Evaluating treatment protocols to prevent antibiotic resistance

The spread of bacteria resistant to antimicrobial agents calls for population-wide treatment strategies to delay or reverse the trend toward antibiotic resistance. Here we propose new criteria for the evaluation of the population-wide effects of treatment protocols for directly transmitted bacterial infections and discuss different usage patterns for single and multiple antibiotic therapy. A mathematical model suggests that the long-term benefit of single drug treatment from introduction of the antibiotic until a high frequency of resistance precludes its use is almost independent of the pattern of antibiotic use. When more than one antibiotic is employed, sequential use of different antibiotics in the population (“cycling”) is always inferior to treatment strategies where, at any given time, equal fractions of the population receive different antibiotics. However, treatment of all patients with a combination of antibiotics is in most cases the optimal treatment strategy.

Differences in frontal cortical activation by a working memory task after substitution of risperidone for typical antipsychotic drugs in patients with schizophrenia

Antipsychotic drug treatment of schizophrenia may be complicated by side effects of widespread dopaminergic antagonism, including exacerbation of negative and cognitive symptoms due to frontal cortical hypodopaminergia. Atypical antipsychotics have been shown to enhance frontal dopaminergic activity in animal models. We predicted that substitution of risperidone for typical antipsychotic drugs in the treatment of schizophrenia would be associated with enhanced functional activation of frontal cortex. We measured cerebral blood oxygenation changes during periodic performance of a verbal working memory task, using functional MRI, on two occasions (baseline and 6 weeks later) in two cohorts of schizophrenic patients. One cohort (n = 10) was treated with typical antipsychotic drugs throughout the study. Risperidone was substituted for typical antipsychotics after baseline assessment in the second cohort (n = 10). A matched group of healthy volunteers (n = 10) was also studied on a single occasion. A network comprising bilateral dorsolateral prefrontal and lateral premotor cortex, the supplementary motor area, and posterior parietal cortex was activated by working memory task performance in both the patients and comparison subjects. A two-way analysis of covariance was used to estimate the effect of substituting risperidone for typical antipsychotics on power of functional response in the patient group. Substitution of risperidone increased functional activation in right prefrontal cortex, supplementary motor area, and posterior parietal cortex at both voxel and regional levels of analysis. This study provides direct evidence for significantly enhanced frontal function in schizophrenic patients after substitution of risperidone for typical antipsychotic drugs, and it indicates the potential value of functional MRI as a tool for longitudinal assessment of psychopharmacological effects on cerebral physiology.

HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4+ T cell counts ≥ 350 cells/μl and viral load below the limits of detection for ≥1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2–3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log10 copies) day−1, which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log10 copies) day−1 (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4+ T cells.

Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells—at plausible peripheral blood mononuclear cell-to-target ratios—with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per μl, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

Influence of follicular dendritic cells on decay of HIV during antiretroviral therapy

Drug treatment of HIV type 1 (HIV-1) infection leads to a rapid initial decay of plasma virus followed by a slower second phase of decay. To investigate the role of HIV-1 retained on follicular dendritic cells (FDCs) in this process, we have developed and analyzed a mathematical model for HIV-1 dynamics in lymphoid tissue (LT) that includes FDCs. Analysis of clinical data using this model indicates that decay of HIV-1 during therapy may be influenced by release of FDC-associated virus. The biphasic character of viral decay can be explained by reversible multivalent binding of HIV-1 to receptors on FDCs, indicating that the second phase of decay is not necessarily caused by long-lived or latently infected cells. Furthermore, viral clearance and death of short-lived productively infected cells may be faster than previously estimated. The model, with reasonable parameter values, is consistent with kinetic measurements of viral RNA in plasma, viral RNA on FDCs, productively infected cells in LT, and CD4+ T cells in LT during therapy.

p53-dependent and -independent responses to cisplatin in mouse testicular teratocarcinoma cells

Testicular cancers respond favorably to chemotherapy with the platinum-containing drug cis-diamminedichloroplatinum(II) (cisplatin). One factor that could explain the efficacy of cisplatin is the low frequency of p53 mutations observed in this tumor type. The present study examines the p53-mediated responses in murine testicular teratocarcinoma cells exposed to the drug. Cisplatin treatment of teratocarcinoma cells with a wild-type p53 gene resulted in accumulation of the p53 protein through posttranscriptional mechanisms; induction of p53-target genes was also observed. Drug treatment resulted in rapid apoptosis in p53-wild-type cells but not in p53−/− teratocarcinoma cells. In the latter cells, cisplatin exposure caused prolonged cell cycle arrest accompanied by induction of the p21 gene. Clonogenic assays demonstrated that the p53 mutation did not confer resistance to cisplatin. These experiments suggest that cisplatin inhibits cellular proliferation of testicular teratocarcinoma cells by two possible mechanisms, p53-dependent apoptosis and p53-independent cell cycle arrest.

