Pharmacokinetic and pharmacodynamic modelling of the novel human granulocyte colony-stimulating factor derivative Maxy-G34 and pegfilgrastim in rats

Objectives:  This study aims to compare pharmacokinetics and pharmacodynamics of pegfilgrastim, a pharmaceutical recombinant human granulocyte colony-stimulating factor (rhG-CSF), with that of a newly developed reagent, Maxy-G34. This comparison was performed using rat experiments and biomathematical modelling of granulopoiesis.
Methods:  Healthy rats and those with cyclophosphamide-induced neutropenia were treated with either pegfilgrastim or Maxy-G34 under various schedules. Time courses of absolute neutrophil count (ANC) and G-CSF serum level were measured and we constructed a combined pharmacokinetic/pharmacodynamic model of both drugs. Neutropenic episodes were assessed by experimental data and model simulations.
Results:  Both Pegfilgrastim and Maxy-G34 showed strong dose-dependent efficacy in reducing neutropenic episodes. However, time courses of ANC and G-CSF serum levels were markedly different. The biomathematical model showed good agreement with these data. We estimated that differences between the two drugs could be explained by lower bioavailability and reduced elimination of Maxy-G34. Based on the data and model interpolations, we estimated that Maxy-G34 is superior in reducing neutropenic episodes. Also, we predicted that G-CSF administration 48 h after cyclophosphamide would be superior to its administration after 2 or 24 h, for both derivatives.
Conclusion:  Maxy-G34 is a highly potent drug for stimulation of neutrophil production in rats. By our modelling approach, we quantified differences between Maxy-G34 and pegfilgrastim, related to pharmacokinetic parameters. Model simulations can be used to estimate optimal dosing and timing options in the present preclinical rat model.     

A pharmacokinetic model of filgrastim and pegfilgrastim application in normal mice and those with cyclophosphamide-induced granulocytopaenia

Objectives:  Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used as treatment for granulocytopaenia during cytotoxic chemotherapy; however, optimal scheduling of this pharmaceutical is unknown. Biomathematical models can help to pre-select optimal application schedules but precise pharmacokinetic properties of the pharmaceuticals are required at first. In this study, we have aimed to construct a pharmacokinetic model of G-CSF derivatives filgrastim and pegfilgrastim in mice.
Methods:  Healthy CD-1 mice and those with cyclophosphamide-induced granulocytopaenia were studied after administration of filgrastim and pegfilgrastim in different dosing and timing schedules. Close meshed time series of granulocytes and G-CSF plasma concentrations were determined. An ordinary differential equations model of pharmacokinetics was constructed on the basis of known mechanisms of drug distribution and degradation.
Results:  Predictions of the model fit well with all experimental data for both filgrastim and pegfilgrastim. We obtained a unique parameter setting for all experimental scenarios. Differences in pharmacokinetics between filgrastim and pegfilgrastim can be explained by different estimates of model parameters rather than by different model mechanisms. Parameter estimates with respect to distribution and clearance of the drug derivatives are in agreement with qualitative experimental results.
Conclusion:  Dynamics of filgrastim and pegfilgrastim plasma levels can be explained by the same pharmacokinetic model but different model parameters. Beause of a strong clearance mechanism mediated by granulocytes, granulocytotic and granulocytopaenic conditions must be studied simultaneously to construct a reliable model. The pharmacokinetic model will be extended to a murine model of granulopoiesis under chemotherapy and G-CSF application.     

