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Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies 1-3 . Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity 4,5 , its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (T reg ) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on T reg cells. Accordingly, in mouse models, PI3Kδi decreased the number of T reg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 T reg cells, accompanied by expansion of pathogenic T helper 17 (T H 17) and type 17 CD8 + T (T C 17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity., (© 2022. The Author(s).)

Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but they have exacerbated the disease. Mycoplasma pulmonis causes a chronic inflammatory lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets promote resistance to mycoplasma disease and infection, whereas Th2 responses contribute to immunopathology. The purpose of the present study was to evaluate the capacity of cytokine-differentiated dendritic cell (DC) populations to influence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma Ag-pulsed bone marrow-derived DCs could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with Ag-pulsed DCs resulted in enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeficient SCID mice with Ag-pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with Ag-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either Ag-pulsed bone marrow-derived DCs or pulmonary DCs were shown to be IL-13(+) Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DCs most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-specific Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Mycoplasma respiratory diseases have a significant impact on the economy, health and wildlife. The hallmark of these diseases is the persistence of the mycoplasma infections and chronic inflammatory responses associated with the airways. There is still much that needs to be understood about the immune mechanisms involved in mycoplasma disease and resistance from infection. It is clear that immune responses can contribute to the generation of inflammatory lesions in mycoplasma respiratory disease, as well as provide protection from infection and extrapulmonary dissemination of the organisms. The evolution of this lung disease is under the control innate immune mechanisms and the contrasting effects of different T cell populations. The mechanisms of immunity involved in mycoplasma diseases are multifaceted, and a fascinating story of its complexity is being uncovered. Research in mycoplasma respiratory diseases have underscored the idea that immunity along the respiratory tract against infectious agents is a dynamic process and involves a network of cellular and cytokine signals that determine the type of responses generated, and ultimately, the outcome of infection. The aim of this article is to present on overview of our work on mycoplasma disease and immunity, focusing on the interactions and regulation of T cell responses that influence disease pathogenesis. We will first provide an overview of immune mechanisms involved in controlling infection and participate in the generation of T cell responses, and the role of T cell populations in generating protection and contributing to lesion development will be discussed.

Polymerase chain reaction analysis of Amblyomma americanum adults, nymphs, and larvae from Aberdeen Proving Ground, MD (APG), revealed a very high prevalence of a spotted fever group (SFG) rickettsia. Restriction fragment length polymorphism (RFLP) and sequence analysis identified "Rickettsia amblyommii." This organism is not yet described or well studied, and its pathogenicity is unknown; however, investigations of the organism are warranted because of its high prevalence in A. americanum. This tick is extremely abundant at military training facilities in the south, central, and Mid-Atlantic United States, and many soldiers experience multiple concurrent tick bites. Bites by R. amblyommii-infected A. americanum may account for rates of SFG rickettsia seropositivity that are higher than reported rates of Rocky Mountain spotted fever (RMSF) cases from the same location. Seroconversion to SFG rickettsia following bites of A. americanum may suggest that R. amblyommii is infectious in humans. Subclinical infection in the numerous A. americanum tick bite victims could contaminate donated blood and compromise immunodeficient recipients. Detection of R. amblyommii in questing A. americanum larvae suggests transovarial transmission. The absence of R. rickettsii, the agent of RMSF, in A. americanum may be due to transovarial interference by R. amblyommii. The likelihood of pathogen transmission by larvae is magnified by their habit of mass attack. The very small size of the larvae is also a risk factor for pathogen transmission. High R. amblyommii prevalence in populations of A. americanum presage co-infection with other A. americanum-borne pathogens. A. americanum nymphs and adults from APG were found to be co-infected with R. amblyommii and Borrelia lonestari, Ehrlichia chaffeensis and Ehrlichia ewingii, respectively, and larval pools were infected with both R. amblyommii and B. lonestari. Co-infections can compound effects and complicate diagnosis of tick-borne disease.

Aims: The aim of this study was to evaluate a population model for epirubicin clearance using internal and external validation techniques., Methods: Jackknife samples were used to identify outliers in the population dataset and individuals influencing covariate selection. Sensitivity analyses were performed in which serum aspartate transaminase (AST) values (a covariate in the population model) or epirubicin concentrations were randomly changed by +/-10%. Cross-validation was performed five times, on each occasion using 80% of the data for model development and 20% to assess the performance of the model. External validation was conducted by assessing the ability of the population model to predict concentrations and clearances in a separate group of 79 patients., Results: Structural parameter estimates from all jackknife samples were within 7.5% of the final population estimates and examination of log likelihood values indicated that the selection of AST in the final model was not due to the presence of outliers. Alteration of AST or epirubicin concentrations by +/-10% had a negligible effect on population parameter estimates and their precision. In the cross-validation analysis, the precision of clearance estimates was better in patients with AST concentrations>150 U l-1. In the external validation, epirubicin concentrations were over-predicted by 81.4% using the population model and clearance values were also poorly predicted (imprecision 43%)., Conclusions: The results of internal validation of population pharmacokinetic models should be interpreted with caution, especially when the dataset is relatively small.

