Strain-related Differences Research Articles

Super pharmacological levels of calcitriol (1,25-(OH)2 D3) inhibits mineral deposition and decreases cell proliferation in a strain dependent manner in chicken mesenchymal stem cells undergoing osteogenic differentiation in vitro

The biologically active form of vitamin D3, calcitriol (1,25-(OH)2 D3), plays a key role in mineral homeostasis and bone formation and dietary vitamin D3 deficiency is a major cause of bone disorders in poultry. Supplementary dietary cholecalciferol (25-hydroxyvitamin D, 25-OH), the precursor of calcitriol, is commonly employed to combat this problem; however, dosage must be carefully determined as excess dietary vitamin D can cause toxicity resulting in a decrease in bone calcification, hypercalcinemia and renal failure. Despite much research on the therapeutic administration of dietary vitamin D in humans, the relative sensitivity of avian species to exogenous vitamin D has not been well defined. In order to determine the effects of exogenous 1,25-(OH)2 D3 during avian osteogenesis, chicken bone marrow-derived mesenchymal stem cells (BM-MSCs) were exposed to varying doses of 1,25-(OH)2 D3 during in vitro osteogenic differentiation and examined for markers of early proliferation and osteogenic induction. Similar to humans and other mammals, poultry BM-MSCs were found to be highly sensitive to exogenous 1,25-(OH)2 D3 with super pharmacological levels exerting significant inhibition of mineralization and loss of cell proliferation in vitro. Strain related differences were apparent, with BM-MCSs derived from layers strains showing a higher level of sensitivity to 1,25-(OH)2 D3 than those from broilers. These data suggest that understanding species and strain specific sensitivities to 1,25-(OH)2 D3 is important for optimizing bone health in the poultry industry and that use of avian BM-MSCs are a useful tool for examining underlying effects of genetic variation in poultry.

Patterns of Spontaneous Local Network Activity in Developing Cerebral Cortex: Relationship to Adult Cognitive Function.

Detecting neurodevelopμental disorders of cognition at the earliest possible stages could assist in understanding them mechanistically and ultimately in treating them. Finding early physiological predictors that could be visualized with functional neuroimaging would represent an important advance in this regard. We hypothesized that one potential source of physiological predictors is the spontaneous local network activity prominent during specific periods in development. To test this we used calcium imaging in brain slices and analyzed variations in the frequency and intensity of this early activity in one area, the entorhinal cortex (EC), in order to correlate early activity with level of cognitive function later in life. We focused on EC because of its known role in different types of cognitive processes and because it is an area where spontaneous activity is prominent during early postnatal development in rodent models of cortical development. Using rat strains (Long-Evans, Wistar, Sprague-Dawley and Brattleboro) known to differ in cognitive performance in adulthood we asked whether neonatal animals exhibit corresponding strain-related differences in EC spontaneous activity. Our results show significant differences in this activity between strains: compared to a high cognitive-performing strain, we consistently found an increase in frequency and decrease in intensity in neonates from three lower performing strains. Activity was most different in one strain considered a model of schizophrenia-like psychopathology. While we cannot necessarily infer a causal relationship between early activity and adult cognition our findings suggest that the pattern of spontaneous activity in development could be an early predictor of a developmental trajectory advancing toward sub-optimal cognitive performance in adulthood. Our results further suggest that the strength of dopaminergic signaling, by setting the balance between excitation and inhibition, is a potential underlying mechanism that could explain the observed differences in early spontaneous activity patterns.

Cardiac mRNA Expression in Inbred Rat Strains Divergent in Survival Time after Hemorrhage

