is enough to calculate the residual layer thickness as the height of the printed resist feature. Good accordance has been obtained between the theoretical approach and experimental results. Moreover, the impact of the pattern symmetry breakdown on mould deformation is clearly shown in this paper in the printed areas as well as in the unprinted areas.

Influence of the molecular weight and imprint conditions on the formation of capillary bridges in nanoimprint lithography.

Sub-100 nm patterns can be duplicated by nanoimprint lithography with high reproducibility, even on 200 mm wafers. Nevertheless, several problems have to be solved before this technique reaches a mature state for industrial applications. Several kinds of defect appear frequently in printed polymers. Some of them are induced by capillary effects and are related to mould deformation. Capillary bridges are observed on the flat surfaces around the pattern areas, or inside the printed structures. In this paper, the influence of the polymer molecular weight (M(w)) on the capillary bridge distribution is presented. It will be shown that for smaller M(w), they appear first around the pattern areas and move towards the structures more rapidly. It is also demonstrated that this evolution depends directly on the printing temperature and pattern filling related to the feature density and the film thickness. Finally, it is shown that the influence of these parameters is related to the polymer viscosity, which is the dominant property of the capillary effects, and a trade-off has to be made between the limitation due to the capillary bridges, the decrease of the temperature, which is important to reduce the cycle time, and the sticking defects.

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons.

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.

Tailoring size effects on the exchange bias in ferromagnetic-antiferromagnetic <100 nm nanostructures.

The hysteresis loop shift in sub-100 nm ferromagnetic- (FM-)antiferromagnetic (AFM) nanostructures can be either enhanced or reduced with respect to continuous films with the same composition, with varying the AFM layer thickness. An enhancement of the coercivity and a reduction of the blocking temperature are also observed. These effects are mainly ascribed to the physical limitations that the dot sizes impose on the AFM domain size and the concomitant weakening of the pinning strength exerted by the AFM during magnetization reversal of the FM.

Induction of cholinergic function in cultured sympathetic neurons by periosteal cells: cellular mechanisms.

Periosteum, the connective tissue surrounding bone, alters the transmitter properties of its sympathetic innervation during development in vivo and after transplantation. Initial noradrenergic properties are downregulated and the innervation acquires cholinergic and peptidergic properties. To elucidate the cellular mechanisms responsible, sympathetic neurons were cultured with primary periosteal cells or osteoblast cell lines. Both primary cells and an immature osteoblast cell line, MC3T3-E1, induced choline acetyltransferase (ChAT) activity. In contrast, lines representing marrow stromal cells or mature osteoblasts did not increase ChAT. Growth of periosteal cells with sympathetic neurons in transwell cultures that prevent direct contact between the neurons and periosteal cells or addition of periosteal cell-conditioned medium to neuron cultures induced ChAT, indicating that periosteal cells release a soluble cholinergic inducing factor. Antibodies against LIFRbeta, a receptor subunit shared by neuropoietic cytokines, prevented ChAT induction in periosteal cell/neuron cocultures, suggesting that a member of this family is responsible. ChAT activity was increased in neurons grown with periosteal cells or conditioned medium from mice lacking either leukemia inhibitory factor (LIF) or LIF and ciliary neurotrophic factor (CNTF). These results provide evidence that periosteal cells influence sympathetic neuron phenotype by releasing a soluble cholinergic factor that is neither LIF nor CNTF but signals via LIFRbeta.

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice.

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361-376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers,VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.

Catecholamines are required for the acquisition of secretory responsiveness by sweat glands.

