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To examine the ability of normal and autoimmune individuals to produce circulating anti-TNF alpha antibodies, plasma samples from 10 RA patients, 10 SLE patients and 5 normal subjects were assessed for anti-TNF alpha antibody. While every individual tested demonstrated circulating IgM anti-TNF alpha antibody, IgG anti-TNF alpha autoantibody was seen predominantly in autoimmune patients. Only 1 of 5 normal individuals, but 15 of 20 autoimmune individuals had plasma IgG anti-TNF alpha antibodies. To examine the ability of normal and autoimmune individuals to produce anti-TNF alpha autoantibody from their circulating lymphocytes, EBV transformation was performed. Oligoclonal immortal cell lines were successfully established from 13 patients and each one secreted detectable IgM anti-TNF alpha autoantibody. Transformed cells from only 1 of 5 normal individuals secreted IgM anti-TNF alpha autoantibody. These results indicate a higher prevalence of anti-TNF alpha autoantibody production among autoimmune individuals although normal individuals are also capable of producing these autoantibodies.

Neonatal myocardium is distinctly more sensitive to extracellular calcium levels than is mature myocardium. This has been ascribed to the poorly developed sarcoplasmic reticulum of neonatal myocardium. Recent evidence has suggested that there is an increased dependence of neonatal myocardium on the sodium-calcium exchange current, and that sodium-calcium exchange may be a major source of calcium influx in neonatal myocardial cells. We determined the effect of halothane on the sodium-calcium exchange current on single neonatal (2- to 5-day-old) rabbit ventricular myocytes by means of the whole cell voltage clamp. Lower (1.5%) halothane decreased sodium-calcium exchange current by 49%, from 29 +/- 3 to 15 +/- 6 pA. Higher (3%) halothane decreased this current by 66%, from 50 +/- 9 to 17 +/- 9 pA. Thus halothane has a reversible inhibition of sodium-calcium exchange current in neonatal myocardium. Inhibition of sodium-calcium exchange current would be expected to have a magnified effect on contractility in neonatal as opposed to adult myocardium, and could theoretically ameliorate reperfusion injury due to influx of calcium via the sodium-calcium exchanger.

We evaluated the effects of clinically relevant concentrations of halothane (1%) and ketamine (10(-4) M) on activation, inactivation, and recovery from inactivation of voltage-gated sarcolemmal calcium current (ICa) in single guinea pig ventricular myocytes, using the whole cell voltage clamp. Both anesthetics had qualitatively similar effects. The potential at half-activation was shifted from -18 to -23 mV for halothane (p < 0.03) and from -17 to -21 mV for ketamine (p = 0.005). There was no change in the slope of the activation curve for either anesthetic. The potential at half-inactivation was shifted from -29 to -40 mV with exposure to halothane (p < 0.001) and from -27 to -33 mV (p < 0.001) with exposure to ketamine. There was no change in the slope of the inactivation curve with either agent. The changes in time constant of recovery from steady-state inactivation with halothane did not reach statistical significance (178 vs. 207 ms, p = 0.20) and was significantly prolonged with exposure to ketamine (106 vs. 157 ms, p = 0.005). The two anesthetics show parallel shifts in activation, inactivation, and recovery from inactivation of ICa in ventricular myocardial cells. These findings in normal ventricular myocytes may help interpret the interactions of these anesthetics with other types of heart muscle, such as ischemic and immature myocardium.

Several monoclonal human IgM antibodies to recombinant human tumor necrosis factor-alpha (rhTNF alpha) have been generated and partially characterized. The F78-1A10-B5 monoclonal antibody (mAb) (B5) binds to rhTNF alpha with a titer comparable to three high-affinity neutralizing mouse mAbs, when tested by ELISA. However, the B5 mAb binds relatively weakly to soluble rhTNF alpha. It appears to bind to epitopes on rhTNF alpha distinct from those bound by the mouse mAbs for three reasons. First, preincubation of plate-bound rhTNF alpha with mouse mAbs does not decrease or compete subsequent B5 mAb binding. Second, rhTNF alpha complexed to the mouse mAbs can still be bound by B5 mAb. Third, the mouse mAbs neutralize TNF alpha cytotoxicity whereas the B5 mAb does not. Binding analyses indicate that this human IgM autoantibody binds to both human and mouse recombinant TNF alpha, but not to other antigens commonly recognized by polyreactive natural IgM autoantibodies. The high level of amino acid identity between the human and mouse TNF alpha molecules suggest that the B5 mAb is monospecific for a given epitope shared by these two forms of TNF alpha. This spectrum of characteristics makes B5 a novel mAb.

