Clinical decision making in prostate cancer care-evaluation of EAU-guidelines use and novel decision support software.

Keeping up to date with the latest clinical advances in prostate cancer can be challenging. We investigated the impact of guideline use on quality of treatment decisions as well as the impact of a novel, CE-certified clinical decision support tool (Siemens AIPC software) on the amount of time clinicians spend on decision-making in a multicenter setting. Ten urologists assessed ten clinical cases (screening and localized prostate cancer) in three settings: without support, using a digital version of the EAU guidelines, and with the AIPC tool, resulting in 300 clinical decisions. Comparison involved time spent, decision correct- and completeness. Using AIPC compared to digital guidelines led to a significant reduction of expenditure of time at a per case level (3.57 min and 0:14 min, p < 0.01) and for overall time per urologist (39.45 min and 02:20 min, p < 0.01). Decision options without guidelines support, online guideline usage and usage of AIPC were complete in 61%, 80% and 100%, respectively (p < 0.01). Decision making without guidelines support, online guideline usage and usage of AIPC was correct including all options in 28%, 66% and 100%, respectively (p < 0.01).Clinical decision support systems have the potential to reduces decision-making time and to enhance decision quality.

Metallothioneins are upregulated in symptomatic mice with astrocyte-targeted expression of tumor necrosis factor-alpha.

Transgenic mice expressing TNF-alpha under the regulatory control of the GFAP gene promoter (GFAP-TNFalpha mice) exhibit a unique, late-onset chronic-progressive neurological disorder with meningoencephalomyelitis, neurodegeneration, and demyelination with paralysis. Here we show that the metallothionein-I + II (MT-I + II) isoforms were dramatically upregulated in the brain of symptomatic but not presymptomatic GFAP-TNFalpha mice despite TNF-alpha expression being present in both cases. In situ hybridization analysis for MT-I RNA and radioimmunoassay results for MT-I + II protein revealed that the induction was observed in the cerebellum but not in other brain areas. Increased MT-I RNA levels occurred in the Purkinje and granular neuronal layers of the cerebellum but also in the molecular layer. Reactive astrocytes, activated rod-like microglia, and macrophages, but not the infiltrating lymphocytes, were identified as the cellular sources of the MT-I + II proteins. In situ hybridization for MT-III RNA revealed a modest increase in the white matter of the cerebellum, which was confirmed by immunocytochemistry. MT-III immunoreactivity was present in cells which were mainly round or amoeboid monocytes/macrophages. The pattern of expression of the different MT isoforms in the GFAP-TNFalpha mice differed substantially from that described previously in GFAP-IL6 mice, demonstrating unique effects associated with the expression of each cytokine. The results suggest that the MT expression in the CNS reflects the inflammatory response and associated damage rather than a direct role of the TNF-alpha in their regulation and support a major role of these proteins during CNS injury.

Transgenic expression of TNF by astrocytes increases mechanical allodynia in a mouse neuropathy model.

It has been hypothesized that increased expression of proinflammatory cytokines mediate a variety of central nervous system disorders such as multiple sclerosis, Alzheimer's disease, cerebral ischemia, spinal cord injury, HIV encephalopathy and chronic pain. In order to further examine the central role of TNF in neuropathic pain, transgenic mice were used in which expression of murine TNF was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF fusion gene. Spinal nerve (L5) transection was performed in either the GFAP-TNF transgenic or wild type mice. Mechanical allodynia was significantly enhanced in the GFAP-TNF transgenic mice compared with the wild type mice. These data support a central role of glial expression of TNF in the generation of neuropathic pain.

Late-onset chronic inflammatory encephalopathy in immune-competent and severe combined immune-deficient (SCID) mice with astrocyte-targeted expression of tumor necrosis factor.

