Aerosol-induced brucellosis increases TLR-2 expression and increased complexity in the microanatomy of astroglia in rhesus macaques

Brucella melitensis, a bacterial pathogen and agent of epizootic abortion causes multiple pathologies in humans as well as a number of agriculturally important animal species. Clinical human brucellosis manifests as a non-specific, chronic debilitating disease characterized by undulant fever, arthropathies, cardiomyopathies and neurological sequelae. These symptoms can occur acutely for a few weeks or persist for months to years. Within the brain, endothelial and glial cells can be infected leading to downstream activation events including matrix metalloprotease (MMP) and cytokine secretion and Toll-like receptor (TLR) signaling. These events are likely to lead to tissue remodeling, including morphologic changes in neuronal and glial cells, which are linked to neurological complications including depressive behavior, immune activation and memory loss. Our hypothesis was that B. melitensis infection and neurobrucellosis would lead to activation of astrocytes through upregulation of TLR2 and stimulate concurrent changes in the microanatomy. All six animals were infected via inhalation route. TLR2 expression was approximately doubled in white matter astrocytes of infected rhesus macaques. There was also a 50% increase in the number of astrocytes per unit area in subcortical white matter tracts suggesting increased innate immune activation. This coincided with dramatic increases in the length and complexity of the cell arbor of hypertrophic astrocytes in both cortical gray and white matter. Thus, aerosol-induced brucellosis results in dramatically increased innate immune activation of astrocytes in the absence of widespread neuroinflammation

Carcinoma Matrix Controls Resistance to Cisplatin through Talin Regulation of NF-kB

Extracellular matrix factors within the tumor microenvironment that control resistance to chemotherapeutics are poorly understood. This study focused on understanding matrix adhesion pathways that control the oral carcinoma response to cisplatin. Our studies revealed that adhesion of HN12 and JHU012 oral carcinomas to carcinoma matrix supported tumor cell proliferation in response to treatment with cisplatin. Proliferation in response to 30 µM cisplatin was not observed in HN12 cells adherent to other purified extracellular matrices such as Matrigel, collagen I, fibronectin or laminin I. Integrin β1 was important for adhesion to carcinoma matrix to trigger proliferation after treatment with cisplatin. Disruption of talin expression in HN12 cells adherent to carcinoma matrix increased cisplatin induced proliferation. Pharmacological inhibitors were used to determine signaling events required for talin deficiency to regulate cisplatin induced proliferation. Pharmacological inhibition of NF-kB reduced proliferation of talin-deficient HN12 cells treated with 30 µM cisplatin. Nuclear NF-kB activity was assayed in HN12 cells using a luciferase reporter of NF-kB transcriptional activity. Nuclear NF-kB activity was similar in HN12 cells adherent to carcinoma matrix and collagen I when treated with vehicle DMSO. Following treatment with 30 µM cisplatin, NF-kB activity is maintained in cells adherent to carcinoma matrix whereas NF-kB activity is reduced in collagen I adherent cells. Expression of talin was sufficient to trigger proliferation of HN12 cells adherent to collagen I following treatment with 1 and 30 µM cisplatin. Talin overexpression was sufficient to trigger NF-kB activity following treatment with cisplatin in carcinoma matrix adherent HN12 cells in a process disrupted by FAK siRNA. Thus, adhesions within the carcinoma matrix create a matrix environment in which exposure to cisplatin induces proliferation through the function of integrin β1, talin and FAK pathways that regulate NF-kB nuclear activity

Astrocyte atrophy and immune dysfunction in self-harming macaques.

BACKGROUND: Self-injurious behavior (SIB) is a complex condition that exhibits a spectrum of abnormal neuropsychological and locomotor behaviors. Mechanisms for neuropathogenesis could include irregular immune activation, host soluble factors, and astrocyte dysfunction. METHODS: We examined the role of astrocytes as modulators of immune function in macaques with SIB. We measured changes in astrocyte morphology and function. Paraffin sections of frontal cortices from rhesus macaques identified with SIB were stained for glial fibrillary acidic protein (GFAP) and Toll-like receptor 2 (TLR2). Morphologic features of astrocytes were determined using computer-assisted camera lucida. RESULTS: There was atrophy of white matter astrocyte cell bodies, decreased arbor length in both white and gray matter astrocytes, and decreased bifurcations and tips on astrocytes in animals with SIB. This was combined with a five-fold increase in the proportion of astrocytes immunopositive for TLR2. CONCLUSIONS: These results provide direct evidence that SIB induces immune activation of astrocytes concomitant with quantifiably different morphology

The carcinoma matrix mediates cisplatin induced tumor cell proliferation.