A human IgM monoclonal antibody (B5) recognizing human TNF alpha was established from peripheral blood lymphocytes by transformation with Epstein-Barr virus and subsequent cell fusion. The B5 monoclonal antibody (mAb) binds to cell surface TNF alpha (csTNF alpha) on human T cells, B cells, and monocytes. In addition, this autoantibody binds to csTNF alpha on a variety of lymphoid and monocyte lineage cell lines of human origin, as well as astrocytomas, a breast carcinoma, and a melanoma. Interestingly, the B5 mAb also binds to chimpanzee lymphocytes and to mouse T lymphoma cell line csTNF alpha. Many neutralizing mouse anti-TNF alpha mAbs do not exhibit comparable binding to csTNF alpha. This is consistent with the previous demonstration that B5 recognizes an epitope on TNF alpha distinct from those recognized by three neutralizing mouse anti-TNF alpha mAbs. B5 binding to csTNF alpha is specific since it can be inhibited by TNF alpha. No inhibition of B5 binding was seen by a neutralizing mouse anti-TNF alpha mAb. The B5 autoantibody appears to recognize the transmembrane form of TNF alpha and most likely also recognizes TNF alpha associated with its receptor. The unique specificity of this B5 autoantibody provides some additional insight into the complex physiology of cell surface-associated TNF alpha.

The properties of the ATP-sensitive potassium (KATP) current were studied in freshly isolated rabbit ventricular myocytes using the patch clamp technique. Removing ATP from the bath (intracellular) solution activated a large K+ conductance in patches from neonatal cells with properties similar to those of KATP channels in other preparations. In membrane patches from neonatal ventricular myocytes, the density of KATP channels was higher than the density of inwardly rectifying K+ channels and the mean patch KATP current was approximately 10 times that of the inwardly rectifying K+ current, at a patch membrane potential of -60 mV. Glibenclamide (10 microM) in the bath solution decreased the number of functional KATP channels, the open-state probability, and the mean patch membrane current. The single-channel conductance of the KATP channel was dependent on the external K+ concentration, and the relationship between channel conductance and external K+ concentration was fit by an exponential equation. In addition, the voltage dependence, channel density, and open-state probability of this channel were compared between neonatal and adult isolated ventricular myocytes. The single-channel conductance and channel density of the KATP channel in neonatal myocytes were significantly smaller than in adult cells. These results suggest that age-related changes occur in the properties of KATP channels.

Recent studies suggest that amiodarone's acute clinical effects in infants and children are related predominantly to its class I antiarrhythmic activity. However, the effects of amiodarone on Na+ currents have not been investigated directly in immature cardiac cells. Accordingly, the tight seal whole cell voltage clamp technique was used to measure time- and voltage-dependent Na+ currents in acutely isolated neonatal ventricular myocytes from 2- to 5-day-old rabbits, before and after addition of amiodarone (0.1-10 microM). To evaluate the class I antiarrhythmic activity of amiodarone in this age group, the effects of amiodarone on Na+ currents were compared with those of procainamide. Similar to procainamide, amiodarone significantly decreased peak inward Na+ current in neonatal ventricular myocytes. Moreover, both amiodarone and procainamide shifted the steady-state inactivation curve to more negative membrane potentials and delayed recovery of the Na+ current from inactivation. Thus, the effects of amiodarone on the Na+ current in immature myocardium are qualitatively similar to those of procainamide, suggesting that amiodarone may act acutely as a class I antiarrhythmic agent in the newborn heart.

