Microfluidics enables multiplex evaluation of the same cells for further studies.

OBJECTIVE: The continuous discovery of biomarkers and their evolving use for the diagnosis and guidance of therapy for patients with cancer has increased awareness of the need to triage biospecimens properly. On occasion, cytology samples are the only type of biospecimen available for analysis. Often, the current approach for these latter specimens is cytopathology-centric, with cells limited to examination by bright field microscopy. When specimens are paucicellular, there is often insufficient material for ancillary testing. Therefore, a need exists to develop an alternative approach that allows for the multiplexed analysis of cells when they are limited in number. In recent previous publications, we demonstrated that clinically derived cells from tissue are suitable for evaluation in a microfluidic device. In our current endeavour, we seek to expand upon those findings and determine if those same cells can be recovered for further analysis. METHODS: A microfluidic channel was designed, fabricated and tested using cytology specimens generated from tissue specimens. The cytological features of the cells tested were examined prior to entering the channel; they were then compared to similar cells while in the channel, and upon recovery from the channel. Recovery of DNA and proteins were also tested. RESULTS: The morphology of the tested cells was not compromised in either the channel or upon recovery. More importantly, the integrity of the cells remained intact, with the recovery of proteins and high molecular weight DNA possible. CONCLUSIONS: We developed and tested an alternative approach to the processing of cytopathology specimens that enables multiplexed evaluation. Using microfluidics, cytological examination of biopecimens can be performed, but in contrast to existing approaches, the same cells examined can be recovered for downstream analysis.

Reducing short-acting beta-agonist use in asthma: Impact of national incentives on prescribing practices in England and the findings from SENTINEL Plus early adopter sites.

Short-acting beta-agonist (SABA) over-use in asthma is harmful for patients and the environment. The Investment and Impact Fund (IIF) 2022/2023 financially rewarded English primary care networks that achieved specific targets, including reducing SABA over-use (RESP-02) and lowering the mean carbon footprint per salbutamol inhaler prescribed (ES-02). SENTINEL Plus is a co-designed quality improvement package that aims to improve asthma outcomes and reduce asthma's environmental impact by addressing SABA over-use. We investigated the impact of (i) the IIF incentives and (ii) SENTINEL Plus implementation on asthma prescribing. Using Openprescribing.net data, we demonstrate that IIF 2022-2023 had no significant impact on the total number of SABA prescribed in England (25,927,252 during 12-months pre- and 25,885,213 12-months post-IIF; 0.16% decrease; p=NS), but lower carbon footprint SABA inhaler use increased (Salamol™ prescribing increased from 5.1% to 19% of SABA prescriptions, p < 0.01). In contrast, SENTINEL Plus sites significantly reduced SABA prescribing post-implementation (5.43% decrease, p < 0.05).

A systematic review of treatment guidelines for metastatic colorectal cancer.

AIM: A systematic review of treatment guidelines for metastatic colorectal cancer (mCRC) was performed to assess recommendations for monoclonal antibody therapy in these guidelines. METHOD: Relevant papers were identified through electronic searches of MEDLINE, MEDLINE In Process, EMBASE and the Cochrane Library; through manual searches of reference lists; and by searching the Internet. RESULTS: A total of 57 relevant guidelines were identified, 32 through electronic database searches and 25 through the website searches. The majority of guidelines were published between 2004 and 2010. The country publishing the most guidelines was the USA (12), followed by the UK (10), Canada (eight), France (eight), Germany (three), Australia (two), Spain (two) and Italy (one). In addition, eight European and three international guidelines were identified. As monoclonal antibody therapy for mCRC was not introduced until 2004, no firm recommendations for monoclonal antibody therapy were made in guidelines published between 2004 and 2006. Recommendations for monoclonal antibody therapy first appeared in 2007 and evolved as more data became available. The most recent international, European and US guidelines recommend combination chemotherapy with the addition of a monoclonal antibody for the first-line treatment of mCRC. Second-line treatment depends on the first-line regimen used. For chemoresistant mCRC, cetuximab or panitumumab are recommended as monotherapy in patients with wild-type KRAS tumours. CONCLUSION: The study indicates that recent treatment guidelines have recognized the role of monoclonal antibodies in the management of mCRC, and that treatment guidelines should be updated in a timely manner to reflect the most recently available data.

Genomic and proteomic analysis of the effects of cannabinoids on normal human astrocytes.