Multistability in an age-structured model of hematopoiesis: Cyclical neutropenia

Cyclical neutropenia (CN) is a rare hematopoietic disorder in which the patient's neutrophil level drops to extremely low levels for a few days approximately every three weeks. CN is effectively treated with granulocyte colony stimulating factor (G-CSF), which is known to interfere with apoptosis in neutrophil precursors and to consequently increase the circulating neutrophil level. However, G-CSF treatment usually fails to eliminate the oscillation. In this study, we establish an age-structured model of hematopoiesis, which reduces to a set of four delay differential equations with specific forms of initial functions. We numerically investigate the possible stable solutions of the model equations with respect to changes in the parameters as well as the initial conditions. The results show that the hematopoietic system possesses multistability for parameters typical of the normal healthy state. From our numerical results, decreasing the proliferation rate of neutrophil precursors or increasing the stem cell death rate are two possible mechanisms to induce cyclical neutropenia, and the periods of the resulting oscillations are independent of the changing parameters. We also discuss the dependence of the model solution on the initial condition at normal parameter values corresponding to a healthy state. Using insight from our results we design a hybrid treatment method that is able to abolish the oscillations in CN.     

Granulopoeisis and leukemogenesis: lessons from congenital neutropenia

Congenital neutropenia are extremely rare diseases, defined by a permanent or cyclic decrease of blood neutrophils. Molecular basis of several congenital neutropenia has been recently determined, involving gene coding for the neutrophil elastase gene (ELA2), GFI1, WAS protein and mitochondrial HAX1 protein. These mutations, dominant (ELA2, GFI1), X-linked (WAS) and autosomal recessive (HAX1), result in instability of the contents of the granules- particularly the neutrophil elastase- or in abnormalities of the cytoskeleton, and possibly, in an increased apoptosis. ELA2 mutations resulting both in profound and permanent neutropenia, and in cyclic--pseudo sinusoidal--neutropenia lead to consider that time pattern is very close in the two apparently distinct phenotypes. This observation suggests that temporal variations of neutrophils could be represented by non linear functions. Congenital neutropenia, specifically ELA2 mutated, are also characterized by a high rate of leukemia (about 15% at 20 years of age). Leukemia risk does not appear to be related to an oncogenic effect of ELA2 mutations, but much likely to the deepness of the neutropenia, and the intensity of G-CSF therapy.     

Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation

Objectives:  The key to fertility in adult males is production of mature spermatogenic cells. Spermatogonial stem cells (SSC) have the dual capacity of self-renewal and of differentiation into mature sperm. SSC transplantation may provide potential treatment for specific male infertilities. However, until now, there has been no evidence of offspring produced by transplantation of adult SSC line cells in humans or other mammals.
Materials and methods:  A new line of SSCs from adult C57BL/6 mouse was established by using magnetic-activated cell sorting. The cell line was characterized by immunocytochemistry, karyotype analysis and telomeric repeat amplification protocol (TRAP) telomerase activity assay. Spermatogenic function was examined by allograft into germ cell-ablated recipient mice.
Results:  For more than 14 months with more than 65 maintenance passages, the cell line showed a normal karyotype (40, XY) and high telomerase activity. It represented a Thy-1+, Oct4+, SSEA-1-, c-kit- (99 ± 1%) cell subpopulation. We cryopreserved these SSCs and successfully produced normal offspring after transplanting them into testes of busulphan-sterilized mice.
Conclusions:  We established and long-term maintained an adult SSC line with normal spermatogenic function, without the need of genetic modification; thus, this study provides a model system for basic research and clinical application.     

Administration of recombinant human stem cell factor (rhSCF) and granulocyte-colony stimulating factor (rhG-CSF) to maternal and fetal rhesus monkeys (Macaca mulatta).

