SPECT changes and their correlation with EEG changes in tuberculous meningitis

OBJECTIVE: In tuberculous meningitis (TBM) blood flow may be altered due to associated vasculitis, hydrocephalus and raised intracranial pressure. Electroencephalography (EEG) and single photon emission computed tomography (SPECT) provide information about electrical activity and regional cerebral blood flow respectively. This study aims at the correlation of EEG and SPECT changes in patients with TBM. METHOD: Sixteen patients with TBM whose age ranged between 5 and 62 years and 3 of whom were females were subjected to clinical, radiological (CT and/or MRI), EEG and SPECT studies using 99mTc ethylene cystine dimer (ECD). Ten patients were in stage III and 3 each in stage II and stage I meningitis. Cranial CT scan was carried out in 15 and MRI in 4 patients. Hydrocephalus was present in 9, infarction in 7 and tuberculoma in 5 patients. RESULTS: SPECT studies were abnormal in all except 2 patients revealing basal ganglionic hypoperfusion in 14 and focal cortical hypoperfusion in 9 patients. The EEG was abnormal in 11 patients which included delta slowing in 5, theta slowing in 6, frontal intermittent rhythmic delta activity (FIRDA) in 3 and epileptiform discharges in 2 patients. All the patients with abnormal EEG had abnormal SPECT study except 1. In 4 patients, EEG was normal although there was subcortical hypoperfusion on SPECT. In spite of high frequency of focal cortical hypoperfusion (9 patients), EEG revealed focal abnormality in 3 patients only. CONCLUSION: It can be concluded that the SPECT reveals more frequent abnormalities compared to EEG and CT scan. Cortical hypoperfusion with or without basal ganglia hypoperfusion is associated with FIRDA and diffuse delta slowing on EEG.     

Raft.1, a monoclonal antibody raised against the raft microdomain,recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line

SUMMARY: Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.     

Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo

Heat shock protein 60 (HSP60) from Chlamydia pneumoniae was described to trigger in vitro inflammatory and cytokine responses including TNF and IL-12p40. Although it can be found in atherosclerotic plaques of patients, the stimulatory potential of chlamydial and other HSP60 in vivo is unclear. We now report that chlamydial HSP60 fails to induce TNF expression in vivo, and significant serum levels of IL-12p40 are only found upon intraperitoneal injection of high doses of HSP60 or after intravenous application. Upon purification of chlamydial HSP60 with polymyxin B-agarose columns, its ability to induce TNF secretion in vitro is much reduced. However, purified chlamydial HSP60 causes increased serum levels of the CXC chemokines KC and MIP2 in vivo, as well as a strong accumulation of polymorphonuclear neutrophils (PMN) in the peritoneal cavity upon intraperitoneal challenge. With respect to PMN accumulation, chlamydial HSP60 is more potent than endotoxin or the CpG oligonucleotide 1668. The responses observed are completely abolished in Toll-like receptor (TLR)2/4-double-deficient mice, while single-deficient mice respond almost normally. Furthermore, KC induction and PMN accumulation are largely dependent on MyD88. In conclusion, HSP60 from C. pneumoniae triggers inflammatory responses in vivo that differ from responses induced by endotoxin or CpG oligonucleotides and are dependent on TLR2 and 4.     

Induction of antibody synthesis: Effect of blocking defined determinants of an antigen

Rabbit antibody specific for bovine γ-globulin (BGG) and for the dinitrophenyl group (DNP) was used to block defined determinants of BGG—DNP, and the immunogenicity of antibody-complexed BGG—DNP in rabbits was compared with that of BGG—DNP. Antibody-complexed BGG—DNP was less immunogenic, eliciting less antibody reactive with BGG—DNP. However, BGG-specific antibody, in contrast to DNP-specific antibody, suppressed primarily the induction of antibody specific for the BGG determinants of BGG—DNP. This finding suggests: (1) that induction of antibody specific for a given determinant of an antigen requires the active participation of that determinant as an inducer, and (2) that the immunosuppressive action of antibody is determinant-specific and not antigen-specific. The immunogenicity of antibody-complexed sheep erythrocytes was also studied in mice. Rabbit antibody to sheep erythrocytes, in different amounts, was used to block determinants on the surface of sheep erythrocytes. The agglutinin response was inversely proportional to the amount of antibody used to complex the erythrocytes. This finding supports the role of determinants as inducers of antibody formation and indicates that the immunogenicity of an antigen is a function of the number of determinants it carries in an accessible reactive state.     