Delta-9-tetrahydrocannabinol (Delta(9)-THC), the main psychoactive component of marijuana, is known to dysregulate various immune responses. Cannabinoid (CB)-1 and -2 receptors are expressed mainly on cells of the central nervous system (CNS) and the immune system. The CNS is the primary target of cannabinoids and astrocytes are known to play a role in various immune responses. Thus we undertook this investigation to determine the global molecular effects of cannabinoids on normal human astrocytes (NHA) using genomic and proteomic analyses. NHA were treated with Delta(9)-THC and assayed using gene microarrays and two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) to elucidate their genomic and proteomic profiles respectively. Our results show that the expression of more than 20 translated protein gene products from NHA was differentially dysregulated by treatment with Delta(9)-THC compared to untreated, control NHA.

Spray characterization of thermal fogging equipment typically used in vector control.

Droplet size spectra from different sprayers used to generate insecticide-laden fogs for controlling flying insects were measured by a laser diffraction instrument and Teflon-coated slides. The objectives of this work were to present not only information on spray-system droplet size generated by different sprayers, but to compare methodologies by which other similar systems can be evaluated and give applicators sprayer-system performance data. Data from 45 replicated spray tests, comprising 11 sprayers and 5 pesticides, showed a wide range in the droplet size spectra produced. The volume median diameter measurements ranged from 2.6 to 75.5 microm for diesel-diluted sprays and from 27.9 to 59.9 microm for water-diluted sprays. Similarly, the percent volume <20 microm ranged between 12.0-100% and 8.5-30.7%, for diesel- and water-diluted sprays, respectively. The droplet sizes measured by the swinging slide and laser diffraction methods were not consistent. The information presented aids users in sprayer selection and operation to produce the specific droplet size spectra required for a particular application.

Pyocyanin and 1-hydroxyphenazine produced by Pseudomonas aeruginosa inhibit the beating of human respiratory cilia in vitro.

Pseudomonas aeruginosa culture filtrates varied in their ability to slow human ciliary beat frequency (7-71%). This activity did not correlate with known virulence factors. However, a close correlation (r = 0.97) existed between ciliary slowing and pigment content. In a prolonged culture, the increase in activity correlated (r = 0.94) with pigment accumulation. Gel filtration of lyophilized filtrate yielded a single peak of activity corresponding to the pigment fraction. Pyocyanin extracted from an active strain, and 1-hydroxyphenazine were purified by high performance liquid chromatography, and characterized by ultraviolet absorbance spectra and mass spectrometry. Both slowed cilia in a dose-dependent manner, and were synthesized and shown to be indistinguishable from the biological compounds. Pyocyanin caused gradual onset of slowing and ultimate widespread ciliostasis with epithelial disruption. 1-hydroxyphenazine caused rapid onset of ciliary slowing associated with dyskinesia and ciliostasis. Pyocyanin assayed within filtrates accounted for a significant proportion of the bioactivity present.

Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor-dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression.

The genes encoding Hoxa9 and Meis1 are transcriptionally coactivated in a subset of acute myeloid leukemia (AML) in mice. In marrow reconstitution experiments, coexpression of both genes produces rapid AML, while neither gene alone generates overt leukemia. Although Hoxa9 and Meis1 can bind DNA as heterodimers, both can also heterodimerize with Pbx proteins. Thus, while their coactivation may result from the necessity to bind promoters as heterodimers, it may also result from the necessity of altering independent biochemical pathways that cooperate to generate AML, either as monomers or as heterodimers with Pbx proteins. Here we demonstrate that constitutive expression of Hoxa9 in primary murine marrow immortalizes a late myelomonocytic progenitor, preventing it from executing terminal differentiation to granulocytes or monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3. This immortalized phenotype is achieved in the absence of endogenous or exogenous Meis gene expression. The Hoxa9-immortalized progenitor exhibited a promyelocytic transcriptional profile, expressing PU.1, AML1, c-Myb, C/EBP alpha, and C/EBP epsilon as well as their target genes, the receptors for GM-CSF, G-CSF, and M-CSF and the primary granule proteins myeloperoxidase and neutrophil elastase. G-CSF obviated the differentiation block of Hoxa9, inducing neutrophilic differentiation with accompanying expression of neutrophil gelatinase B and upregulation of gp91phox. M-CSF also obviated the differentiation block, inducing monocytic differentiation with accompanying expression of the macrophage acetyl-low-density lipoprotein scavenger receptor and F4/80 antigen. Versions of Hoxa9 lacking the ANWL Pbx interaction motif (PIM) also immortalized a promyelocytic progenitor with intrinsic biphenotypic differentiation potential. Therefore, Hoxa9 evokes a cytokine-selective block in differentiation by a mechanism that does not require Meis gene expression or interaction with Pbx through the PIM.

