Behavior of F-response and determination of actively involved motoneurons.

Fifteen nerves were examined in 10 healthy subjects, using a collision technique with 2 stimulation pulses of different intensity on the same point. The F-waves which occurred after the antidromic activation of the motor neuron cells by the first supramaximal pulse were blocked on their return pathway by the collision with the depolarization induced by the second pulse. The progressive decrement of the voltage of the second stimulus allowed, the transmission of the recurrent discharge along motor fibers with a lower depolarization threshold. The amplitude and the persistence of the F-waves increased (p < 0.05 and p < 0.001 respectively), while their latency decreased (p < 0.01) concurrent with the liberation of additional motor neurons with a lower threshold of depolarization. These findings suggest that the F-wave may be elicited in motoneuron of different depolarization threshold but primarily in larger and faster nerve fibers, with a lower threshold of depolarization.

Effect of hyperglycemia and the aldose reductase inhibitor tolrestat on sural nerve biochemistry and morphometry in advanced diabetic peripheral polyneuropathy. The Tolrestat Study Group.

Tolrestat is a well tolerated nonhydantoin aldose reductase inhibitor that has been reported to improve nerve conduction in diabetic animals and humans. Its effects on nerve biochemistry and structure have not been studied in patients with diabetic neuropathy. Patients with advanced diabetic neuropathy treated with long-term open-label tolrestat were randomly assigned to continuation on drug treatment or to placebo-controlled drug withdrawal for 12 months. At the end of this period, sural nerve biopsies were obtained for measurement of glucose, sorbitol, and fructose content, and for detailed morphometric analysis. Tolrestat ameliorated the glucose-mediated increase in sorbitol and fructose in sural nerve tissue. No statistically significant differences in nerve morphometry emerged between the two groups; however, both treatment groups exhibited increased nerve-fiber regeneration and normalization of axo-glial dysfunction and segmental demyelination following long-term tolrestat treatment. These findings are similar to those previously reported in a placebo-controlled sequential nerve biopsy study with the aldose reductase inhibitor sorbinil. Thus tolrestat is a biochemically effective aldose reductase inhibitor in human diabetic nerve with potential therapeutic efficacy for diabetic neuropathy.

Withdrawal of the aldose reductase inhibitor tolrestat in patients with diabetic neuropathy: effect on nerve function. The Tolrestat Study Group.

A double-blind, placebo-controlled clinical trial was conducted to study the effects of discontinuing tolrestat, an aldose reductase inhibitor, on peripheral sensorimotor diabetic neuropathy. After an average of 4.2 years of continuous tolrestat use, 372 patients were randomly assigned to either placebo or continued tolrestat therapy and were followed for 52 weeks. After 3 months, patients who perceived worsening of symptoms of neuropathy were allowed to switch once to the alternate treatment group while maintaining the double-blind. Patients assigned to placebo had significant deterioration in motor nerve conduction velocity (MNCV) while those maintained on tolrestat did not (p < 0.05). The 28 patients who were randomly assigned to tolrestat and elected to switch to placebo had a significant deterioration in MNCV while the 36 assigned to placebo who switched to tolrestat had a significant improvement (p < 0.05). Treatment differences in favor of tolrestat were observed for sensation in the toes as well as for pain (p < 0.05). These data indicate that withdrawal from long-term treatment with tolrestat has a detrimental effect on several measures of diabetic neuropathy, whereas continuation of treatment is associated with stabilization of these measures, suggesting a continued role for polyol pathway activity in late neuropathy.

Possibilities of interference with the immune system of tumor bearers by non-lymphoid Fc gamma RII expressing tumor cells.

The ectopic expression of Fc gamma RII by PyV transformed 3T3 cells derived from tumors of long latency has been established. It was suggested that this expression is one of several changes conferring upon the cells an increased capacity for survival. We found that in one case cells expressing a very high level of Fc gamma RII had also a very high metastatic phenotype as compared to FcR negative cells. Direct evidence that Fc gamma RIIbl functions as a progression factor was provided by transfection experiments. The transfected gene conferred an increased malignancy and invasive phenotype upon PyV or c-Ha-ras transformed cells. In the present study we tested the possibility that Fc gamma RII expressing tumor cells could interfere with the immune system. The following subjects were investigated: 1) The ability of Fc gamma R on the tumor cells to bind the ligand and/or release IBF. 2) The effect of a local accumulation of ligand and/or IBF (assumed to take place in situ in the tumor) on Fc gamma RII expressing T cells. It was found that both tumor-derived receptor positive and beta l transfected PyV transformed cells were capable of binding aggregated mouse IgG. The binding of bivalent ligand was followed by an increase in membrane Fc gamma RII expression. Also both types of cells were capable of releasing IBF. We then tested the possibility that a local accumulation of IgG within the tumor could effect Fc gamma R expressing T cells. It was found that aggregated mouse IgG (as well as IgGl) could stimulate the proliferation of the T cell hybridoma (T2D4) and other Fc gamma RII expressing T cells. We also found that the expression of beta Fc gamma RII specific mRNA peaked at the logarithmic phase of T2D4 cultures, in parallel with their maximal potential to release IBF. Several pathways for interference with the immune system are suggested.

