Bryostatin/ionomycin-activated T cells mediate regression of established tumors.

We have shown that adoptive transfer of tumor-sensitized lymphocytes activated in vitro with bryostatin-1 and ionomycin (B/I), and expanded in culture, can induce regression of small established tumors. We set out to determine whether similar treatment would be effective against larger tumors and what cells mediate this effect. We also attempted to shorten the ex vivo culture period with the ultimate aim of developing a more clinically useful protocol. BALB/c mice were injected in one footpad with IL-2-transfected 4T07 mammary tumor cells. Ten days later, popliteal draining lymph nodes (DLN) were harvested and activated with B/I for 18 h. Mice with either 3-day or 10-day 4T07 flank tumors were treated with cyclophosphamide (100 mg/ kg ip, CYP) alone or CYP followed the next day by infusion of either B/I-activated lymphocytes transferred immediately or activated cells that had been expanded in vitro for 3 or 10 days. In some experiments, mice were also treated with rat anti-mouse CD4 monoclonal antibody (GK1.5) or anti-CD8 antibody (2.43). All mice receiving CYP alone or CYP + sensitized, nonactivated DLN cells demonstrated progressive tumor growth. One hundred percent (6/6) of mice treated with CYP + AIT with B/I-activated,10-day expanded cells had complete regression of 3-day flank tumors. Treatment with activated, nonexpanded cells, induced tumor regression in a majority of mice, but was not as reliable as AIT with expanded cells. We developed a protocol with a shortened expansion period (3-day) that was efficacious for treatment of 4T07 when adoptively transferred to either 3 or 10 day tumor-bearing mice. In vivo depletion of CD4(+) cells had no effect on regression of 3-day tumors, but treatment with anti-CD8 antibody abrogated the effect of immunotherapy. Adoptive transfer of B/I-activated cells, with or without long-term expansion, induced regression of early and late stage 4T07 tumors and is dependent on CD8(+) but not CD4(+) T cells.

Systems neuroscience: the slowly sleeping slab and slice.

The physiological functions of structured spontaneous activity in the brain, such as the slow oscillations characteristic of certain sleep states, remain unclear, but new studies using brain slices or slabs are starting to shed light on the underlying neural mechanisms.

The computation of directional selectivity in the retina occurs presynaptic to the ganglion cell.

Directional selectivity is a response that is greater for a visual stimulus moving in one (PREF) direction than for the opposite (NULL) direction, and its computation in the vertebrate retina is a classical issue in functional neurophysiology. To date, most quantitative experimental studies have relied on extracellular responses for identifying properties of the directionally selective circuit. Here I describe an intracellular analysis using whole-cell patch recordings of the synaptic events underlying the spike response in directionally selective ganglion cells of the turtle retina. These quantitative measurements allowed me to distinguish among various explicit classes of circuit models that can, in principle, account for ganglion cell directional selectivity. I found that ganglion cell directional selectivity is due to an excitatory input that itself is directionally selective, and that the crucial shunting inhibition implicated in this computation must act on cells presynaptic to the ganglion cell.

Protective role of IL-2 during activation of T cells with bryostatin 1.

Pharmacologic agents such as bryostatin 1 (bryostatin) can regulate cell activation, growth, and differentiation by modulating the activities of protein kinase C isoenzymes. Inhibition of growth of tumor cells and activation of T lymphocytes in vitro are the most recognized consequences of bryostatin treatment. The effect of bryostatin on T cells ranges from induction of apoptotic cell death to T cell activation, expansion, and acquisition of antigen-specific effector functions. Here, we describe the conditions under which these wide ranging effects occur. Mouse mammary tumor 4TO7-IL-2-primed lymph node cells exposed ex vivo to bryostatin upregulated CD25 expression but lost the ability to secrete IL-2. Most of these cells died by apoptosis unless IL-2 was provided for the duration of bryostatin treatment. Analysis of T cell repertoire by screening of T cells for the expression of different Vbeta T cell receptor (TCR) families revealed that bryostatin-induced T cell death was unbiased and Vbeta-nonspecific. Within particular Vbeta clones, only CD25(+) T cells survived exposure to bryostatin and IL-2. Treatment of 4TO7 tumor-bearing mice with a single injection of low dose bryostatin followed by multiple low doses of IL-2, but not with bryostatin alone, delayed tumor growth. These results indicate that activation of T cells with bryostatin should be carried out under protection of exogenous IL-2 to ensure survival and expansion of T cells that may exhibit anti-tumor activity.

