The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy.

Replication-competent HIV-1 can be isolated from infected patients despite prolonged plasma virus suppression by anti-retroviral treatment. Recent studies have identified resting, memory CD4+ T lymphocytes as a long-lived latent reservoir of HIV-1 (refs. 4,5). Cross-sectional analyses indicate that the reservoir is rather small, between 103 and 107 cells per patient. In individuals whose plasma viremia levels are well suppressed by anti-retroviral therapy, peripheral blood mononuclear cells containing replication-competent HIV-1 were found to decay with a mean half-life of approximately 6 months, close to the decay characteristics of memory lymphocytes in humans and monkeys. In contrast, little decay was found in a less-selective patient population. We undertook this study to address this apparent discrepancy. Using a quantitative micro-culture assay, we demonstrate here that the latent reservoir decays with a mean half-life of 6.3 months in patients who consistently maintain plasma HIV-1 RNA levels of fewer than 50 copies/ml. Slower decay rates occur in individuals who experience intermittent episodes of plasma viremia. Our findings indicate that the persistence of the latent reservoir of HIV-1 despite prolonged treatment is due not only to its slow intrinsic decay characteristics but also to the inability of current drug regimens to completely block HIV-1 replication.

Risk assessment of recent Egyptian H5N1 influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014-2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

Protein evolution on partially correlated landscapes.

We extend an earlier model of protein evolution on a rugged landscape to the case in which the landscape exhibits a variable degree of correlation (i.e., smoothness). Correlation is introduced by assuming that a protein is composed of a set of independent blocks or domains and that mutation in one block affects the contribution of that block alone to the overall fitness of the protein. We study the statistical structure of such landscapes and apply our theory to the evolution by somatic hypermutation of antibody molecules composed of framework and complementarity-determining regions. We predict the expected number of replacement mutations in each region.

A multistage model for the action of cytotoxic T lymphocytes in multicellular conjugates.

We propose a multistage stochastic model to explain data on the kinetics of target cell lysis by cytotoxic T lymphocytes in multicellular conjugates. A novel feature of our model is that we explicitly consider both the lethal hitting stage and the target cell disintegration stage of the cytolytic process. Further, we allow for the possibility that target cell disintegration is itself a complex process composed of many events. The comparison of our model with the data of other investigators suggests that cytotoxic T cells deliver lethal hits at random to undamaged target cells. Having received a lethal hit, the target cell disintegrates over a variable length of time. The disintegration times of target cells from different conjugates appear to be randomly distributed and to be consistent with a model in which disintegration occurs by at least two major, sequential, rate-limiting events. For conjugates containing one lymphocyte and multiple target cells, the mean rate at which a lethally hit target cell disintegrates is found to be independent of the total number of target cells in the conjugate. Our model predicts that in such multicellular conjugates, individual target cells lyse one by one, on average at approximately 30-min intervals, thus agreeing closely with previously reported experimental observations.

Aggregation of cell surface receptors by multivalent ligands.

Using a combination of branching processes and kinetic equations a solution is provided to the problem of describing the size of aggregates formed on cell surfaces when multivalent ligands bind and cross-link multivalent receptors. A criterion is given for the onset of gelation in infinite 2-dimensional systems, which may be relevant to the phenomenon of ligand-induced receptor patching.

Protein evolution on rugged landscapes.

We analyze a mathematical model of protein evolution in which the evolutionary process is viewed as hill-climbing on a random fitness landscape. In studying the structure of such landscapes, we note that a large number of local optima exist, and we calculate the time and number of mutational changes until a protein gets trapped at a local optimum. Such a hill-climbing process may underlie the evolution of antibody molecules by somatic hypermutation.

Renewal theory, Geiger counters, and the maximum number of receptors bound to a randomly haptenated polymer chain.

Polymers containing small chemical groups (haptens) covalently attached at random along the chain are commonly used to initiate an immune response. Properties of the polymer such as its length, the spacing of the haptens, and the total number of haptens along the chain, correlate with its immune reactivity. Here we model the ability of many finite-sized cell surface receptors to bind simultaneously the haptens conjugated to a polymer chain. The binding sites on two different receptors or on separate parts of a multivalent receptor cannot be arbitrarily close to one another; so, in general, not all haptens along a polymer chain can be simultaneously bound by receptors. We develop an analogy between the steric hindrance among receptors detecting randomly placed haptens and the temporary locking of a Geiger counter that has detected a radioactive decay. Using renewal theory, we compute the probability distribution, and its moments, for the maximum number of haptens that can be simultaneously bound by monovalent receptors. We also model flexible bivalent receptors and obtain the mean and variance of the maximum number of receptors bound to randomly haptenated polymers, and the mean and variance of the maximum number of haptens bound. We demonstrate the importance of our results by applying them to immunological data and showing that, for polymers used in immunology, the effective valence of a polymer may be as much as 50% smaller than its nominal valence.

Effects of sulfuric acid and nitrogen dioxide on airway responsiveness of the guinea pig.

Hartley guinea pigs were exposed for 1 h to either NO2 gas of H2SO4 aerosol and examined for changes in airway responsiveness to inhaled histamine. Concentrations ranged from 7 to 146 ppm NO2 and 4 to 40 mg/m3 H2SO4. One group of animals exposed to filtered air served as controls. Histamine challenges were performed on unanesthetized animals 2 h before pollutant or air exposure (baseline) and 10 min and 2 and 19 h after exposure. No2-exposed animals had increased histamine sensitivities 10 min after exposure, and the magnitude of the increase was directly dependent on the NO2 concentration. Most of the NO2-exposed animals demonstrated a dramatic return toward baseline values by 2 h after exposure; however, several animals had not returned to baseline by 19 h after exposure. Some animals exposed to H2SO4 developed severe labored breathing during exposure, and major increases in histamine sensitivity were observed only in those animals. These results suggest that both No2 and H2SO4 alter airway sensitivity to histamine, but apparently by different mechanisms. Changes produced by NO2 exposures appeared primarily concentration-dependent, while changes produced by H2SO4 exposures appeared related to dyspnea developed during exposure.

