Judith Weidenhofer

A comparative examination of the anti-inflammatory effects of SSRI and SNRI antidepressants on LPS stimulated microglia

Selective serotonin and serotonin norepinephrine reuptake inhibitors (SSRI; SNRI) are the first choice pharmacological treatment options for major depression. It has long been assumed that the primary therapeutic mechanism of action of these drugs involves the modulation of monoaminergic systems. However, contemporary investigations have revealed that depression is linked with inflammation, and that SSRI/SNRIs possess significant anti-inflammatory actions. While these anti-inflammatory properties initially only related to work undertaken on cells of the peripheral immune system, it has recently become apparent that these drugs also exert anti-inflammatory effects on microglia, the principal cells within the CNS that regulate and respond to inflammatory factors. The aim of the current study was to compare SSRI/SNRIs in terms of their anti-inflammatory potency, and to determine the specific mechanisms through which these effects are mediated. Accordingly, the current study evaluated the ability of five different SSRIs (fluoxetine, sertraline, paroxetine, fluvoxamine and citalopram) and one SNRI (venlafaxine) to suppress microglial responses to an inflammatory stimulus. Specifically, we examined their ability to alter tumour necrosis factor-α (TNF-α) and nitric oxide (NO) production after 4 and 24 h stimulation with lipopolysaccharide. Our results indicated that the SSRIs potently inhibited microglial TNF-α and NO production. We then investigated whether these effects might involve either β-adrenoceptor or cAMP signalling. Using the protein kinase A inhibitor Rp-CAMPs, we found evidence to suggest that cAMP signalling is involved in regulating the anti-inflammatory response. These findings suggest that antidepressants may owe at least some of their therapeutic effectiveness to their anti-inflammatory properties.

Preliminary investigation of gene expression profiles in peripheral blood lymphocytes in schizophrenia.

Schizophrenia is a heterogenous disorder that is phenomenologically characterised by a combination of negative, positive, and cognitive symptoms with variable expression in the course of illness. Here, we investigated differential gene expression in relation to age to address the heterogeneity of this disorder We used 6000 gene cDNA microarrays to generate gene expression profiles from peripheral blood lymphocytes from 14 individuals with schizophrenia and 14 non-psychiatric controls. Genes showing altered expression were identified and 18 genes with brain-related functions were altered, 4 of which, endothelial differentiation gene 2 (Edg-2), ezrin-radixin-moesin phosphoprotein 50 (EBP50), Myc-associated zinc finger protein (MAZ) and Tumor Necrosis Factor Receptor 2 (TNFR2), were confirmed by relative real-time PCR. Dendrograms were constructed using genes that showed significantly different expression (p<0.05) between groups based on median split of age dividing the matched pairs into distinct subclasses. Our findings suggest that distinct gene expression profiles in peripheral blood lymphocytes associated with schizophrenia phenotypes may provide a first step towards the biological classification of schizophrenia subtypes. The validity of this approach may lead to better methods of defining this enigmatic disease.

Altered gene expression in the amygdala in schizophrenia: up-regulation of genes located in the cytomatrix active zone.

The amygdala is implicated in the pathophysiology of schizophrenia through its function in the processing of emotions. However, the genes involved in the dysfunction of the amygdala in schizophrenia are yet to be identified. This study examined gene expression in the amygdala in postmortem tissue from seven matched pairs of schizophrenia and non-psychiatric control subjects, using oligonucleotide-microarrays representing 19,000 gene transcripts and real-time PCR confirmation of gene expression changes in eleven matched pairs. Genes involved in presynaptic function, myelination and cellular signalling were identified as being consistently dysregulated in this cohort of subjects with schizophrenia. In particular, the expression of three genes involved in the cytomatrix active zone, Regulating membrane exocytosis 2, Regulating membrane exocytosis 3 and Piccolo, was up-regulated. These results implicate for the first time the dysfunction of the cytomatrix active zone of synapses in the amygdala in the pathophysiology of schizophrenia.