Studies with recombinant human stem cell factor (rhSCF) and granulocyte-colony stimulating factor (rhG-CSF) have suggested significant clinical utility although little is known regarding the effect of these cytokines when administered during pregnancy. rhSCF (25 microg/kg/day)+/-rhG-CSF (50 microg/kg/day) were administered chronically to gravid rhesus monkeys ( n =12) or directly to the rhesus fetus ( n=2) during the second and third trimesters. Maternal/fetal blood samples were collected to assess circulating SCF/G-CSF levels and complete blood counts compared to non-treated animals (n=40). Fetal endogenous SCF levels were four-fold greater than the dam (fetus approximately 2500 pg/ml, dam approximately 500 pg/ml), whereas circulating G-CSF was similar in the fetal/maternal compartments ( approximately 50-100 pg/ml). There were no adverse effects detected in the fetus or dam as a result of SCF+/-G-CSF administration. Although high levels of SCF and G-CSF were achieved in the maternal circulation with maternal administration (SCF: 7000-15 000 pg/ml; G-CSF: 7000-54 000 pg/ml), there was little evidence of placental transport or effects on fetal haematopoiesis. In contrast, direct fetal administration of SCF+G-CSF resulted in a rapid rise in fetal neutrophil counts. These studies have shown the monkey to be an excellent model for studying haematopoietic interventions during gestation, and suggest the best approach for achieving haematopoietic changes in the fetus and newborn is by direct in utero administration.     

Hereditary neutropenia: dogs explain human neutrophil elastase mutations

Mutations in ELA2, the gene encoding neutrophil elastase (NE), cause the human diseases cyclic neutropenia (CN) and severe congenital neutropenia (SCN). Numerous mutations are known, but their lack of consistent biochemical effect has proven puzzling. The recent finding that mutation of AP3B1, which encodes the beta subunit of adaptor protein complex 3 (AP3), is the cause of canine CN suggests a model for the molecular basis of hereditary neutropenias, involving the mistrafficking of NE: AP3 recognizes NE as a cargo protein, and their interaction implies that NE is a transmembrane protein. Computerized algorithms predict two NE transmembrane domains. Most CN mutations fall within predicted transmembrane domains and lead to excessive deposition of NE in granules, whereas SCN mutations usually disrupt the AP3 recognition sequence, resulting in excessive transport to the plasma membrane.     

Mathematical model for G-CSF administration after chemotherapy

Granulocyte-colony stimulating factor (G-CSF) is used clinically for treating chemotherapy-induced neutropenia (low neutrophil levels). Here we present a delay differential equation model for the regulation of neutrophil production that accounts for the effects of G-CSF. Using a combination of analysis and numerical simulations, we use this model to study the effects of delaying G-CSF treatment following chemotherapy for two recombinant forms of G-CSF (filgrastim and pegfilgrastim). We also examine the consequences of varying the duration of filgrastim treatment. We found that varying the starting day or the duration of G-CSF treatment can lead to different qualitative responses in the neutrophil count. These changes can be explained by the coexistence of two stable solutions in the mathematical model.     

Rapid detection of lactobacillus and yeast concentrations using a particle size distribution analyser

Aims:  To see the possibility of particle size distribution analyser (PSDA) in detecting concentration of lactobacillus contaminants in yeast fermentation.
Methods and Results:  A PSDA was used to rapidly determine the size and concentration of lactobacillus and Saccharomyces cerevisiae . Data showed that the aerodynamic diameters of Lactobacillus casei and S. cerevisiae cells were around 0·63 and 2·9 μm, respectively, with both cultures showing a linear relationship between cell density and particle count on a size distribution curve of PSDA. In addition, Lactobacillus fermentum showed high similarity in bacterial size distribution and particle count numbers with L. casei . The PSDA also rapidly detected (within 1 min) the cell concentrations of S. cerevisiae and L. casei in a mixed sample with different concentration ratios with 10⁷–10⁹ cells ml⁻¹ of detection range.
Conclusions:  PSDA was demonstrated to be useful for the rapid detection of lactobacillus and S. cerevisiae concentrations.
Significance and Impact of the Study:  This is the first report concerning PSDA to detect the concentration of bacteria and yeast. This method can be useful in the actual field during ethanol fermentation because of relatively easy handling and rapid detection.     