To examine the role of tumor necrosis factor (TNF)-alpha in the pathogenesis of degenerative disorders of the central nervous system (CNS), transgenic mice were developed in which expression of murine TNF-alpha was targeted to astrocytes using a glial fibrillary acidic protein (GFAP)-TNF-alpha fusion gene. In two independent GFAP-TNFalpha transgenic lines (termed GT-8 or GT-2) adult (>4 months of age) animals developed a progressive ataxia (GT-8) or total paralysis affecting the lower body (GT-2). Symptomatic mice had prominent meningoencephalitis (GT-8) or encephalomyelitis (GT-2) in which large numbers of B cells and CD4+ and CD8+ T cells accumulated at predominantly perivascular sites. The majority of these lymphocytes displayed a memory cell phenotype (CD44high, CD62Llow, CD25-) and expressed an early activation marker (CD69). Parenchymal lesions contained mostly CD45+ high, MHC class II+, and Mac-1+ cells of the macrophage microglial lineage with lower numbers of neutrophils and few CD4+ and CD8+ T cells. Cerebral expression of the cellular adhesion molecules ICAM-1, VCAM-1, and MAdCAM as well as a number of alpha- and beta-chemokines was induced or upregulated and preceded the development of inflammation, suggesting an important signaling role for these molecules in the CNS leukocyte migration. Degenerative changes in the CNS of the GFAP-TNFalpha mice paralleled the development of the inflammatory lesions and included primary and secondary demyelination and neurodegeneration. Disease exacerbation with more extensive inflammatory lesions that contained activated cells of the macrophage/microglial lineage occurred in GFAP-TNFalpha mice with severe combined immune deficiency. Thus, persistent astrocyte expression of murine TNF-alpha in the CNS induces a late-onset chronic inflammatory encephalopathy in which macrophage/microglial cells but not lymphocytes play a central role in mediating injury.

Transgenic models to study the actions of cytokines in the central nervous system.

To better understand the actions of cytokines in the mammalian central nervous system (CNS), we have developed transgenic mice in which the expression of various cytokines including interleukin (IL)-3, IL-6, IL-12, interferon-alpha or tumor necrosis factor-alpha was targeted to astrocytes under the transcriptional control of the glial fibrillary acidic protein (GFAP) promoter. Transgenic lines displaying low astrocyte expression of the respective cytokine were developed and characterized. The findings indicate that expression of these different cytokines in the intact CNS produces divergent inflammatory responses which are associated with the development of wide-ranging and progressive molecular, cellular and functional CNS impairments. These transgenic mice provide a powerful tool which we are now exploiting further to define novel mechanisms that might underlie the individual cytokine-driven neuroinflammatory responses. To date the results clearly show there are distinct model-associated patterns of cerebral expression of key molecules involved in the inflammatory response including the cellular adhesion molecules, chemokines, major histocompatibility complex molecules and the matrix metalloproteinases. In conclusion, these GFAP-cytokine transgenic mice highlight the potent and diverse array of actions mediated by cytokines when expressed in the CNS and provide a valuable resource to further our knowledge of the mechanisms by which cytokines exert their effects.

Distribution of human herpesvirus 8 DNA in tumorous and nontumorous tissue of patients with acquired immunodeficiency syndrome with and without Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) DNA is present in virtually all Kaposi's sarcomas (KSs). Conflicting results, however, exist with respect to the presence of HHV-8 in nontumorous tissue samples. To define the specificity and predictive value of HHV-8 DNA detection in KS, we analyzed autopsy-derived tissue samples from patients with acquired immunodeficiency syndrome (AIDS) with and without KS for the presence of HHV-8 DNA, using single-step and nested polymerase chain reaction. Semiquantitative analysis of HHV-8 DNA was performed by endpoint dilution assays. HHV-8 DNA was detected in 41 (100%) tumor tissue samples of KSs. According to nested polymerase chain reaction results, HHV-8 DNA was also present in 16 (32%) of 50 nontumorous specimens of patients with AIDS patients with KS and in 3 (2.7%) of 113 specimens of patients with AIDS without KS; it was absent in 26 autopsy tissues and 15 transurethral resected prostatic specimens of patients without AIDS. By use of a second, unrelated primer set, the presence of HHV-8 DNA was confirmed in 12 (63.2%) of 19 nontumorous samples and detected in another 6 (17.7%) of 34 samples tested. Significantly higher titers of HHV-8 DNA were found in tumorous than in nontumorous tissues samples (1.9 x 10(4) vs. 1.2 x 10(2); P < .05). Specificity and positive predictive values for the diagnosis of KS by detecting HHV-8 DNA in a given tissue sample were 56 and 65.1% in patients with manifest KS and 97.4 and 100% in patients without previously known KS. An increased specificity and a positive predictive value were observed when the presence of KS anywhere in a given patient was considered (92.9 and 77.8%, respectively). In conclusion, the detection of HHV-8 DNA is a sensitive test for the diagnosis of KS. Its specificity, however, might be lower because HHV-8 can be detected in histologically unaffected tissue of patients with KS.

Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states.

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of inflammatory disorders of the central nervous system (CNS) whereas the contribution of the major endogenous counter-regulators of MMPs, the tissue inhibitors of the matrix metalloproteinases (TIMPs), is unclear. We investigated the temporal and spatial expression patterns in the CNS of nine MMP genes and three TIMP genes in normal mice, in mice with EAE, and in transgenic mice with astrocyte (glial fibrillary acidic protein)-targeted expression of the cytokines interleukin-3 (macrophage/microglial demyelinating disease), interleukin-6 (neurodegenerative disease), or tumor necrosis factor-alpha (lymphocytic encephalomyelitis). In normal mice, the MMPs MT1-MMP, stromelysin 3, and gelatinase B were expressed at low levels, whereas high expression of TIMP-2 and TIMP-3 was observed predominantly in neurons and in the choroid plexus, respectively. In EAE and the transgenic mice, significant induction or up-regulation of various MMP genes was observed, the pattern of which was somewhat specific for each of the models, and there was significant induction of TIMP-1. In situ localization experiments revealed a dichotomy between MMP expression that was restricted to leukocytes and possibly microglia within inflammatory lesions and TIMP-1 expression that was observed in activated astrocytes circumscribing the lesions. These findings demonstrate specific spatial and temporal regulation in the expression of individual MMP and TIMP genes in the CNS in normal and inflammatory states. The distinct localization of TIMP-1 and MMP expression during CNS inflammation suggests a dynamic state in which the interplay between these gene products may determine both the size and resolution of the destructive inflammatory focus.

Diagnosis of pulmonary Kaposi's sarcoma by detection of human herpes virus 8 in bronchoalveolar lavage.

Human herpes virus 8 (HHV8) DNA has recently been detected in sarcoma tissue of patients with Kaposi's sarcoma. HHV8 DNA could also be found in bronchoalveolar lavage (BAL) fluid of patients with tracheobronchial Kaposi's sarcoma. To determine the specificity, sensitivity and predictive values of HHV8 DNA detection in the BAL for the diagnosis of pulmonary Kaposi's sarcoma, 100 consecutive BAL were prospectively analyzed for the presence of HHV8 DNA using a nested PCR assay. In addition, 19 BAL samples of 14 AIDS patients with cutaneous or visceral Kaposi's sarcoma were retrospectively investigated. The prospective group consisted of 79 BAL performed in immunocompromised and of 21 BAL in nonimmunocompromised patients. Four patients of the prospectively analyzed group undergoing six BAL showed tracheobronchial Kaposi's sarcoma at five bronchoscopies. All of the five BAL samples performed in these patients with endoscopically visible Kaposi's sarcoma were positive for HHV8 DNA. Following chemotherapy and antiretroviral treatment tracheobronchial Kaposi's sarcoma was no longer detectable at a subsequent bronchoscopy and HHV8 DNA in BAL became negative in one patient. One BAL sample of a HIV-positive patient with no evidence of Kaposi's sarcoma was HHV8 DNA-positive. The sensitivity, specificity, positive and negative predictive values of HHV8 detection for the diagnosis of tracheobronchial Kaposi's sarcoma were 100%, 98.9%, 83.3%, and 100%, respectively. Twelve of 19 BAL samples of the retrospective group were HHV8 DNA-positive. In this group, 10 patients undergoing a total of 14 BAL suffered from pulmonary Kaposi's sarcoma. HHV8 DNA was documented in 10 of these 14 BAL samples. In three BAL of this group HHV8 DNA was positive, but pulmonary Kaposi's sarcoma was diagnosed at a later stage. In conclusion, the detection of HHV8 DNA in BAL is restricted to patients with Kaposi's sarcoma and is highly sensitive and specific for pulmonary involvement of Kaposi's sarcoma.

