Inhibition of plasmin attenuates murine acute graft-versus-host disease mortality by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and effector cell trafficking.

The systemic inflammatory response observed during acute graft-versus-host disease (aGVHD) is driven by proinflammatory cytokines, a 'cytokine storm'. The function of plasmin in regulating the inflammatory response is not fully understood, and its role in the development of aGVHD remains unresolved. Here we show that plasmin is activated during the early phase of aGVHD in mice, and its activation correlated with aGVHD severity in humans. Pharmacological plasmin inhibition protected against aGVHD-associated lethality in mice. Mechanistically, plasmin inhibition impaired the infiltration of inflammatory cells, the release of membrane-associated proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and Fas-ligand directly, or indirectly via matrix metalloproteinases (MMPs) and alters monocyte chemoattractant protein-1 (MCP-1) signaling. We propose that plasmin and potentially MMP-9 inhibition offers a novel therapeutic strategy to control the deadly cytokine storm in patients with aGVHD, thereby preventing tissue destruction.

Effects of bosentan on the skin lesions: an observational study from a single center in Japan.

Effects of a dual endothelin receptor antagonist, bosentan on peripheral circulatioin and skin lesions as well as pulmonary arterial hypertension (PAH) were investigated in Japanese patients with connective tissue diseases (CTD). Fifteen patients with PAH associated with CTD [systemic sclerosis (SSc) 13, mixed connective tissue disease (MCTD) 2] were treated with bosentan for 40-96 weeks, and changes of exercise capacity (6-min walk distance and Borg's dyspnea scale), cardio-pulmonary hemodynamics (right ventricular pressure, specific activity scale and cardiac index), Raynaud's phenomenon, digital ulcers and dermal sclerosis were observed. Bosentan improved exercise capacity, had a positive effect on hemodynamic parameters, and was well tolerated as previously reported. After a median 8 weeks of treatment, 13 out of 15 patients had improved Raynaud's phenomenon. Digital ulcers also improved after a median 12 weeks' treatment in all of 8 patients. Modified Rodnan total skin score decreased from 21.0 +/- 5.9 to 11.5 +/- 3.9 in diffuse cutaneous SSc and from 17.0 +/- 6.5 to 9.5 +/- 4.5 in limited cutaneous SSc after 24 months' treatment, reaching significance after 6 months in both groups. These data suggest that bosentan is effective for both PAH and peripheral vascular diseases in Japanese patients with CTD. The pathological background to the improvement in dermal sclerosis observed in this study should be further investigated.

Activity-dependent conduction block in multifocal motor neuropathy: magnetic fatigue test.

BACKGROUND: Multifocal motor neuropathy (MMN) is often misdiagnosed as motor neuron disease, especially when overt evidence of conduction block (CB) is lacking. Activity-dependent CB (ADCB), defined as transient CB induced by brief exercise, has been recently found in MMN but not in ALS. METHODS: To test the diagnostic utility of ADCB for differentiating MMN from ALS, the authors recorded the compound muscle action potentials (CMAPs) from small hand muscles by magnetically stimulating nerve roots before and after 1 minute of maximal voluntary contraction (magnetic fatigue test). They examined nine patients with MMN with unequivocal clinical responses to IV immunoglobulins (IVIgs), yet lacked CB according to the conventional criteria. RESULTS: Six MMN patients had postexercise CB/temporal dispersion maximum in the immediate postexercise period. ADCB in an MMN patient improved after IVIg. Further analysis revealed that prolongation of the duration from the onset to the positive peak of the CMAP was the most sensitive indicator for MMN, presumably because the phase cancellation obscures the abnormalities of the other parameters. CONCLUSION: The magnetic fatigue test is useful in detecting mild conduction block presumably located in a proximal nerve segment in patients with multifocal motor neuropathy who do not fulfill its conventional electrodiagnostic criteria.

Macaque ventral premotor cortex exerts powerful facilitation of motor cortex outputs to upper limb motoneurons.