Rapid odor conditioning in newborn rabbits: amnesic effect of hypothermia.

Rabbit pups are completely dependent on a maternal pheromone for the release of suckling behavior. However, if the mother is perfumed prior to nursing, pups will learn to respond to the novel odor with the characteristic nipple-search behavior in just one 3-4 min nursing episode. In a first investigation of the processes underlying this recently developed learning paradigm, time-dependent effects of hypothermia on retention of the task could be demonstrated. Thus pups whose whole body was cooled to a mouth temperature of 7 degrees C immediately after conditioning (n = 10) and tested 24 h later for 3 min on a perfumed fur did not differ significantly in their search response from naive, untreated controls (n = 10). In contrast, pups cooled 4 h after conditioning (n = 10) demonstrated clear retention of the learned response and searched as vigorously as conditioned but uncooled animals (n = 10). As pups of all groups demonstrated normal nipple-search behavior when tested on a lactating doe, the deficits associated with immediate cooling appear to have been specific to the learning task and time of treatment.     

Inhibitory role of dentate hilus neurons in guinea pig hippocampal slice

1. Current and voltage-clamp recording of CA3/CA4 pyramidal neurons, hilar neurons, and granule cells or pairs of these neurons were used to study the generation of Cl-dependent and K-dependent inhibitory postsynaptic potentials (IPSPs) in the guinea pig hippocampal slice preparation. 2. A sequence of an early Cl-dependent and a late K-dependent IPSP was evoked in CA3 neurons by electrical stimulation from the stratum moleculare of the dentate gyrus, the hilus, and the stratum oriens/alveus. Blockade of glutamatergic excitation by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)-2-amino-5-phosphonovaleric acid (APV, 30 microM) abolished IPSPs evoked from the stratum moleculare of the dentate gyrus, but IPSPs could still be evoked from the hilus and the stratum oriens/alveus. 3. Repetitive giant IPSPs, which consisted of Cl-dependent and K-dependent components, were evoked by bath application of 4-aminopyridine (4-AP, 10-50 microM) in CA3 neurons and in granule cells. Giant IPSPs were blocked by bath-applied tetrodotoxin (TTX). In addition, 4-AP hyperpolarized CA3 neurons in a Cl-dependent and picrotoxin-sensitive way. 4. Focal application of TTX to the dentate gyrus or the hilus considerably reduced the amplitude of giant IPSPs evoked by 4-AP in CA3 neurons. In hilar neurons, 4-AP evoked repetitive bursts, eventually, but not necessarily intermingled with giant IPSPs. Bursts were observed in hilar neurons in presence as well as absence of CNQX and APV. 5. In paired recordings, bursts in hilar neurons induced by 4-AP occurred simultaneously to giant IPSPs in granule cells and CA3 neurons, and giant IPSPs in granule cells occurred simultaneously to giant IPSPs in CA3 neurons. Blockade of glutamatergic excitation by CNQX and APV did not abolish this synchrony. 6. 4-AP-evoked Cl- and K-dependent IPSPs were, unlike electrically evoked IPSPs, not strictly coupled: some 20% of large IPSPs and up to 90% of small IPSPs were either Cl or K dependent. In granule cells K-dependent components either preceded or followed Cl-dependent components. 7. K-dependent IPSPs only could be evoked in CA3 neurons by focal application of 4-AP (1 mM) to the hilus, the stratum lacunosum moleculare or the stratum pyramidale. Wash out of Ca for 15-20 min blocked the Cl-dependent but not the K-dependent component of giant IPSPs evoked by bath-applied 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transforming growth factor-beta 1, 2, and 3 can inhibit epithelial tissue outgrowth on smooth and microgrooved substrates

