Age-adjustment in experimental animal data and its application to lung cancer in radon-exposed rats

Procedures for age-adjustment of cancer fractions are proposed which do not require fixed age intervals. The full available information on survival times can then be used, which is especially important in small treatment groups. For incidental cancers a non-decreasing prevalence function and for fatal cancers the Kaplan-Meier estimator is used. In the latter case, the estimated competing risk of the control population is standardized, not its true survival. This makes the technique also applicable to treatment groups with high incidence, which otherwise may give adjusted rates above 100%. In the application part these age-adjustment techniques are used here to study lung cancer in radon-exposed Wistar and Sprague-Dawley rats. The data include a classification in fatal and incidental lung cancers. For fatal lung cancer, the lifetime excess absolute risk (LEAR) at 1 WLM averaged over all exposed groups is 0.67×10^–4 for the Wistar rats, while for the Sprague-Dawley rats it is 0.40×10^–4. For the Sprague-Dawley rats, there are several groups exposed later in life. When the averaging is restricted to animals with start of exposure prior to 150 days of age, the weighted average risk among the Sprague-Dawley rats is 0.79×10^–4. Compared to groups with similar exposures as young adults (up to about 150 days), animals exposed later in life have substantially lower lifetime risks. The Wistar rats include groups with roughly equal exposure rates and ages at start of exposure, but with increasing exposure duration. Within these groupings the LEAR at 1 WLM does not decrease with additional exposure at higher age, as would be expected if the risk from exposures at different ages would be additive.     

Psoralen plus near ultraviolet light inactivation of cultured Chinese hamster cells and its relation to DNA cross-links

The survival curve of Chinese hamster cells exposed to near ultraviolet (NUV) light in the presence of 4,5′,8-trimethylpsoralen exhibits a pronounced shoulder, i.e. cells accumulate sublethal damage before survival decreases exponentially. During incubation between fractionated exposures, the cells are able to recover their capacity to accumulate sublethal damage, although at a slower rate than after X-irradiation. Synchronized cells demonstrate a pronounced age-response variation, similar to that of X-irradiated cells, in which maximum resistance occurs in the second half of the S phase. During a second NUV exposure without psoralen, following a first treatment consisting of psoralen-plus-NUV, only DNA cross-links are produced. Using this procedure a correlation was found between cell killing and the production of DNA cross-links.     

The relationship between cell volume,ploidy, and functional activity in differentiating hepatocytes

Liver cells were isolated by collagenase perfusion from rats of 1 day, and 1, 3, 5, and 12 weeks of age, fractionated by velocity sedimentation at 1g (STAPUT), and the major cell types were identified in terms of specific functions. Alphafetoprotein and albumin were used as markers of differentiating hepatocytes and these functional activities were evaluated in a quantitative manner using a radio-immunoassay. The capacity of this cell type to store³⁵S-BSP, an indicator of bile formation, was also evaluated. Sinusoidal cells and hematopoietic cells were identified on the basis of their ability to take up^99mTC-colloid sulfur and to incorporate⁵⁹Fe, respectively. The fractionation procedure allowed a good separation of sinusoidal cells from hepatocytes at all postnatal ages and also of erythroid cells still present during the first week after birth. With increasing age, alphafetoprotein-producing hepatocytes exhibited changes in sedimentation velocities that parallelled those of albumin-producers. In turn, the latter hepatocyte subpopulation underwent gradual shifts in modal peak velocities similar to those of bile-forming hepatocytes. The fractionated hepatocytes obtained at different ages were further analyzed in terms of cell volume and nuclear ploidy using a Coulter counter system. This quantitative analysis obtained at the cellular level demonstrated that during the age-related differentiation of hepatocytes, which occurs during the postnatal period and results in the gradual appearance of cells of higher ploidy levels, the extent of albumin production and bile formation can be correlated with the hepatocyte volume.     

