The Role of Osmotic Therapy in Hemispheric Stroke.

BACKGROUND: Decompressive hemicraniectomy (DHC) can be lifesaving in hemispheric stroke complicated by cerebral edema. Conversely, osmotic agents have not been shown to improve survival, despite their widespread use. It is unknown whether medical measures can similarly confer survival in certain patient subgroups. We hypothesized that osmotic therapy (OT) without DHC may be associated with a greater likelihood of survival in particular populations depending on demographic, radiologic, or treatment characteristics. METHODS: We performed a retrospective cohort analysis of patients with large anterior circulation strokes with an NIH stroke scale (NIHSS) ≥10 who received OT. We compared clinical, radiologic, and treatment characteristics between two groups: (1) those who survived until discharge with only OT (medical management success) and (2) those who required either DHC or died (medical management failure). RESULTS: Thirty patients met eligibility criteria. Median NIHSS was 19 [interquartile range (IQR) 13-24], and median GCS was 10 [IQR 8-14]. Forty-seven percent of the medical management cohort survived to discharge. Demographic characteristics associated with medical management success included NIHSS (p = 0.009) and non-black race (p = 0.003). Of the various interventions, the administration of OT after 24 hours and a smaller hypertonic saline dose was also associated with survival to discharge (p = 0.038 and 0.031 respectively). CONCLUSION: Our results suggest that patients with moderate size hemispheric infarcts on presentation and those who do not require OT within the first 24 h of stroke may survive until discharge with medical management alone. Black race was also associated with conservative management failure, a finding that may reflect a cultural preference toward aggressive management. Further prospective studies are needed to better establish the utility of medical management of hemispheric edema in the setting of moderate size hemispheric infarcts.

Role of EIF5A2, a downstream target of Akt, in promoting melanoma cell invasion.

BACKGROUND: Cutaneous melanoma is a life-threatening skin cancer because of its poorly understood invasive nature and high metastatic potential. This study examines the importance of eukaryotic translation initiation factor 5A2 (EIF5A2) in melanoma pathogenesis. METHODS: We examined EIF5A2 expression in 459 melanocytic lesions using tissue microarray. In addition, melanoma cell lines were subjected to invasion and cell proliferation assays, zymography, FACS and real-time PCR to investigate the role of EIF5A2 in cancer progression. RESULTS: Positive EIF5A2 staining increased from dysplastic naevi to primary melanomas (PMs; P=0.001), and further increased in metastatic melanomas (P=0.044). Eukaryotic translation initiation factor 5A2 expression was correlated with melanoma thickness (P<0.001) and was inversely correlated with the 5-year survival of PM patients especially those with tumour ≤2 mm thick. Strikingly, none of the latter died within 5 years in EIF5A2-negative staining group. Cox regression analysis revealed that EIF5A2 is an independent prognostic marker. Further, we found that EIF5A2 is a novel downstream target of phosphorylated Akt. Both melanoma cell invasion and MMP-2 activity increased and decreased with EIF5A2 overexpression and knockdown, respectively. CONCLUSION: We for the first time showed that EIF5A2, as a target of PI3K/Akt, promotes melanoma cell invasion and may serve as a promising prognostic marker and a potential therapeutic target for melanoma.

The influence of growth hormone/insulin-like growth factor deficiency on prostatic dysplasia in pbARR2-Cre, PTEN knockout mice.

BACKGROUND: Elevated insulin-like growth factor-I (IGF-I) serum levels and phosphatase and tensin homolog (PTEN) loss are prostate cancer (PCa) risk factors that enhance androgen-responsive and castration-resistant PCa xenografts growth. METHODS: The impact of suppressed growth hormone (GH)/IGF-I levels on neoplastic initiation of PTEN-deficient prostate epithelia was assessed histologically and by epithelial-to-mesenchymal marker expression in Ghrhr D60G homozygous (lit/lit) and heterozygous (lit/+) pbARR2-Cre, PTEN(fl/fl) (PTEN-/-) mice. How suppressed GH/IGF-I levels impacted growth of PTEN-/- mouse-derived prostate cells (MPPK) was examined by growth and survival signaling of cells cultured in lit/+ or lit/lit serum. RESULTS: Body weight, prostate weight and serum GH and IGF-I levels were reduced in lit/lit relative to lit/+ PTEN-/- littermates. While the anterior lobes of lit/+ PTEN-/- prostates consistently presented swollen, indicative of ductal blockage, the degree of prostatic dysplasia in 15- and 20-week-old lit/lit and lit/+ PTEN-/- mice was indistinguishable as measured by normalized prostatic weight, tissue histology, or probasin, PSP94, E-cadherin, N-cadherin and vimentin expression. However, growth and AKT activation of MPPK cells was decreased when cultured in lit/lit serum as compared with lit/+ serum and restored in lit/lit serum supplemented with IGF-I and, to a lesser extent, GH. CONCLUSIONS: These results suggest that initiation of prostate carcinogenesis by loss of PTEN is not influenced by germline variation of genes encoding signaling molecules in the GH/IGF-I axis, but suggests that these factors may affect the progression of dysplastic phenotype and supports previous studies, indicating that the GH/IGF milieu does impact the growth of PTEN-deficient dysplastic prostatic cells once transformed.

Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch.

Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression.

Pten (phosphatase and tensin homologue gene) haploinsufficiency promotes insulin hypersensitivity.

AIMS/HYPOTHESIS: Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake. MATERIALS AND METHODS: Insulin sensitivity in Pten heterozygous (Pten(+/-)) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten(+/-) mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3beta (GSK3beta), a substrate of PKB/Akt, was determined by western immunoblotting. RESULTS: Following i.p. insulin challenge, blood glucose levels in Pten(+/-) mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten(+/-) mice. Enhanced glucose uptake was observed both in Pten(+/-) myocytes and in skeletal muscle of Pten(+/-) mice by PET. PKB and GSK3beta phosphorylation was enhanced and prolonged in Pten(+/-) myocytes. CONCLUSIONS/INTERPRETATION: Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten(+/-) mice.

Key Feature Extraction for Fatigue Identification using Random Forests.

Electroencephalogram (EEG) might be the most predictive and reliable physiological indicator of mental fatigue. However, the extraction of key features from massive EEG data for mental fatigue identification remains a challenge. The objective of this study is to identify the key EEG features in relationship to mental fatigue, from a broad pool of EEG features generated by quantitative EEG (qEEG) techniques, using Random Forests (RF), which is a recently developed machine learning algorithm. The method is applied to key EEG feature extraction for 5-level mental fatigue identification using the five subjects' EEG data recorded in 25-hour fatigue experiments. RF produces significant feature reduction with little compromise of the classification performance. The identified key EEG features also indicate that electrode locations in frontal and occipital regions of the brain are most important for adequate representation of the deactivation of functional lobes of the brain, which is consistent with the anatomical areas known to be involved in mental fatigue. It is also interesting to discover that the four frequency bands are all important for the mental fatigue identification.

A role for T helper 2 cells in mediating skin fibrosis in tight-skin mice.

Mice heterozygous for the tight-skin (Tsk) mutation develop skin fibrosis. Previous studies have implicated a role for the immune system and, specifically, CD4(+) T cells, in the etiology of skin fibrosis in Tsk/+ mice. We have recently shown that the administration of neutralizing anti-IL-4 antibodies to Tsk/+ mice prevented the development of skin fibrosis in these mice. Since IL-4 is a major cytokine produced by T helper 2 (Th2) cells, we investigated the role of Th2 cells in mediating skin fibrosis in Tsk/+ mice. Previous studies have shown that the development of Th2 cells in non-Tsk mice is abrogated in mice with null mutation for either the IL-4 or the Stat6 gene. In this study we showed that the polarization of CD4(+) T cells from Tsk/+ mice toward the Th2 lineage is also dependent on a functioning IL-4 or Stat6 gene. More importantly, the development of skin fibrosis in Tsk/+ mice was abrogated by the IL4(-/-) or the Stat6(-/-) mutation. We also determined whether alteration of the TCR repertoire in Tsk/+ mice, achieved by the introduction of TCR transgenes, was able to prevent the development of skin fibrosis in Tsk/+ mice. We found that the exclusive usage of the Vbeta8.2 gene segment by T cells was sufficient to prevent skin fibrosis in Tsk/+ mice. This result suggests that the exclusive use of this Vbeta gene segment by T cells may have prevented the development of fibrosis-causing Th2 cells.

Receptor-specific allelic exclusion of TCRV alpha-chains during development.