Lipopolysaccharide-induced IL-12 expression in the central nervous system and cultured astrocytes and microglia.

We examined whether the cytokine IL-12 could be induced locally in the brain or in glial cell cultures following LPS treatment. In the brain, expression of IL-12 p35 mRNA was constitutive and did not alter following i.p. injection of LPS. In contrast, IL-12 p40 mRNA was only detectable in the brain of mice given two staggered injections of LPS. Dual labeling in situ analysis revealed IL-12 p40 RNA-positive cells scattered throughout the brain parenchyma, with a small number of these cells being identified as astrocytes, while the majority of IL-12 p40 RNA-expressing cells appeared to be microglia. In cultured microglia or astrocytes, LPS and to a much lesser degree IL-1beta, but not IFN-gamma or TNF-alpha, induced the expression of IL-12 p40 mRNA. Numerous glial fibrillary acidic protein-immunopositive cells colabeled for IL-12 p40 RNA; indicating that LPS-stimulated astrocytes expressed IL-12 in vitro. Immunoblot analysis of lysates from LPS-treated astrocytes revealed the presence of multiple species of 40, 43, 75, and 120 kDa containing the IL-12 p40 protein. Finally, secretion of the IL-12 p75 heterodimer was detectable by ELISA from astrocytes treated with LPS plus IFN-gamma, but not with LPS alone. The findings indicate that IL-12 gene expression can be activated in the brain, with the resident glial cells being a prodigious source of this cytokine. The localized production of IL-12 may have a significant impact on the development of cell-mediated immune responses within the central nervous system.

Delayed molecular responses to brain irradiation.

The chance of life-threatening complications occurring late after brain irradiation limits the efficacy of this form of cancer therapy. The molecular and cellular events that trigger radiation-induced brain damage are still unknown, but since they have the potential to serve as valuable targets for therapeutic intervention they are worth delineating. In this murine study, the effect of irradiation on the expression of molecules which are known to contribute to brain damage in other model systems was examined. Expression of genes encoding cytokines (TNF-alpha/beta, IL-1 alpha/beta, IL-2, IL-3, IL-4, IL-5, IL-6 and IFN-gamma), cytokine receptors (TNF-Rp55 and p75, IL-1R- p60 and p80, IFN-gamma R, and IL-6R), the cell adhesion molecule (ICAM-1), inducible nitric oxide synthetase (iNOS), anti-chymotrypsin (EB22/5.3), and the gliotic marker (GFAP) was evaluated over a 6-month period using a sensitive RNase protection assay (RPA). We had previously demonstrated that within 24 h of brain irradiation there is an acute transitory molecular response involving TNF-alpha, IL-1, ICAM-1, EB22/5.3 and GFAP. This study shows re-elevation of TNF-alpha, EB22/5.3 and GFAP mRNA levels at 2-3 months, but only TNF-alpha mRNA was overexpressed at 6 months. These time points are when neurological abnormalities are seen after higher doses. The data suggest that TNF-alpha may be involved in late brain responses to irradiation and could contribute to clinical symptoms.

[Detection of human herpesvirus 8 (HHV 8) in Kaposi sarcomas of the gastrointestinal tract]. / Nachweis von humanem Herpesvirus 8 (HHV 8) in Kaposi-Sarkomen des Magen-Darm-Traktes.

Involvement of Kaposi's sarcoma in the gastrointestinal tract is common in AIDS patients. The disease is, however, usually asymptomatic and, due to the tumor growth primarily in the submucosa, biopsy diagnosis is possible in under 25%. The recently described human herpes virus 8 (HHV8) is closely associated with all forms of Kaposi's sarcoma. Detection of HHV8 in the tissue samples may therefore improve the diagnosis of gastrointestinal Kaposi's sarcoma. In the present study we analyze autopsy samples of tumor and non-tumor tissue from the gastrointestinal tract in patients with and without Kaposi's sarcoma for the presence of HHV8 DNA using a nested polymerase chain reaction (PCR) assay. HHV8 DNA was present in all 15 tissues with Kaposi's sarcoma. In contrast, HHV8 DNA was present only in 3 (18.8%) of 16 gastrointestinal tissues of patients with Kaposi's sarcoma but without histologically detectable tumor. No HHV8 DNA was present in 15 tissue samples of AIDS patients without Kaposi's sarcoma. Our data show that detection of HHV8 DNA using a nested PCR assay is a highly sensitive and specific diagnostic test for Kaposi's sarcoma in autopsy tissue samples from the gastrointestinal tract. It should therefore be possible to use detection of HHV8 DNA in biopsy material as an assay for the diagnosis of Kaposi's sarcoma.

