MEG response to median nerve stimulation correlates with recovery of sensory and motor function after stroke.

OBJECTIVE: Hemiparesis due to damage by stroke in primary motor cortex (MI) or its underlying projections presents a problem for functional neuroimaging technologies that attempt to evaluate the neurophysiological basis for restoration of motor function. Traditional assessments of MI function require patients to move their fingers, hands, or limbs, which can be either impossible or markedly compromised after stroke. We recently demonstrated in normal subjects that magnetoencephalography (MEG), a non-invasive neuromagnetic functional imaging technique, detects neuronal response elicited by electrical median nerve stimulation in MI, as well as primary somatosensory cortex (SI). In the present study, we used the MEG response from median nerve stimulation to investigate the recovery of primary motor and somatosensory in acute ischemic stroke patients. METHODS: Twelve patients with unilateral ischemic strokes that affected sensorimotor functions of their hand were studied in the acute stage (4.4+/-1.2 days, mean+/-SD) and during a 1-month follow-up (38.6+/-5.6 days, except for one patient's follow-up done 6 month after stroke). RESULTS: Among the multiple cortical sources localized after median nerve stimulation, one source localized to SI and another localized to the vicinity of MI. Changes in the source strengths of the first component post-stimulus of MI and SI correlated with the extent of recovery of sensorimotor functions as determined by neurological exams. CONCLUSIONS: This study provides a novel way of indirectly assessing MI function using MEG during the acute stroke phase, when many patients often cannot perform motor tasks due to paralysis.

Task relevance enhances early transient and late slow-wave activity of distributed cortical sources.

The primary purpose of these studies was to link together concepts related to attention/working memory and feedforward/feedback activity using MEG response profiles obtained in humans. Similar to recent studies of attention in monkeys, we show early "spike-like" activity (<200 ms poststimulus), most likely reflecting an early transient excitatory response mixed with feedback influences, followed by "slow-wave" activity (>200 ms poststimulus) in MEG cortical response profiles evoked by a visual working memory task. We experimentally dissociated the early transient activity from the later sustained activity (predominantly feedback) by conducting an auditory size classification task. Words, representing common objects, evoked activity in occipital cortex (presumably due to imagery) even though visual stimuli were not present in this task. The initial "spike" was absent from the response profile obtained from occipital cortex, leaving only "slow-wave" activity, thereby allowing us to characterize or profile feedback activity in this situation. Attention or task relevance enhanced the initial "spike" and "slow-wave" activity in visually responsive areas. Prefrontal activity, along the superior frontal sulcus, evoked by the working memory task, was active later in time than initial activity in visual cortex and later than the earliest effect of attention modulation in visual cortex.

Sources on the anterior and posterior banks of the central sulcus identified from magnetic somatosensory evoked responses using multistart spatio-temporal localization.

A Multi-Start Spatio-Temporal (MSST) multidipole localization algorithm was used to study sources on the anterior and posterior banks of the central sulcus localized from early somatosensory magnetoencephalography (MEG) responses. Electrical stimulation was applied to the right and left median nerves of 8 normal subjects. Two sources, one on the anterior and one on the posterior bank of the central sulcus, were localized from 16 data sets (8 subjects, 2 hemispheres). Compared with the more traditional practice of single-dipole fits to peak latencies, MSST provided more reliable source locations. The temporal dynamics of the anterior and posterior central sulcus sources, obtained using MSST, showed considerable temporal overlap. In some cases, the two sources appeared synchronous. On the other hand, in the traditional single-dipole peak-latency fit approach, there is no time course other than a focal dipole moment activated only at the selected peak latency. The same group of subjects also performed a motor task involving index-finger lifting; the anterior central sulcus source obtained from electrical median nerve stimulation localized to the same or similar region in the primary motor area identified from the finger-lift task. The physiological significance of the anterior central sulcus source is discussed. The findings suggest that one can test the integrity of cortical tissue in the region of primary motor cortex using electrical somatosensory stimulation.

Multistart algorithms for MEG empirical data analysis reliably characterize locations and time courses of multiple sources.

We applied our newly developed Multistart algorithm (M. Huang et al., 1998, Electroencephalogr. Clin. Neurophysiol. 108, 32-44) to high signal-to-noise ratio (SNR) somatosensory responses and low SNR visual data to demonstrate the reliability of this analysis tool for determining source locations and time courses of empirical multisource neuromagnetic data. This algorithm performs a downhill simplex search hundreds to thousands of times with multiple, randomly selected initial starting parameters from within the head volume, in order to avoid problems of local minima. Two subjects participated in two studies: (1) somatosensory (left and right median nerves were stimulated using a square wave pulse of 0.2 ms duration) and (2) visual (small black and white bull's-eye patterns were presented to central and peripheral locations in four quadrants of the visual field). One subject participated in both of the studies mentioned above and in a third study (i.e., simultaneous somatosensory/visual stimulation). The best-fitting solutions were tightly clustered in high SNR somatosensory data and all dominant regions of activity could be identified in some instances by using a single model order (e.g., six dipoles) applied to a single interval of time (e.g., 15-250 ms) that captured the entire somatosensory response. In low SNR visual data, solutions were obtained from several different model orders and time intervals in order to capture the dominant activity across the entire visual response (e.g. , 60-300 ms). Our results demonstrate that Multistart MEG analysis procedures can localize multiple regions of activity and characterize their time courses in a reliable fashion. Sources for visual data were determined by comparing results across several different models, each of which was based on hundreds to thousands of different fits to the data.