Congenital neutropenias

The term congenital neutropenia (CN) has been used for a group of hematologic disorders characterized by severe neutropenia with absolute neutrophil counts (ANC) below 0.5 x 10(9)/L associated with increased susceptibility to bacterial infections. This group of diseases includes primary bone marrow failure syndromes with isolated neutropenias and neutropenias associated with metabolic or immunologic disorders or with a complex syndrome. To avoid confusion, we prefer using the term CN only for the most severe disorder among this group: severe neutropenia characterized by an early stage maturation arrest of myelopoiesis leading to bacterial infections from early infancy. This disease has originally been described as Kostmann syndrome with an autosomal recessive inheritance. Recent pathogenetic investigations have demonstrated that this clinical phenotype includes also autosomal dominant and sporadic cases with different point mutations in the neutrophil elastase gene in a subgroup of patients. Data on over 400 patients with CN collected by the Severe Chronic Neutropenia International Registry demonstrate that independent from the CN-subtype more than 90% of these patients respond to recombinant human granulocyte-colony stimulating factor (rHuG-CSF filgrastim, lenograstim) with ANC that can be maintained around 1.0 x 10(9)/L. Adverse events include mild splenomegaly, moderate thrombocytopenia, osteoporosis and malignant transformation into myelodysplastic syndrome/leukemia. Development of additional genetic aberrations, e.g., G-CSF-receptor gene mutations, monosomy 7 or ras mutations during the course of the disease indicate an underlying genetic instability leading to an increased risk of malignant transformation. If and how G-CSF treatment impacts on these adverse events remains unclear since there are no historical controls for comparison. Hematopoietic stem cell transplantation is still the only available treatment for patients refractory to G-CSF treatment.     

Unravelling Escherichia coli dynamics close to the maximum growth temperature through heterogeneous modelling

Aims:  Previous work showed that the exponential phase of Escherichia coli K12 MG1655, grown in Brain Heart Infusion broth at temperatures close to its maximum growth temperature, is disturbed. Based on plate count data, microscopic images and literature, the existence of a heat-resistant subpopulation was hypothesized. Here, this hypothesis is mathematically explored via a heterogeneous model.
Methods and Results:  A heat-sensitive and a heat-resistant subpopulation are considered. A large fraction of the population is inactivated, while the remaining smaller fraction is able to resist (or adapt to) the inimical temperature and grows. A heterogeneous model that encloses a growth model (resistant population) and an inactivation model (sensitive population) is used to describe the global population dynamics. Most experimental data can be predicted when taking parameter uncertainty via Monte Carlo simulation into account.
Conclusions:  The heterogeneous model accurately describes disturbed growth curves at superoptimal temperatures, except for high initial cell densities.
Significance and Impact of the Study:  This study strengthens the hypothesis of the existence of a (small) heat-resistant subpopulation in typical inoculum cultures of E. coli K12 MG1655.     

Phenotypic plasticity in host-plant specialisation in Aphis fabae

Abstract.  1. The study of phenotypic plasticity in host utilisation is crucial for predicting evolutionary patterns of insect – plant interactions. The presence of sufficient variation in plasticity may facilitate host race formation and sympatric speciation.
2.  Aphis fabae genotypes showed high levels of phenotypic plasticity in the intrinsic rate of natural increase ( r _m), relative growth rate (RGR), birth weight (BW), adult weight (AW), fecundity ( F ), and development time (1/ d ).
3. Thirteen A. fabae genotypes reared both on broad bean and nasturtium exhibited statistically significant genotypic variability in phenotypic plasticity.
4. Some genotypes displayed fitness improvement on novel host plants.
5. Differences in genotypic correlation among fitness components between the two hosts and increased variance on nasturtium indicated different genomic expression on nasturtium.
6. The results indicated that phenotypic plasticity in a novel environment may be a major determinant of the evolutionary trajectory of a parasitic species and might support the idea that speciation starts with phenotypic plasticity.     