Distribution of tumor necrosis factor receptor messenger RNA in normal and herpes simplex virus infected trigeminal ganglia in the mouse.

PURPOSE: to investigate the distribution of p55 and p75 tumor necrosis factor (TNF) receptor mRNA in normal murine trigeminal ganglia, and in murine trigeminal ganglia acutely infected with McKrae strain herpes simplex virus (HSV). METHODS: in situ hybridization with antisense 35S-labeled riboprobes for mRNA encoding both the p55 and p75 TNF receptor (TNFR) subtypes was used in normal and HSV-infected murine trigeminal ganglia. Sense riboprobes were used as controls. RESULTS: in situ hybridization with both p55 and p75 riboprobes produced a strong autoradiographic signal over many, but not all, trigeminal sensory neurons. Signal for mRNA encoding both TNFR subtypes was also present over the arachnoid layers surrounding trigeminal ganglia. Acute ocular HSV infection was accompanied by an intense leukocytic infiltrate into the ophthalmic portion of the trigeminal ganglia, and, in this setting, increased p55 and p75 mRNA signal was closely related to the location and number of infiltrating white blood cells. The distribution and number of trigeminal sensory neurons expressing mRNA for the two TNFR subtypes did not appear to change following infection. Signal over control sections hybridized with sense p55 and p75 TNFR cRNA probes was comparable to background. CONCLUSIONS: the observed distribution of p55 and p75 TNFR mRNA over trigeminal sensory neurons and over the arachnoid layers surrounding trigeminal ganglia supports suggestions that TNF has a direct effect on neurons, either as a neuromodulator or neurotrophic factor, and that TNF may play a central role in blood-brain barrier regulation. Increased signal for TNFR mRNA in acutely infected trigeminal ganglia appears to reflect infiltration by receptor-bearing white blood cells.

Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin.

The signalling mechanisms that regulate epidermal permeability barrier homeostasis are not known. Previous Northern blot analysis showed that both acute and chronic barrier disruption increase mRNA levels of several cytokines in murine epidermis. To further characterize the epidermal response to barrier abrogation, we used more sensitive, multi-probe RNase protection assays to measure the mRNA levels of additional cytokines, as well as cytokine receptors in acute and chronic models of barrier disruption. Normal mouse epidermis expressed interleukin (IL)-1 alpha, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and IL-6 mRNAs. Following tape-stripping, only the mRNA levels for TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 increased at 2.5 and 7 h, and returned toward normal levels by 18 h. No mRNAs encoding TNF-beta, IL-2, IL-3, IL-4 or IL-5, were detected in the epidermis either under basal conditions or after tape-stripping. Similarly, in a chronic model, essential fatty acid deficiency, epidermal levels of TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 mRNAs, but not IFN-gamma mRNA, were elevated over controls; and again, mRNAs for the remaining probed cytokines were not detected. In contrast, in the dermis, only IL-1 beta mRNA levels increased 2.5 h after tape-stripping, and remained elevated at 18 h. mRNAs encoding the IL-1 (p60), IFN-gamma and IL-6 receptors were present in epidermis, but their levels remained unchanged following either acute or chronic barrier disruption. In contrast, epidermal TNF (p55) receptor mRNA levels were increased by 87% (P < 0.01) at 2.5 h, returned to control levels at 7 h and were increased by 68% (P < 0.03) at 18 h after tape-stripping. The increase at 2 h was confirmed by Northern blot analysis and was not prevented by latex occlusion performed immediately after tape-stripping mRNAs for the IL-1 (p80) receptor and TNF (p75) receptor were not detected in epidermis. Low levels of TNF (p55) receptor mRNA were present in the dermis, and they remained unchanged after tape-stripping. The presence of specific receptor mRNAs in the epidermis and dermis suggests that these tissues are capable of responding in an autocrine and/or paracrine fashion to the cognate cytokines. These results suggest that epidermal cytokines produced after barrier disruption may initiate a cytokine cascade which could regulate cytokine and cytokine receptor production and/or inflammatory responses.

