Michael Bette

Effects of Antidepressant Drugs on the Behavioral and Physiological Responses to Lipopolysaccharide (LPS) in Rodents

Antidepressants produce various immunomodulatory effects, as well as an attenuation of the behavioral responses to immune challenges, such as lipopolysaccharide (LPS). To explore further the effects of antidepressants on neuroimmune interactions, rats were treated daily with either fluoxetine (Prozac) or saline for 5 weeks, and various behavioral, neuroendocrine, and immune functions were measured following administration of either LPS or saline. Chronic fluoxetine treatment significantly attenuated the anorexia and body weight loss, as well as the depletion of CRH-41 from the median eminence and the elevation in serum corticosterone levels induced by LPS. Chronic treatment with imipramine also attenuated LPS-induced adrenocortical activation. In rats and in mice, which normally display a biphasic body temperature response to LPS (initial hypothermia followed by hyperthermia), chronic treatment with fluoxetine completely abolished the hypothermic response and facilitated and strengthened the hyperthermic response. The effects of antidepressants on the responsiveness to LPS are probably not mediated by their effects on peripheral proinflammatory cytokine production, because LPS-induced expression of TNFα and IL-1β mRNA in the spleen (assessed by semiquantitative in situ hybridization) was not altered following chronic treatment with either fluoxetine or imipramine. The effects of antidepressants on the acute phase response may have important clinical implications for the psychiatric and neuroendocrine disturbances that are commonly associated with various medical conditions.

A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity.

ABSTRACT Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu333 remained unchanged after five mouse passages, an Asn194→Lys194 mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn194 with Lys194 in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn194→Lys194 mutation does not arise when Asn194 is exchanged with Ser194 (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.

The Central Nervous System Inflammatory Response to Neurotropic Virus Infection Is Peroxynitrite Dependent

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO−), a product of the free radicals NO· and superoxide (O2·−). To determine whether this is a reflection of the physiological contribution of ONOO− to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO−. The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO− or its products on the blood-brain barrier.

Constitutive expression of p55TNFR mRNA and mitogen-specific up-regulation of TNFα and p75TNFR mRNA in mouse brain

Serum tumor necrosis factor (TNF) functions as a mediator of the immune‐to‐brain axis. Numerous TNF receptor‐mediated effects on the nervous system are described but the knowledge about the regional and cellular expression of TNF receptor p55TNFR and p75TNFR in vivo is far from being complete. It is unclear whether TNF mediates its neuroimmune effects alone or in combination with other factors, e.g., bacterial mitogens. Here, we investigated the distribution of TNFα, p55TNFR, and p75TNFR in normal mouse brain and examined the stimulus‐specific effects of lipopolysaccharide (LPS) and staphylococcal enterotoxin B (SEB) on the expression of the cerebral TNF system. Both mitogens caused enhanced TNFα serum levels and induced c‐fos mRNA in the paraventricular nucleus but exhibited different effects on the cerebral gene expression of the TNF system. LPS but not SEB rapidly induced TNFα mRNA in circumventricular organs (CVOs) followed by spreading of TNFα mRNA into brain parenchyma close to the CVOs. The p55TNFR gene was constitutively expressed in many neurons with high levels in brainstem motor nuclei and in neurons of the sensory mesencephalic trigeminal nucleus. Moderate levels of p75TNFR mRNA were seen in single cells scattered throughout the brain in a pattern resembling microglia. Neither LPS nor SEB modulated the p55TNFR gene expression in any region or cell type of the brain, and LPS but not SEB induced p75TNFR mRNA in the CVOs. Thus, enhanced TNF serum levels able to stimulate c‐fos mRNA expression in the paraventricular nucleus did not necessarily result in a modulation of the cerebral TNF system. J. Comp. Neurol. 465:417–430, 2003.

Overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice.

ABSTRACT The effect of tumor necrosis factor alpha (TNF-α) on rabies virus (RV) infection of the mouse central nervous system (CNS) was studied, using recombinant RV engineered to express either soluble TNF-α [SPBN-TNF-α(+)] or insoluble membrane-bound TNF-α [SPBN-TNF-α(MEM)]. Growth curves derived from infections of mouse neuroblastoma NA cells revealed significantly less spread and production of SPBN-TNF-α(+) than of SPBN-TNF-α(MEM) or SPBN-TNF-α(−), which carries an inactivated TNF-α gene. The expression of soluble or membrane-bound TNF-α was not associated with increased cell death or induction of alpha/beta interferons. Brains of mice infected intranasally with SPBN-TNF-α(+) showed significantly less virus spread than did mouse brains after SPBN-TNF-α(−) infection, and none of the SPBN-TNF-α(+)-infected mice succumbed to RV infection, whereas 80% of SPBN-TNF-α(−)-infected mice died. Reduced virus spread in SPBN-TNF-α(+)-infected mouse brains was paralleled by enhanced CNS inflammation, including T-cell infiltration and microglial activation. These data suggest that TNF-α exerts its protective activity in the brain directly through an as yet unknown antiviral mechanism and indirectly through the induction of inflammatory processes in the CNS.