The sympathetic innervation of sweat glands undergoes a developmental change in transmitter phenotype from catecholaminergic to cholinergic. Acetylcholine elicits sweating and is necessary for development and maintenance of secretory responsiveness, the ability of glands to produce sweat after nerve stimulation or agonist administration. To determine whether catecholamines play a role in the development or function of this system, we examined the onset of secretory responsiveness in two transgenic mouse lines, one albino and the other pigmented, that lack tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Although both lines lack TH, their catecholamine levels differ because tyrosinase in pigmented mice serves as an alternative source for catecholamine synthesis (Rios et al., 1999). At postnatal day 21 (P21), 28 glands on average are active in interdigital hind footpads of albino TH wild-type mice. In contrast, fewer than one gland is active in albino TH null mice, which lack catecholamines in gland innervation. Treatment of albino TH null mice with DOPA, a catecholamine precursor, from P11 to P21 increases the number of active glands to 14. Pigmented TH null mice, which have faint catecholamine fluorescence in the developing gland innervation, possess 12 active glands at P21, indicating that catecholamines made via tyrosinase, albeit reduced from wild-type levels, support development of responsiveness. Gland formation and the appearance of cholinergic markers occur normally in albino TH null mice, suggesting that catecholamines act directly on gland cells to trigger their final differentiation and to induce responsiveness. Thus, catecholamines, like acetylcholine, are essential for the development of secretory responsiveness.

Using focus groups to assess primary care patients' satisfaction.

The present report describes the use of patient focus groups by a primary health care facility. We review our rationale for using focus groups and the process we used to prepare for and conduct them. We then highlight the results and lessons learned through this experience. Focus groups can be an excellent method for primary care practices to assess the complexities of patient satisfaction issues and engage patients in the continuous quality improvement process. Focus groups can uncover unanticipated issues that surveys fail to identify. Our experience demonstrated that this benefit can be critical in identifying and prioritizing quality of care improvements and that focus group results can be used to make immediate improvements in the quality of care, even though this type of study is not intended to generalize.

Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum.

During the development of sweat gland innervation, interactions with the target tissue induce a change from noradrenergic to cholinergic and peptidergic properties. To determine whether the change in neurotransmitter properties that occurs in the sweat gland innervation occurs more generally in sympathetic neurons, we identified a new target of cholinergic sympathetic neurons in rat, the periosteum, which is the connective tissue covering of bone, and characterized the development of periosteal innervation of the sternum. During development, sympathetic axons grow from thoracic sympathetic ganglia along rib periosteum to reach the sternum. All sympathetic axons displayed catecholaminergic properties when they reached the sternum, but these properties subsequently disappeared. Many axons lacked detectable immunoreactivities for vesicular acetylcholine transporter and vasoactive intestinal peptide when they reached the sternum and acquired them after arrival. To determine whether periosteum could direct changes in the neurotransmitter properties of sympathetic neurons that innervate it, we transplanted periosteum to the hairy skin, a noradrenergic sympathetic target. We found that the sympathetic innervation of the transplant underwent a noradrenergic to cholinergic and peptidergic change. These results suggest that periosteum, in addition to sweat glands, regulates the neurotransmitter properties of the sympathetic neurons that innervate it.

The p75 neurotrophin receptor influences NT-3 responsiveness of sympathetic neurons in vivo.

To determine the role of the p75 neurotrophin receptor (p75NTR) in sympathetic neuron development, we crossed transgenic mice with mutations in p75NTR, nerve growth factor (NGF) and neurotrophin-3 (NT-3). Neuron number is normal in sympathetic ganglia of adult p75NTR-/- mice. Mice heterozygous for a NGF deletion (NGF+/-) have 50% fewer sympathetic neurons. In the absence of p75NTR (p75NTR-/- NGF+/-), however, neuron number is restored to wild-type levels. When NT-3 levels are reduced (p75NTR-/- NGF+/- NT3 +/-), neuron number decreases compared to p75NTR-/- NGF+/- NT3+/+. Thus, without p75NTR, NT3 substitutes for NGF, suggesting that p75 alters the neurotrophin specificity of TrkA in vivo.

Treatment of community-acquired pneumonia in the elderly: the role of cefepime, a fourth-generation cephalosporin.