Transgenic models to assess the pathogenic actions of cytokines in the central nervous system.

In order to better understand the actions of proinflammatory cytokines in the mammalian CNS, a transgenic approach was employed in which the expression of IL-6, IL-3 or TNF-alpha was targeted to astrocytes in the intact CNS of mice. Transgenic mice exhibited distinct chronic-progressive neurological disorders with neurodegeneration and cognitive decline due to IL-6 expression, macrophage/microglial-mediated primary demyelination with motor impairment due to IL-3 expression and lymphocytic meningoencephalomyelitis with paralysis induced by TNF-alpha expression. Thus, expression of specific cytokines alone in the intact CNS results in unique neuropathological alterations and functional impairments, thereby directly implicating these mediators in the pathogenesis of CNS disease.

Localization of tumor necrosis factor receptor messenger RNA in normal and herpes simplex virus-infected mouse eyes.

PURPOSE: To investigate the distribution of p75 and p55 tumor necrosis factor receptor (TNFR) mRNA in normal mouse eyes and in mouse eyes acutely infected with McKrae strain herpes simplex virus (HSV). METHODS: In situ hybridization with antisense 35S-labeled riboprobes for p55 and p75 TNFR subtypes was used in uninfected and HSV-infected mouse eyes. Controls included the use of sense riboprobes and corneas inoculated with vehicle alone. RESULTS: In uninfected and infected mouse eyes, in situ hybridization produced an autoradiographic signal for mRNA, encoding both p75 and p55 over the corneal endothelium, iris, ciliary body, choroid, and arachnoid layers of the optic nerve sheath. In addition, the signal was observed over scattered cells at the vitreoretinal interface. Signal for p75, but not p55, was observed over cells in the retinal ganglion cell layer. Acute HSV infection was accompanied by an intense leukocytic infiltrate in the conjunctiva, the corneal subepithelium and stroma, the anterior and posterior chambers, the iris root and ciliary body, and the vitreous cavity. In this setting, increased p75 and p55 mRNA signal was correlated closely with the number and location of receptor-bearing white blood cells. Signal over control sections hybridized with sense p75 and p55 TNFR cRNA probes was comparable to background. Signal over control eyes inoculated with sterile vehicle showed slight increased signal in the immediate vicinity of the traumatic keratitis, but otherwise it was comparable to that observed in uninfected animals. CONCLUSIONS: The observed distribution of p75 and p55 TNFR mRNA in normal and acutely infected mouse eyes, and particularly over the heavily vascularized uveal tract and over cells at the vitreoretinal interface, supports a role for TNF as a mediator of intraocular inflammation, perhaps as a key regulator of the blood-ocular barrier.

Altered tissue distribution of viral replication and T cell spreading is pivotal in the protection against fatal lymphocytic choriomeningitis in mice after neutralization of IFN-alpha/beta.

IFN-alpha/beta have been shown to play a central role in the development of lymphocytic choriomeningitis and increasing attention has been focused on this group of cytokines as early regulatory factors directing T lymphocyte responses. In the present study, injection of antiserum to IFN-alpha/beta prevented the development of lymphocytic choriomeningitis, was associated with the absence of detectable expression of early 2'-5' oligo-adenylate synthetase mRNA and coincided with viremia of lymphocytic choriomeningitis virus (LCMV) followed by establishment of a persistent infection. The LCMV-specific cytotoxic T lymphocyte response was unchanged in cervical lymph nodes but decreased in the spleen of anti-IFN-alpha/beta-treated animals. The expression of cytokine mRNA (particularly IFN-gamma) in organs of LCMV-infected mice treated with anti-IFN-alpha/beta coincided with infiltration of lymphocytes and tissue destruction. Furthermore, a reduced number of infiltrating leukocytes in the brain and cervical lymph nodes and a low expression of cytokine mRNA in the brain was observed in anti-IFN-alpha/beta-treated animals. In total, the findings support the view that neutralization of IFN-alpha/beta leads to extensive LCMV replication in the viscera. The therapeutic effects of anti-IFN-alpha/beta antiserum seem to be independent of the functional capacity of T cells but probably result in a dispersion of activated T cells throughout the body of LCMV-infected mice. Absence of IFN-alpha/beta expression in the central nervous system is proposed as the mechanism behind the IFN-alpha/beta-dependent targeting of T cells to the brain.