The ventral premotor area (F5) is part of the cortical circuit controlling visuomotor grasp. F5 could influence hand motor function through at least two pathways: corticospinal projections and corticocortical projections to primary motor cortex (M1). We found that stimulation of macaque F5, which by itself evoked little or no detectable corticospinal output, could produce a robust modulation of motor outputs from M1. Arrays of fine microwires were implanted in F5 and M1. During terminal experiments under chloralose anesthesia, single stimuli delivered to M1 electrodes evoked direct (D) and indirect (I1,I2, and I3) corticospinal volleys. In contrast, single F5 shocks were ineffective; double shocks (3 msec separation) evoked small I waves but no D wave. However, when the test (T) M1 shock was conditioned (C) by single or double F5 shocks, there was strong facilitation of I2 and I3 waves from M1, with C-T intervals of <1 msec. Intracellular recordings from 79 arm and hand motoneurons (MNs) revealed no postsynaptic effects from single F5 shocks. In contrast, these stimuli produced a robust facilitation of I2 and I3 EPSPs evoked from M1 (60% of MNs); this was particularly marked in hand muscle MNs (92%). Muscimol injection in M1 reduced I waves from F5 and abolished the F5-induced facilitation of late I waves from M1, and of EPSPs associated with them. Thus, some motor effects evoked from F5 may be mediated by corticocortical inputs to M1 impinging on interneurons generating late corticospinal I waves. Similar mechanisms may allow F5 to modulate grasp-related outputs from M1.

Facilitation from ventral premotor cortex of primary motor cortex outputs to macaque hand muscles.

We demonstrate that in the macaque monkey there is robust, short-latency facilitation by ventral premotor cortex (area F5) of motor outputs from primary motor cortex (M1) to contralateral intrinsic hand muscles. Experiments were carried out on two adult macaques under light sedation (ketamine plus medetomidine HCl). Facilitation of hand muscle electromyograms (EMG) was tested using arrays of fine intracortical microwires implanted, respectively, in the wrist/digit motor representations of F5 and M1, which were identified by previous mapping with intracortical microstimulation. Single pulses (70-200 microA) delivered to F5 microwires never evoked any EMG responses, but small responses were occasionally seen with double pulses (interval: 3 ms) at high intensity. However, both single- and double-pulse stimulation of F5 could facilitate the EMG responses evoked from M1 by single shocks. The facilitation was large (up to 4-fold with single and 12-fold with double F5 shocks) and occurred with an early onset, with significant effects at intervals of only 1-2 ms between conditioning F5 and test M1 stimuli. A number of possible pathways could be responsible for these effects, although it is argued that the most likely mechanism would be the facilitation, by cortico-cortical inputs from F5, of corticospinal I wave activity evoked from M1. This facilitatory action could be of considerable importance for the coupling of grasp-related neurons in F5 and M1 during visuomotor tasks.

A model for predicting sediment-water partition of toxic chemicals in aquatic environments.

In order to investigate the characteristics of sediment-water partition of chemicals in aquatic environments using published data, we developed a model for predicting the sediment-water partition coefficient (Kp) as the sum of sorption to sediment organic matter and sorption to sediment inorganic matter. This model is so successful that the differences between Kp (median for a variety of Japanese water bodies) and pre-Kp (predicted K) are within one order of magnitude in 24 out of 28 chemicals.

Disynaptic inhibition of omnipause neurons following electrical stimulation of the superior colliculus in alert cats.

We investigated the synaptic organization responsible for the inhibition of omnipause neurons (OPNs) following stimulation of the superior colliculus (SC) in alert cats. Stimulation electrodes were implanted bilaterally in the rostral and caudal SC where a short-pulse train induced small and large saccades, respectively. Effects of single-pulse stimulation on OPNs were examined with intracellular and extracellular recordings. In contrast to monosynaptic excitatory postsynaptic potentials, which were induced by rostral SC stimulation, inhibitory postsynaptic potentials were induced with disynaptic latencies (1.3--1.9 ms) from both the rostral and caudal SC in most OPNs. Analysis of a larger extracellular sample complemented intracellular observations. Monosynaptic activation of OPNs was elicited more frequently from rostral sites than from caudal sites, whereas spike suppression with disynaptic latencies was induced by caudal as well as rostral stimulation with similar frequencies. The results imply that disynaptic inhibition is produced by activation of SC cells that are distributed over wide regions related to saccades of different sizes. We suggest that signals from these neurons initiate a saccadic pause of OPNs through single inhibitory interneurons.

DYT1 mutation in Japanese patients with primary torsion dystonia.

A GAG deletion at position 946 in the DYT1 gene has been identified as one of the gene mutations responsible for autosomal dominant primary torsion dystonia. We examined 178 Japanese patients with various forms of dystonia, and found the mutation in six patients (3.4%) from three families. Five of them had early clinical onset (before age 12) with initial involvement of a limb. To our knowledge, this is the first report of the frequency and the clinical features of DYT1 mutation in oriental patients, and the clinical presentation of the mutation in these patients was similar to that of Jewish or non-Jewish Caucasian patients.

