Extracellular matrix-associated cytokines regulate CD4+ effector T-cell responses in the human intestinal mucosa.

Extracellular matrix (stroma) regulation of mucosal T-cell function is incompletely understood. In this study, we uncovered a role for intestinal stromal products in the innate regulation of effector T cells. Stroma-conditioned media (S-CM) derived from the normal human intestinal stroma (transforming growth factor-ß (TGF-ß)(hi)/interleukin (IL)-6(lo)/IL-1ß(lo)) significantly downregulated T-cell proliferation and interferon-γ (IFN-γ) production compared with S-CM derived from the inflamed Crohn's mucosa (TGF-ß(hi)/IL-6(hi)/IL-1ß(hi)). Antibody neutralization studies showed that TGF-ß in normal S-CM inhibited T-cell proliferation and IFN-γ production, whereas IL-6 plus IL-1ß in Crohn's S-CM promoted T-cell proliferation, and IL-1ß alone promoted IFN-γ and IL-17 release. Importantly, normal S-CM inhibited T-bet expression, whereas Crohn's S-CM activated signal transducer and activator of transcription 3, suggesting that discordant T-cell responses are regulated at the transcription factor and signaling levels. These findings implicate stromal TGF-ß in the downregulation of T-cell 2 responses in the normal intestinal mucosa, and stromal IL-6 and IL-1ß in the promotion of Th1 and Th17 responses in the inflamed Crohn's mucosa, suggesting an innate regulatory function for the intestinal extracellular matrix.

Olanzapine activates the rat locus coeruleus: in vivo electrophysiology and c-Fos immunoreactivity.

BACKGROUND: Activation of central noradrenergic pathways by atypical antipsychotics has been hypothesized to play a role in their efficacy in treating the negative symptoms and cognitive impairment of schizophrenia. Because acute administration of the atypical antipsychotic olanzapine has been shown to increase extracellular levels of norepinephrine in the medial prefrontal cortex, we examined the effects of olanzapine on the noradrenergic cells of the locus coeruleus (LC). METHODS: The effects of olanzapine (0.25-16 mg kg(-1), IV) on the firing rates and patterns of LC neurons were determined by extracellular, single-unit recordings in chloral hydrate-anaesthetized rats. The effects of olanzapine and clozapine on c-Fos expression in the LC, nucleus subcoeruleus part alpha (SubCA), and nucleus A5 (A5) were studied by immunohistochemistry. RESULTS: Olanzapine increased LC cell firing rates, de-regularized firing, and induced burst firing. Induction of c-Fos expression in the LC by olanzapine and clozapine was confirmed and was also found in the SubCA, but not in A5. CONCLUSIONS: Acute administration of olanzapine activates the noradrenergic neurons of the rat LC. This increased activity of LC neurons may play an important role in the efficacy of olanzapine and clozapine in treating both the negative symptoms and cognitive impairment observed in schizophrenic patients.

Potentiation of responses to AMPA on central neurones by LY392098 and LY404187 in vivo.

Enhancement of AMPA receptor mediated synaptic excitation has the potential to aid in the treatment of several psychiatric conditions. To test such claims there is a need to develop more potent compounds than those presently available and to demonstrate that they cross the blood-brain barrier to affect responses at central AMPA receptors. We have now completed in vivo tests with two such compounds, the newly discovered biarylpropylsulfonamides, LY392098 and LY404187, on spinal and hippocampal neurones in anaesthetised rats. In the initial study on spinal neurones, LY392098 (30-1000 microg/kg i.v.) dose-dependently increased responses to iontophoretically administered AMPA but not those to NMDA. Subsequently in a more detailed follow-up study on hippocampal neurones, LY392098 (1-100 microg/kg i.v.) and LY404187 (1-100 microg/kg i.v.) enhanced in a dose-dependent manner responses to AMPA. Responses to NMDA were also enhanced but to a less extent. Such enhanced responses to NMDA, but not those to AMPA, were reduced by the NMDA antagonist, ketamine (0.5-1 mg/kg i.v.) whereas the selective AMPA antagonist, LY300168 (GYKI53655; 1 mg/kg i.v.), reduced responses to both NMDA and AMPA. LY392098 also potentiated the synaptic excitation of dentate granule cells following perforant path stimulation. These combined data show that, at doses not dissimilar to those affecting behavioural responses (1-1000 microg/kg; see accompanying papers), the two new drugs cross the blood-brain barrier to affect directly the sensitivity of central AMPA receptors and enhance synaptic excitation in vivo.

Mosaic tetrasomy 8q: inverted duplication of 8q23.3qter in an analphoid marker.