We previously identified inbred rat strains differing in survival time after severe controlled hemorrhage (StaH). To identify early biomarkers of hemorrhagic shock and genes that might explain differences in STaH, rats from three strains [Brown Norway (BN); Dark Agouti (DA), and Fawn Hooded Hypertensive (FHH)] with different StaH (DA = FHH > BN; 4-5 rats/group) were assigned to one of three treatment groups: non-operated controls (CNTL), surgically catheterized rats (CATH), or rats catheterized and bled (~46% hemorrhage) 24 h post-surgery (BLD). Rats were euthanized 30 min after handling or 30 min after start of a 26 min bleed. After euthanasia, hearts were removed and frozen in liquid nitrogen. Total RNA was isolated, fluorescently labeled, hybridized to Agilent rat whole genome microarrays, and statistically analyzed with BRB Array Tools. After controlling for multiple comparisons via the False Discovery Rate (<10%), mRNA expression for 65 known genes differed (P<.01). There were no inbred rat strain-related differences in mRNA expression (P>.05). Expression of 26 genes changed after BLD (20 decreased, 6 increased). These changes ranged from 5 to 556-fold. Genes with hemorrhage-associated changes included: Hif1α (22-fold decrease; oxygen homeostasis); Map4 (45-fold decrease; mitochondrial permeabilization); Mmp21 (24-fold decrease; Ca++ binding); Tubb5 (556-fold decrease; phagocytosis and protein complex binding); and Rnase10 (70-fold increase; nucleic acid binding). In the absence of inbred rat strain differences in mRNA expression, our data do not identify cardiac genes associated with differences in STaH. However, the data do provide new information concerning cardiac mRNA expression that rapidly changes after hemorrhage. Funding: US Army MRMC.

Scopolamine-induced convulsions in fasted animals after food intake: Sensitivity of C57BL/6J mice and Sprague-Dawley rats

Food intake triggers convulsions in fasted BALB/c mice and Wistar albino rats treated with antimuscarinic drugs, scopolamine or atropine. Inbred strain studies have yielded considerable information regarding genetic influences on seizure susceptibility and factors contribute to epileptogenesis in rodents. This study, therefore, investigated sensitivity to antimuscarinic-induced seizures in C57BL/6J mice and Sprague-Dawley rats. Food deprivation for 48h in mice and 52h in rats did not produce strain differences in body weight loss. Fasted animals treated i.p. with 3mg/kg scopolamine developed convulsions after food intake. The incidence of convulsions was indifferent in comparison to BALB/c mice and Wistar albino rats. Number of animals developing stage 5 was more and onset of convulsions was longer in C57BL/6J mice than in BALB/c mice. Strain-related differences in sensitivity to seizures in C57BL/6J mice may need further evaluation for investigating genetic influences on scopolamine-induced seizures.

Comparison of spatial learning in the partially baited radial-arm maze task between commonly used rat strains: Wistar, Spargue-Dawley, Long-Evans, and outcrossed Wistar/Sprague-Dawley.

Strain-related differences in animals' cognitive ability affect the outcomes of experiments and may be responsible for discrepant results obtained by different research groups. Therefore, behavioral phenotyping of laboratory animals belonging to different strains is important. The aim of the present study was to compare the variation in allothetic visuospatial learning in most commonly used laboratory rat strains: inbred Wistar (W) and Sprague-Dawley (SD), outcrossed Wistar/Sprague-Dawley (W/SD), and outbred Long Evans (LE) rats. All rats were trained to the arbitrary performance criterion of 83 % correct responses in the partially baited 12-arm radial maze allowing for simultaneous evaluation of both working and reference memory. In the present study, testing albino versus pigmented and inbred versus outcrossed rats revealed significant strain-dependent differences with the inbred SD rats manifesting lower performance on all learning measures compared to other strains. On the other hand, the outcrossed W/SD rats showed a lower frequency of reference memory errors and faster rate of task acquisition compared to both LE and W rats, with W rats showing a lower frequency of working memory errors compared to other strains. In conclusion, albinism apparently did not reduce the animals' performance in the allothetic visuospatial learning task, while outcrossing improved the spatial learning. A differential effect of strain on the contribution of each error type to the animals' overall performance was observed. The strain-dependent differences were more pronounced between subpopulations of learning-deficient individuals ("poor" learners), and generally the reference memory errors contributed more to the final behavioral output than did the working memory errors.

A histological procedure to study fungal infection in the wax moth Galleria mellonella.

The invertebrate model Galleria mellonella is a widely used factitious host to study the microbial pathogenesis in vivo. However, a specific procedure for the recovery and the processing of the infected tissues, important for a better understanding of the host-pathogen interactions, has not been reported to our knowledge. In the present study we describe a new procedure of fixation and processing of larval tissue that allows studying the larval topographic anatomy and assessing the morphological changes due to the fungal infection. Lepidopteran larvae were infected with Candida albicans strains displaying various biofilm-forming abilities. The whole larvae were then examined for tissue changes by histological techniques. We show that comparing cutting planes, serial transversal sections of paraffin-embedded larva result in better accuracy and information recovering. Using this technique, it was possible to preserve the integrity of G. mellonella internal structures allowing the detailed analysis of morphological differences in different experimental groups (i.e., healthy vs infected larvae). We were also able to study strain-related differences in the pathogenesis of C. albicans by observing the immune response elicited and the invasiveness of two isolates within the larval tissues.In general, by processing the whole larva and optimizing routinely histochemical stainings, it is possible to visualize and analyse infected tissues. Various degrees of pathogenicity (strain- or inoculum-related), and the infection time course can be described in details. Moreover, the host immune response events can be followed throughout the infectious process leading to a comprehensive picture of the studied phenomenon.