Aims: To develop a limited sampling strategy for estimation of epirubicin clearance., Methods: The data set comprised 1051 concentrations measured in 105 patients with advanced or metastatic breast cancer treated with epirubicin alone. Ten limited sampling designs comprising two or three blood samples were proposed, taken at times identified by D-optimality from population pharmacokinetic parameter estimates. The data set was then truncated to include the sampling times for each of the designs. MAP Bayesian estimates of clearance were generated for each design and compared with clearance estimates obtained using all the data. The limited sampling designs were also validated using a separate data set obtained from 18 patients with either breast cancer or hepatocellular carcinoma. The sensitivity of the best limited sampling designs to sample time recording errors of 0-10% or 10-20% was then assessed using a simulated data set including 200 patients., Results: The optimum sampling times were: end of the injection and 18 min, 40 min, 3 h, 10 h and 48 h after the start of the injection. The best three-sample design included samples at 40 min, 3 h and 48 h and gave unbiased estimates of clearance with an imprecision of 9.1% (95% CI 7.3, 10.5). The best two sample design included samples at 3 and 48 h and gave unbiased estimates of clearance with an imprecision of 12.4% (95% CI 9.6, 14.6). Using the validation data set, these two and three sample designs gave unbiased estimates of clearance with an imprecision of 5.6% (95% CI 3.7, 7.0) and 4.2% (95% CI 2.6, 5.3), respectively. Simulations that included 0-10% or 10-20% errors in the recording of the blood sampling times had negligible effects on the bias and imprecision of clearance estimates., Conclusions: Limited sampling designs have been identified and validated that estimate epirubicin clearance with adequate precision and without bias from two or three blood samples. These designs also allow flexibility in blood sample collection and are robust with regard to sample time recording errors.

We used a nested PCR with Borrelia flagellin gene (flaB) primers and DNA sequencing to determine if Borrelia lonestari was present in Amblyomma americanum ticks removed from military personnel and sent to the Tick-Borne Disease Laboratory of the U.S. Army Center for Health Promotion and Preventive Medicine. In our preliminary investigation, we detected Borrelia sequences in 19 of 510 A. americanum adults and nymphs from Ft. A. P. Hill, Va. During the 2001 tick season, the flaB primers were used to test all A. americanum samples as they were received, and 29 of 2,358 A. americanum samples tested individually or in small pools were positive. PCRs with 2,146 A. americanum samples in 2002 yielded 26 more Borrelia-positive samples. The positive ticks in 2001 and 2002 were from Arkansas, Delaware, Kansas, Kentucky, Maryland, New Jersey, North Carolina, Tennessee, and Virginia. The last positive sample of the 2001 season was a pool of larvae. To further investigate larval infection, we collected and tested questing A. americanum larvae from Aberdeen Proving Ground, Md.; 4 of 33 pools (40 larvae per pool) were positive. Infection of unfed larvae provides evidence of the maintenance of B. lonestari by means of transovarial transmission. Sequence analysis revealed that the amplicons were identical to sequences of the B. lonestari flaB gene in GenBank. Despite the low prevalence of infection, the risk of B. lonestari transmission may be magnified because A. americanum is often abundant and aggressive, and many tick bite victims receive multiple bites.

Purpose: To use a population approach to identify readily available clinical or biochemical characteristics that influence the pharmacokinetics of epirubicin and to develop new dosage guidelines based on these results., Methods: Data were available from 109 patients with advanced breast cancer, 72 of whom were known to have liver metastases. They were treated with single-agent epirubicin 12.5 to 120 mg/m(2). Analysis was performed using the software package NONMEM and a three-compartment model was fitted to the data., Results: Individual clearance (CL) estimates ranged from 4 to 86 l/h and the final model included CL as a function of aspartate aminotransferase (AST): CL (l/h)=72.9-(72.9x0.135xlnAST). Inclusion of this factor reduced the interindividual variability in CL from 49% to 39%. Using a target AUC of 4000 ng.h/ml, the following doses were predicted to achieve this exposure with the greatest precision: AST <150 IU/l 125 mg; AST 150-250 IU/l 90 mg; AST 250-500 IU/l 60 mg; AST >500 IU/l 30 mg. These new guidelines were compared with three other guidelines based on serum bilirubin or AST concentrations and body surface area (BSA). The new guidelines achieved the target with greater precision (root mean squared error, rmse, 39.0%) than the current UK guidelines, current USA guidelines or an earlier equation based on AST (rmse 63%, 62% and 59%, respectively)., Conclusions: The proposed dosing guidelines should reduce variability in systemic exposure to epirubicin more effectively than traditional approaches. In addition, as they do not require adjustment according to BSA, they could reduce dosage preparation time and the potential for prescribing and dispensing errors.