Additional efficient computation of branched nerve equations: adaptive time step and ideal voltage clamp.

Various improvements are described for the simulation of biophysically and anatomically detailed compartmental models of single neurons and networks of neurons. These include adaptive time-step integration and a reordering of the circuit matrix to allow ideal voltage clamp of arbitrary nodes. We demonstrate how the adaptive time-step method can give equivalent accuracy as a fixed time-step method for typical current clamp simulation protocols, with about a 2.5 reduction in runtime. The ideal voltage clamp method is shown to be more stable than the nonideal case, in particular when used with the adaptive time-step method. Simulation results are presented using the Surf-Hippo Neuron Simulation System, a public domain object-oriented simulator written in Lisp.

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCgamma1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed.

Functional independence and interdependence of the Src homology domains of phospholipase C-gamma1 in B-cell receptor signal transduction.

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line. PLCgamma1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCgamma1 with the adapter protein, BLNK. Forcing PLCgamma1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCgamma1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCgamma1 activation.

Phenylephrine induces delayed cardioprotection against necrosis without amelioration of stunning.

BACKGROUND: Alpha-adrenergic stimulation induces protection in reperfused ischemic (I/R) myocardium 24 hours later. We tested the hypothesis that phenylephrine improves dysfunction after global I/R by limiting cell death not stunning. METHODS: Rabbits were pretreated with either phenylephrine or vehicle. Twenty-four hours later, isolated hearts underwent either 45 (infarction protocol) or 20 minutes (stunning protocol) of global ischemia before 2 hours of reperfusion (n = 6 per group). Cell death was determined by triphenyl tetrazolium chloride staining (infarction) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (apoptosis). RESULTS: Compared with vehicle, phenylephrine pretreatment improved post-I/R-developed pressures in hearts after infarction (53.2 +/- 4.0 vs 35.8 +/- 4.1 mm Hg, p = 0.01) but not stunning protocol (64.3 +/- 8.9 vs 57.7 +/- 6.2 mm Hg, p = NS). The improved developed pressure was due to better diastolic recovery. Systolic pressures were similar between groups. Phenylephrine markedly decreased infarction (9.0 +/- 1.9% vs 40.8 +/- 1.8% for vehicle, p < 0.001) and TUNEL-positive staining. Stunned hearts of either group had less than 3% infarction and no apoptosis. CONCLUSIONS: Phenylephrine pretreatment 24 hours before global I/R improves function by limiting infarction but not stunning.

Delayed myocardial preconditioning by alpha1-adrenoceptors involves inhibition of apoptosis.

OBJECTIVE: Previous studies have demonstrated that alpha1-adrenoceptor activation increases myocardial resistance to ischemic injury 24 hours later. Here we tested the hypothesis that delayed protection is associated with limited infarction and involves altered expression of pro-apoptotic and/or anti-apoptotic proteins. METHODS: Rabbits were treated with phenylephrine or an equivalent volume of vehicle (n = 6 per group). Twenty-four hours after pretreatment, isolated hearts were perfused with a bovine erythrocyte suspension in modified Krebs solution, subjected to 45 minutes of global ischemia (37 C), and reperfused for 120 minutes. Infarct size was determined by triphenyltetrazolium chloride staining. Apoptosis was quantified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. Left ventricular tissue from separate groups of animals (n = 5 per group), 24 hours after pretreatment with phenylephrine or vehicle but without ischemia and reperfusion, was analyzed by Western blotting for content of the anti-apoptotic protein, bclx, and pro-apoptotic protein, bax. RESULTS: Isolated hearts after phenylephrine pretreatment had increased end-reperfusion developed pressures (56.8 +/- 4.9 vs 36.2 +/- 3.9 mm Hg for vehicle, P =.008) and decreased elevated end-diastolic pressures (26.7 +/- 4.5 vs 42.3 +/- 5.0 mm Hg for vehicle, P =.04). Phenylephrine pretreatment abrogated infarction (9.9 +/- 2.4% vs 42.6 +/- 6.3% for vehicle, P =.002) and reduced the number of apoptotic nuclei (24 +/- 4.8 vs 51 +/- 4.6 for vehicle, P = .038). Analysis by Western blotting showed that the ratio of bclx to bax protein increased in phenylephrine-pretreated hearts (2.65 +/- 0.5 vs 1.0 +/- 0.1 for vehicle, P =.008). CONCLUSION: Delayed myocardial protection to infarction mediated by alpha1-adrenoceptor activation involves an increased bclx/bax ratio, thereby limiting apoptotic cell death.