Contributions of depressive mood and circulating inflammatory markers to coronary heart disease in healthy European men: the Prospective Epidemiological Study of Myocardial Infarction (PRIME).

BACKGROUND: Data on the possible association between depressive disorders and inflammatory markers are scarce and inconsistent. We investigated whether subjects with depressive mood had higher levels of a wide range of inflammatory markers involved in coronary heart disease (CHD) incidence and examined the contribution of these inflammatory markers and depressive mood to CHD outcome. METHODS AND RESULTS: We built a nested case-referent study within the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study of healthy middle-aged men from Belfast and France. We considered the baseline plasma sample from 335 future cases (angina pectoris, nonfatal myocardial infarction, coronary death) and 670 matched controls (2 controls per case). Depressive mood characterized men whose baseline depression score (13-item modification of the Welsh depression subscale) was in the fourth quartile (mean score, 5.75; range, 4 to 12). On average, men with depressive mood had 46%, 16%, and 10% higher C-reactive protein, interleukin-6, and intercellular adhesion molecule-1 levels, respectively, independently of case-control status, social characteristics, and classic cardiovascular risk factors; no statistical difference was found for fibrinogen. The odds ratios of depressive mood for CHD were 1.35 (95% CI, 1.05 to 1.73) in univariate analysis and 1.50 (95% CI, 1.04 to 2.15) after adjustment for social characteristics and classic cardiovascular risk factors. The latter odds ratio remained unchanged when each inflammatory marker was added separately, and in this analysis, each inflammatory marker contributed significantly to CHD event risk. CONCLUSIONS: These data support an association of depressive mood with inflammatory markers and suggest that depressive mood is related to CHD even after adjustment for these inflammatory markers.

Characterization of patients with an inadequate clinical outcome from osteoporosis therapy: the Observational Study of Severe Osteoporosis (OSSO).

BACKGROUND: Osteoporotic fractures remain a major public health problem. Currently available osteoporosis therapies significantly reduce the risk of fractures, but up to 50% of patients have an inadequate clinical outcome to therapy. AIM: To describe the clinical and quality of life (QOL) of a study population meeting a proposed definition of inadequate clinical outcome to osteoporosis therapy, recruited for the Observational Study of Severe Osteoporosis (OSSO). DESIGN: Cross-sectional, observational study. METHODS: Post-menopausal women with osteoporosis (n = 2314) were divided into Group 1 (those who had previously experienced a fragility fracture despite osteoporosis drug therapy for at least 12 months) (n = 1309, 57%), or Group 2 (those who had previously discontinued osteoporosis drug therapy due to non-compliance or side-effects) (n = 1005; 43%). Baseline clinical characteristics, quality of life (QOL) and osteoporosis/falls risk factors were analysed. RESULTS: The overall population had low BMD (mean +/- SD T-score at lumbar spine -3.1 +/- 1.1), and risk factors for fracture such as previous fractures (67.8%), family history (15.1%), and prolonged glucocorticoid use (17.5%). QOL was poor: total QUALEFFO and EQ-5D scores were 46.8 +/- 18.7, and 0.50 +/- 0.33, respectively. Patients in Group 1 had higher age and body mass index, fewer hours of exercise, more previous fragility fractures and falls, and poorer QOL scores. DISCUSSION: Our definition of inadequate clinical outcome from osteoporosis drug therapy identifies a severe osteoporosis cohort with poor QOL and increased fracture risk. Using such a definition may lead to earlier recognition of inadequate clinical outcome to osteoporosis therapy, and improved interventions and results.

The truncated glucocorticoid receptor in the P1798 mouse lymphosarcoma is associated with resistance to glucocorticoid lysis but not to other glucocorticoid-induced functions.

Glucocorticoid receptors in lines of the P1798 mouse lymphosarcoma either sensitive or resistant to glucocorticoid-induced lysis have been characterized and their functional significance determined. The glucocorticoid receptor from the cortisol-sensitive tumor is an Mr approximately 98,000 protein with a Stokes radius of 7.4 nm in the oligomeric, non-DNA-binding state and 5.6 nm in the transformed, DNA-binding state. This receptor binds glucocorticoid and reacts with the BUGR-2 monoclonal antibody. In contrast, two abnormal receptor species were identified in the cortisol-resistant tumor. One is an Mr approximately 98,000 non-steroid-binding but immunologically reactive protein. The other is an Mr approximately 45,000 species which contains both steroid- and DNA-binding sites but exhibits little or no reactivity with BUGR-2, suggesting that its NH2 terminus is truncated in a region within or adjacent to the BUGR epitope. This species had Stokes radii of 5.8 and 3.5 nm in nontransformed and transformed states, respectively. In both tumor lines, glucocorticoids stimulated the activities of glutamine synthetase and 5'-nucleotidase and the synthesis of glucocortin. However, glucocorticoid-induced tumor regression occurred only in the cortisol-sensitive tumor. Additionally, the glucocorticoid inducibility of a specific protein in the sensitive, but not in the resistant, tumor was demonstrated, as well as the presence of a protein specific to the resistant line. Taken together, these results suggest that the truncated glucocorticoid receptor in the P1798 lymphosarcoma is functional, although possibly in a more restricted gene-specific manner, and that the lysis defect, while possibly resulting from a truncated receptor, may also result from the inability of glucocorticoids to induce a critical protein in the pathway of programmed cell death and/or from the presence of a protein which inhibits the lytic response.

Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis.

A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh(-120/+)) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh(-120/+) mice. We also find a much larger fibril strain/tissue strain ratio in Crh(-120/+) mice (~1.5) compared to the wild-type mice (~0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.

HoxB8 requires its Pbx-interaction motif to block differentiation of primary myeloid progenitors and of most cell line models of myeloid differentiation.

HoxB8 was the first homeobox gene identified as a cause of leukemia. In murine WEHI3B acute myeloid leukemia (AML) cells, proviral integration leads to the expression of both HoxB8 and Interleukin (IL-3). Enforced expression of HoxB8 blocks differentiation of factor-dependent myeloid progenitors, while IL-3 co-expression induces autocrine proliferation and overt leukemogenicity. Previously, we demonstrated that HoxB8 binds DNA cooperatively with members of the Pbx family of transcription factors, and that HoxB8 makes contact with the Pbx homeodomain through a hexameric sequence designated the Pbx-interaction motif (PIM). E2a-Pbx1, an oncogenic derivative of Pbx1, both retains its ability to heterodimerize with Hox proteins and arrest myeloid differentiation. This observation prompts the question of whether E2a-Pbx1 and Hox oncoproteins use endogenous Hox and Pbx proteins, respectively, to target a common set of cellular genes. Here, we use four different models of neutrophil and macrophage differentiation to determine whether HoxB8 needs to bind DNA or Pbx cofactors in order to arrest myeloid differentiation. The ability of HoxB8 to bind DNA or to bind Pbx was essential (1) to block differentiation of factor-dependent myeloid progenitors from primary marrow; (2) to block IL-6-induced monocytic differentiation of M1-AML cells; and (3) to block granulocytic differentiation of GM-CSF-dependent ECoM-G cells. However, while DNA-binding was required, the HoxB8 Pbx-interaction motif was unnecessary for preventing macrophage differentiation of ECoM-M cells. We conclude that HoxB8 prevents differentiation by directly influencing cellular gene expression, and that the genetic context within a cell dictates whether the effect of HoxB8 is dependent on a physical interaction with Pbx proteins.

Substrate recognition of isopeptidase: specific cleavage of the epsilon-(alpha-glycyl)lysine linkage in ubiquitin-protein conjugates.

The structural chromatin protein A24 (uH2A) is a conjugate of histone H2A and a non-histone protein, ubiquitin. Eukaryotic cells contain an enzyme, generically termed isopeptidase, which can cleave A24 stoichiometrically into H2A and ubiquitin in vitro. Isopeptidase, free of proteinase activity, has been partially purified from calf thymus by ion-exchange chromatography, gel filtration and affinity chromatography, and analyzed for its substate specificity. There are three major types of isopeptide bonds besides the epsilon-(alpha-glycyl)lysine bond between H2A and ubiquitin; namely, the disulfide bridge, the aldol and aldimide bonds and the epsilon-(gamma-glutamyl)lysine crosslink. Under conditions where A24 was completely cleaved into H2A and ubiquitin, none of these naturally occurring isopeptide bonds was cleaved by isopeptidase. Furthermore, the bonds formed in vitro by transglutaminase reaction between casein and putrescine, through the gamma-NH2 of glutamine residue and the NH2 of putrescine, were not cleaved by the enzyme. The enzyme also failed to cleave the glycyl-lysyl and other orthodox peptide linkages within proteins. Among various proteins examined, the substrates for isopeptidase reaction were confined to conjugates between ubiquitin and other proteins, formed through epsilon-(alpha-glycyl)lysine bonds. Since ubiquitin released by isopeptidase is re-usable for an ATP-dependent conjugation with other proteins, its carboxyl terminal -Gly-Gly-COOH most likely is preserved intact, and is not blocked. These results suggest that isopeptidase specifically recognizes and cleaves the epsilon-(alpha-glycyl)lysine bond. A possible biological significance of this enzyme is discussed.