Experiments were performed to examine the nature of agents which could induce IL-5 responsiveness in small, resting splenic B lymphocytes. First, IL-5 increased plaque forming cell responses to the TI-1 antigen TNP-LPS. A second set of experiments using anti-IgM + LPS which allowed limiting dilution analysis showed induction of IL-5 responsiveness in about 20% of the resting B cell population. In the same system, IL-4 increased the percentage of proliferating cells by about 40%. A third system using the TI-2 analog conjugate anti-IgD-dextran (anti-delta-dextran) also rendered small, resting B cells responsive to IL-5. An additional system employing anti-IgM plus dextran sulfate, which also allowed limiting dilution analysis, induced IL-5 responsiveness in at least 10% of resting B cells. The features common to all four systems inducing B cell IL-5 responsiveness are at least twofold. Each system directly accesses the B cell antigen receptor and causes crosslinking. Second, each system also provides an additional polyclonal activating moiety, some of which may be similar to those in thymus independent antigens. These results suggest that some resting B cells may become IL-5 receptive after perception of at least two kinds of signals one of which perturbs sIg and the second being nonspecific and polyclonally activating.

In addition to its ability to enhance antibody secretion, Interleukin 5 (IL-5) enhances murine B lymphocyte proliferation. This so-called growth factor activity has been amply demonstrated by many laboratories assessing thymidine incorporation or cell recovery. Attempts to actually quantitate the fraction of fresh splenic B cells responding to IL-5, by limiting dilution analysis or other means, with few exceptions have yielded disappointingly small numbers--generally between 1 and 5%, or perhaps less. We have recently identified the peritoneal cavity as a reservoir rich in IL-5-responsive B cells. In this report, we provide independent corroboration of this high IL-5 reactivity by means of cell cycle analysis. Low-density peritoneal B cells, more than 90% of which are in G0 and G1 phases, were stimulated with polyclonal activators in the presence of mitotic inhibitors. Frequencies of IL-5-responsive B cells were measured by observing the differences in the proportions of cultured cells entering S and later phases in the presence, compared to the absence, of IL-5. Some 10 to 20% more of the low-density peritoneal B cells from normal mice entered S phase when IL-5 was present with LPS + DXS. A similar IL-5-mediated elevation in the frequency of S phase entry was seen with peritoneal B cells from the autoimmune mouse strain NZB. Furthermore, a measurable fraction of peritoneal B cells from these mice were even capable of responding to IL-5 alone. These IL-5-induced increases could be blocked by anti-IL-5 mAb. About 30% of the BCL1 leukemic B cell line initiated DNA replication when stimulated with IL-5 alone. Hence, IL-5-responsive B cell fractions have been measured for some normal, autoimmune strain and transformed leukemic B cell phenotypes. In addition to quantitating the proportion of IL-5-responsive B cells, these experiments formally demonstrate that IL-5 can act in the G1 phase to increase S phase entry.

Single sucrose gap and isolated myocyte voltage-clamp techniques were used to study the effects of diltiazem on calcium current (ICa) and tension generation in isolated ventricular myocytes and right ventricular papillary muscles from neonatal New Zealand White rabbits. Diltiazem was shown to significantly shorten the duration of isolated myocyte action potentials with no effect on overshoot potential or resting membrane potential. Diltiazem blocked but did not completely abolish ICa in these neonatal cells. Addition of diltiazem to the solution bathing papillary muscles resulted in a similar reduction in action potential duration accompanied by a reduction in twitch tension. When the duration of depolarization was controlled employing the single sucrose gap voltage clamp, the decrease in tension development caused by diltiazem was abolished despite a significant decrease in twitch tension in the same muscles. In another series of experiments it was demonstrated that the magnitude of developed tension in neonatal papillary muscles is dependent upon the duration of depolarization. Taken together, the results of this investigation suggest that in neonatal myocardium when ICa is blocked by diltiazem, the resulting reduction in developed tension is caused in part by reduction of action potential duration. The calcium carried into the neonatal heart cell by ICa does not appear to be the only source of extracellular Ca2+ for excitation-contraction coupling. Finally, the action potential appears to act as a gate for calcium movement into the neonatal heart cell.