Acute brain death alters left ventricular myocardial gene expression.

OBJECTIVES: The depressed myocardial function observed in brain dead organ donors has been attributed to massive sympathetic discharge and catecholamine cardiotoxicity. Because elevated catecholamines are associated with altered myocardial gene expression, we investigated whether acute brain death from increased intracranial pressure alters the expression of myocardial gene products important in contractility. METHODS: A balloon expansion model was used to increase intracranial pressure in rabbits (n = 22). At timed intervals after brain death, mean arterial pressure, heart rate, electrocardiograms, histologic myocardial injury, and systemic catecholamines were assessed. Messenger RNA levels encoding myofilaments, adrenergic receptors, sarcoplasmic reticulum proteins, transcription factors, and stress-induced programs were measured with blot hybridization of total left ventricular RNA. RESULTS: Increased intracranial pressure induced an immediate pressor response that temporally coincided with diffuse electrocardiographic ST segment changes. Systemic epinephrine and norepinephrine levels concurrently increased (5- to 8-fold within 1 minute), then fell below baseline within 2 hours, and remained depressed at 4 hours. By 1 hour, histologic injury was evident. Four hours after the induction of increased intracranial pressure, levels of messenger RNA-encoding skeletal and cardiac alpha-actins, egr-1, and heat shock protein 70 were significantly increased. Sham-operated animals did not exhibit these changes. CONCLUSIONS: Select changes in myocardial gene expression occur in response to increased intracranial pressure and implicate ventricular remodeling in the myocardial dysfunction associated with acute brain death.

A phenomenological model of visually evoked spike trains in cat geniculate nonlagged X-cells.

The visual information that first-order cortical cells receive is contained in the visually evoked spike trains of geniculate relay cells. To address functional issues such as the ON/OFF structure of visual cortical receptive fields with modelling studies, a geniculate cell model is needed where the spatial and temporal characteristics of the visual response are described quantitatively. We propose a model simulating the spike trains produced by cat geniculate nonlagged X-cells, based on a review of the electrophysiological literature. The level of description chosen is phenomenological, fitting the dynamics and amplitude of phasic and tonic responses, center/surround antagonism, surround excitatory responses, and the statistical properties of both spontaneous and visually evoked spike trains. The model, which has been constrained so as to reproduce the responses to centered light spots of expanding size and optimal light and dark annuli, predicts responses to thin and large bars flashed in various positions of the receptive field. The switching gamma renewal process method has been introduced for modelling spontaneous and visually evoked spike trains within the same mathematical framework. The statistical structure of the spike process is assumed to be more regular during phasic than tonic visual responses. On the whole, this model generates more realistic geniculate input to cortex than the currently used retinal models.

Sequences surrounding the Src-homology 3 domain of phospholipase Cgamma-1 increase the domain's association with Cbl.

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency.

Visual input evokes transient and strong shunting inhibition in visual cortical neurons.