Phenotypic properties of 3T3 cells transformed in vitro with polyoma virus and passaged once in syngeneic animals.

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) acquired a higher tumorigenicity phenotype after a single in vivo passage. Some of the in vivo passaged cells (CTC cells) exhibited also a higher metastatic phenotype than cells from the same clones that were maintained only in culture (C cells). A phenotypic comparison between CTC and C cells was performed. It was found that most CTC lines exhibited a higher binding to laminin compared to their clonal C cell ancestors. Some CTC cells were less sensitive to the cytotoxic effects of TNF-alpha than the corresponding C cells. CTC cells originating from tumors which appeared after a long latency period (late tumors) tended to express Fc gamma RII while CTC cells originating from tumors which appeared after a short latency period (early tumors) as well as the corresponding C cells tended not to express Fc gamma RII. The expression of a membrane epitope recognized by a monoclonal antibody expressing specificity towards PyV transformed cells, was down-regulated on late tumor cells compared to early tumor cells. Transfection of cloned PyV-transformed BALB/c 3T3 cells with the beta 1Fc gamma RII gene augmented the tumorigenicity and metastatic phenotype of the transfectants compared to control transfectants.

Diagnostic value of computer analysis of multipeaked EMG spikes.

Computer analysis of absolute number of peaks per second and the number of peaks in the multipeaked spikes/sec was carried out in the EMG interference recordings of 5 healthy, 6 myopathic and 8 neuropathic subjects. The purpose of the study was to detect diagnostically different patterns. A spike was considered multipeaked if it had 6 or more peaks. The amplitude of elimination (baseline) was examined at 1: 5, 1: 10 and 1: 15 of the average amplitude per second. Both the number of peaks and the number of peaks in multipeaked spikes in the neuropathic and myopathic muscles showed statistically significant differences when compared to healthy muscles. This technique could give an indication for the differential diagnosis of myopathic, neuropathic or healthy state of the muscle.

In vivo tumorigenicity and in vitro sensitivity to tumor-necrosis-factor alpha mediated killing of c-Ha-ras-transformed cells.

Cellular subclones of high and low tumorigenicity obtained from a mouse c-Ha-ras-transformed clone, were examined for their sensitivity to tumor-necrosis-factor (TNF)-mediated cytotoxicity. Cells of the highly tumorigenic subclones showed a significantly enhanced resistance to the cytotoxic effect of TNF plus cyclohexamide (CHI) as compared to cells of the low-tumorigenic subclones. The enhanced resistance to TNF+CHI was not due to a lower expression of TNF receptors on the cells. The c-Ha-ras-transfected cells were transformed and maintained in culture only (C cells). In vivo passage of cells of the initially low-tumorigenic c-Ha-ras subclones through the mouse significantly enhanced the tumorigenic potential of these CTC cells (culture/tumor/culture). In correlation with their enhanced tumorigenicity, the CTC cells were highly resistant to TNF-mediated cytotoxicity as compared to C cells of the same subclone. Furthermore, the involvement of TNF in determining the tumorigenic phenotype of the c-Ha-ras-transformed cells was demonstrated in a more direct manner. Cells of a c-Ha-ras-transformed low-tumorigenic, highly TNF-sensitive subclone were selected by repeated cycles of in vitro exposure to TNF alpha. As a result, a stable, highly TNF-resistant population of cells emerged. These TNF-resistant cells caused more tumors in mice as compared to their original TNF-sensitive cells. These results show that the resistance to the cytotoxic effect of TNF plus cyclohexamide may be involved, at least partially, in the tumorigenic potential of c-Ha-ras-transformed cells and suggest a possible role for TNF in the enhancement of the tumorigenic potential of these cells in mice.