Identification of key gene products required for acquisition of plasmin-like enzymatic activity by group A streptococci.

Group A streptococci incubated in human plasma can acquire a plasmin-like enzymatic activity. This process involves at least two bacterial proteins and two human protein cofactors. In this study, the key bacterial proteins were identified by using a series of isogenic mutants of group A isolate, CS101. These studies confirm a key role for the secreted plasminogen activator, streptokinase, and identify the major surface fibrinogen-binding protein as the product of the mrp gene. The requirement for human fibrinogen and plasminogen as key cofactors was also confirmed.

Role of staphylokinase in the acquisition of plasmin(ogen)-dependent enzymatic activity by staphylococci.

In this study, the role of the staphylococcal plasminogen activator, staphylokinase (SAK), was analyzed for its ability to mediate acquisition of cell-associated plasmin-like activity by staphylococci in the presence of a source of human plasminogen. A panel of staphylococcal strains isolated from humans was tested for the presence of the SAK gene, secretion of the plasminogen activator, and the ability to acquire enzymatic activity when incubated with purified human plasminogen or serum. When SAK was compared with the eukaryotic plasminogen activators, urokinase and tissue plasminogen activator, only SAK could mediate acquisition of cell-associated enzymatic activity by staphylococci without first generating significant fluid-phase plasmin. These studies provide evidence for a novel mechanism by which SAK-producing S. aureus can acquire an unregulatable host plasmin-like activity that might contribute to their invasive potential.

Binding of human serum amyloid P-component to phosphocholine.

Human serum amyloid P-component (SAP) and C-reactive protein (CRP) are structurally similar pentraxins composed of five identical subunits in a disc-like configuration and display Ca(2+)-dependent binding reactivity to a variety of unrelated ligands. CRP is generally classified and defined as a phosphocholine (PC)-binding protein, whereas SAP is identified as a polysaccharide-binding protein. We examined the PC-binding activity of human SAP and compared it to human CRP since many of the biological activities of CRP are triggered upon PC-binding. SAP was able to bind to immobilize PC in a saturable, Ca(2+)-dependent manner but with lower avidity than CRP in direct competitive binding assays. The affinity of the binding of SAP to soluble [14C]PC was slightly lower than the affinity of CRP; however, the valence of SAP was only one PC-binding site/pentraxin or 2/protein vs 5 such sites per CRP molecule. Both SAP and CRP displayed a similar binding preference for PC vs phosphoethanolamine (PE). Two monoclonal antibodies (mAb) generated against the PC-binding site of SAP also reacted with the PC-binding site of CRP and inhibited PC-binding by both pentraxins. A mAb specific for the PC-binding site on CRP also inhibited SAP binding to PC. SAP was also recognized by two anti-idiotypic mAb that shared reactivity with the TEPC-15 PC-binding myeloma protein and the PC-binding site of CRP. Both pentraxins could be isolated from human serum by affinity chromatography on either PC- or PE-substituted agarose beads. The findings indicate that SAP is also a PC-binding protein.

Specificity of the binding interaction between human serum amyloid P-component and immobilized human C-reactive protein.

C-reactive protein (CRP) and serum amyloid P-component (SAP) are two members of a group of plasma proteins termed pentraxins, which are composed of five identical noncovalently linked subunits that display Ca(2+)-dependent binding to a wide variety of substrates. Purified human SAP binds to CRP, only when the latter is immobilized, in a Ca(2+)-dependent manner under physiological conditions. Externally labeled SAP rapidly binds to two distinct forms of immobilized CRP (direct and phosphorylcholine captured) with a relatively high affinity (KD = 5 nM) at a molar ratio of specifically bound SAP/CRP = 0.3. Studies of binding inhibition using monoclonal antibodies to CRP or synthetic peptides of CRP revealed that residues 134-148 and the COOH-terminal region (residues 191-206) were recognized by SAP. A fragment of CRP consisting of the COOH-terminal 60 residues within each subunit was also selectively bound by SAP. The ability of immobilized CRP to bind SAP was distinguished from CRP's lectin-like binding reactivity since deglycosylated SAP retained its binding reactivity for CRP and sugars that inhibit CRP's lectin-like binding activity failed to inhibit binding. A peptide from trypsin digested SAP composed of residues 144-199 retained CRP binding activity, implicating the COOH-terminal region of SAP as the CRP recognition site.

Acid phosphatase activity in Coxiella burnetii: a possible virulence factor.