Mechanism of cell-mediated cytotoxicity at the single cell level. VIII. Kinetics of lysis of target cells bound by more than one cytotoxic T lymphocyte.

We measured the effects of having multiple cytotoxic T lymphocytes (CTL) bound to one target cell by using the single-cell cytotoxicity in agarose assay. We found that even though there is variability in the time at which individual target cells are lysed, we can identify a general trend: the mean rate of lysis increases with the number of CTL bound per target cell, reaching a maximum when the CTL-target cell ratio is three. Combining a quantitative model for the rate of lethal hitting in multicellular conjugates with a multi-event model for the rate of target cell disintegration, we developed a new multistage kinetic model for predicting the rate of target cell lysis in multiple lymphocyte-target cell conjugates. The variability in the time at which target cells are hit and the variability in the time until they disintegrate are incorporated into the model. By analyzing our measured data in the context of the multistage kinetic model, we were able to estimate via nonlinear least squares regression the target cell disintegration rate, but not the lethal hitting rate. Lethal hitting appeared to be too fast, when compared with disintegration, to significantly affect the time of target cell lysis. By using previously determined values of the lethal hitting rate for single lymphocyte-target cell conjugates and by postulating that lymphocytes act independently of each other in delivering lethal hits, we were able to estimate the rate at which target cells are hit in multiple-lymphocyte single target cell conjugates. By using this estimate of the lethal hitting rate and the regression estimate of the disintegration rate, the multistage kinetic model gave a quantitative fit to our data. From this analysis, we found that the rate at which a target cell disintegrates after being lethally hit increases with the number of CTL per conjugate. This result is quite surprising, because once the first hit has been received, a target cell can disintegrate in a killer cell-independent manner. Under the conditions of our experiment, it appears as if target cell disintegration is not killer cell-independent. Furthermore, our analysis of the time course of target cell disintegration suggests that the process is not governed by simple first order kinetics, but rather by a more complex multistep mechanism.

Effects of sulfuric acid aerosols on pulmonary function of guinea pigs.

Forty-seven Hartley guinea pigs were exposed for 1 h to approximately 1-micrometer (mass median aerodynamic diameter) sulfuric acid aerosols at concentrations that ranged from 1.2 to 48.3 mg/m3. Ten animals (controls) were exposed to filtered room air only. Eight H2SO4-exposed animals exhibited marked increases in total pulmonary resistance and marked decreases in dynamic compliance. Four of these eight "responsive" animals died during exposure. All other H2SO4-exposed animals exhibited no major difference from controls and were termed nonresponsive. The proportion of responsive to nonresponsive animals increased with exposure concentration, but the magnitude of pulmonary function change was similar for all responsive animals regardless of concentration. Compared to nonresponders, responsive animals had higher preexposure values of tidal transpulmonary pressure excursions and total pulmonary resistance and lower values of dynamic compliance. Preexposure transpulmonary pressure excursions were positively correlated with minute volume only for nonresponsive animals; transpulmonary pressure excursions were positively correlated with total pulmonary resistance in responsive animals. The results suggest that the Hartley guinea pig reacts to inhaled H2SO4 with an essentially all-or-none airway constrictive response and that an animal's sensitivity to this response may be related to its preexposure airway caliber.

Persistence of HIV-1 transcription in peripheral-blood mononuclear cells in patients receiving potent antiretroviral therapy.

BACKGROUND AND METHODS: Although potent antiretroviral therapy can control infection with human immunodeficiency virus type 1 (HIV-1), a long-lived reservoir of infectious virus persists in CD4+ T cells. We investigated this viral reservoir by measuring the levels of cell-associated viral DNA and messenger RNA (mRNA) that are essential for HIV-1 replication. Approximately every 6 months, we obtained samples of peripheral-blood mononuclear cells from five men with long-standing HIV-1 infection who had had undetectable levels of plasma HIV-1 RNA for 20 months or more during treatment with potent antiretroviral drugs. RESULTS: Before treatment, plasma levels of HIV-1 RNA correlated with the levels of cell-associated unintegrated HIV-1 DNA and unspliced viral mRNA. After treatment, plasma levels of HIV-1 RNA fell by more than 2.7 log to undetectable levels. The decrease in cell-associated integrated and unintegrated HIV-1 DNA and mRNA occurred in two phases. The first phase occurred during the initial 500 days of treatment and was characterized by substantial decreases in the levels of DNA and mRNA, but not to undetectable levels. The concentrations of cell-associated unintegrated viral DNA, integrated proviral DNA, and unspliced viral mRNA decreased by 1.25 to 1.46 log. The second phase occurred during the subsequent 300 days or more of treatment and was characterized by a plateau in the levels of HIV-1 DNA and unspliced mRNA. After an initial rapid decline, the ratio of unspliced to multiply spliced viral mRNA (a measure of active viral transcription) stabilized and remained greater than zero at each measurement. CONCLUSIONS: Despite treatment with potent antiretroviral drugs and the suppression of plasma HIV-1 RNA to undetectable levels for 20 months or more, HIV-1 transcription persists in peripheral-blood mononuclear cells. Unless the quasi-steady state levels of HIV DNA and mRNA eventually disappear with longer periods of therapy, these findings suggest that HIV-1 infection cannot be eradicated with current treatments.