Genetic diversity influences the response of the brain to developmental lead exposure.

Although extrinsic factors, such as nutritional status, and some intrinsic genetic factors may modify susceptibility to developmental lead (Pb) poisoning, no studies have specifically examined the influence of genetic background on outcomes from Pb exposure. In this study, we used gene microarray profiling to identify Pb-responsive genes in rats of different genetic backgrounds, including inbred (Fischer 344 (F344)) and outbred (Long Evans (LE), Sprague Dawley (SD)) strains, to investigate the role that genetic variation may play in influencing outcomes from developmental Pb exposure. Male and female animals received either perinatal (gestation through lactation) or postnatal (birth through weaning) exposure to Pb in food (0, 250, or 750 ppm). RNA was extracted from the hippocampus at day 55 and hybridized to Affymetrix Rat Gene 1.0 ST Arrays. There were significant strain-specific effects of Pb on the hippocampal transcriptome with 978 transcripts differentially expressed in LE rats across all experimental groups, 269 transcripts differentially expressed in F344 rats, and only 179 transcripts differentially expressed in SD rats. These results were not due to strain-related differences in brain accumulation of Pb. Further, no genes were consistently differentially regulated in all experimental conditions. There was no set of "Pb toxicity" genes that are a molecular signature for Pb neurotoxicity that transcended sex, exposure condition, and strain. These results demonstrate the influence that strain and genetic background play in modifying the brain's response to developmental Pb exposure and may have relevance for better understanding the molecular underpinnings of the lack of a neurobehavioral signature in childhood Pb poisoning.

Strain differences in fatigue and depression after experimental stroke.

Fatigue and depression are common symptoms after stroke. Animal models of poststroke fatigue (PSF) and poststroke depression (PSD) would facilitate the study of these symptoms. Spontaneous locomotor activity is as an objective measure of fatigue and learned helplessness an accepted correlate of depression. We used different rat strains to evaluate stroke-induced changes in behavior in hopes that interstrain differences would provide insights into the biological basis of these symptoms. Male Lewis, Wistar, and Sprague-Dawley (SD) rats underwent experimental stroke. Spontaneous activity was assessed continually after stroke (for up to 50 days). In a subset of animals, the forced swim test was performed prior to and 1 month after stroke to assess learned helplessness; blood was obtained at sacrifice for cytokine assay. Stroke induced strain-related differences in activity; Lewis rats increased spontaneous activity during the dark cycle, while Wistar and SD rats increased activity during the light cycle. The velocity of movement decreased during the dark cycle in Wistar and SD rats and during the light cycle in Lewis rats. Stroke also led to an increase in learned helplessness in Lewis rats. In summary, different patterns of behaviors emerge in different rat strains after stroke. Lewis rats displayed behavior consistent with depression but not fatigue, while Wistar and SD rats displayed behavior consistent with fatigue but not depression. These data argue that PSF and PSD are different biological constructs and suggest that analysis of strain-related differences may provide insight into symptom pathophysiology.

Genetic strain differences in the development of peritoneal fibroproliferative processes in mice.

Fibroproliferative processes are regulated by a wide variety of tissue components and genetic factors. However, whether there are genetic differences in peritoneal fibroproliferative tissue formation, with consequent differences in response to drug treatment, is unclear. We characterize the influence of the genetic background on peritoneal fibroproliferative tissue induced by sponge implants in DBA/1, Swiss, C57BL/6, and BALB/c mouse strains. In addition, responses to dipyridamole in the implants were evaluated. Angiogenesis, assessed by intra-implant hemoglobin content, was highest in Swiss mice, whereas levels of vascular endothelial growth factor were highest in C57BL/6 mice. The levels of pro-inflammatory cytokines and of inflammatory enzymes (myeloperoxidase- and N-acetyl-β-D-glucosaminidase) were also strain-related. The pro-fibrogenic markers transforming growth factor beta-1 and collagen were lowest in implants placed in DBA/1 mice, whereas those in C57BL/6 mice had the highest levels. Differential sensitivity to dipyridamole was also observed, with this compound being pro-angiogenic in implants placed in DBA/1 mice but antiangiogenic in implants placed in Swiss. An overall anti-inflammatory response was observed in the inbred strains. Antifibrogenic effects were observed only in implants placed in C57BL/6 mice. These important strain-related differences in the development of peritoneal fibrosis and in response to dipyridamole must be considered in the design and analysis of studies on fibrogenesis in mice.