In this study, we describe the influence of parallel surface microgrooves, and of TGF-beta, on the outgrowth of corneal epithelial tissue. Microgrooves (depth 1 microm, width 1-10 microm) were made in polystyrene culturing surfaces. These surfaces were left untreated, or loaded with TGF-beta 1, 2, or 3 (6.0 ng/cm(2)). Subsequently, epithelial explants from bovine corneas were placed on the experimental surfaces. After 9 days of culturing, tissue outgrowth was evaluated. Furthermore, the tissue cultures were analyzed histologically. It was shown that epithelial tissue grew from the explants over all experimental surfaces. On microgrooved surfaces outgrowth proceeded in the direction of the grooves, rather than perpendicular to the grooves. The addition of each type of TGF-beta resulted in a reduction of outgrowth. However, outgrowth remained directed by the grooves. Further, the explants had shrunk after TGF treatment. Histology showed that this shrinkage was not related to alpha-smooth muscle actin expression in the explants. We conclude that microgrooves can direct, and TGF-betas can inhibit the outgrowth of epithelial tissue. This finding could be useful in biomaterial applications where the growth of epithelial tissue needs to be discouraged.     

A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins.

One of the consequences of the heat shock response is a shutdown of pre-mRNA splicing, a phenomenon that can be reproduced in extracts prepared from heat-shocked cells. The block in splicing occurs before the covalent modifications that generate spliced mRNA at the level of spliceosome formation. We have used extracts prepared from heat-shocked cells as a complementation system to characterize and partially purify a protein factor that is inactivated during the in vivo heat shock. The activity functions in the formation of the active spliceosome by assembling U4/U6 and U5 snRNPs into a triple snRNP particle. The factor appears to be different from previously isolated splicing factors and is functionally equivalent to several polypeptides that are specifically associated with the purified triple snRNP but not with individual U4/U6 or U5 snRNPs. Our data confirm the hypothesis that U4/U6 and U5 snRNPs enter the spliceosome as a triple snRNP complex and show for the first time a function of specific snRNP-associated polypeptides in the mammalian splicing pathway.     

Malignant effusions are sources of fibronectin and other promigratory and proinvasive components

Metastatic dissemination is the primary cause of death in ovarian cancer (OvCa) patients, and dissemination to pleural and peritoneal effusions is a common clinical event. Effusion samples were collected from 15 OvCa patients. Twenty-six samples were collected prospectively, two were archival, and eight were taken from patients with other malignancies. Twenty-nine samples were from malignant ascites, and seven specimens were pleural fluids. In addition, six ascites and two pleural fluids from noncancer patients were studied as effusion controls. Effusion supernatants were tested for migration-stimulation activity, using A2058 human melanoma cells as the index responder cell. Malignant samples induced a 400-1200% increase in migration. Sixty percent of the migration was inhibited by incubation of the malignant fluid with antifibronectin (FN) antibody, in contrast to 75% inhibition of control fluid-stimulated migration (P = 0.017). Gelatin zymography and Western blot analyses showed that latent and activated MMP-2 and MMP-9 collagenases, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were present in all malignant fluids. Serial samples were taken from several patients, and a trend for correlation between MMPs and clinical behavior of the tumors was shown. Free TIMP-2 correlated with CA-125 levels in two patients for whom serial samples were available. The demonstration of promigratory and proinvasive activity in malignant effusions is consistent with their association with other metastatic disease in OvCa patients and their function as a haven for metastatic cells.     

Two unusual periosteal exotoses

After maceration of the skeleton of 2 persons we found 2 different solitary exostoses; one at the sciatic tuber, in the other at the sciatic tuber, in the other at the ventral side of the distal fibula. The exostoses reach a length of 2 cm. Prescher (1987) described a cartilaginous exostosis with a length of 2 cm at the proximal tibia. In our cases the exostoses belong to the periosteal form. We consider them to be bony origins of ligaments: at the sciatic tuber--the bony origin of the sacrotuberal ligament, at the distal fibula--the bony origin of the peroneal compartment of the retinaculum mm extensorum inferius.