High frequency spikes in long period blood cell oscillations

Several hematological diseases are characterised by oscillations of various blood cell populations. Two of these are a variant of chronic myelogenous leukemia (CML) and cyclical neutropenia (CN). These oscillations typically have long periods ranging from 20 to 60 days, despite the fact that the stem cell cycling time is thought to be of the order of 2–3 days. Clinical data from humans and laboratory data from the grey collie animal model of CN is suggestive of the idea that these long period oscillations may also contain higher frequency spiky oscillations. We show how such oscillations can be understood in the context of slow periodic stem cell oscillations, by analysing a two component differential-delay equation model of stem cell and neutrophil populations.For Karl Hadeler, on his 70th birthday, leader, teacher, colleague and friend.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Dual-Priming Oligonucleotide-Based Multiplex PCR for the Detection of Helicobacter pylori and Determination of Clarithromycin Resistance with Gastric Biopsy Specimens

Background:  Assessment of Helicobacter pylori ( H. pylori ) clarithromycin resistance has rarely been performed routinely despite an increasing resistance rate. Our aim was to develop and evaluate the use of dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) to detect point mutations in the 23S rRNA gene responsible for clarithromycin resistance of H. pylori.
Materials and Methods:  Gastric biopsy specimens from 212 untreated patients with dyspepsia were examined by culture, histology, and DPO-based multiplex PCR. A disk diffusion test and E-test were used for performing phenotypic antibiotic susceptibility tests.
Results:  Among the biopsy specimens tested, 22.2% (47/212), 42.5% (90/212), and 41.5% (88/212) of the specimens were classified as H. pylori positive by culture, histology, and DPO-based multiplex PCR, respectively. Among 96 strains identified by either culture or DPO-based multiplex PCR, 80 strains were clarithromycin-susceptible and 16 strains (16.7%) were clarithromycin-resistant. There was 94.1% (32/34) concordance between phenotypic susceptibility tests and DPO-based multiplex PCR. In two patients with discrepant results, only DPO-based multiplex PCR detected clarithromycin-resistant strains. DPO-based multiplex PCR identified additional 49 clarithromycin-resistant or clarithromycin-susceptible H. pylori among 165 culture-negative specimens.
Conclusions:  DPO-based multiplex PCR can be used as a practical method for the detection of H. pylori infection and the determination of clarithromycin susceptibility in addition to phenotypic antimicrobial susceptibility tests.     

Interactions of Salmonella enterica with lettuce leaves

Aims:  To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors.
Methods and Results:  Salmonella strains were tested for their ability to form biofilm in various growth media using a polystyrene plate model. Strong biofilm producers were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Confocal microscopy and viable count studies revealed preferential attachment of Salmonella to cut-regions of the leaf after 2 h at 25°C, but not for 18 h at 4°C. Storage of intact lettuce pieces contaminated with Salmonella for 9 days at 4°C resulted only in small changes in population size. Exposure of lettuce-associated Salmonella cells to acidic conditions (pH 3·0) revealed increased tolerance of the attached vs planktonic bacteria.
Conclusions:  Biofilm formation on polystyrene may provide a suitable model to predict the initial interaction of Salmonella with cut Romaine lettuce leaves. Association of the pathogen with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance.
Significance and Impact of the Study:  Understanding the interactions between foodborne pathogens and lettuce might be useful in developing new approaches to prevent fresh produce-associated outbreaks.     

Predicting virulence of Aeromonas isolates based on changes in transcription of c-jun and c-fos in human tissue culture cells

Aims:  To screen for the virulence potential of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2.
Methods and Results:  Aeromonas cells were added to Caco-2 cells at a ratio of approx. 1 : 1. After 1-, 2- and 3-h incubation at 37°C, mRNA was extracted from the cells and gene expression of two host genes, c-jun and c-fos, quantified. Aeromonas isolates which were pathogenic in the neonatal mouse model demonstrated up-regulation of c-jun and c-fos compared to avirulent isolates.
Conclusions:  Human cell culture results showed that c-jun and c-fos were predictive of Aeromonas virulence.
Significance and Impact of the Study:  An Aeromonas relative virulence scale is proposed for use in the testing of Aeromonas drinking water isolates.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).