Abstract T P81: Long-Term Activity Levels Vary by Rat Strain Following Stroke: A Disruption of the Circadian Rhythm?

Background: Assessment of long-term behavioral outcome after experimental cerebral ischemia is important for evaluating potential therapeutic interventions. In this study, we explored strain related differences in baseline behavior and in response to stroke. Methods: Spontaneous cage activity was monitored (cm moved per hr) and analyzed before and after 2 hour middle cerebral artery occlusion (MCAO) in male Lewis, Wistar, and Sprague Dawley (SD) rats using the Noldus PhenoTyper® cages and EthoVision® Software system. Stroke severity was assessed using the neuroscore, foot fault errors, and performance on the rotarod. Infarct volume at 24 hours was determined in a second cohort of animals. Animals were sacrificed 56 days after MCAO. Data are analyzed using non-parametric statistics. Results: Prior to stroke, the median distance moved per hour during the dark was similar among the 3 strains, but Lewis rats were more active during the light cycle (P=0.001). Neuroscores did not differ between strains at 3 hrs after MCAO nor did infarct volumes at 24 hours after MCAO. Lewis rats, however, performed worse on the rotarod in the month following MCAO (P&lt;0.05 at each time point). Foot fault errors were similar throughout the study period. After stroke, Lewis rats became more active during the dark cycle while Wistar and SD rats became more active during the light cycle (Figure). Summary: The 3 strains of rats evaluated in this study had different patterns in the change in activity after MCAO. Lewis rats showed an increase in activity during the dark cycle while Wistar and SD rats showed an increase in activity during the light cycle. This observation suggests that there are genetic differences in the response to stroke that may alter the circadian rhythm after stroke.

Nociception and conditioned fear in rats: strains matter.

When using rats in pain research, strain-related differences in outcomes of tests for pain and nociception are acknowledged. However, very little is known about the specific characteristics of these strain differences. In this study four phylogenetically distant inbred rat strains, i.e. Wistar Kyoto (WKY), Fawn Hooded (FH), Brown Norway (BN) and Lewis (LE), were investigated in different tests related to pain and nociception. During Pavlovian fear conditioning, the LE and WKY showed a significantly longer duration of freezing behaviour than the FH and BN. Additionally, differences in c-Fos expression in subregions of the prefrontal cortex and amygdala between rat strains during retrieval and expression of conditioned fear were found. For example, the BN did not show recruitment of the basolateral amygdala, whereas the WKY, FH and LE did. During the hot plate test, the WKY and LE showed a lower thermal threshold compared to the BN and FH. In a follow-up experiment, the two most contrasting strains regarding behaviour during the hot plate test and Pavlovian fear conditioning (i.e. FH and WKY) were selected and the hot plate test, Von Frey test and somatosensory-evoked potential (SEP) were investigated. During the Von Frey test, the WKY showed a lower mechanical threshold compared to the FH. When measuring the SEP, the FH appeared to be less reactive to increasing stimulus intensities when considering both peak amplitudes and latencies. Altogether, the combined results indicate various differences between rat strains in Pavlovian fear conditioning, nociception related behaviours and nociceptive processing. These findings demonstrate the necessity of using multiple rat strains when using tests including noxious stimuli and suggest that the choice of rat strains should be considered. When selecting a strain for a particular study it should be considered how this strain behaves during the tests used in that study.