The function and nature of inhibition of neurons in the visual cortex have been the focus of both experimental and theoretical investigations. There are two ways in which inhibition can suppress synaptic excitation. In hyperpolarizing inhibition, negative and positive currents sum linearly to produce a net change in membrane potential. In contrast, shunting inhibition acts nonlinearly by causing an increase in membrane conductance; this divides the amplitude of the excitatory response. Visually evoked changes in membrane conductance have been reported to be nonsignificant or weak, supporting the hyperpolarization mode of inhibition. Here we present a new approach to studying inhibition that is based on in vivo whole-cell voltage clamping. This technique allows the continuous measurement of conductance dynamics during visual activation. We show, in neurons of cat primary visual cortex, that the response to optimally orientated flashed bars can increase the somatic input conductance to more than three times that of the resting state. The short latency of the visually evoked peak of conductance, and its apparent reversal potential suggest a dominant contribution from gamma-aminobutyric acid ((GABA)A) receptor-mediated synapses. We propose that nonlinear shunting inhibition may act during the initial stage of visual cortical processing, setting the balance between opponent 'On' and 'Off' responses in different locations of the visual receptive field.

Cbl-mediated regulation of T cell receptor-induced AP1 activation. Implications for activation via the Ras signaling pathway.

The functional role of Cbl in regulating T cell receptor (TCR)-mediated signal transduction pathways is unknown. This study uses Cbl overexpression in conjunction with a Ras-sensitive AP1 reporter construct to examine its role in regulating TCR-mediated activation of the Ras pathway. Cbl overexpression in Jurkat T cells inhibited AP1 activity after TCR ligation. However, AP1 induction by 4beta-phorbol 12-myristate 13-acetate, which up-regulates Ras activity in a protein kinase C-dependent, TCR/tyrosine kinase-independent manner, was not affected by Cbl overexpression. Cbl overexpression also did not affect AP1 induction by an activated Ras protein or a membrane-bound form of the guanine nucleotide exchange factor Sos. In addition, activation of the mitogen-activated protein kinase Erk2 was decreased by Cbl overexpression. Therefore, Cbl regulates events that are required for full TCR-mediated Ras activation, and data are presented to support a model whereby Cbl regulates events required for Ras activation via its association with Grb2.

Is the input to a GABAergic synapse the sole asymmetry in turtle's retinal directional selectivity?

We examined the effects of picrotoxin and pentylenetetrazol (PTZ) on the responses to motions of ON-OFF directionally selective (DS) ganglion cells of the turtle's retina. These drugs are antagonists of the inhibitory neurotransmitter GABA. For continuous motions, picrotoxin markedly reduced the overall directionality of the cells. In 21% of the cells, directional selectivity was lost regardless of speed and contrast. However, other cells maintained their preferred direction despite saturating concentrations of picrotoxin. And in most cells, loss, maintenance, or even reversal of preferred and null directions could occur as speed and contrast were modulated. In 50% of the cells, reversal of preferred and null directions occurred at some condition of visual stimuli. However, picrotoxin did not tend to alter the preferred-null axis for directional selectivity. For apparent motions, picrotoxin made motion facilitation, which normally occurs exclusively in preferred-direction responses, to become erratic and often occur during null-direction motions. Finally, PTZ had effects similar to picrotoxin but with less potency. The results in this paper indicated that models of directional selectivity based solely on a GABAergic implementation of Barlow and Levick's asymmetric-inhibition model do not apply to the turtle retina. Alternative models may comprise multiple directional mechanisms and/or a symmetric inhibitory one, but not asymmetric facilitation.

Practice based health needs assessment: use of four methods in a small neighbourhood.

OBJECTIVE: To explore the use of four methods to define health needs in a community, with a view to formulating guidelines for practice based assessment of health needs. DESIGN: Collection of data on health needs for a specific neighbourhood with four complementary methods: rapid participatory appraisal, postal survey, analysis of routinely available small area statistics, and collation of practice held information. SETTING: Council estate of 670 homes in Edinburgh. MAIN OUTCOME MEASURES: Description and comparison of health needs found by the different methods. RESULTS: Each method yielded particular insights into both health and health care needs. CONCLUSIONS: An extended primary care team with public health support can assess health and health care needs in a neighbourhood by means of a mixture of quantitative and qualitative methods. Different methods may be more suitable to assess different health needs or to explore potential service provision in the community or in primary or secondary care. A composite method may be most informative.