High-frequency SEP components generated in the somatosensory cortex of the monkey.

To investigate the origin of high-frequency somatosensory evoked potential (SEP) components, we recorded median nerve SEPs from the scalp and the depth in six monkeys. Laminar field potentials were analyzed in area 3b (N10; corresponding to human N20) and area 1 (P12; corresponding to human P25). After digital filtering (300-900 Hz), 4-6 components were identified, and the 1st to 4th peaks in area 3b (7-11 ms in latency) and the 3rd to 5th in area 1 (9-13 ms) showed clear polarity reversals between the surface and the depth of the cortex. These results provide direct evidence for intracortical origin of early high-frequency components in area 3b and of late ones in area 1.

Abnormal premovement gating of somatosensory input in writer's cramp.

One characteristic of focal dystonia is the sensory trick, by which sensory input to a certain area of the body can reduce abnormal contractions in muscles nearby. This suggests that adjusting the link between sensory input and movement allows motor commands to be issued more effectively from the brain. To explore this sensorimotor link, we studied the attenuation (gating) of somatosensory evoked potentials (SEPs) before and during hand movements in patients with writer's cramp. For premovement gating, 10 patients and 11 age-matched normal subjects were given a warning sound followed 1s later by an electric stimulus to the right median nerve at the wrist. The latter served both as a reaction signal to start a finger extension task and as the input to evoke SEPs over the scalp. Because reaction times always exceeded 70 ms, short-latency SEPs thus obtained were unaffected by the afferents activated by the movement. The amplitudes of frontal N30 components were significantly decreased over the frontal leads compared with SEPs elicited at rest (P: < 0.002) in the normal group, whereas significant gating was found not for N30 but for frontal P22 (P: = 0. 002) in the patient group. For midmovement gating studies, SEPs to the right median nerve stimulation were recorded in 16 patients and 12 age-matched normal subjects at rest, and during active and passive finger extension-flexion movements. In contrast to the premovement SEPs, the frontal N30 was equally gated during active and passive movements both in the patient (P: < or = 0.002) and the normal group (P: < or = 0.003). These findings indicate that in writer's cramp the sensitivity of sensory input channels from the hand is wrongly set by the central command to move. Perhaps the sensory trick, by supplying additional input not usually present during unobstructed movement, is a manoeuvre to correct this imbalance. Dystonia may result not only from abnormalities in the central motor command but also from disturbed central processing of sensory input.

Pre-movement gating of short-latency somatosensory evoked potentials.

Somatosensory evoked potentials (SEPs) are reduced in amplitude during movement (gating). The mechanism involves central gating of afferent input and competition from other afferents activated by the movement. We distinguished these two by giving 11 normal subjects a warning sound followed 1 s later by an electric stimulus to the right median nerve at the wrist. The latter served both as a cue to start a finger movement and as stimulation to evoke SEPs. Gating effects were widespread in frontal (N30) and central (N60) areas, but were also seen, albeit to a lesser extent, in the recordings at P3 (P30). Since finger movement began after the stimulus, such gating must have been purely central in origin, presumably reflecting motor preparation.

Saccade-related inhibitory input to pontine omnipause neurons: an intracellular study in alert cats.

Omnipause neurons (OPNs) are midline pontine neurons that are thought to control a number of oculomotor behaviors, especially saccades. Intracellular recordings were made from OPNs in alert cats to elucidate saccade-associated postsynaptic events in OPNs and thereby determine what patterns of afferent discharge impinge on OPNs to cause their saccadic inhibition. The membrane potential of impaled OPNs exhibited steep hyperpolarization before each saccade that lasted for the whole period of the saccade. The hyperpolarization was reversed to depolarization by intracellular injection of Cl- ions, indicating it consisted of temporal summation of inhibitory postsynaptic potentials (IPSPs). The duration of the saccade-related hyperpolarization was almost equal to the duration of the concurrent saccades. The time course of the hyperpolarization was similar to that of the radial eye velocity except for the initial phase. During the falling phase of eye velocity, the correlation between the instantaneous amplitude of hyperpolarization and the instantaneous eye velocity was highly significant. The amplitude of hyperpolarization at the eye velocity peak was correlated significantly with the peak eye velocity. The time integral of the hyperpolarization was correlated with the radial amplitude of saccades. The initial phase disparity between the hyperpolarization and eye velocity was due to the relative constancy of peak time (approximately 20 ms) of the initial steep hyperpolarization regardless of the later potential profile that covaried with the eye velocity. The initial steep hyperpolarization led the beginning of saccades by 15.9 +/- 3.8 (SD) ms, which is longer than the lead time for medium-lead burst neurons. These results demonstrate that the pause of activity in OPNs is caused by IPSPs initiated by an abrupt, intense input and maintained, for the whole duration of the saccade, by afferents conveying eye velocity signals. We suggest that the initial sudden inhibition originates from central structures such as the superior colliculus and frontal eye fields and that the eye velocity-related inhibition originates from the burst generator in the brain stem.