Genetic factors modulate the impact of pubertal androgen excess on insulin sensitivity and fertility.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. The syndrome is caused by a combination of environmental influences and genetic predisposition. Despite extensive efforts, the heritable factors contributing to PCOS development are not fully understood. The objective of this study was to test the hypothesis that genetic background contributes to the development of a PCOS-like reproductive and metabolic phenotype in mice exposed to excess DHEA during the pubertal transition. We tested whether the PCOS phenotype would be more pronounced on the diabetes-prone C57BL/6 background than the previously used strain, BALB/cByJ. In addition, we examined strain-dependent upregulation of the expression of ovarian and extra-ovarian candidate genes implicated in human PCOS, genes containing known strain variants, and genes involved with steroidogenesis or insulin sensitivity. These studies show that there are significant strain-related differences in metabolic response to excess androgen exposure during puberty. Additionally, our results suggest the C57BL/6J strain provides a more robust and uniform experimental platform for PCOS research than the BALB/cByJ strain.

Lower Omega-3 Index Is a Marker of Increased Propensity of Hypertensive Rat Heart to Malignant Arrhythmias

Polyunsaturated omega-3 fatty acids (omega-3 PUFA) are important components of cell membrane affecting its function and their deficiency is deleterious to health. We have previously shown that spontaneously hypertensive rats (SHR) are prone to life-threatening arrhythmias that are reduced by omega-3 PUFA intake. Purpose of this study was to explore plasma and red blood cells (RBC) profile of omega-3 and omega-6 PUFA as well as to determine omega-3 index, a risk factor for sudden cardiac death, in aged SHR and the effect of omega-3 PUFA intake. Male and female 12-month-old SHR and age-matched Wistar rats fed with omega-3 PUFA (200 mg/kg BW/day/2 month) were compared with untreated rats. Composition of omega-3 PUFA: alpha linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as omega-6 PUFA: linoleic acid and arachidonic acid was analyzed by gas chromatography. Results showed sex- and strain-related differences of basal omega-3 and omega-6 PUFA levels in plasma and RBC as well as in response to omega-3 PUFA intake. Comparing to Wistar rats omega-3 index, expressed as a percentage of EPA+DHA of total fatty acids, was lower in SHR and it increased due to consumption of omega-3 PUFA. Findings support our hypothesis that lower omega-3 index may be also a marker of increased propensity of the hypertensive rat heart to malignant arrhythmias.

Psychosocial work characteristics and sleep – a well-known but poorly understood association

Psychosocial work characteristics and sleep – a well-known but poorly understood association

Association of job strain with working hours, shift-dependent perceived workload, sleepiness and recovery

We explored the relationship of job strain with working hours, shift-dependent perceived workload, sleepiness and recovery. Nurses/nursing assistants (n = 95) were recruited from wards that belonged to either the top (high-strain group, HJS) or the bottom (low-strain group, LJS) job strain quartiles of a Job Content Questionnaire survey of employees in five health care districts and four cities in Finland. Three-week field measurements during naturally occurring shift schedules and a subset of pre-selected shift arrangements consisted of the Karolinska Sleepiness Scale, perceived workload and recovery. The HJS group (n = 42) had more single days off and quick returns than the LJS group (n = 53, p < 0.01), and both mental workload and physical workload were rated as higher (p < 0.01). During naturally occurring shift arrangements, severe sleepiness was more common in the HJS group only in quick returns (p = 0.04) and the HJS group recovered on average more poorly from work after all shifts (p = 0.01) and morning shifts (p = 0.02). During pre-selected shift arrangements, the differences between the groups were only minor. In conclusion, job strain-related differences in sleepiness and recovery were mostly attributable to differences in shift arrangements.

The effects of CX3CR1 deficiency and irradiation on the homing of monocyte-derived cell populations in the mouse eye.

This study examined whether CX3CR1 deficiency altered monocytic cell replenishment dynamics in ocular tissues in the context of radiation chimeras. Long-term effects of irradiation and effects of sublethal irradiation on ocular macrophages were also assessed. Bone marrow from BALB/c Cx 3 cr1 +/gfp or Cx 3 cr1 gfp/gfp mice was used to reconstitute full body irradiated WT mice and donor cell densities in the uveal tract were compared at 4 and 8 weeks post-transplantation. BALB/c and C57BL/6J chimeric mice were examined at 6 months of age to determine strain-related differences in microglial replenishment and radiation sensitivity. A separate cohort of mice were sublethally irradiated (5.5 Gy) and retinal tissue assessed 8 and 12 weeks later. CX3CR1 deficiency altered the early replenishment of monocytes in the posterior iris but not in the iris stroma, choroid or retina. In six month old chimeric mice, there were significantly higher GFP+ cell densities in the uveal tract when compared to non-irradiated 8-12 week old Cx 3 cr1 +/gfp mice. Additionally, MHC Class II expression was upregulated on hyalocytes and GFP+ cells in the peripheral retina and the repopulation of microglia appeared to be more rapid in C57BL/6J mice compared to BALB/c mice. Transient expression of MHC Class II was observed on retinal vasculature in sublethally irradiated mice. These data indicate CX3CR1-deficiency only slightly alters monocyte-derived cell replenishment in the murine uveal tract. Lethal irradiation leads to long-term increase in monocytic cell density in the uveal tract and retinal microglial activation, possibly as a sequelae to local irradiation induced injury. Microglial replenishment in this model appears to be strain dependent.