Functionally undefined gene, yggE, alleviates oxidative stress generated by monoamine oxidase in recombinant Escherichia coli

Real-time PCR analysis showed that yggE gene was about two and three times up-regulated in Escherichia coli cells exposed to UVA irradiation and thermal elevation, respectively, suggesting that this gene is responsive to physiological stress. The yggE gene was introduced into E. coli BL21 cells, together with a monoamine oxidase (MAO) gene as a model source for oxidative stress generation. The distribution of independently isolated transformants (two dozen isolates) was examined in terms of MAO activity and cell vitality. In the case of control strain expressing MAO alone, the largest number of transformants existed in the low range of MAO activity less than 2 units mg⁻¹ and the number significantly decreased at increased MAO activity. On the other hand, the distribution of MAO/YggE-coexpressing transformants shifted to higher MAO activity with frequent appearance in the activity range of 4–8 units mg⁻¹. The yggE gene product therefore has a possible function for alleviating the stress generated in the cells.     

Effect of light intensity on chlorophyll fluorescence in wheat leaves: Application of PAM-fluorometry

Application of pulse-amplitude-modulation (PAM) fluorometers for measuring slow stages of chlorophyll fluorescence induction (CFI) is considered. With an example of Triticum aestuvum L. plants grown under continuous illumination at a photon flux density of 600 μmol/(m² s) photosynthetically active radiation (PAR), the CFI curves were analyzed with leaves of various ages as a function of actinic light intensity. The fluorometer PAM-2100 was applied for measurements of CFI curves. The characteristic peaks of CFI curves in wheat leaves were most conspicuous and had the largest amplitudes at 600–800 μmol/(m² s) PAR, which corresponds to the middle range of actinic light intensities employed in PAM-2100 fluorometers. In plants exposed to favorable and stressful conditions, the developmental stages may proceed at different rates; thus, the comparison of fluorescence parameters for leaves of equal calendar age but having different physiological states may provide ambiguous data. Therefore, the feasibility of recording CFI curves of different types is quite important for rapid diagnostics of the age and state of plant leaves, as well as for adequate physiological conclusions.     

Recovery Patterns of Moina Macrocopa Exposed Previously to Different Concentrations of Cadmium and Methyl Parathion: Life-Table Demography and Population Growth Studies

In most toxicity studies using Cladocera, bioassays are routinely done to determine median lethal concentration (LC₅₀) or the responses to sublethal exposure. However, information on the patterns of recovery of cladocerans exposed to different concentrations of toxicants is scarce. This is important because cladocerans exposed to toxicants for a short duration may later recuperate under favourable conditions. Using the life table demographic and population growth, the present study was conducted to evaluate the recovery patterns of Monia macrocopa exposed to five different concentrations (0, 25, 50, 75 and 100% of 24 h LC₅₀ for CdCl₂ or methyl parathion) and then returned to toxicant-free medium containing alga (Chlorella vulgaris) at low (0.25 × 10⁶cells ml^–1), medium (0.5 × 10⁶cells ml^–1) or high (1 × 10⁶cells ml^–1) levels. We measured selected life history variables such as average lifespan, life expectancy at birth, gross and net reproductive rates, generation time and the rate of population increase. Results indicated that regardless of food concentration, surviving individuals of M. macrocopa exposed to a median lethal concentration did not recover. The effect of food level was significant at 25 and 50% of the median lethal concentration for cadmium or methyl parathion. Age-specific fecundity curves showed that exposure to either toxicant for a duration as short as 24 h at one-fourth of the LC₅₀ showed reduced output of offspring, especially at a lower food level. At and above exposures of 0.037 mgl^–1 of methyl parathion, no reproduction occurred. The highest gross and net reproductive rates (127 and 55 offspring female^–1) were obtained in controls at the high (1 × 10⁶ cells ml^–1) algal food level. The rate of population increase obtained from life table data was around 0.7 per day in controls but decreased when exposed to toxicant concentrations. The rates of population increase per day derived from population growth data varied from 0.22 to 0.33 per day for the controls, depending on the food levels.     

Toward reconciling inferences concerning genetic variation in senescence in Drosophila melanogaster.