Vascular tone in patients with hemorrhagic shock.

BACKGROUND: In hemorrhagic shock, the alterations in arterial vascular tone, which are primarily regulated by adrenosympathetic influences are compensatory responses to bleeding. OBJECTIVE: The aim of this study was to evaluate vascular tone expressed by the volume elastic modulus (Ev) as a clinical monitor to detect the hypovolemic state. METHODS: Thirteen patients with hemorrhagic shock were studied. The initial Ev measurement was performed at arrival, and subsequent measurements were obtained 4 and 12 hours after arrival. Patients were divided into two groups by cluster analysis by using the Ev values at arrival and 4 hours after arrival. Circulatory parameters, the clinical course, and fluid were compared. RESULTS: The Ev values were identical at admission (cluster I vs. II: 456.4+/-197.1 vs 566.1+/-234.1 mm Hg, mean +/- SD). After 4 hours of fluid resuscitation, all patients were recovered from shock. In cluster I, the Ev remained high at 4 hours (523.4+/-75.1 mm Hg) and invasive treatments for hemostasis were required. In cluster II, the Ev significantly decreased at 4 hours (182.8+/-70.7 mm Hg, p < 0.01) and clinical courses were eventless thereafter. During 4 to 12 hours, more fluid was given in cluster I (p < 0.05). At 12 hours after arrival, the Ev values were identical in both clusters. CONCLUSION: These data demonstrate that the Ev increases in hemorrhagic shock. Furthermore, normotensive hypovolemic conditions generated by persistent bleeding can be detected by measuring the Ev.

Reproductive toxicity, mutagenicity and antigenicity of pamiteplase (genetical recombination).

Pamiteplase (genetical recombination), YM866, is a novel recombinant modified human tissue-type plasminogen activator developed by Yamanouchi Pharmaceutical Co. Ltd., Tokyo, Japan. An intended route of administration in the clinical use of this drug is intravenous administration. We conducted an intravenous fertility and general reproduction studies of this drug in male and female rats and teratology study of this drug in rabbits at the dose levels of 0 (vehicle control), 0.1, 0.3 or 1 mg/kg/day. In the rat, no treatment-related abnormalities were observed up to the maximum dose in parental animals and their offspring. In the teratology study in rabbits, prolonged coagulation time at the injection site was observed at 0.3 mg/kg or more. One death and one abortion occurred at 1 mg/kg on days 22 and 23 of pregnancy, respectively. No toxic effects on the litters were observed up to the maximum dose. Results of evaluation of the mutagenicity of YM866 and its ability to induce chromosome aberrations using the L5178Y TK+/- mouse lymphoma assay, human lymphocyte chromosome aberration assay and the micronucleus assay in mice were negative. Evaluation of the immunogenicity of YM866 by repeated intravenous injection in chimpanzees elicited no confirmed antibody titers.

The community health center and family practice residency training.

Community health centers (CHCs), sponsored by US Public Health Service (USPHS) Section 330, represent successful models of the application of community-oriented primary care principles. Recently, increased interest has been shown in conducting medical education programs in CHCs. The USPHS has tried to facilitate this interest, particularly through its support of establishing linkages between CHCs and family practice residency programs. In this paper, we describe an integrated CHC-family practice residency continuity training clinic program. We discuss the challenges inherent to conducting family practice residency training in the CHC, including the educational content of clinical experiences, the impact of provider productivity expectations, the academic and operational governance of the program, and the financial considerations pertinent to the integrated function of the program. We conclude that while the clinical experiences available in the CHC differ somewhat from mainstream family practice, successful adaptations can be made, and a CHC offers a rich educational environment. We also conclude that the challenges inherent to integrated CHC-family practice residency programs can be successfully addressed. Of great concern, however, are financial considerations relevant to the operation of such integrated programs. These considerations underscore the urgent need for a reassessment of the funding of ambulatory clinical medical education.