<p>The proliferation of HN12 cells adherent to matrix proteins following treatment with vehicle DMSO or cisplatin for 48 hours is shown. A. Proliferation of HN12 cells adherent to BSA (0.1%), collagen I (0.5, 5.0 and 50.0 µg/mL) or carcinoma matrix. Proliferation is normalized to DMSO-treated cells for each substratum. B. Ki67 staining of HN12 cells adherent to collagen and treated with DMSO or cisplatin (1 and 30 µM). Shown are representative fluorescent images of the anti-Ki67 and control secondary antibody staining. The nuclear compartment was visualized by co-staining with DAPI. C. Shown in the bar graph is the mean percentage of HN12 nuclei that are positive for Ki67 in HN12 cells adherent to collagen and treated with vehicle DMSO or cisplatin (1 and 30 µM). Error bars indicate standard deviations. D. Proliferation of HN12 cells adherent to BSA (0.1%), matrigel, laminin I, fibronectin or collagen I (10 µg/mL) and treated with vehicle DMSO or 30 µM cisplatin. Error bars on the mean proliferation are standard deviations. These experiments were performed in three separate trials in quadruplicate. Student's t-test <i>p</i> values, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Schematic of oral carcinoma response to cisplatin when adherent to the carcinoma matrix.

<p>Cisplatin treatment of oral carcinoma adherent to carcinoma matrix induces proliferation through integrin β₁ dependent pathways. Treatment with cisplatin regulates NF-kB activity of talin overexpressing oral carcinoma via a FAK-dependent mechanism.</p

Role of integrin β₁ in cisplatin induced oral carcinoma proliferation.

<p>Proliferation of JHU012 and JHU012 integrin β₁ deficient cells adherent to A. carcinoma matrix and B. collagen I (10 µg/mL). Cells were treated for 48 hours with DMSO or cisplatin (0.05, 1, 30 µM) as indicated. Shown in the bar graph is the mean proliferation and error bars that indicate standard error. The horizontal line indicates the proliferation of control JHU012 cells after treatment with 30 µM cisplatin. Statistical comparisons for each substratum, a = JHU012/DMSO, b = JHU012/0.05 µM, c = JHU012/1 µM, d = JHU012/30 µM. C. Analysis of surface expression levels of integrin β₁, α₂ and α₆ subunits in JHU012 and JHU012 β₁ deficient cells by flow cytometry. Shown are overlayed histograms, grey is the isotype control and black is the indicated integrin subunit. MFI: Mean Fluorescence Intensity. D. Proliferation of HN12 cells adherent to carcinoma matrix. Control and integrin β₁ siRNA transfected cells were treated for 48 hours with DMSO or 1 µM cisplatin as indicated. Shown in the bar graph is mean proliferation and error bars indicate standard error. Student's t-test <i>p</i> values, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Talin and p130Cas control cisplatin induced proliferation of oral carcinoma.

<p>HN12 cell transfectants (plasmids containing cDNA expression vectors either talin, talin432G, p130Cas, p130CasDM (D416E, D748E) and cDNA3.1) were seeded onto A. carcinoma matrix and B. collagen I. Proliferation was measured following treatment with DMSO or cisplatin (1 and 30 µM) for 48 hours. The mean proliferation is shown with error bars indicating standard error. The data was normalized to cDNA 3.1 control transfectants treated with DMSO on each substratum. This experiment was performed 4 times. Statistical comparisons a = control/DMSO, b = control/1 µM, c = control/30 µM. Student's t-test <i>p</i> values, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Adhesion to carcinoma matrix and talin overexpression regulate NF-kB activity after treatment with cisplatin.

<p>A. HN12 cell transfectants ( (NF-kB)₄ firefly luciferase together with renilla luciferase) adherent to carcinoma matrix or collagen I were treated for 24 hours with 30 µM cisplatin or vehicle DMSO. Shown in the bar graph is the mean renilla normalized firefly luciferase activity in arbitrary light units with error bars indicating standard deviation. B. HN12 cell transfectants (firefly and renilla luciferase constructs with plasmids containing cDNA expression vectors either cDNA3.1, talin or talin432G) adherent to carcinoma matrix and treated with DMSO for 24 hours. The mean renilla normalized firefly luciferase activity in arbitrary light units with error bars indicating standard deviation is shown in the bar graph. C. HN12 cells transfected with talin cDNA expression vector together with (NF-kB)₄ firefly luciferase and renilla luciferase were plated onto carcinoma matrix or collagen. Adherent cells were treated with DMSO or 30 µM cisplatin for 24 hrs and renilla normalized firefly luciferase activity is shown in the bar graph with error bars indicating standard error. D. HN12 cells transfected with talin cDNA expression vector together with tk-luciferase (lacking the NF-kB regulatory element) and renilla luciferase adherent to carcinoma matrix were treated with DMSO and 30 µM cisplatin. Renilla normalized firefly luciferase activity is shown in the bar graph with error bars indicating standard error. E. HN12 cells transfected with (NF-kB)₄ firefly luciferase, renilla luciferase and talin cDNA in combination with either control or FAK siRNA. Carcinoma matrix adherent cells were treated with DMSO or 1 µM cisplatin and renilla normalized firefly luciferase activity is shown in the bar graph with error bars indicating standard error. Student's t-test <i>p</i> values *<i>p</i><0.05, **<i>p</i><0.01.</p