In a prospective, multicentre double-blind trial, 151 patients over the age of 65 years were randomly assigned to receive either cefepime 2 g every 12 h for a minimum of 3 days and up to 14 days or ceftriaxone 1 g every 12 h for a minimum of 3 days and up to 14 days. Antibiotics were maintained until 48 h after fever had resolved; no other antibiotics were permitted. The average age in each group exceeded 77 years and significant co-morbidity was found in the majority of patients. The mean total duration of therapy was 5.8+/-2.4 days for the cefepime group and 6.7+/-2.7 days for the ceftriaxone group (P = 0.06). The clinical success rate at the end of therapy was 79.1% with cefepime and 75.4% with ceftriaxone (P = 0.62). At the end of follow-up, 91.7% of the cefepime-treated patients and 86.5% of the ceftriaxone patients had a satisfactory clinical response (P = 0.38). In 35 bacteriological evaluable patients, potential pathogens were eradicated in all but one patient receiving cefepime. Seven patients in each group died during the study period but in each case the death was unrelated to study drug. The commonest side-effect was diarrhoea (cefepime, five patients; ceftriaxone, two patients). The clinical and microbiological efficacy of cefepime is similar to that of ceftriaxone in elderly patients with community-acquired pneumonia requiring hospitalization. Cefepime is an appropriate choice for the treatment of community-acquired respiratory tract infections in the elderly.

NT-3, like NGF, is required for survival of sympathetic neurons, but not their precursors.

Superior cervical ganglia of postnatal mice with a targeted disruption of the gene for neurotrophin-3 have 50% fewer neurons than those of wild-type mice. In culture, neurotrophin-3 increases the survival of proliferating sympathetic precursors. Both precursor death (W. ElShamy et al., 1996, Development 122, 491-500) and, more recently, neuronal death (S. Wyatt et al., 1997, EMBO J. 16, 3115-3123) have been described in mice lacking NT-3. Consistent with the second report, we found that, in vivo, neurogenesis and precursor survival were unaffected by the absence of neurotrophin-3 but neuronal survival was compromised so that only 50% of the normal number of neurons survived to birth. At the time of neuron loss, neurotrophin-3 expression, assayed with a lacZ reporter, was detected in sympathetic target tissues and blood vessels, including those along which sympathetic axons grow, suggesting it may act as a retrograde neurotrophic factor, similar to nerve growth factor. To explore this possibility, we compared neuron loss in neurotrophin-3-deficient mice with that in nerve growth factor-deficient mice and found that neuronal losses occurred at approximately the same time in both mutants, but were less severe in mice lacking neurotrophin-3. Eliminating one or both neurotrophin-3 alleles in mice that lack nerve growth factor does not further reduce sympathetic neuron number in the superior cervical ganglion at E17.5 but does alter axon outgrowth and decrease salivary gland innervation. Taken together these results suggest that neurotrophin-3 is required for survival of some sympathetic neurons that also require nerve growth factor.

Amino acid substitutions at the subunit interface of dimeric Escherichia coli alkaline phosphatase cause reduced structural stability.

The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild-type and altered alkaline phosphatases show comparable catalytic behavior with k(cat) values between 51.3 and 69.5 s(-1) and Km values between 14.8 and 26.3 microM. The ultracentrifugation profiles indicate that the wild-type enzyme is more stable than all the interface-modified enzymes. The wild-type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface-modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild-type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild-type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.

Catecholamine synthesis is mediated by tyrosinase in the absence of tyrosine hydroxylase.

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to L-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

Cellular and molecular determinants of sympathetic neuron development.

The development of the sympathetic nervous system can be divided into three overlapping stages. First, the precursors of sympathetic neurons arise from undifferentiated neural crest cells that migrate ventrally, aggregate adjacent to the dorsal aorta, and ultimately differentiate into catecholaminergic neurons. Second, cell number is refined during a period of cell death when neurotrophic factors determine the number of neuronal precursors and neurons that survive. The final stage of sympathetic development is the establishment and maturation of synaptic connections, which for sympathetic neurons can include alterations in neurotransmitter phenotype. Considerable progress has been made recently in elucidating the cellular and molecular mechanisms that direct each of these developmental decisions. We review the current understanding of each of these, focusing primarily on events in the peripheral nervous system of rodents.