We observed an analphoid marker chromosome stable through cell division in a 16-year-old girl with developmental delay, short stature, limb contractures, and ovaries containing multiple cysts. She also developed myasthenia gravis at 15 years. The marker chromosome, present in 75% of metaphases (and in 90% of transformed lymphoblastoid cells), was C-band negative, and had no pan alpha-satellite sequences detectable by fluorescence in situ hybridization (FISH). The 8q origin of the marker was determined by use of subtelomeric probes and was confirmed by chromosome 8 painting probes. The marker was shown to be an inversion duplication of 8q when subtelomeric, telomeric, and c-myc FISH probes hybridized to both ends of the marker. The karyotype was 47,XX,+inv dup(8)(qter--> q23.3::q23.3-->[neocen]-->qter), resulting in tetrasomy for 8q23.3qter. The parents had normal karyotypes. Centromeric proteins CENP-C and CENP-E were present, but alpha associated centromere protein CENP-B was absent at a position defining a neocentromere.

Commercial recombinant human beta-nerve growth factor and adult rat dorsal root ganglia contain an identical molecular species of nerve growth factor prohormone.

Examination of commercial recombinant human beta-nerve growth factor (rh-beta-NGF) preparations with polyclonal antibodies specific to 13-kDa NGF and pro-NGF-specific domains revealed the presence of high-molecular-mass immunoreactive proteins, including a 60-kDa NGF prohormone. On incubation with a mixture of N- and O-specific glycosidases, the 60-kDa NGF pro-hormone generated a 32-kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF- and pro-NGF domain-specific antibodies also revealed the presence of high-molecular-mass proteins, including the 60-kDa NGF prohormone. Based on the presence of the 60-kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate.

Sudden unexpected death associated with HHV-6 in an adolescent with tuberous sclerosis.

A 14-year-old female with tuberous sclerosis and history of seizures was found dead in bed at home 3 days after she had been assessed as doing well at a routine neurology clinic appointment. She had been treated with an antiepileptic drug, felbamate, for 36 months and had been seizure-free except for one seizure episode 5 months before death. Postmortem examination revealed cerebral edema, with uncal and tonsillar herniation, and pulmonary edema, consistent with seizure-induced apnea. Multiple microglial nodules with mature perivascular lymphocytic cuffing and diffuse infiltrates were identified around subependymal tuberous sclerosis giant cell nodules. Immunostaining and electron microscopy revealed human herpesvirus-6-infected macrophages, astrocytes, lymphocytes, and endothelial cells in the subependymal tuberous sclerosis lesions and choroid plexus. Subacute human herpesvirus-6 encephalitis is postulated to have precipitated a seizure and thus sudden unexpected death in epilepsy in this otherwise stable adolescent patient.

When are students most at risk of encountering academic difficulty? A study of the 1992 matriculants to U.S. medical schools.

The authors carried out the study reported here to assess which variables are most predictive of the risk of medical students' experiencing academic difficulties and to assess when these students are most susceptible to encountering those difficulties. The entering class of 1992 was chosen as the study population because it was the first matriculating class in which the majority of students (88%) applied to medical school with scores from the revised Medical College Admission Test (MCAT), first implemented in 1991. The primary event of interest in this study was the first occurrence of one of the following events because of academic difficulty: withdrawal, leave of absence, dismissal, or delay of graduation date. The variables examined were MCAT scores undergraduate science GPA, undergraduate institutional selectivity, undergraduate major, racial-ethnic background, sex, and age upon entering medical school. Survival analysis was used to assess which variables were most predictive of the risk of academic difficulty and when students with different characteristics were most at risk. The results of the survival analysis indicated that (1) while the risk and timing of academic difficulty varied across the groups studied, a majority of the students who experienced academic difficulty eventually graduated from medical school and (2) students with non-science undergraduate majors did not have a greater risk of academic difficulty. The results confirm previous findings that increased risk of academic difficulty is associated with low MCAT scores, low science GPA, low undergraduate institutional selectivity, being a woman, being a member of a racial-ethnic underrepresented minority, or being older. The study findings can be generalized to help in early identification of students who are more likely to be at risk of experiencing academic difficulty. Knowing when these students are more likely to be at risk can help medical schools develop targeted remedial and enrichment programs. Further studies are needed to investigate school-related factors associated with risk.

Physiologic MRI of a tumefactive multiple sclerosis lesion.

Structural and physiologic MRI were performed after subacute onset of left hemiparesis in a patient with MS. MRI showed a large ring-enhancing lesion with surrounding edema and mass effect; differential diagnosis included a neoplasm or a large MS plaque. Physiologic MRI showed reduced blood flow and magnetization transfer, as well as increased diffusion, in the large lesion. Because these findings suggested a tumefactive MS plaque rather than a neoplasm, the patient received steroid treatment for acute MS exacerbation. Three months later the patient improved clinically and on MRI.

Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

Evaluation of a cassette-screen-film combination for radiation therapy portal localization imaging with improved contrast.