Action potentials and voltage clamp-induced ionic currents were recorded in acutely isolated neonatal rabbit cardiac myocytes using the whole-cell voltage clamp technique. Time- and voltage-dependent Ca2+ currents in neonatal myocytes were elicited by depolarizations from a holding potential of -80 mV to various clamp potentials. The maximal measured inward Ca2+ current was 206 +/- 10 pA (mean +/- SEM, n = 51). The peak current occurred at a mean membrane potential of 7.8 +/- 1.3 mV (n = 51). The Ca2+ current voltage relation was shifted 26 mV in the positive direction when the external Ca2+ concentration was increased 10-fold. Ca2+ current rundown was observed with a half-time of approximately 20 min. Cells dialyzed with solution containing the Ca2+ chelating agent, EGTA (0.04 mM), had action potential durations similar to those previously reported in papillary muscle. In contrast, a higher concentration of EGTA (14 mM) prolonged the action potential duration. Control of the cell internal ionic composition was achieved by dialysis of the cell with a time constant for Na+ ions of 1.2 to 2.6 min. Tetrodotoxin (10 microM), included in some experiments to block Ca2+ entry via Na+ channels, was shown to be more than 98% effective. These results characterize the whole-cell voltage clamp technique as applied to immature heart cells.

We have compared transsarcolemmal Ca2+ currents in acutely isolated neonatal (1- to 5-d-old) and adult rabbit cardiac myocytes prepared using similar enzymatic techniques. Time- and voltage-dependent inward Ca2+ currents were measured using the whole-cell voltage clamp technique. In neonatal myocytes, peak Ca2+ currents measured 114 +/- 10 pA (mean +/- SEM, n = 18) as compared with 2014 +/- 403 pA in adult myocytes (n = 5, p less than 0.001). Although adult myocytes had a larger surface area (estimated from cell capacitance) than neonatal cells (113 +/- 15 x 10(-6) versus 28 +/- 2 x 10(-6) cm2, p less than 0.001), the calculated peak current density was also significantly larger in adult cells (17.9 +/- 2.5 compared to 4.3 +/- 0.4 microA/cm2 for neonatal cells, p less than 0.001). The voltage dependence of the peak Ca2+ current was similar in neonatal and adult myocytes. Early transient (T-type) Ca2+ currents were also studied by comparing the current induced by depolarization to -20 mV from holding potentials of -40 and -80 mV. T-type Ca2+ channels were present in 91% of the adult cells but were evident in only 39% of the neonatal cells. In summary, voltage-gated Ca2+ current amplitude, current density, and T-type Ca2+ channel prevalence all increase with maturation. These data suggest that neonatal myocytes may be relatively deficient in Ca2+ channel activity when compared to adult myocytes.

Properties of the inwardly rectifying K+ channel, which contributes to the maintenance of the resting membrane potential, were studied in neonatal rabbit ventricular myocytes using the patch-clamp technique. Inward rectification was evident in single-channel current-voltage (I-V) relations at potentials positive to the potassium equilibrium potential (Ek = 0 mV with [K+]o = [K+]i = 150 mM, [Mg2+]i = 2 mM). The single-channel conductance was 3.2 +/- 0.1 pS in physiological (5.4 mM) [K+]o. The zero-current potential shifted 48.4 +/- 2.4 mV for a ten-fold change in [K+]o in neonatal cells. External Ba2+ blocked the current in a dose-dependent manner. The voltage dependence, open-state probability and channel density of this channel were compared between neonatal and adult ventricular myocytes isolated by similar techniques. The open-state probability of the channel was approximately the same in neonatal (0.39 +/- 0.06, n = 13) as in adult cells (0.4 +/- 0.05, n = 11). However, in symmetrical transmembrane K+ concentration [( K+]o = [K+]i = 150 mM), the single channel conductance was significantly smaller in neonatal (25 +/- 0.3 pS, n = 25) as compared with adult cells (31 +/- 0.4 pS, n = 12). In addition, the relationship between resting membrane potential and [K+]o was measured in neonatal and adult myocytes. The resting membrane potential in the neonate was less dependent on [K+]o than in the adult. These results are consistent with an age-related change in resting membrane K+ permeability which may result from a developmental change in the single-channel conductance properties of the inwardly rectifying K+ channel.