Fourier series analysis of the electrophysiological pattern of fatigue in healthy human beings, after curare administration.

Real time computer analysis of the electrophysiological development of muscular fatigue after small doses of d-tubocurarine (TC), has been examined in anesthetized human beings. As compared to a decrease of frequency in the control measurements, previous studies have shown an increase of the frequency of spikes after TC administration. The present experiments were carried out on the biceps brachii of 8 healthy human volunteers maintained in isometric contraction against a constant counter load until complete fatigue occurred. The Fourier spectrum analysis showed a statistically significant shift to lower frequencies before, and a milder statistically non significant shift after TC. These results may indicate that under mild curarization the early phase of muscular contraction requires a higher number of large motor units and thus, at a later stage of the contraction the pool of available large motor units becomes smaller. This conclusion supports the hypothesis that mild curarization causes a state of initial muscular fatigue.

Manometry and electromyography of the pharyngeal muscles in patients with dysphagia.

Simultaneous recording of the electromyographic activity of the pharyngeal muscles and the intraluminal pressure in the upper sphincter zone was performed routinely in patients with swallowing problems for the first time, to our knowledge. This technique was found to be very useful for the localization of the "site of lesion." The procedure is safe, easy to master, and causes minimal inconvenience. It can reveal, in the most direct way, whether the disturbance is in the hypopharyngeal musculature (represented by the inferior constrictor muscle), in the cricopharyngeal muscle (spasm or lack of relaxation), or in the synchronization between them. Simultaneous recording of intraluminal pressure adds valuable information about the mechanical events associated with electromyographic activity. It was found that in pathologic cases there is quite often no correlation between the electrical and mechanical events. Thus, simultaneous recording of both electrical and mechanical events is essential for the understanding of the pathophysiology of disturbances of deglutition.

Electromyography of the inferior constrictor and cricopharyngeal muscles during swallowing.

Electromyography (EMG) of the inferior pharyngeal constrictor (IC) and the cricopharyngeal (CP) muscle was recorded in 18 patients with swallowing and/or aspiration problems who were candidates for cricopharyngeal myotomy. The EMG recordings were compared to those of 13 "normal" subjects who did not suffer from such problems. Differences in EMG activity between the control group and the patient group were considered with respect to the clinical symptoms in the patient group. Recording of EMG in the CP and IC muscles is relatively safe, useful, and easily mastered. The technique may provide important information regarding the function of some of the muscles involved in deglutition.

Comparison of the electrophysiological pattern of fatigue between athletes required to perform explosive and endurance sports.

The electrophysiological behavior of an isometric contraction sustained to fatigue, was examined in 6 long distance runners and 9 athletes involved in explosive (burst) sports, by on line computer analysis of the electrical activity of vastus medialis, rectus femoris and vastus lateralis. The experiments were carried out with a counterload of 50% of the maximal strength of the muscle. The duration of spike increased and the frequency decreased in the 3 examined muscles, in both types of sport. In the burst sports the changes of value of both parameters were statistically significant in the 3 muscles. In endurance sports the variations of duration were not significant and the changes of frequency were statistically significant only in the vastus lateralis. These results could be explained by the gradual activation of motor units of more strikingly different sizes in burst sports. Thus it may be speculated that prolonged training in burst sports may result in the automatic mobilization of higher number of small motor units, for the initiation of contraction while in endurance sports the onset of contraction is more gradual and carried out by large motor units.

Effect of filter setting on the electromyographic parameters of muscles contracting to fatigue.

The effects of various filter settings on the electrophysiological behavior of the development of muscular fatigue were studied. Eleven healthy volunteers were examined during isometric contraction of biceps brachii and rectus femoris against a constant load until fatigue occurred. The electrical activity was taped and computer processing was carried out at the basic setting of 15-5000 cycles and at low (15-200 Hz) and high (200-5000 Hz) frequence filter. The results support the hypothesis that in the low range of frequencies there is a high density of large slow motor units, while in the high range of frequencies there are numerous small fast motor units.

Some immunological properties of high and low tumorigenic cellular variants of c-H-ras transformed 3T3 cells.