Standard models for senescence predict an increase in the additive genetic variance for log mortality rate late in the life cycle. Variance component analysis of age-specific mortality rates of related cohorts is problematic. The actual mortality rates are not observable and can be estimated only crudely at early ages when few individuals are dying and at late ages when most are dead. Therefore, standard quantitative genetic analysis techniques cannot be applied with confidence. We present a novel and rigorous analysis that treats the mortality rates as missing data following two different parametric senescence models. Two recent studies of Drosophila melanogaster, the original analyses of which reached different conclusions, are reanalyzed here. The two-parameter Gompertz model assumes that mortality rates increase exponentially with age. A related but more complex three-parameter logistic model allows for subsequent leveling off in mortality rates at late ages. We find that while additive variance for mortality rates increases for late ages under the Gompertz model, it declines under the logistic model. The results from the two studies are similar, with differences attributable to differences between the experiments.     

Neuropeptides Control the Dynamic Behavior of Airway Mucosal Dendritic Cells

The airway mucosal epithelium is permanently exposed to airborne particles. A network of immune cells patrols at this interface to the environment. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated the impact of neuronal signals on key functions of dendritic cells (DC). Using two-photon microscopic time-lapse analysis of living lung sections from CD11c-EYFP transgenic mice we studied the influence of neuropeptides on airway DC motility. Additionally, using a confocal microscopic approach, the phagocytotic capacity of CD11c⁺ cells after neuropeptide stimulation was determined. Electrical field stimulation (EFS) leads to an unspecific release of neuropeptides from nerves. After EFS and treatment with the neuropeptides vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP), airway DC in living lung slices showed an altered motility. Furthermore, the EFS-mediated effect could partially be blocked by pre-treatment with the receptor antagonist CGRP_8–37. Additionally, the phagocytotic capacity of bone marrow-derived and whole lung CD11c⁺ cells could be inhibited by neuropeptides CGRP, VIP, and Substance P. We then cross-linked these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model altered neuropeptide amounts and DC motility in the airways could be measured. In summary, our data suggest that neuropeptides modulate key features motility and phagocytosis of mouse airway DC. Therefore altered neuropeptide levels in airways during allergic inflammation have impact on regulation of airway immune mechanisms and therefore might contribute to the pathophysiology of asthma.     

Sublethal effect of a weak intermittent magnetic field on the development of Xenopus laevis (Daudin) tadpoles

In three repeated experiments with three different litters of Xenopus laevis (Daudin) tadpoles, three cohorts were reared in an aquarium under the "saw-tooth" magnetic field produced by a television set. Their maturation times are compared with those of three corresponding control cohorts grown in an unexposed aquarium. In the exposed aquarium, the magnetic field amplitude was less than 25 T and the frequency in the extremely-low-frequency and very-low-frequency wavebands. Neither the exposed nor the unexposed cohorts suffered significant mortality and malformations. However, the exposed tadpoles took about 5 days more than the unexposed ones to reach metamorphosis. The differences in mean maturation times between the exposed and control cohorts were extremely significant (P < 0.001). The results show that a biological population can suffer a sublethal effect when exposed to the magnetic field of a TV set for a long time in the course of juvenile life stages, and that this effect can consist of a delay in reaching the adult stage.     

Effects of atmospheric small negative ions on the oxygen consumption of mouse liver cells

The effects of small negative air ions on the oxygen uptake of isolated mouse liver cells were studied by exposing the liver cells to varying ion concentrations. For concentrations of the order of 1–2 × 10⁵ ions/cm³, the oxygen uptake was always higher than in the normal atmospheric conditions of 3–8 × 10²/ions/cm³. For intermediate concentrations varying effects of activation and inhibition were observed. A statistical analysis showed that the oxygen uptake increased by approximately 14% when liver cells were exposed to ion concentrations of values 1–9 times the normal, by approximately 9% when exposed to 10–99 times the normal, and by approximately 38% when exposed to 100–999 times the normal. The significance and possible implications of the results are discussed.     