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR alphabeta transgenes that allelic exclusion at the TCR alpha locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV alpha-chain. TCRV alpha allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV alpha+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV alpha-chain, but lose the nonfunctional receptor. Whereas activation of TCRV beta-chains or CD3epsilon leads to receptor internalization, TCRV alpha ligation promotes retention of the TCR on the cell surface. Although both TCRV alpha- and TCRV beta-chains trigger phosphotyrosine signaling, only the TCRV beta-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV alpha-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.

CD28-induced cytokine production and proliferation by thymocytes are differentially regulated by the p59fyn tyrosine kinase.

CD28 is a 44-kDa homodimeric receptor that is expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with phorbol ester (PMA) induces the production of cytokines and the proliferation of resting T cells via signal transduction pathways independent of the TCR. Evidence is provided herein that CD28 signals leading to cytokine production do not require the p59fyn (Fyn) tyrosine kinase, whereas CD28-mediated proliferation is dependent on the presence of the Fyn kinase in thymic, but not lymph node, cells. The defect in proliferation is not due to failure of IL-2R signaling, since addition of high concentrations of exogenous IL-2 can overcome the proliferative defect. Analysis of CD28-directed induction of the IL-2R alpha (CD25)-chain, which confers high affinity binding to IL-2, showed that Fyn-deficient thymocytes, but not lymph node cells, failed to up-regulate CD25 expression following anti-CD28 and PMA stimulation. Thus, the Fyn tyrosine kinase is critically required for thymic CD28-mediated CD25 expression and proliferation but not for CD28-mediated cytokine production.

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.

T cell development in mice expressing splice variants of the protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is expressed in multiple isoforms as a result of alternative splicing of variable exons encoding the extracellular domain. CD45 expression is critical for T cell development, and thymocyte maturation is blocked at the immature CD4+ CD8+ double-positive stage in CD45 gene-deficient (CD45 -/-) mice. Moreover, splicing of variable CD45 exons changes during thymocyte selection. To test the role of CD45 extracellular splice variants in T cell selection and development, we introduced CD45RO (a low-m.w. splice variant lacking exons 4, 5, and 6) and CD45ABC (a high-m.w. isoform containing all exons) transgenes under the control of a thymocyte-specific promoter into a CD45 -/- background, generating CD45RO transgene-positive CD45 -/- (CD45RO) and CD45ABC transgene-positive CD45 -/- (CD45ABC) mice. We demonstrate that both CD45 splice isoforms can rescue development of CD4+ and CD8+ TCR-alphabeta+ thymocytes. Neither CD45 isoform rescued positive selection of H-Y TCR transgene thymocytes, and these cells were blocked at a HSA(high) CD69- CD5(low) stage of development. Peripheral T cells from CD45RO and CD45ABC mice proliferated in response to allogeneic stimulator cells and anti-CD3epsilon cross-linking. However, only CD45RO mice, not CD45ABC mice, generated cytotoxic T cell responses and neutralizing, Th cell-dependent IgG Abs after viral infections. In addition, we show that T cells from CD45RO and CD45ABC mice accumulate in lymph nodes but not in the spleen, liver, or skin, indicating that the CD45 phosphatase may control the homing behavior and trafficking of T cells.

Distinct differentiative stages of CD4+CD8+ thymocyte development defined by the lack of coreceptor binding in positive selection.

Cortical CD4+CD8+ thymocytes mature into CD4+ or CD8+ thymocytes through a process termed positive selection. To better define differentiative stages of CD4+CD8+ thymocyte development in positive selection, we performed a phenotypic analysis of CD4+CD8+ thymocytes from H-Y mice mated to various genetic backgrounds. We have previously shown that coordinate binding of the H-Y TCR and the CD8 coreceptor to the restricting Db MHC class I molecule is required for the efficient positive selection of this TCR. In this study we have used TCR, CD5, and CD45 expression levels as markers for thymocyte maturation. Lack of CD8/Db interaction was achieved by introducing a mutation that abrogates CD8 binding in the alpha 3 domain of Db. We found that the absence of coreceptor ligation prevented TCR up-regulation in CD4+CD8+ thymocytes and resulted in a developmental arrest characterized by low levels of TCR and CD45. We have previously shown that deletion of CD4+CD8+ thymocytes expressing the H-Y TCR is facilitated by CD8 coreceptor ligation. Here we show that expression of the deleting ligand in the absence of coreceptor ligation caused CD5 up-regulation without concomitant TCR or CD45 up-regulation in CD4+CD8+ thymocytes. In a beta 2-microglobulin null background, introduction of the H-Y TCR caused the majority of CD4+CD8+ thymocytes to express an unusually low level of of the CD5 activation marker, suggesting that a low-affinity or noncognate TCR/MHC interaction may be required for initial CD5 up-regulation to intermediate levels. Collectively, these observations favor a maturational process in positive selection in which CD5 up-regulation precedes CD45 and TCR up-regulation.