Simultaneous analysis of multiple cytokine receptor mRNAs by RNase protection assay in LPS-induced endotoxemia.

In order to examine the regulation of cytokine receptor gene expression an RNase protection assay (RPA) was developed that allows the simultaneous and semiquantitative measurement of mRNAs encoding for the IL-1 p60 and p80, TNF p55 and p75, IFN-gamma, and IL-6 receptors. Titration experiments revealed that this method was very sensitive allowing the detection of the target cytokine receptor mRNAs down to at least 0.01 microgram of spleen poly(A)+ RNA. The cytokine receptor RPA was used to examine the expression of the receptor genes in various organs from normal mice and mice that had been injected with LPS. In normal mice expression of the IL-1R p80, TNFR p55 and p75, IFN-gamma, and IL-6R but not the IL-1R p60 transcripts was readily detectable in spleen, liver, kidney, and brain. Following LPS treatment, there was an induction of the IL-1R p60 mRNA in all organs and an up-regulation of the IL-1R p80, TNFR p55 and p75, IFN-gamma, and IL-6 receptor mRNAs particularly in spleen, liver, and kidney. Interorgan differences were observed in the regulation of these receptor mRNAs, indicating an organ-specific response to the LPS challenge. Our findings indicate the cytokine receptor RPA is a powerful and versatile tool for the simultaneous analysis of multiple cytokine receptor mRNAs in tissue samples. This technique will prove valuable in further evaluating the coordinated regulation of the expression of these genes, which are pivotal in the biology of cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactive gliosis as a consequence of interleukin-6 expression in the brain: studies in transgenic mice.

Gliosis is a characteristic pathologic state in many CNS disorders. Cytokines are considered to be effectors of gliosis. In order to explore the role of IL-6 in gliosis, the temporal and spatial expression of the IL-6 gene and its consequent effects on the brain were studied in a GFAP-IL6 transgenic mouse model. In GFAP-IL6 mice, IL-6 transgene expression was detectable in the brain at 1 week postnatally and increased to maximal levels by 3 months of age before declining at 8 and 12 months. Enhanced glial fibrillary acidic protein (GFAP) (marker for astrocytes) and Mac-I (marker for microglia) mRNA expression were first prominent at 1 month, increased to maximum levels by 3 months and remained significantly elevated through 12 months of age. Western blot analysis revealed that the enhanced GFAP mRNA expression in these transgenic mice was accompanied by increased GFAP protein levels. Immunostaining for Mac-I demonstrated that in addition to an increased staining intensity, the number of cells expressing the microglial/macrophage marker was also apparently increased, particularly in the cerebellum and brain stem. Concurrent with IL-6 transgene mRNA expression and reactive gliosis, upregulation of IL-1 alpha/beta, TNF alpha, ICAM-1 and EB22/5.3 (acute-phase reactant) but not inducible nitric oxide synthase gene expression was also observed. EB22/5.3 mRNA expression was most prominent and increased progressively with age. Expression of the IL-6, GFAP and EB22/5.3 RNAs was found to have similar distribution in the brain being found predominantly in the cerebellum, brain stem and sub-cortical regions. In conclusion, the constitutive expression of IL-6 in the brain induced the development of a pronounced and lifelong reactive gliosis affecting both astrocytes and microglia. The altered state of these cells may contribute to the functional and structural CNS impairment exhibited by the GFAP-IL6 mice. Finally, in these mice, expression of the EB22/5.3 gene correlated closely with the progression of neuropathy indicating that this acute-phase response gene was a good marker for and may be involved in the pathogenesis of CNS injury mediated by the expression of IL-6.