Specific protein synthesis in cellular differentiation: VI. Temporal expression of chorion gene families in Bombyx mori strain C108

Detailed patterns of expression for putative members of 5 major chorion gene families have been obtained by separating labeled proteins using two-dimensional polyacrylamide gel electrophoresis. Proteins fall into 4 temporal cohorts called early, early middle, middle, and late on the basis of when they initiate and terminate synthesis. Proteins synthesized during the early, early middle, and late periods are highly synchronous, exhibiting abrupt onset times and relatively uniform termination times. Middle proteins begin synthesis in small groups at staggered times over a relatively long period, but most cease translation as the late proteins turn on. This data is correlated with a previous follicle staging system based on separation of newly synthesized chorion proteins by isoelectric focusing alone. The absolute timing of choriogenesis was determined in vivo, using trypan blue dye to mark vitellogenic follicles. The relative age difference between chorionating follicles was 2.2–2.6 hr; chorion biosynthesis took 4 days in all. These data are discussed in terms of patterns of activity of chorion gene families, the functions of the temporal cohorts, and regulation of the chorion multigene system.     

Effects of exposure to electromagnetic field from 915 MHz radiofrequency identification system on circulating blood cells in the healthy adult rat

We investigated whether exposure to the 915 MHz radiofrequency identification (RFID) signal affected circulating blood cells in rats. Sprague–Dawley rats were exposed to RFID at a whole‐body specific absorption rate of 2 W/kg for 8 h per day, 5 days per week, for 2 weeks. Complete blood counts were performed after RFID exposure, and the CD4⁺/CD8⁺ ratio was determined by flow cytometry. The number of red blood cells (RBCs) and the values of hemoglobin, hematocrit, and RBC indices were increased in the RFID‐exposed group compared with those in the cage‐control and sham‐exposed groups (P < 0.05). However, the RBCs and platelet numbers were within normal physiologic response ranges. The number of white blood cells, including lymphocytes, was decreased in RFID‐exposed rats. However, there was no statistically significant difference between the sham‐exposed and RFID‐exposed groups in terms of T‐cell counts or CD4⁺/CD8⁺ ratio (P > 0.05). Although the number of circulating blood cells was significantly altered by RFID exposure at a whole‐body specific absorption rate of 2 W/kg for 2 weeks, these changes do not necessarily indicate that RFID exposure is harmful, as they were within the normal physiological response range. Bioelectromagnetics. 39:68–76, 2018. © 2017 Wiley Periodicals, Inc.     

Modeling the time dependent biodistribution of Samarium-153 ethylenediamine tetramethylene phosphonate using compartmental analysis

Aim

The main purpose of this work was to develop a pharmacokinetic model for the bone pain palliation agent Samarium-153 ethylenediamine tetramethylene phosphonate ([¹⁵³Sm]-EDTMP) in normal rats to analyze the behavior of the complex.

Background

The use of compartmental analysis allows a mathematical separation of tissues and organs to determine the concentration of activity in each fraction of interest. Biodistribution studies are expensive and difficult to carry out in humans, but such data can be obtained easily in rodents.

Materials and methods

We have developed a physiologically based pharmacokinetic model for scaling up activity concentration in each organ versus time. The mathematical model uses physiological parameters including organ volumes, blood flow rates, and vascular permabilities; the compartments (organs) are connected anatomically. This allows the use of scale-up techniques to predict new complex distribution in humans in each organ.

Results

The concentration of the radiopharmaceutical in various organs was measured at different times. The temporal behavior of biodistribution of ¹⁵³Sm-EDTMP was modeled and drawn as a function of time.

Conclusions

The variation of pharmaceutical concentration in all organs is described with summation of 6–10 exponential terms and it approximates our experimental data with precision better than 2%.     