NIH 3T3 cells transformed in vitro with the c-H-ras oncogene were subcloned. The resulting subclones were assayed for in vivo tumorigenicity in nude and in immunocompetent mice. The response of two high tumorigenic and two low tumorigenic clones to mediators of natural immunity was analyzed. The clones did not differ in sensitivity to NK cell-mediated lysis. However, compared to low tumorigenic clones, the high tumorigenic ones had a down-regulated expression of a membrane determinant recognized by a certain monoclonal naturally occurring antibody. The determinants recognized by other monoclonal naturally occurring antibodies available in the laboratory were equally expressed on the high and low tumorigenic clones. The high tumorigenic cells showed an increased resistance to cytotoxicity mediated by lymphotoxin. These results suggest that naturally occurring antibodies and lymphotoxin may participate in controlling the tumorigenicity of transformed cells. The high tumorigenic clones but not the low tumorigenic ones contained a novel 3.5-kb ras mRNA.

Low density lipoprotein (LDL) inhibits histamine release from human mast cells.

We examined the effect of low density lipoprotein (LDL) on histamine release from purified human lung mast cells. LDL inhibited anti-IgE- induced histamine release in a dose-dependent manner, with 100 micrograms/ml LDL-protein inhibiting histamine release by 53 +/- 8% (mean +/- SEM); half-maximal inhibition occurred at 40-80 micrograms/ml. LDL also inhibited calcium ionophore A23187-induced histamine release in a dose-dependent manner, with 1 mg/ml of LDL inhibiting histamine release by 83 +/- 9%; half maximal inhibition occurred at 220-280 micrograms/ml. Inhibition by LDL was time-dependent: half-maximal inhibition of anti-IgE- induced histamine release by LDL occurred at 30-50 minutes of incubation. The inhibitory effect of LDL was independent of buffer calcium concentrations (0-5 mM) or temperature (0-37 degrees C). These data are consistent with a newly defined immunoregulatory role for LDL.

Immunogenicity of malondialdehyde-modified low density lipoproteins. Studies with monoclonal antibodies.

Malondialdehyde (MDA)-modified low density lipoprotein (LDL) can stimulate the accumulation of cholesteryl esters in cultured macrophages through its interaction with specific scavenger receptors. It has been speculated that such interaction occurs in vivo thus contributing to the formation of foam cells within atherosclerotic lesions. This report describes the development of new tools in the form of a specific assay for MDA-LDL to investigate this hypothesis. We have immunized BALB/c mice with malondialdehyde mouse low density lipoproteins and antibodies against malondialdehyde human low density lipoproteins were generated. Monoclonal antibodies were produced using hybridoma techniques and one particular clone (EB 7-3) was expanded for further studies. The immunoreactivity of several antigens was tested using antibody EB 7-3 in an enzyme-linked immunosorbent assay (ELISA). In a typical assay malondialdehyde human LDL (with at least 40% of lysines modified) was coated (2 micrograms/ml, 100 microliter) in 96-well microtiter plates. Antibody plus one of several antigens were then added and the interaction between the antibody and coated antigen was measured using alkaline phosphatase-conjugated affinity purified goat anti-mouse immunoglobulin. The binding of antibody EB 7-3 to wells coated with malondialdehyde-LDL was competitively inhibited by malondialdehyde-LDL added in solution, with half maximal inhibition occurring at 150 +/- 80 ng/ml. In addition, the ability of malondialdehyde-LDL to inhibit this interaction was proportional to the degree of modification: the more lysines were modified the more did malondialdehyde-LDL inhibit the binding of antibody EB 7-3 to coated malondialdehyde-LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme linked immunosorbent assay for the measurement of nonenzymatically glucosylated proteins in serum and in tissues.

We have developed an enzyme linked immunosorbent assay (ELISA) for glucosylated proteins. The polyclonal antiserum was prepared against reduced glucosylated lipoproteins and was specific for the glucose-lysine bond. The antiserum recognized, in a dose-dependent manner, all reduced glucosylated proteins tested, including albumin, fibrinogen, low density lipoprotein, high density lipoprotein and hemoglobin, yet had no affinity for native proteins or to glucosylated but nonreduced proteins. The sensitivity of the assay was in the order of 1-5 pmol glucosylated lysine/ml and half maximal displacement occurred at 8-24 pmol glucosylated lysine/ml. The inter- and intraassay variables were 10.8% and 13.5%, respectively. Serum proteins from diabetic patients (n = 30) contained 84 +/- 6 picomoles of glucosylated lysine/mg protein, compared to 28 +/- 3 in controls (n = 20), and the concentration of glycosylated proteins correlated with fasting blood glucose (r = 0.56, p less than 0.02), but not with glucosylated hemoglobin levels (r = 0.29, p greater than 0.1). Proteins from diabetic glomeruli and aortae similarly contained more glucosylated lysine residues than controls.