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.

Intestinal cell apoptosis and Bcl-2 expression.

Programmed cell death (apoptosis) is a normal characteristic of cells with a limited life span like the enterocyte and the usual mode of death for proliferative crypt cells subjected to radiation or chemotherapy. The Bcl-2 proto-oncogene is considered a major regulator of apoptosis. We investigated the relationship of enterocyte apoptosis and Bcl-2 expression in rat intestine and tissue culture cells. Fragmentation of DNA and levels of Bcl-2 transcripts were evaluated in rat enterocyte fractions of the crypt-to-villus axis of differentiation and in IEC tissue culture cells. A low percentage of isolated nuclei from each enterocyte fraction showed features of DNA fragmentation, including crypt cells. Detectable DNA fragmentation was seen in IEC cells only when cells were subjected to long-term confluent culture conditions. Bcl-2 mRNA was not detected in isolated rat intestinal cells but was detected in IEC cells where its level increased with serum deprivation and long-term culture. We conclude that increased Bcl-2 expression may be important in rescue of proliferative enterocytes subjected to stress.

Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

BACKGROUND AND PURPOSE: Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. EXPERIMENTAL APPROACH: Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. KEY RESULTS: The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. CONCLUSIONS AND IMPLICATIONS: These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes.

Galectin-1 suppresses methamphetamine induced neuroinflammation in human brain microvascular endothelial cells: Neuroprotective role in maintaining blood brain barrier integrity.

Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.

Nanotherapeutic approach for opiate addiction using DARPP-32 gene silencing in an animal model of opiate addiction.

Opiates act on the dopaminergic system of the brain and perturb 32 kDa dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein (DARPP-32) function. The DARPP-32 mediated inhibition of protein phosphatase-1 (PP-1) and modulation of transcriptional factor CREB is critical to the changes in neuronal plasticity that result in behavioral responses during drug abuse. To investigate the role of DARPP-32 mediated signaling on withdrawal behavior in a rat model of opiate addiction, we used intracerebral administration of gold nanorods (GNR) complexed to DARPP-32 siRNA to silence DARPP-32 gene expression and measure its effects on the opiate withdrawal syndrome. We hypothesized that DARPP-32 siRNA will suppress the neurochemical changes underlying the withdrawal syndrome and therefore prevent conditioned place aversion by suppressing or removing the constellation of negative effects associated with withdrawal, during the conditioning procedure. Our results showed that opiate addicted animals treated with GNR-DARPP-32 siRNA nanoplex showed lack of condition place aversive behavior consequent to the downregulation of secondary effectors such as PP-1 and CREB which modify transcriptional gene regulation and consequently neuronal plasticity. Thus, nanotechnology based delivery systems could allow sustained knockdown of DARPP-32 gene expression which could be developed into a therapeutic intervention for treating drug addiction by altering reward and motivational systems and interfere with conditioned responses.

Granulocyte-macrophage colony-stimulating factor and B7-2 combination immunogene therapy in an allogeneic Hu-PBL-SCID/beige mouse-human glioblastoma multiforme model.

Glioblastoma multiforme is the most common primary central nervous system neoplasm. Its dismal prognosis has led to investigation of new treatment strategies such as immunogene therapy. We transduced the human glioblastoma cell line D54MG in vitro with genes encoding the proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the T cell co-stimulatory molecule B7-2, or both (in a bicistronic vector) via retroviral vectors. Therapeutic gene expression by D54MG was high after transduction and selection (30 ng/10(6) cells/day for GM-CSF and > 2 orders of magnitude fluorescence shift on flow cytometry for B7-2). The effect of GM-CSF and/or B7-2 transduction on D54MG tumor growth in vivo was monitored in a novel allogeneic human peripheral blood lymphocyte-severe combined immunodeficiency mouse (Hu-PBL-SCID) model. GM-CSF- or B7-2-transduced tumors showed growth suppression in hu-PBL-reconstituted mice compared to untransduced and/or unreconstituted controls. Growth suppression was greatest for B7-2. Furthermore, vaccination with irradiated GM-CSF/B7-2-transduced tumor cells markedly inhibited growth of wild-type tumors at distant sites. Thus, this study illustrates a potential gene therapy strategy for glioblastoma multiforme patients using GM-CSF and/or B7-2 transduced tumor vaccines. Although extension of these allogeneic studies to an autologous system is critical, this is the first demonstration of in vivo efficacy of combination GM-CSF and B7-2 immunogene therapy for human glioblastoma multiforme.