Nonredundant functions for tumor protein D52-like proteins support specific targeting of TPD52

Purpose:Tumor protein D52 (TPD52 or D52) is frequently overexpressed in breast and other cancers and present at increased gene copy number. It is, however, unclear whether D52 amplification and overexpression target specific functional properties of the encoded protein. Experimental Design: The expression of D52-like genes and MAL2 was compared in breast tissues using quantitative reverse transcription-PCR. The functions of human D52 and D53 genes were then compared by stable expression in BALB/c 3T3 fibroblasts and transient gene knockdown in breast carcinoma cell lines. In situ D52 and MAL2 protein expression was analyzed in breast tissue samples using tissue microarray sections. Results: The D52 (8q21.13), D54 (20q13.33), and MAL2 (8q24.12) genes were significantly overexpressed in breast cancer tissue (n = 95) relative to normal breast (n = 7; P ≤ 0.005) unlike the D53 gene (6q22.31; P = 0.884). Subsequently, D52-expressing but not D53-expressing 3T3 cell lines showed increased proliferation and anchorage-independent growth capacity, and reduced D52 but not D53 expression in SK-BR-3 cells significantly increased apoptosis. High D52 but not MAL2 expression was significantly associated with reduced overall survival in breast carcinoma patients (log-rank test, P < 0.001; n = 357) and was an independent predictor of survival (hazard ratio, 2.274; 95% confidence interval, 1.228-4.210; P = 0.009; n = 328). Conclusion: D52 overexpression in cancer reflects specific targeting and may contribute to a more proliferative, aggressive tumor phenotype in breast cancer.

Investigation of the expression of genes affecting cytomatrix active zone function in the amygdala in schizophrenia: effects of antipsychotic drugs.

The cytomatrix active zone (CAZ) is a specialized cellular structure regulating release of vesicles. We reported previously increased expression of three CAZ genes, piccolo, RIMS2 and RIMS3 in the amygdala in schizophrenia. This study determined the levels of gene and protein expression for components of the active zone including two additional CAZ genes in the amygdala from subjects with schizophrenia and non-psychiatric controls, as well as the effects of antipsychotic drugs. Whilst relative real-time PCR analysis did not identify significant change in the expression of six additional active zone genes, Western blot analysis showed increased piccolo and RIMS2 protein expression in the amygdala in schizophrenia. In vitro analysis suggests antipsychotic drug treatment was unlikely to have caused the changes in RIMS2, RIMS3 and piccolo expression observed in the amygdala in schizophrenia. Therefore, this study provides further evidence suggesting that piccolo, RIMS2, RIMS3, but not the entire components of the active zone are involved in the neurobiology of schizophrenia.

Immunohistochemical localisation of the NK1 receptor in the human amygdala : Preliminary investigation in schizophrenia

The amygdala has a role in the modulation of moods and emotion, processes that are known to be affected in people with psychiatric disorders such as schizophrenia and depression. The tachykinin NK(1) receptor is known to be expressed in the amygdala. However to date, there is limited knowledge of the distribution of the NK(1) receptor in this region. This study used immunohistochemistry to analyse the distribution of the NK(1) receptor in fixed human amygdala tissue in control subjects with no history of psychiatric illness and matched subjects with a diagnosis of schizophrenia (n=4 pairs). The NK(1) receptor was observed sparsely distributed in cell bodies in all amygdaloid nuclei with the basolateral and lateral having a greater relative density of NK(1) receptor-immunoreactive cell bodies than the other nuclei. Double labelling with antibodies to microtubule associated protein and the NK(1) receptor revealed that the NK(1) receptor is expressed by large pyramidal, small stellate and large bipolar neurons. Interestingly, the basal nucleus of Meynert, which is just dorsal to the amygdala, was observed to have a significantly higher relative density of NK(1) receptor-immunoreactive cell bodies compared to any of the amygdaloid nuclei. Preliminary analysis of the density of NK(1) receptor-immunoreactive cell bodies in the major amygdaloid nuclei and the basal nucleus of Meynert revealed no significant differences between schizophrenia and control subjects. Real-time PCR showed that the mRNA for both the short and long isoforms of the NK(1) receptor was expressed at low levels in fresh frozen human amygdala tissue from control subjects and that this was not different in matched subjects with schizophrenia (n=11 pairs). In conclusion, this study has demonstrated that the NK(1) receptor is widely distributed in the amygdala, and has shown for the first time a high relative density of NK(1) receptor-immunoreactive cell bodies in the basal nucleus of Meynert.

Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model

BackgroundTetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with β1 and β4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems.MethodsHere we investigated the effect of Cd151 deletion on mammary tumorigenesis by crossing Cd151-deficient mice with a spontaneously metastasising transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV).ResultsCd151 deletion did not affect the normal development and differentiation of the mammary gland. While there was a trend towards delayed tumor onset in Cd151−/− PyMT mice compared to Cd151+/+ PyMT littermate controls, this result was only approaching significance (Log-rank test P-value =0.0536). Interestingly, Cd151 deletion resulted in significantly reduced numbers and size of primary tumors but did not appear to affect the number or size of metastases in the MMTV/PyMT mice. Intriguingly, no differences in the expression of markers of cell proliferation, apoptosis and blood vessel density was observed in the primary tumors.ConclusionThe findings from this study provide additional evidence that CD151 acts to enhance tumor formation initiated by a range of oncogenes and strongly support its relevance as a potential therapeutic target to delay breast cancer progression.

Phenolic Compounds, Antioxidant and Anti-Cancer Properties of the Australian Maroon Bush Scaevola spinescens (Goodeniaceae)

Scaevola spinescens (Goodeniaceae) has been traditionally used by indigenous Australians to treat various ailments including cancer, thus it is necessary to identify optimum extraction conditions for bioactive components from this plant. This study investigated the effects of different extraction conditions on Total Phenolic Content (TPC), antioxidant capacity (ABTS, DPPH, CUPRAC, FRAP assays) and anti-cancer activity (MTT assay) of S. spinescens. The results showed that optimal extraction conditions for TPC using water were 80°C, 15 min and ratio of 20:1 mL/g. However, the aqueous extract prepared under optimal conditions had lower TPC and less antioxidant capacity than those of the organic solvent extracts. The acetone extract displayed the greatest TPC as well as the highest antioxidant capacity and anti-cancer activity against a panel of cancer cell lines, including cancers of the pancreas, breast, lung, brain, skin, colon and ovary. Therefore, further investigations should be conducted to identify key bioactive compounds as potential anti-cancer agents.

Extracellular vesicles with altered tetraspanin CD9 and CD151 levels confer increased prostate cell motility and invasion

To facilitate intercellular communication, cells release nano-sized, extracellular vesicles (EVs) to transfer biological cargo to both local and distant sites. EVs are enriched in tetraspanins, two of which (CD9 and CD151) have altered expression patterns in many solid tumours, including prostate cancer, as they advance toward metastasis. We aimed to determine whether EVs from prostate cells with altered CD9 and CD151 expression could influence cellular behaviour and increase the metastatic capabilities of non-tumourigenic prostate cells. EVs were isolated by ultrafiltration and characterised for their tetraspanin expression and size distribution. iTRAQ was used to identify differences between RWPE1 and tetraspanin-modified RWPE1 EV proteomes, showing an enrichment in protein degradation pathways. Addition of EVs from RWPE1 cells with reduced CD9 or increased CD151 abundance resulted in increased invasion of RWPE1 cells, and increased migration in the case of high CD151 abundance. We have been able to show that alteration of CD9 and CD151 on prostate cells alters the proteome of their resultant EVs, and that these EVs can enhance the migratory and invasive capabilities of a non-tumourigenic prostate cellular population. This work suggests that cellular tetraspanin levels can alter EVs, potentially acting as a driver of metastasis in prostate cancer.

Animal models of pancreatic cancer and their application in clinical research

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