Thymic CD45 tyrosine phosphatase regulates apoptosis and MHC-restricted negative selection.

The acquisition of immunologic self-tolerance is governed, in part, by selection mechanisms that occur during intrathymic T cell ontogeny. Although considerable data exist for the molecular basis of mature T cell signal transduction, the enzymes that participate in thymic TCR selection processes have remained unidentified. We report that augmented thymic expression of the CD45R0 protein tyrosine phosphatase increased the efficacy of TCR-mediated apoptosis and MHC-restricted negative selection of HY TCRs in vivo. Additionally, augmented CD45R0 expression resulted in the activation of endogenous p56lck tyrosine kinase in CD4+CD8+ thymocytes. These results identify a cellular enzyme, the CD45R0 protein tyrosine phosphatase, involved in the regulation of apoptosis and TCR selection mechanisms during CD4+CD8+ thymocyte differentiation.

Specific CD45 isoforms differentially regulate T cell receptor signaling.

Multiple isoforms of T cell CD45 tyrosine phosphatase are expressed as a result of alternative RNA splicing among extracellular exons. To discern the presence and identity of distinct functions among CD45 isoforms, we compared thymic T cell activation responses by elevating expression of two CD45 isoforms normally found on quiescent T cells. We report that CD45RABC significantly increased CD4+ thymic T cell proliferation in both a mixed lymphocyte reaction and following anti-T cell receptor (TCR) antibody stimulation. Additionally, CD45RABC enhanced Ca2+ mobilization and phosphotyrosine accumulation, and suppressed the inhibitory effect of anti-CD4 antibodies. By contrast, CD45R0 did not enhance TCR signaling or phosphotyrosine levels in CD4+ thymic T cells and required a TCR co-stimulus to augment cellular proliferation. These studies provide genetic evidence that alternative CD45 isoforms are functionally distinct and disclose a unique mechanism by which T cell immunologic responsiveness can be modified.

Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus.

Caulobacters attach to surfaces in the environment via their holdfasts, attachment organelles located at the base of the flagellum in swarmer cells and later at the end of the cellular stalk in the stalked cells which develop from the swarmer cells. There seems to be little specificity with respect to the types of surfaces to which holdfasts adhere. A notable exception is that the holdfast of one cell does not adhere to the cell surface of another caulobacter, except by joining holdfasts, typically forming "rosettes" of stalked cells. Thus, the localized adhesion of the holdfasts to the cells is in some way a specialized attachment. We investigated this holdfast-cell attachment by developing an adhesion screening assay and analyzing several mutants of Caulobacter crescentus CB2A selected to be defective in adhesion. One class of mutants made a normal holdfast by all available criteria, yet the attachment to the cell was very weak, such that the holdfast was readily shed. Another class of mutants made no holdfast at all, but when mixed with a wild-type strain, a mutant of this class participated in rosette formation. The mutant could also attach to the discarded holdfast produced by a shedding mutant. In addition, when rosettes composed of holdfast-defective and wild-type cells were examined, an increase in the number of holdfast-defective cells was correlated with a decrease in the ability of the holdfast material at the center of the rosette to bind colloidal gold particles. Gold particles are one type of surface to which holdfasts adhere well, suggesting that the stalk end and the colloidal gold particles occupy the same sites on the holdfast substance. Taken together, the data support the interpretation that there is a specialized attachment site for the holdfast at the base of the flagellum which later becomes the end of the stalk, but not a specialized region of the holdfast for attachment to this site. Also, attachment to the cell is accomplished by bond formations that occur not only at the time of holdfast production. Thus, we propose that the attachment of the holdfast to the cell is a true adhesion process and that the stalk tip and base of the flagellum must have compositions distinctly different from that of the remainder of the caulobacter cell surface.