High-speed supernatant fluids derived from sonicated Coxiella burnetii contained considerable acid phosphatase activity when assayed by using 4-methylumbelliferylphosphate; they also contained a factor that blocked superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe. The pH optimum of the enzyme was approximately 5.0. The level of phosphatase activity detected in several isolates of C. burnetii implicated in acute (Nine Mile) and chronic (S Q217, PRS Q177, K Q154) Q fever was 25 to 60 times greater than that reported in other microorganisms, including Leishmania and Legionella spp. The enzyme was found in rickettsiae grown in different hosts (L929 cells and embryonated eggs) and, in the case of L929 cells, for both short periods (less than a month) and the long term (years). Cytochemical techniques coupled with electron microscopy localized the phosphatase activity to the periplasmic gap in the parasite. Ion-exchange chromatography revealed a major species of the enzyme and showed that the enzyme of the parasite was distinct from that of the host cell (L929 fibroblasts); its apparent molecular weight was 74,000. Phosphatase inhibitors (i.e., molybdate heteropolyanions) had differential effects on the phosphatases of the parasite and host cell. C. burnetii supernatant fluid inhibited superoxide anion production by formyl-Met-Leu-Phe-stimulated human neutrophils; molybdate inhibitors reversed the inhibition. Treatment of C. burnetii-infected L929 cells with one of the molybdate compounds (complex B') significantly reduced the level of infection and did not affect the viability or growth of the host cell. These data suggest that the acid phosphatase of the parasite may be a major virulence determinant, allowing the agent to avoid being killed during uptake by phagocytes and subsequently in the phagolysosome.

Human serum amyloid P-component (SAP) selectively binds to immobilized or bound forms of C-reactive protein (CRP).

The two homologous human pentraxins, C-reactive protein (CRP) and serum amyloid P-component (SAP), specifically bind to each other only when the CRP is in an immobilized form bound to one of its ligands or to an antibody. CRP did not bind to immobilized SAP. The binding of SAP to immobilized forms of CRP was Ca(2+)-dependent and of sufficient affinity to occur in the presence of serum or purified serum proteins. SAP bound preferentially to a synthetic peptide corresponding to the Ca(2+)-binding region of CRP. Monoclonal antibodies to a synthetic peptide corresponding to the Ca(2+)-binding region selectively inhibited the binding interaction. Proteolytic cleavage of CRP between residues 146 and 147 within the Ca2+ binding region abolished the SAP-binding site; however, the intact subunits of the pentameric CRP were capable of binding SAP. The significance of the binding interaction is that it may serve as the basis for localization of SAP to sites of tissue damage or repair, sites where CRP is selectively deposited.

A cell attachment peptide from human C-reactive protein.

The serum acute phase reactant, C-reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27-38 present in each of the five identical subunits of CRP mediated cell attachment activity in vitro. Although the CRP-derived peptide contains a Tuftsin (TKPR)-like sequence at its amino-terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell-attachment activity to the CRP-derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP-derived peptide cell attachment activity. However, the addition of the RGDS and RGDSPASSLP cell-binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell-binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P-component (SAP), displayed nearly identical cell-attachment activity. Several monoclonal antibodies (mAb) specific for the CRP-derived cell-binding peptide neutralized its cell-attachment activity. These mAbs reacted with intact CRP and neutralized the cell-binding activity of CRP itself. The findings suggest that a peptide with cell-binding activity could be generated from the breakdown of CRP and then contribute directly to cellular events leading to tissue repair.

Differential effect of restraint stress on MHC class II expression by murine peritoneal macrophages.

The effect of restraint stress on the expression of MHC class II glycoproteins by peritoneal macrophages was evaluated. Restraint suppressed the expression of I-A by macrophages from mice that are susceptible to Mycobacterial infection. In contrast, restraint did not affect I-A expression by macrophages from resistant mice. The suppression of MHC class II expression required at least 8 h of restraint and recovered within 4 h after stress. The amount of restraint necessary to suppress I-A expression also resulted in higher levels of plasma corticosterone. Changes in I-A expression were under circadian rhythm control. The differences in the effect of restraint stress on expression of I-A by peritoneal macrophages from resistant and susceptible mice may, in part, be due to differences in the effect of corticosterone in MHC class II expression.

Restraint stress-induced suppression of major histocompatibility complex class II expression by murine peritoneal macrophages.

The macrophage plays a central role in the development of immune responses. Macrophages take up and process antigen which is presented to antigen-responsive T lymphocytes in association with major histocompatibility complex class II (Ia) glycoproteins. We have investigated the effect of restraint stress on Ia expression by murine peritoneal macrophages. Stress resulted in a suppression of Ia expression which coincided with an increase in plasma corticosterone levels. In vitro experiments indicate that suppression of Ia expression can occur within 2 h after exposure to corticosterone. The suppression of this important aspect of macrophage function by stressors has important implications regarding the possible immunosuppressive effects of stress on the response of lymphocytes to antigens that depend on intact macrophage function.