A Regulatory Polymorphism in HAVCR2 Modulates Susceptibility to HIV-1 Infection

The HAVCR2 gene encodes TIM-3, an immunoglobulin superfamily member expressed by exhausted CD8+ T cells during chronic viral infection. We investigated whether genetic variation at HAVCR2 modulates the susceptibility to HIV-1 acquisition; specifically we focused on a 3′ UTR variant (rs4704846, A/G) that represents a natural selection target. We genotyped rs4704846 in three independent cohorts of HIV-1 exposed seronegative (HESN) individuals with different geographic origin (Italy and Spain) and distinct route of exposure to HIV-1 (sexual and injection drug use). Matched HIV-1 positive subjects and healthy controls were also analyzed. In all case-control cohorts the minor G allele at rs4704846 was more common in HIV-1 infected individuals than in HESN, with healthy controls showing intermediate frequency. Results from the three association analyses were combined through a random effect meta-analysis, which revealed no heterogeneity among samples (Cochrane''s Q, p value =  0.89, I² =  0) and yielded a p value of 6.8 ×10⁻⁴. The minor G allele at rs4704846 was found to increase HAVCR2 expression after in vitro HIV-1 infection. Thus, a positively selected polymorphism in the 3′ UTR, which modulates HAVCR2 expression, is associated with the susceptibility to HIV-1 infection. These data warrant further investigation into the role of TIM-3 in the prevention and treatment of HIV-1/AIDS.     

Repeated Schistosoma japonicum Infection Following Treatment in Two Cohorts: Evidence for Host Susceptibility to Helminthiasis?

Background

In light of multinational efforts to reduce helminthiasis, we evaluated whether there exist high-risk subpopulations for helminth infection. Such individuals are not only at risk of morbidity, but may be important parasite reservoirs and appropriate targets for disease control interventions.

Methods/Principal Findings

We followed two longitudinal cohorts in Sichuan, China to determine whether there exist persistent human reservoirs for the water-borne helminth, Schistosoma japonicum, in areas where treatment is ongoing. Participants were tested for S. japonicum infection at enrollment and two follow-up points. All infections were promptly treated with praziquantel. We estimated the ratio of the observed to expected proportion of the population with two consecutive infections at follow-up. The expected proportion was estimated using a prevalence-based model and, as highly exposed individuals may be most likely to be repeatedly infected, a second model that accounted for exposure using a data adaptive, machine learning algorithm. Using the prevalence-based model, there were 1.5 and 5.8 times more individuals with two consecutive infections than expected in cohorts 1 and 2, respectively (p<0.001 in both cohorts). When we accounted for exposure, the ratio was 1.3 (p = 0.013) and 2.1 (p<0.001) in cohorts 1 and 2, respectively.

Conclusions/Significance

We found clustering of infections within a limited number of hosts that was not fully explained by host exposure. This suggests some hosts may be particularly susceptible to S. japonicum infection, or that uncured infections persist despite treatment. We propose an explanatory model that suggests that as cercarial exposure declines, so too does the size of the vulnerable subpopulation. In low-prevalence settings, interventions targeting individuals with a history of S. japonicum infection may efficiently advance disease control efforts.     

Adjuvant cytokine-induced killer cell immunotherapy improves long-term survival in patients with stage I–II non-small cell lung cancer after curative surgery

Background aimsNon-small cell lung cancer (NSCLC) remains the most common cancer worldwide, with an annual incidence of around 1.3 million. Surgery represents the standard treatment in early-stage NSCLC when feasible. However, because of cancer recurrence, only approximately 53% of patients with stage I and II NSCLC survive 5 years after radical surgery. The authors performed a retrospective study to investigate the impact of cytokine-induced killer (CIK) cell immunotherapy on the long-term survival of patients with stage I–II NSCLC after curative resection.MethodsFifty-seven patients with NSCLC were included in the study, with 41 and 16 in the control and CIK groups, respectively. Clinical characteristics were compared using a t-test and χ² test. Survival analysis of patients with NSCLC was performed using the Kaplan–Meier method. The phenotypes and anti-tumor functions of CIK cells were evaluated by flow cytometry.ResultsPatients in the CIK group exhibited significantly longer overall survival (OS) and better disease-free survival (DFS) than those in the control group. Subgroup analysis indicated that patients with a higher risk of recurrence benefited more from CIK treatment and attained longer OS and DFS compared with those in the control group. No severe adverse events related to CIK treatment occurred. CIK cells contained a higher proportion of CD3⁺CD56⁺ natural killer (NK) T cells and CD3⁺ and CD8⁺ T cells and a lower proportion of CD3^–CD56⁺ NK cells compared with peripheral blood mononuclear cells. CIK cells exhibited potent tumor-killing ability, with longer contact times with tumor cells and a greater number of cells exposed to tumor cells.ConclusionsThe authors’ data suggest that adjuvant CIK cell therapy is a safe and effective therapeutic strategy for improving OS and DFS in patients with stage I–II NSCLC after curative resection.     