This study aimed to develop an epirubicin dose modification scheme in women with breast cancer and liver dysfunction. We first identified target areas under the concentration-time curve (AUCs) of 2400 and 1600 ng/ml.h from pharmacokinetic studies in 15 women with normal liver tests. In a second group of 16 women with abnormal liver biochemistry, the relationship between raised asparate aminotransferase (AST) and epirubicin clearance was: dose=AUC (97.5-34.2xlog AST). Adaptive dosing was evaluated prospectively in a third group of 41 women with serum AST > or =2xnormal+/-raised bilirubin. The median AUCs were 2444 and 1608 ng/ml.h, close to the high and low target AUCs, respectively. Variability in AUC was lower with adaptive dosing than in a fourth group given an unadjusted dose of epirubicin (coefficient of variation=25.8, 30.0 and 46.5%, respectively; P=0.06). Epirubicin dosing based on AST is safe and may reduce pharmacokinetic variability.

We studied variability in doxorubicin pharmacokinetics in 24 patients with abnormal liver biochemistry tests. Blood samples were collected after the first cycle of single-agent doxorubicin given as an i.v. bolus and plasma levels were measured by high-performance liquid chromatography. The relationship between doxorubicin clearance (dose/AUC) and liver biochemistry tests (AST, bilirubin, albumin, alkaline phosphatase and indocyanine green clearance) was investigated. Patients with a raised bilirubin level had reduced doxorubicin clearance, but there was no clear relationship between the extent of this elevation and the reduction in doxorubicin clearance. Doxorubicin clearance was lower in patients with an isolated increase in AST than in those with normal liver biochemistry, but this difference was not statistically significant. Nevertheless, there was a significant correlation between reduced doxorubicin clearance and both raised serum AST levels and low indocyanine green clearance. These pharmacokinetic data suggest that current dose reductions based solely on the extent to which bilirubin is elevated may not be optimal.

We studied the variability in doxorubicin pharmacokinetics in 27 patients, all of whom had normal liver biochemistry tests. Blood samples were collected after the first cycle of single-agent doxorubicin given as an i.v. bolus and plasma levels were measured by high-performance liquid chromatography (HPLC). The relationship of doxorubicin clearance (dose/AUC) with biochemical tests (AST, bilirubin, alkaline phosphatase, albumin, creatinine) and physical characteristics (age, gender, height, weight, tumour type) was investigated. The 6 men had a significantly higher doxorubicin clearance than did the 21 women (median values, 59 and 27 lh-1 m-2, respectively; P = 0.002). Doxorubicin clearance was significantly lower in patients with breast cancer than in those with other tumours (median values, 26 and 53 lh-1 m-2, respectively; P = 0.0008). The other biochemical and physical parameters did not correlate with doxorubicin clearance. However, in multivariate analysis, gender was the only factor predicting doxorubicin clearance (r2 = 40%). The ratio of the AUCs for doxorubicinol and doxorubicin (R) was higher in the men than in the women (median values, 0.62 and 0.36, respectively; P = 0.03). We conclude that gender may be an important determinant of doxorubicin clearance in patients with normal liver biochemistry.

We have studied the metabolism of compounds containing 31P in normal breast using magnetic resonance spectroscopy (MRS). Spectra were acquired from non-lactating pre-menopausal breast (n = 14 women), lactating breast (n = 8) and post-menopausal breast (n = 8). The standard acquisition protocol used a 5.5 cm surface coil with the volunteer prone to minimize chest wall signal contamination. In pre-menopausal non-lactating women the phosphocreatine (PCr) peak area, expressed relative to the sum of all 31P peak areas, was negatively correlated with breast size (r = -0.56, p = 0.02) suggesting that much of the PCr signal originated from the chest wall. The phosphodiester (PDE) relative peak area was positively correlated with breast size (r = 0.71; p = 0.002). Spectra could be acquired at all phases of the menstrual cycle. In sequential examinations of five women not taking the oral contraceptive pill (OCP), phosphomonoester (PME) relative peak area was significantly lower on Week 2 than other weeks of the cycle (p = 0.03). Among pre-menopausal women no clear difference was apparent between the spectra from women taking the OCP and those not taking the OCP. Lactating breast had significantly higher PME relative peak area than non-lactating pre-menopausal breast (p = 0.02), probably reflecting the higher proportion of epithelial tissue in lactation; the lower PCr relative peak area in lactating breast (p = 0.05) is probably due to the greater size of the breast during lactation. Spectra were acquired from post-menopausal women but with a relatively low signal-to-noise ratio. The only significant difference between 31P relative peak areas of breast spectra acquired from pre- and post-menopausal women was that less PCr was detected in the post-menopausal volunteers (p = 0.03), probably as a result of differences in breast size.