Intake of n-3 Polyunsaturated Fatty Acids Increases Omega-3 Index in Aged Male and Female Spontaneously Hypertensive Rats.

The purpose of this study was to examine whether n-3 PUFA intake affects n-3 and n-6 FA levels in plasma and red blood cells as well as omega-3 index in old male and female spontaneously hypertensive (SHR) and healthy rats. Plasma linoleic acid and eicosapentaenoic acid increased due to n-3 PUFA intake in SHR and healthy rats. Comparing to healthy rats the levels of PUFA in red blood cells of SHR were lower in males and higher in females with exception of arachidonic acid, which was high in males and low in females. Feeding of rats with n-3 PUFA resulted in increase of red blood cells levels of eicosapentaenoic acid and/or docosahexaenoic acid in a sex- and strain-dependent manner. Moreover, n-3 PUFA intake decreased arachidonic acid in healthy female rats but increased it in SHR and did not affect it in males. Omega-3 index was lower in SHR comparing to healthy rats and it increased due to the consumption of n-3 PUFA. Results point out sex- and strain-related differences in red blood cells levels of n-3 and n-6 PUFA in basal conditions as well as in response to n-3 PUFA intake.

Regarding "Clostridium Difficile Ribotype Does Not Predict Severe Infection"

To the Editor—We read with interest the recent article from Walk et al, “Clostridium difficile Ribotype Does Not Predict Severe Infection” [1]. However, their interpretation makes 2 epidemiologic errors, rendering their conclusions unreliable. First, they treat “no evidence of difference” as “evidence of no difference”: These are equivalent if, and only if, a study is adequately powered to exclude the possibility of a modest effect, for example, based on a 95% confidence interval (CI) (as in noninferiority trials). The comparison of 027/078 vs other ribotypes for severe disease had an unadjusted odds ratio (OR) of 2.33 (95% CI, 1.03–5.02; P = .035). Adjusting for age, Charlson comorbidity index, hematocrit, or platelets reduced the OR only slightly (still remaining >2.1) with P values of .06–.07 and 95% CIs of .92–4.78 (ie, essentially unchanged). The .05 P value cutoff is an arbitrary threshold from days when significance tables had to be calculated laboriously by hand [2]. Statistically, there is little difference in the strength of evidence provided from findings with P values of .035 and .07, as evidenced by the CIs in Table 3 [1], which all extend above 4, well above any plausible margin that might be considered noninferior. The lowest OR is for tcdC deletion (1.26 [95% CI, .38–4.94]); however, the meaning of this estimate is unclear as this deletion is ubiquitous among ribotype 027 isolates, and hence is completely confounded with the majority of the “hypervirulent” strains included in the dataset. Unfortunately, replicating “no evidence of difference” in a separate validation cohort [1] does not transform it into “evidence of no difference”—it simply means that 2 underpowered studies have been conducted. Another problem is adjusting for factors (biomarkers at diagnosis) on the causal pathway between an exposure (C. difficile ribotype) and an outcome (disease severity), sometimes denoted mediation or overadjustment bias [3]. Consider a pathogen in which the entire mechanism for causing disease is through raising white blood cell (WBC) count. Suppose there are only 2 strains, 1 of which causes twice the WBC count rise. By definition, all the differential mortality between these 2 strains will be due to their differential WBC count rise—adjusting for this will reduce the WBC count–adjusted effect of a strain to zero. Essentially, if there is any strain effect on a biomarker (not analyzed by Walk et al [1]), then the biomarker-adjusted OR for a strain represents an effect that is not mediated through biomarkers. It cannot be interpreted as the causal effect of strain, because it overadjusts for strain-related biomarker differences. This study shows that WBC count and albumin are strong predictors of C. difficile severity and that there is an unadjusted association between strain and severity. However, unfortunately it does not address the key question as to the causal impact of strain including biomarker-mediated and biomarker-independent effects. Our larger study of 1893 enzyme immunoassay–positive, culture-positive, multilocus sequence–typed strains shows definitively that strains vary in their overall impact on mortality (adjusted for multiple potential confounders) and in their overall impact on biomarkers (predominantly those associated with inflammatory pathways), and that residual variation in mortality risk remains even after adjusting for biomarker-mediated effects [4].