Growth rates of young-of-year shovelnose sturgeon in the Upper Missouri River

Information on growth during the larval and young‐of‐year life stages in natural river environments is generally lacking for most sturgeon species. In this study, methods for estimating ages and quantifying growth were developed for field‐sampled larval and young‐of‐year shovelnose sturgeon Scaphirhynchus platorynchus in the upper Missouri River. First, growth was assessed by partitioning samples of young‐of‐year shovelnose sturgeon into cohorts, and regressing weekly increases in cohort mean length on sampling date. This method quantified relative growth because ages of the cohorts were unknown. Cohort increases in mean length among sampling dates were positively related (P < 0.05, r² > 0.59 for all cohorts) to sampling date, and yielded growth rate estimates of 0.80–2.95 mm day⁻¹ (2003) and 0.44–2.28 mm day⁻¹ (2004). Highest growth rates occurred in the largest (and earliest spawned) cohorts. Second, a method was developed to estimate cohort hatch dates, thus age on date of sampling could be determined. This method included quantification of post‐hatch length increases as a function of water temperature (growth capacity; mm per thermal unit, mm TU⁻¹), and summation of mean daily water temperatures to achieve the required number of thermal units that corresponded to post‐hatch lengths of shovelnose sturgeon on sampling dates. For six of seven cohorts of shovelnose sturgeon analyzed, linear growth models (r² ≥ 0.65, P < 0.0001) or Gompertz growth models (r² ≥ 0.83, P < 0.0001) quantified length‐at‐age from hatch through 55 days post‐hatch (98–100 mm). Comparisons of length‐at‐age derived from the growth models indicated that length‐at‐age was greater for the earlier‐hatched cohorts than later‐hatched cohorts. Estimated hatch dates for different cohorts were corroborated based on the dates that newly‐hatched larval shovelnose sturgeon were sampled in the drift. These results provide the first quantification of growth dynamics for field‐sampled age‐0 shovelnose sturgeon in a natural river environment, and provide an accurate method for estimating age of wild‐caught individuals. Methods of age determination used in this study have applications to sturgeons in other regions, but require additional testing and validation.     

Investigation on supraphysiological thermal injury in two well-differentiated human hepatoma cell lines,HepG2 and Hep3B

Hyperthermia is a promising treatment for carcinoma cells. The thermal injuries of two hepatoma carcinoma cell lines with the identical cytological grade, HepG2 and Hep3B cell lines, were investigated systematically in the present study. The homemade heating stage was used to provide a constant temperature between 40 and 70 °C for thermal treatment. When the cells were exposed to temperatures ranging from 40 to 45 °C, Hep3B cells had a lower thermotolerance than the HepG2 cells; however, the survival rate of these two cell lines was still high. The differences in thermotolerance between HepG2 and Hep3B cells were more significant at the range of 50–55 °C than those at lower-level temperatures of 40–45 °C. Furthermore, the viability of the cells was less than 10% when they were exposed to a supraphysiological temperature of 60 °C for 5 min; these cell lines suffered from injury saturation under that thermal treatment. The statistical analysis also concluded that Hep3B cells are more susceptible to heat stress than are the HepG2 cells when subjected to the thermal treatment applied in this work, the exception being when thermal injury saturation occurred. The kinematic parameters of the activation energy and frequency factor for HepG2 and Hep3B cells were also quantitatively determined herein. The activation energies (ΔE) for HepG2 and Hep3B cells were 170.17 and 152.44 kJ/mol, respectively. Furthermore, the frequency factors (A) for HepG2 and Hep3B cells were 4.11×10²⁴ and 1.07×10²² s⁻¹, respectively.