Monosynaptic activation of medium-lead burst neurons from the superior colliculus in the alert cat.

1. Extracellular recordings were made from medium-lead burst neurons (MLBNs) in the paramedian pontomedullary reticular formation rostral and caudal to the abducens nucleus in the alert cat. 2. Single-pulse stimulation of the contralateral superior colliculus during intersaccadic intervals evoked no response in most MLBNs. When collicular stimulation was applied at the beginning of saccades, spikes of MLBNs were consistently evoked with short latencies. The shortest latency was 0.8 ms, indicating monosynaptic activation of MLBNs from the superior colliculus. 3. Results suggest that monosynaptic excitatory effects from the colliculus are concealed by inhibitory input from omnipause neurons (OPNs) during intersaccadic intervals and that the monosynaptic collicular activation is disclosed when this inhibition is removed by a pause in OPN activity at the beginning of saccades.

[Single-dose toxicity studies of prulifloxacin (NM441) in mice, rats and dogs and the active metabolite (NM394) in rats].

Single-dose toxicity studies of prulifloxacin, a new antibacterial agent, were conducted in mice, rats and dogs. In addition, a single-dose toxicity study of (+/-)-6-fluoro-1-methyl-4-oxo-7- (1-piperazinyl)-4H-[1,3]thiazeto[3,2-a]quinoline- 3-carboxylic acid (NM394), an active metabolite of prulifloxacin, was performed in rats. Prulifloxacin was administered orally, intraperitoneally (i.p.) or subcutaneously (s.c.) to mice and rats, and orally to dogs. NM394 was administered intravenously (i.v.) to rats. When prulifloxacin was administered orally or s.c., LD50 values were more than 5000 mg/kg in both sexes of mice and rats; when it was administered i.p., LD50 values were 1757 mg/kg in male mice, 1652 mg/kg in female mice, 915 mg/kg in male rats, and 1076 mg/kg in female rats. The lethal doses of this drug were more than 5000 mg/kg in both sexes of dogs by the oral route. The LD50 values of NM394 were 226 mg/kg in male rats and 238 mg/kg in female rats by the i.v. route. In mice, the major clinical signs observed following the administration of prulifloxacin were sedation, oligopnea, abnormal gait, piloerection, closed eye and tremor by the i.p. route and a scab at the site of injection by the s.c. route; in rats, decreased spontaneous locomotor activity by any of the three routes, oligopnea, lacrimation, hypothermia, piloerection and abnormal gait by the i.p. route, and a scab at the site of injection by the s.c. route; and in dogs, vomiting, reddening of the skin, and loose stool by the oral route. When NM394 was administered i.v., rats showed clonic convulsion and dyspnea. The site of injection was hyperemic, swollen and necrotic. Mice showed a decrease in body weight or an inhibition in weight gain when prulifloxacin was administered i.p. and rats showed the same effects when prulifloxacin or NM394 was administered by any of the above-mentioned routes. Macroscopic findings detected following the i.p. administration of prulifloxacin in mice were pale color of the liver and spleen, thickening of the liver, and adhesion of intra-abdominal organs; and in rats, hydrothorax, congestion and edema of the lung, adhesion of intra-abdominal organs, swelling of the kidney accompanied by fine yellowish-white foci, and atrophy of the testis. When NM394 was administered i.v. to rats, congestion of the lung was macroscopically observed.

Inhibitory input to pause neurons from pontine burst neuron area in the cat.

Extra- and intracellular recordings were made from pontine pause neurons (PNs) in the cat. Spontaneous spikes of PNs were suppressed after single shock stimulation of excitatory burst neuron (EBN) area immediately rostral to the abducens nucleus. The most effective stimulation site for the suppression was the region where long-lead burst neurons (LLBNs) were predominantly located. Intracellular recordings from PNs showed that stimulation of the LLBN area induced short-latency inhibitory postsynaptic potentials (IPSPs) in PNs and that steep hyperpolarization of PNs associated with quick phases of nystagmus occurred prior to an abrupt change in abducens nerve activity. Results suggest that a pause of PN spikes associated with quick phases is, at least in part, produced by inhibitory action mediated through LLBNs.