Gender and strain contributions to the variability of buprenorphine-related respiratory toxicity in mice

While most deaths from asphyxia related to buprenorphine (BUP) overdose have been reported in males, higher plasma concentrations of BUP and its toxic metabolite norbuprenorphine (NBUP) have been observed in females. We previously demonstrated that P-glycoprotein (P-gp) modulation at the blood–brain barrier (BBB) contributes highly to BUP-related respiratory toxicity, by limiting NBUP entrance into the brain. In this work, we sought to investigate the role of P-gp-mediated transport at the BBB in gender and strain-related variability of BUP and NBUP-induced respiratory effects in mice. Ventilation was studied using plethysmography, P-gp expression using western blot, and transport at the BBB using in situ cerebral perfusion. In male Fvb and Swiss mice, BUP was responsible for ceiling respiratory effects. NBUP-related reduction in minute volume was dose-dependent but more marked in Fvb (p<0.01 at 1mg/kg NBUP and p<0.001 at 3 and 9mg/kg NBUP) than in Swiss mice (p<0.001 at 9mg/kg NBUP). Female Fvb mice were more susceptible to BUP than males with significantly increased inspiratory time (p<0.05) and to NBUP with significantly increased expiratory time (p<0.01). Following BUP administration, plasma BUP concentrations were significantly higher (p<0.01) and plasma NBUP concentrations significantly lower (p<0.001) in Fvb mice compared to Swiss mice. Plasma BUP concentrations were significantly higher (p<0.05) and plasma NBUP concentrations significantly lower (p<0.01) in male compared to female Fvb mice. In contrast, following NBUP administration, comparable plasma NBUP concentrations were observed in both genders and strains. No differences in P-gp expression or BUP and NBUP transport across the BBB were observed between male and female Fvb mice as well as between Swiss and Fvb mice. Our results suggest that P-gp-mediated transport across the BBB does not play a key-role in gender and strain-related variability in BUP and NBUP-induced respiratory toxicity in mice. Both gender- and strain-related differences in respiratory effects of BUP could be attributed to BUP itself rather than to its metabolite, NBUP.

IGF-I 3' untranslated region: strain-specific polymorphisms and motifs regulating IGF-I in osteoblasts.

Reduced IGF-I is associated with low bone mass in humans and mice. C3H/He/J (C3H) mice have higher skeletal IGF-I and greater bone mass than C57BL/6J (B6). We hypothesized that strain-related genotypic differences in Igf1 affected skeletal function. The Igf1 coding region is nonpolymorphic, but its 3' untranslated region (UTR) is polymorphic between C3H and B6. Luciferase-Igf1 3' UTR reporter constructs showed that these polymorphic regions did not affect UTR function. IGF-I splice variants give rise to a common mature IGF-I peptide, but different E peptides. We identified two splice products, exon 4+6 (Ea) and exon 4+5+6 (Eb, mechano-growth factor) and found that their abundance was unchanged during osteoblastic differentiation. The Igf1 3' UTR encoded by exon 6 contains alternative polyadenylation sites. Proximal site use produces a short 3' UTR of approximately 195 bases, whereas distal site usage results in an approximately 6300-base UTR. Although Igf1 mRNA levels did not change during osteoblastic differentiation, distal polyadenylation site usage was increased in B6 cells but not in C3H. The resulting long Igf1 RNA isoform is less stable and has decreased translation efficiency, which may be one mechanism contributing to decreased IGF-I in B6 vs. C3H mice. Although the long UTR contains a conserved [GU](18) repeat, which is a positive regulator of UTR activity, it is also targeted by negative regulators, miR-29 and miR-365. These microRNAs are increased in B6 and C3H cells during osteoblastic differentiation. Differential expression of the long Igf1 3' UTR isoform may be a possible mechanism for enhanced IGF-I regulation in B6 vs. C3H mice.