A traditional limitation with radiation therapy portal images is low image contrast, due in part to the low attenuation of the exposing radiation by the tissues being imaged, and the contrast capabilities of the image receptor. We have developed, and have clinically evaluated, a cassette-screen-film combination for portal localization imaging, which features a copper front screen plus Gd2O2S:Tb fluorescent screens and a slow-speed, fine grain, film emulsion with inherently high contrast coated on both sides of a 7 mil Estar base. The film can be processed in a conventional rapid-process film processor. Sensitometric data indicate that the film contrast (average gradient) for the new combination is approximately 3.5 times higher than the conventional portal localization systems in current use. The new combination has been clinically compared with two conventional systems. The required monitor unit settings were found to be similar. Initial clinical results indicate portal images made with the new combination are superior to those obtained with the conventional combinations. The images have much higher contrast, subjective impressions of lower noise, show clearer definition of structures, and are much easier to read.

Brain infarction in children with diabetic ketoacidosis.

During the last 10 years, five children were treated at Childrens Hospital Los Angeles for acute, persistent neurologic loss during diabetic ketoacidosis (DKA). Four were transferred from local hospitals after the neurologic crisis. Computed tomography (CT) studies showed one or more areas of brain infarction in each patient, and none had evidence of diffuse cerebral edema. As three of the five patients had been treated for cerebral edema before their CT, brain edema may have been present initially. Our findings emphasize the importance of brain infarction as a cause of persistent neurologic loss in children with DKA.

Prolonged response to carboplatin in an infant with brain stem glioma.

Adults and children with brain stem gliomas have a mean survival time of 15 months after radiation therapy (XRT). Infants with this tumor present additional complexities for treatment because of possible neurotoxicity of the radiation to the developing brain. We report a 15-month-old child with biopsy-proven brain stem glioma with clinical and radiographic evidence of disease progression. She was treated with 24 monthly courses of carboplatin without radiation therapy and has had a 39+ month response. The clinical response started after 3 months and the radiographic evidence was documented at 10 months by magnetic resonance imaging. The toxicity was minimal. Longitudinal neuropsychological assessment demonstrated continued improvement at 36 months post diagnosis but with some motor functioning below expected age levels. Cervico-medullary astrocytoma in a young patient may be the appropriate clinical setting for future trials of chemotherapy without XRT.

Progesterone receptors in advanced breast cancer.

Progesterone receptor (PR) assay was performed on tumor specimen obtained from 145 women consisting of 82 women with advanced breast cancer and 73 women with early breast cancer (EBC). Advanced breast cancer patients consisted of 50 (61%) of 82 women with advanced primary breast cancer (APBC) and 32 (39%) women with recurrent or secondary breast cancer (SBC). Of 82 advanced breast cancer, 35 (42.7%) were PR+. These consisted of 21 APBC and 14 SBC patients. 43 (59%) of 73 EBC patients had PR+ tumors. Quantitative value of PR correlated with age in overall and in all groups. There was also a significant difference in PR quantitative value between PR+ tumors and PR- tumors. Finally premenopausal APBC patients with PR+ tumors had a 2:1 survival advantage over those with PR- tumors. PR assay is as an invaluable a tool as ER assay in the management of breast cancer.

Cytostatic effects of 2',3'-dideoxyribonucleosides on transformed human hemopoietic cell lines.

The 2',3'-dideoxy analogues of cytidine, guanosine, adenosine, inosine, and thymidine have been compared for their cytostatic effects on 14 cell lines that include B lymphoblastic, T lymphoblastic, and myeloblastic lines. In all cases 2',3'-dideoxycytidine (ddCyd) was the most toxic nucleoside, with dideoxyguanosine (ddGuo) next, and little cytostatic action by the analogues of thymidine, adenosine, or inosine. The cytostatic action of ddCyd was examined in more detail. The concentration for 50% inhibition of growth over 4 days (IC50) was 0.2 to 3 microM for five T lymphoblastic lines. Although most B lymphoblastic and myeloblastic lines were less sensitive (IC50, 16-70 microM), some were as sensitive as the T lymphoblastic lines. The four lines most sensitive to ddGuo (three T lymphoblastic and one B lymphoblastic) had IC50 values of 47-80 microM. Two lines with high sensitivity to ddCyd had levels of ddCyd triphosphate about 4-7 times higher than those found in the two least sensitive after 1-, 3-, or 24-hr exposure to 3 microM [3H]ddCyd. This was associated with a much higher ratio of the diphosphate to the triphosphate in the least sensitive cells, an observation suggesting slow nucleoside diphosphate kinase action on the diphosphate of ddCyd in the resistant cells. Catabolism of the mono-, di-, and triphosphate was very slow in all cell lines, and in lines of low sensitivity disappearance of the triphosphate had a half life (approximately 19 hr) about twice as long as in sensitive lines. This may be due to continuing slow conversion of diphosphate to triphosphate after removal of nucleoside from the medium.

