The paradox of alloreactivity and self MHC restriction: Quantitative analysis and statistics

Although 1–24% of T cells are alloreactive, i.e., respond to MHC molecules encoded by a foreign haplotype, it is generally believed that T cells cannot recognize foreign peptides binding foreign MHC molecules. We show using a quantitative model that, if T cell selection and activation are affinity-driven, then an alloreactivity of 1–24% is incompatible with the textbook notion that self MHC restriction is absolute. If an average of 1% of clones are alloreactive, then according to our model, at most 20-fold more clones should, on average, be activated by antigens presented on self MHC than by antigens presented on foreign MHC. This ratio is at best 5 if alloreactivity is 5%. These results describe average properties of the murine immune system, but not the outcome of individual experiments. Using supercomputer technology, we simulated 100,000 MHC restriction experiments. Although the average restriction ratio was 7.1, restriction was absolute in 10% of the simulated experiments, greater than 100, although not absolute, in 29%, and below 6 in 24%. This extreme variability agrees with experimental estimates. Our analysis suggests that alloreactivity and average self MHC restriction both cannot be high, but that a low average restriction level is compatible with high levels in a significant number of experiments.

The unpredictability paradox: review of empirical comparisons of randomised and non-randomised clinical trials

Objective To summarise comparisons of randomised clinical trials and non-randomised clinical trials, trials with adequately concealed random allocation versus inadequately concealed random allocation, and high quality trials versus low quality trials where the effect of randomisation could not be separated from the effects of other methodological manoeuvres.

Mycobacterial infection of macrophages results in membrane-permeable phagosomes

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.

Complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive T-cell clones

T cells recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T-cell recognition of two distinct MHC–antigen complexes is due to similarities in the three-dimensional structure of the complexes. Here we report results of experiments examining the crossreactivity of TCRs recognizing the myelin basic protein peptide MBPp85–99 and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T-cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T-cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR–MHC–peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and its remarkable crossreactivity with different MHC–peptide complexes.

Transforming neural computations and representing time

Motifs of neural circuitry seem surprisingly conserved over different areas of neocortex or of paleocortex, while performing quite different sensory processing tasks. This apparent paradox may be resolved by the fact that seemingly different problems in sensory information processing are related by transformations (changes of variables) that convert one problem into another. The same basic algorithm that is appropriate to the recognition of a known odor quality, independent of the strength of the odor, can be used to recognize a vocalization (e.g., a spoken syllable), independent of whether it is spoken quickly or slowly. To convert one problem into the other, a new representation of time sequences is needed. The time that has elapsed since a recent event must be represented in neural activity. The electrophysiological hallmarks of cells that are involved in generating such a representation of time are discussed. The anatomical relationships between olfactory and auditory pathways suggest relevant experiments. The neurophysiological mechanism for the psychophysical logarithmic encoding of time duration would be of direct use for interconverting olfactory and auditory processing problems. Such reuse of old algorithms in new settings and representations is related to the way that evolution develops new biochemistry.

Consequences of placing an intramolecular crosslink in myosin S1

This paper describes the placement of a crosslinking agent (dibromobimane) between two thiols (Cys-522 and Cys-707) of a fragment, “S1,” of the motor protein, myosin. It turns out that fastening the first anchor of the crosslinker is easy and rapid, but fastening the second anchor (Cys-522) is very temperature dependent, taking 30 min at room temperature but about a week on ice. Moreover, crystallography taken at 4°C would seem to predict that the linkage is impossible, because the span of the crosslinking agent is much less than the interthiol distance. The simplest resolution of this seeming paradox is that structural fluctuations of the protein render the linkage increasingly likely as the temperature increases. Also, measurements of the affinity of MgADP for the protein, as well as the magnetic resonance of the P-atoms of the ADP once emplaced, suggest that binding the first reagent anchor to Cys-707 initiates an influence that travels to the rather distant ADP-binding site, and it is speculated what this “path of influence” might be.

Inhibition of advanced glycation endproduct formation by acetaldehyde: Role in the cardioprotective effect of ethanol

Epidemiological studies suggest that there is a beneficial effect of moderate ethanol consumption on the incidence of cardiovascular disease. Ethanol is metabolized to acetaldehyde, a two-carbon carbonyl compound that can react with nucleophiles to form covalent addition products. We have identified a biochemical modification produced by the reaction of acetaldehyde with protein-bound Amadori products. Amadori products typically arise from the nonenzymatic addition of reducing sugars (such as glucose) to protein amino groups and are the precursors to irreversibly bound, crosslinking moieties called advanced glycation endproducts, or AGEs. AGEs accumulate over time on plasma lipoproteins and vascular wall components and play an important role in the development of diabetes- and age-related cardiovascular disease. The attachment of acetaldehyde to a model Amadori product produces a chemically stabilized complex that cannot rearrange and progress to AGE formation. We tested the role of this reaction in preventing AGE formation in vivo by administering ethanol to diabetic rats, which normally exhibit increased AGE formation and high circulating levels of the hemoglobin Amadori product, HbA1c, and the hemoglobin AGE product, Hb-AGE. In this model study, diabetic rats fed an ethanol diet for 4 weeks showed a 52% decrease in Hb-AGE when compared with diabetic controls (P < 0.001). Circulating levels of HbA1c were unaffected by ethanol, pointing to the specificity of the acetaldehyde reaction for the post-Amadori, advanced glycation process. These data suggest a possible mechanism for the so-called “French paradox,” (the cardioprotection conferred by moderate ethanol ingestion) and may offer new strategies for inhibiting advanced glycation.

Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens

This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host–pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

Globular clusters, Hipparcos, and the age of the galaxy

We discuss the impact of the results from the recent Hipparcos astrometric satellite on distance estimates of galactic globular clusters. Recalibrating the clusters not only implies a relatively small change in the distance to the Large Magellanic Cloud, and hence a rescaling of several estimates of the Hubble constant, but also leads to significantly younger cluster ages. Although the data are not yet conclusive, the results so far point to a likely resolution of the apparent paradox of a universe younger than its constituents, without requiring significant modifications to simple cosmological models.

The metabolic implications of intracellular circulation

Two views currently dominate research into cell function and regulation. Model I assumes that cell behavior is quite similar to that expected for a watery bag of enzymes and ligands. Model II assumes that three-dimensional order and structure constrain and determine metabolite behavior. A major problem in cell metabolism is determining why essentially all metabolite concentrations are remarkably stable (are homeostatic) over large changes in pathway fluxes—for convenience, this is termed the [s] stability paradox. For muscle cells, ATP and O2 are the most perfectly homeostatic, even though O2 delivery and metabolic rate correlate in a 1:1 fashion. In total, more than 60 metabolites are known to be remarkably homeostatic in differing metabolic states. Several explanations of [s] stability are usually given by traditional model I studies—none of which apply to all enzymes in a pathway, and all of which require diffusion as the means for changing enzyme–substrate encounter rates. In contrast, recent developments in our understanding of intracellular myosin, kinesin, and dyenin motors running on actin and tubulin tracks or cables supply a mechanistic basis for regulated intracellular circulation systems with cytoplasmic streaming rates varying over an approximately 80-fold range (from 1 to >80 μm × sec−1). These new studies raise a model II hypothesis of intracellular perfusion or convection as a primary means for bringing enzymes and substrates together under variable metabolic conditions. In this view, change in intracellular perfusion rates cause change in enzyme–substrate encounter rates and thus change in pathway fluxes with no requirement for large simultaneous changes in substrate concentrations. The ease with which this hypothesis explains the [s] stability paradox is one of its most compelling features.

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

The organization of seismicity on fault networks.

Although models of homogeneous faults develop seismicity that has a Gutenberg-Richter distribution, this is only a transient state that is followed by events that are strongly influenced by the nature of the boundaries. Models with geometrical inhomogeneities of fracture thresholds can limit the sizes of earthquakes but now favor the characteristic earthquake model for large earthquakes. The character of the seismicity is extremely sensitive to distributions of inhomogeneities, suggesting that statistical rules for large earthquakes in one region may not be applicable to large earthquakes in another region. Model simulations on simple networks of faults with inhomogeneities of threshold develop episodes of lacunarity on all members of the network. There is no validity to the popular assumption that the average rate of slip on individual faults is a constant. Intermediate term precursory activity such as local quiescence and increases in intermediate-magnitude activity at long range are simulated well by the assumption that strong weakening of faults by injection of fluids and weakening of asperities on inhomogeneous models of fault networks is the dominant process; the heat flow paradox, the orientation of the stress field, and the low average stress drop in some earthquakes are understood in terms of the asperity model of inhomogeneous faulting.

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.

Multineuronal codes in retinal signaling.

The visual world is presented to the brain through patterns of action potentials in the population of optic nerve fibers. Single-neuron recordings show that each retinal ganglion cell has a spatially restricted receptive field, a limited integration time, and a characteristic spectral sensitivity. Collectively, these response properties define the visual message conveyed by that neuron's action potentials. Since the size of the optic nerve is strictly constrained, one expects the retina to generate a highly efficient representation of the visual scene. By contrast, the receptive fields of nearby ganglion cells often overlap, suggesting great redundancy among the retinal output signals. Recent multineuron recordings may help resolve this paradox. They reveal concerted firing patterns among ganglion cells, in which small groups of nearby neurons fire synchronously with delays of only a few milliseconds. As there are many more such firing patterns than ganglion cells, such a distributed code might allow the retina to compress a large number of distinct visual messages into a small number of optic nerve fibers. This paper will review the evidence for a distributed coding scheme in the retinal output. The performance limits of such codes are analyzed with simple examples, illustrating that they allow a powerful trade-off between spatial and temporal resolution.