The pharmacokinetics and clinical activity of epirubicin were investigated in 16 patients with hepatocellular carcinoma (HCC) who received epirubicin at 75 mg/m2; the drug was given intravenously to 7 patients and via the hepatic artery to 9 patients (7 of whom also underwent embolisation). Lignocaine (1 mg/kg) was also given intravenously to 15 patients, and the metabolite monoethylglycinexylidide (MEGX) was measured as an indicator of liver function. Epirubicin clearance correlated with serum aspartate aminotransferase (AST), albumin and bilirubin values in patients treated intravenously or intraarterially. Although the route of administration did not affect the median total plasma clearance of epirubicin, early- and intermediate-phase clearance was higher following intraarterial administration. MEGX levels correlated with serum bilirubin levels but there was no correlation with albumin or AST values or epirubicin clearance. The rate of response to epirubicin was 3/13 (23%; 95% confidence interval, 8%-50%). Intravenous epirubicin was tolerated well, but intraarterial treatment was associated with significant morbidity. These data confirm that although current recommended dose adjustments are based primarily on serum bilirubin levels, altered epirubicin pharmacokinetics correlate more strongly with AST and albumin values than with serum bilirubin concentrations. However, at this dose and schedule, epirubicin has only modest activity against HCC.

We have studied the practical implications and acceptability to patients of pharmacokinetic studies in 34 women receiving anthracyclines for advanced breast cancer. The following parameters were recorded: age, ECOG performance status, psychological state (Rotterdam Symptom Checklist), cytotoxic drug and dose, number of venepunctures for treatment and sampling, and time when the sampling cannula was removed. Immediately after finishing pharmacokinetic sampling, patients completed a questionnaire which revealed that (i) all patients understood sampling was for research, (ii) 35% of patients experienced problems with sampling, (iii) benefits from participation were perceived by 56% of patients. Of 20 patients later questioned after completion of their treatment course, 40% recalled difficulties with blood sampling. Factors identifying in advance those patients who tolerate pharmacokinetic studies poorly were not identified but the number of venepunctures should be minimised. Patients may also perceive benefits from 'non-therapeutic' research.

A sensitive and rapid method for measuring epidoxorubicin and its six metabolites by high-performance liquid chromatography using an advanced automated sample processor is described. Plasma samples (1 ml) were extracted using C2 cassettes, and reversed-phase chromatography was performed with an Apex II ODS column. The isocratic mobile phase of acetonitrile-0.019 M NaH2PO4 (pH 4.0) had a flow-rate of 1 ml/min and the fluorescence detector an excitation wavelength of 480 nm with an emission at 580 nm. Linear calibration curves were obtained which were reproducible both within-day and day-to-day (coefficients of variation less than 10%). The extraction efficacy of epidoxorubicin was 88% and ranged from 51 to 88% for the metabolites. This method has been successfully applied to measure the plasma levels of these compounds in patients receiving epidoxorubicin over a wide dose range (12-120 mg/m2) and in patients with disturbed liver biochemistry.

The pharmacokinetics of doxorubicin given according to three different schedules with a similar dose-time intensity have been studied and compared in 16 women with metastatic breast cancer. Six patients were treated with doxorubicin 75 mg/m2 by i.v. bolus repeated every 3 weeks; 5 patients received doxorubicin by 4-day continuous infusion every 3 weeks (4 at 75 mg/m2 and 1 at 60 mg/m2); 5 patients received 25 mg/m2 by i.v. bolus given weekly. Timed blood samples were collected and plasma levels of doxorubicin and its metabolite doxorubicinol were measured by high-performance liquid chromatography with fluorescence detection. Peak plasma concentrations were measured, and areas under the concentration-time curves calculated. Peak plasma levels of doxorubicin were significantly lower with the 4-day infusion than with either of the bolus injections. The 4-day infusion, however, gave significantly greater total exposure to doxorubicin and doxorubicinol, as indicated by area under the concentration-time curve, than weekly or 3-weekly bolus treatment. A single bolus injection of doxorubicin 25 mg/m2 yielded a total exposure to doxorubicin approximately half that achieved with a 75 mg/m2 bolus injection. Over a 3-week period, therefore, total exposure to doxorubicin would be greater with the weekly low-dose schedule than with the 3-weekly administration. We conclude that drug scheduling has significant effects on doxorubicin pharmacokinetics.