Multiple modes of cellular activation and virus transmission in HIV infection: A role for chronically and latently infected cells in sustaining viral replication

CD4+ T cell activation, required for virus replication in these cells, occurs in local microenvironmental domains in transient bursts. Thus, although most HIV originates from short-lived virus-producing cells, it is unlikely that chronic infection is generally sustained in rapid continuous cycles of productive infection as has been proposed. Such continuity of productive infection cycles would depend on efficient long-range transmission of HIV from one set of domains to another, in turn requiring the maintenance of sufficiently high concentrations of cell-free virus across lymphoid tissues at all times. By contrast, long-lived cellular sources of HIV maintain the capacity to infect newly activated cells at close range despite the temporal and spatial discontinuities of activation events. Such proximal activation and transmission (PAT) involving chronically and latently infected cells may be responsible for sustained infection, particularly when viral loads are low. Once CD4 cells are productively infected through PAT, they can infect other activated cells in their immediate vicinity. Such events propagate locally but generally do not spread systemically, unlike in the acute phase of the infection, because of the early establishment of protective anergy. Importantly, antiretroviral drug treatment is likely to differentially impact long-range transmission and PAT.

Effectiveness of treatments for infantile colic: systematic review

Objective: To evaluate the effectiveness of diets, drug treatment, and behavioural interventions on infantile colic in trials with crying or the presence of colic as the primary outcome measure.

Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs

The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.

Viral dynamics in vivo: limitations on estimates of intracellular delay and virus decay.

Anti-viral drug treatment of human immunodeficiency virus type I (HIV-1) and hepatitis B virus (HBV) infections causes rapid reduction in plasma virus load. Viral decline occurs in several phases and provides information on important kinetic constants of virus replication in vivo and pharmacodynamical properties. We develop a mathematical model that takes into account the intracellular phase of the viral life-cycle, defined as the time between infection of a cell and production of new virus particles. We derive analytic solutions for the dynamics following treatment with reverse transcriptase inhibitors, protease inhibitors, or a combination of both. For HIV-1, our results show that the phase of rapid decay in plasma virus (days 2-7) allows precise estimates for the turnover rate of productively infected cells. The initial quasi-stationary phase (days 0-1) and the transition phase (days 1-2) are explained by the combined effects of pharmacological and intracellular delays, the clearance of free virus particles, and the decay of infected cells. Reliable estimates of the first three quantities are not possible from data on virus load only; such estimates require additional measurements. In contrast with HIV-1, for HBV our model predicts that frequent early sampling of plasma virus will lead to reliable estimates of the free virus half-life and the pharmacological properties of the administered drug. On the other hand, for HBV the half-life of infected cells cannot be estimated from plasma virus decay.

Viral dynamics in hepatitis B virus infection.

Treatment of chronic hepatitis B virus (HBV) infections with the reverse transcriptase inhibitor lamivudine leads to a rapid decline in plasma viremia and provides estimates for crucial kinetic constants of HBV replication. We find that in persistently infected patients, HBV particles are cleared from the plasma with a half-life of approximately 1.0 day, which implies a 50% daily turnover of the free virus population. Total viral release into the periphery is approximately 10(11) virus particles per day. Although we have no direct measurement of the infected cell mass, we can estimate the turnover rate of these cells in two ways: (i) by comparing the rate of viral production before and after therapy or (ii) from the decline of hepatitis B antigen during treatment. These two independent methods give equivalent results: we find a wide distribution of half-lives for virus-producing cells, ranging from 10 to 100 days in different patients, which may reflect differences in rates of lysis of infected cells by immune responses. Our analysis provides a quantitative understanding of HBV replication dynamics in vivo and has implications for the optimal timing of drug treatment and immunotherapy in chronic HBV infection. This study also represents a comparison for recent findings on the dynamics of human immunodeficiency virus (HIV) infection. The total daily production of plasma virus is, on average, higher in chronic HBV carriers than in HIV-infected patients, but the half-life of virus-producing cells is much shorter in HIV. Most strikingly, there is no indication of drug resistance in HBV-infected patients treated for up to 24 weeks.

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.