Intraglandular application of botulinum toxin leads to structural and functional changes in rat acinar cells.

Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands.

Absence of spinal response to extracorporeal shock waves on the endogenous opioid systems in the rat

Extracorporeal shock wave therapy (ESWT) seems to be a new therapeutic strategy for chronic pain due to tendopathies. Neurophysiological mechanisms of action for pain relief following ESWT are still unknown. The aim of this study was to investigate if the analgesic effect of ESWT is caused by modulation of the endogenous spinal opioid system. Rats were treated with two different energy flux densities (0.04 and 0.11mJ/mm(2)) and immunohistochemical analysis of met-enkephalin (MRGL) and dynorphin (Dyn) was performed at 4 or 72 h after ESWT. ESWT had no modulatory influence on the expression of the spinal opioid systems. Different energy doses or repetitive treatment did not alter MRGL or Dyn immunoreactivity in the spinal cord. Furthermore, a delayed effect of ESWT at 72 h after treatment was not detectable. We conclude from these findings that the analgesic effects of ESWT treatment are not supported by endogenous opioids.

A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9

Head and neck squamous cell carcinomas (HNSCCs) are the most frequent malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis concludes that CSCs constitute the dangerous tumor cell population due to their ability of self-renewal and being associated with relapse of tumor disease, invasiveness and resistance to chemo(radio)therapy. The aim of this study was to look for CSC candidates and expression of MMP-9 that previously was implicated in HNSCC invasiveness. Immunohistochemical, immunofluorescence and Western blot analysis were performed on HNSCC tumor specimens using antibodies specific for MMP-9, CD44, ALDH1 and CK14. Gelatinolytic activity was assessed by zymography. Pearson correlation analysis was used for statistical comparison. Immunohistochemical analysis found CD44 and MMP-9 to co-localize in tumor cells at the invasive front. Western blot analysis demonstrated a significant correlation (p=0.0047) between CD44 and MMP-9 in the tested tissues. In addition gelatinolytic activity of HNSCC tissues was found to significantly correlate (p=0.0010) with MMP-9 expression. The CD44(+) invasive front of the tumor was also positive for ALDH1 and CK14, all of them being typically expressed by cells in the basal cell layer of normal stratified squamous epithelia that also harbors the epithelial stem cells. The observations point to a role of a MMP-9 positive basal-cell-like cell layer in the process of HNSCC invasiveness. This compartment likely contains CSCs since it is expressing the putative CSC markers CD44, ALDH1 and CK14. This cell layer therefore should be considered a major therapeutic target in the treatment of head and neck cancer.

Treatment with ozone/oxygen-pneumoperitoneum results in complete remission of rabbit squamous cell carcinomas

Head and neck squamous cell carcinomas (HNSCC) represent a group of metastasizing tumors with a high mortality rate in man and animals. Since the biomolecule ozone was found to inhibit growth of various carcinoma cells in vitro we here applied the highly aggressive and lethal VX2 carcinoma HNSCC tumor model of the New Zealand White rabbit to test whether ozone exerts antitumorous effects in vivo. Therapeutic insufflation of medical ozone/oxygen (O3/O2) gas mixture into the peritoneum (O3/O2‐pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits presented full tumor regression and the absence of local or distant lung metastases. Insufflation of pure oxygen (O2) resulted in a survival rate of 3/13 animals accompanied by full tumor remission in 2 of the 3 surviving animals. Of the 14 sham‐treated animals only 1 had spontaneous tumor remission and survived. No adverse effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflations of the O3/O2 or O2 gas. Animals with O3/O2‐induced tumor eradication developed tolerance against reimplantation of the VX2 tumor. This could be reversed by immune suppression with a combination of dexamethasone and cyclosporin A suggesting an antitumorous effect of O3/O2‐mediated activation of the bodys own immunosurveillance. Although the exact mechanisms of action are still unclear the present data point to O3/O2‐pneumoperitoneum as a promising new strategy in anticancer therapy.

Treatment of HNSCC cell lines with the EGFR‐specific inhibitor cetuximab (Erbitux®) results in paradox phosphorylation of tyrosine 1173 in the receptor

Overexpression of the epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequent in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression. Inhibition of EGFR with the kinase inhibitor AG1478 abolished receptor phosphorylation and reduced cell proliferation. However, treatment of HNSCC cells with cetuximab (Erbitux®), a monoclonal antibody designed to block the EGFR ligand binding site, led to paradox EGFR activation due to hyperphosphorylation of tyrosine 1173, however, with a concomitant reduction in Erk1/2 phosphorylation levels. No pronounced influence on cell proliferation levels could be observed after treatment with this antibody. Since cetuximab appears able to activate EGFR in HNSCC cell lines, it is necessary to rethink the exact mechanisms by which cetuximab that recently was approved for the treatment of advanced head and neck cancer, inhibits tumor growth.