Proliferation-related responses in rat astrocytes to epidermal growth factor.

The signals which regulate the proliferation of astrocytes have relevance to both normal developmental processes and abnormal states of gliosis or glial tumor formation. We have extended studies of astrocyte proliferation and related responses in primary cultures of rat telencephalic cortical astrocytes as a result of treatment with epidermal growth factor. Epidermal growth factor stimulates the rate of DNA synthesis five fold and maintains the rate of protein synthesis. The stimulation occurs at a dose of 2 ng/ml and is greater in higher density cultures than in lower density cultures, perhaps representing a relative starvation for the growth factor. The astrocyte response is still present even after being cultured 3 1/2 weeks in serum-free and non-growth factor or hormone-supplemented media. Combined immunofluorescence and thymidine autoradiography disclose that glial fibrillary acidic protein containing cells are the cells synthesizing DNA in response to the growth factor, and combined rhodamine and fluorescein-linked stains disclose that epidermal growth factor is in the glial fibrillary acidic protein containing cells. Proliferation-related 2-deoxyglucose uptake is stimulated at approximately the same dose as DNA synthesis is stimulated, but the time course is relatively slow, maximizing at 48 hr. Ornithine decarboxylase is stimulated in 6 hr indicating more rapid nuclear stimulation by the signal. In conclusion, epidermal growth factor has a clear direct interaction with glial fibrillary acidic protein-containing cells which is greater in higher density cultures, is still present in long-quiescent cells, and includes DNA synthesis, cell cycle progression, hexose uptake, and polyamine synthesis.

Fibroblast growth factor inhibits epidermal growth factor-induced responses in rat astrocytes.

The signals which regulate the proliferation of astrocytes have relevance to normal developmental processes, transformational loss of growth control, and reactive gliosis present in many brain disease states. We have studied, in primary cultures of rat astrocytes, a sequential interaction of two growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF), which may be relevant to the brain in these conditions. EGF is a strong mitogen and stimulator of 2 deoxyglucose (2 DG) transport with no effect on plating of cells, and FGF is a lesser mitogen and 2 DG uptake stimulator. However, when FGF is given to the cells as a pretreatment, FGF strongly inhibits the ability of EGF to stimulate both DNA synthesis and 2 DG uptake. The inhibition of EGF stimulation by FGF is across the EGF dose-response curve, present at high and low culture densities, and stable for at least 3 days. Specificity is indicated by lack of inhibition by PDGF pretreatment and much less inhibition of fetal calf serum-induced stimulations than EGF-induced stimulation. Cell counts confirmed that the FGF pretreatment also inhibits EGF stimulation of cell division. Because of FGF brain derivation and angiogenic and neurotropic functions, it may serve as a regulator of EGF-astrocyte interactions in processes such as development, gliomatous transformation, and neural regeneration.

Chronic confusional state.

An acute confusional state after infarction in the distribution of the right middle cerebral artery has been described. Patient recovery usually is excellent. Some patients, however, do not improve, resulting in a chronic confusional state.

Fibroblast growth factor pretreatment reduces epidermal growth factor-induced proliferation in rat astrocytes.

Epidermal Growth Factor (EGF) is mitogenic for purified rat astrocytes in primary tissue culture. A combined concomitant treatment by EGF and Fibroblast Growth Factor (FGF) does not reduce the proliferation effect of EGF, however when the astrocytes are pretreated with FGF, their response to an EGF stimulation is reduced by 70%. This inhibition of EGF stimulation by FGF pretreatment is consistent across the EGF dose response curve and perhaps represents a mechanism for local modulation of astrocyte mitogenic effects.

The insulin-like growth factor binding protein BP-25 is expressed by human breast cancer cells.

Specific binding proteins are thought to modulate the effects of IGF-I. Previous work has demonstrated that media conditioned by human breast cancer cells contains IGF-I binding activity. Radiolabelled IGF-I incubated with serum-free conditioned media from the breast cancer cell line MDA-MB 231 eluted with an apparent M.W. of 35-40 kDa when analyzed by gel filtration chromatography at pH 7.4. The M.W. of this binding activity corresponded to that of BP-25, a binding protein cloned from the hepatocellular carcinoma cell line HepG2. Two breast cancer cell lines, MDA-MB 231 and Hs578T, were found to express BP-25 RNA. Specific BP-25 radioimmunoassay detected BP-25 production in the conditioned media of these two cell lines. Immunoprecipitation confirmed that metabolically labelled MDA-MB 231 released 30 kDa BP-25 into its medium. This study demonstrates that some breast cancer cells express the IGF-I binding protein, BP-25.