In this paper we show the presence of a B cell growth-promoting activity in T cell replacing factor (TRF) supernatants from a monoclonal T cell line and polyclonally activated splenic T cells. The target cell of this activity is indisputably shown to be the B cell, which indicates that T cell-derived factors can act directly on B cells. The effect of monoclonal TRF-containing supernatant from the C.C3.11.75 Dennert cell line, (DL)TRF, which demonstrates B cell growth-promoting activity, is to increase the frequency of B cell clones stimulated by mitogens as opposed to increasing B cell clone sizes. (DL)TRF B cell growth enhancement is observed when B cells are activated by fetal calf serum mitogens, lipopolysaccharide (LPS), dextran sulfate (DXS), or LPS + DXS. The growth-promoting activity of (DL)TRF appears to be that of a costimulator rather than a classical growth factor because (DL)TRF alone is not sufficient to maintain clonal growth of activated B lymphoblasts.

Interferon-gamma (IFN-gamma) exerts a broad spectrum of activities which affect the responses of mature B-cells. It strongly inhibits B-cell activation, acts as a B-cell growth factor (BCGF), and also induces final differentiation to immunoglobulin (Ig) production. IFN-gamma is deeply involved in the differential control of isotype expression, as it enhances IgG2a production and suppresses both IgG1 and IgE production. Although it is now possible to draw a general scheme of the effects of IFN-gamma on B-cells, a number of paradoxical results still exist in the field. In this manuscript, different experimental systems are analyzed in an attempt to explain these apparent paradoxes.

The driving force for active transport of Na+ in the isolated toad bladder, ENa, was measured as the reciprocal slope of the change in conductance with change in short-circuit current after stimulation with antidiuretic hormone. The base-line short-circuit current was altered by change in ambient Na+ concentration or addition of amiloride, maneuvers which alter availability of Na+ at the site of active transport. In the absence of a chemical gradient for Na+ across the bladder, ENa was found to be inversely related to the rate of Na+ transport, a finding incompatible with the simple electrical analogue that has been proposed for the system. The results provide additional support for the view that ENa measured in this way has both energetic and kinetic components.

The value of intrapartum tcpO2 measurement is discussed, with reference being made to clinical examples. Also mentioned are certain setbacks of the method which, at present, still are limiting factors to general clinical intrapartum use.

An accurate description of the coronary anatomy is desired before anatomic correction of d-transposition of the great arteries. Two-dimensional echocardiography and aortic root angiography are occasionally unable to delineate the coronary arteries. In neonates, the arterial approach for selective coronary arteriography is difficult and may compromise visceral and lower limb perfusion. A venous technique was used in two newborns to define selectively the coronary origins and their distribution. The venous approach for selective coronary arteriography may be effective in the evaluation of newborns with d-transposition of the great arteries when echocardiography inadequately defines the coronary circulation.

The in vitro effects of recombinant interleukin (IL) 5 on proliferation and maturation of mouse Ly-1 B cells were studied. Most freshly isolated peritoneal Ly-1 B cells expressed high levels of IL5 receptor (R). Limiting dilution analyses showed that mitogens could reveal IL5 responsiveness in more than half of low density peritoneal Ly-1 B cells. IL 5 was able not only to increase the proportion of these Ly-1 B cells induced to proliferate, but it also shifted the clone size distribution of proliferating cells towards larger clone sizes. Splenic Ly-1 B cells also proliferated in response to mitogens plus IL5. Spontaneous and polyclonal activator-induced plaque-forming cell responses of Ly-1 B cells were increased by IL5. Furthermore, IL5 increased the frequency of peritoneal Ly-1 B cells induced to secrete certain autoantibodies. IL5 was certainly the agent responsible since its effects on both proliferation and differentiation were inhibited by either anti-IL5R monoclonal antibodies or by anti-IL5 monoclonal antibodies. Hence, Ly-1 B cells, IL5 and the IL5R appear to constitute a system of cellular proliferation, differentiation and some autoantibody production. Strategies specifically targeting the interleukin and receptor elements of this system might afford external control of these cellular responses.