Development of Standardized Insulin Treatment Protocols for Spontaneous Rodent Models of Type 1 Diabetes

Standardized protocols for maintaining near-normal glycemic levels in diabetic rodent models for testing therapeutic agents to treat disease are unavailable. We developed protocols for 2 common models of spontaneous type 1 diabetes, the BioBreeding diabetes-prone (BBDP) rat and nonobese diabetic (NOD) mouse. Insulin formulation, dose level, timing of dose administration, and delivery method were examined and adjusted so that glycemic levels remained within a normal range and fluctuation throughout feeding and resting cycles was minimized. Protamine zinc formulations provided the longest activity, regardless of the source of insulin. Glycemic control with few fluctuations was achieved in diabetic BBDP rats through twice-daily administration of protamine zinc insulin, and results were similar regardless of whether BBDP rats were acutely or chronically diabetic at initiation of treatment. In contrast, glycemic control could not be attained in NOD mice through administration of insulin twice daily. However, glycemic control was achieved in mice through daily administration of 0.25 U insulin through osmotic pumps. Whereas twice-daily injections of protamine zinc insulin provided glycemic control with only minor fluctuations in BBDP rats, mice required continuous delivery of insulin to prevent wide glycemic excursions. Use of these standard protocols likely will aid in the testing of agents to prevent or reverse diabetes.Abbreviations: BBDP, BioBreeding diabetes-prone; BBDR, BioBreeding diabetes-resistant; NOD, nonobese diabetic; PZI, protamine zinc insulin; T1D, type 1 diabetes; VAF, viral-antibody–free; ZT, Zeitgeber timeClinical trials to prevent or reverse type 1 diabetes (T1D) are predicated on preclinical study data obtained from animal models of the disease to determine agents that exhibit efficacy and translational potential. However, according to findings published over the past several years (summarized in references 2, 17, and 31), not all preclinical T1D studies are created equal. Without a standardized screening process, the hundreds of candidate therapeutic agents in development cannot be evaluated critically for translational potential. One parameter that varies considerably from report to report in T1D reversal studies is the insulin treatment provided to diabetic NOD mice. To address the need for standardized preclinical screening of new therapeutics, the National Institute for Diabetes and Digestive and Kidney Diseases has developed the Type 1 Diabetes Preclinical Testing Program.^2,35 Under this program, a central contract testing facility (Biomedical Research Models) bridged the gap between discovery of potential therapeutics and clinical testing for efficacy in prevention or reversal of T1D. Using 2 of the best characterized models of T1D, the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse, we sought to develop standardized protocols for the treatment of diabetes with insulin to provide the best glycemic control throughout the fed and nonfed states. We began by housing these models in a viral-antibody–free (VAF) barrier facility, we created study designs approved by a scientific advisory board consisting of leaders in the field, and we performed studies by using standard operation procedures.The standard of care in patients with T1D is to attempt to maintain near-normal glucose levels, by providing exogenous insulin therapy several times daily via injection or pump after rigorous monitoring of glycemic levels and by appropriately coordinating insulin dosing with food intake. Current blood glucose control in diabetic rodent models focuses on maintaining the diabetic animal in a state of moderate hyperglycemia, with normal weight gain in the absence of severe ketonuria. This state is achieved by once-daily injections of titrated insulin doses or by implantation of continuous release insulin pellets;³⁸ however, insulin types and methods can vary widely between institutions and laboratories, yielding a wide range of glycemic control. Therefore there is marked difference between the stringent glycemic control targeted by humans with diabetes as compared with the relatively loose glycemic control afforded to rodents with diabetes. Despite the many physiologic differences between humans and rodents, glycemic control potentially can be addressed by making insulin treatment in rodents more comparable in terms of glycemic control to what is achieved currently in humans, especially given that patients with T1D will continue to administer insulin during treatment with therapeutic agents (for example, antiCD3).¹¹ The lessening of the frequency, duration, and severity of hyperglycemic events is anticipated to provide the best chance for β cells to rest (function properly) while interventions are tested.²¹ Ideally, for these studies, animals should receive sufficient insulin to maintain glycemic levels close to the normal range in control nondiabetic animals.For these studies, we focused on the 2 most widely used spontaneous rodent models of T1D: the BioBreeding diabetes-prone (BBDP) rat and the nonobese diabetic (NOD) mouse.^1,12 The BBDP strain originated from a colony of outbred Wistar rats that developed spontaneous diabetes at the BioBreeding Laboratories in the 1970s. In the 1980s, the strain was acquired by the University of Massachusetts Medical School. During inbreeding, the BioBreeding diabetes- resistant (BBDR) control strain was established. Both strains are maintained at our facility and represent the most fully inbred (more than 110 generations) and characterized colonies available. BBDP rats develop T1D at 50 to 90 d of age at a frequency of approximately 85% to 90%, with equal frequency in male and female rats; the disease in BBDP rats results from autoimmune insulitis that is mediated primarily by CD4⁺ and CD8⁺ T cells and the development of autoantibodies to islet antigen. This insulitis is similar to that in human patients.¹⁸ Insulin therapy is required shortly after onset of hyperglycemia or death will occur due to ketoacidosis.¹⁹ The Gimap5 mutation in BBDP rats results in a T-cell lymphopenia and is necessary for development of T1D in BBDP rats (along with expression of a MHC class II RT1 B/D^u allele); adoptive transfer of splenocytes or regulatory T cells from BBDR rats before 35 d of age prevents the onset of diabetes in BBDP rats.^9,28,38 Alternatively, depletion of regulatory T cells from BBDR rats (which are nonlymphopenic) induces T1D in that strain.The NOD mouse strain originated from selective inbreeding of the Cataract Shionogi mouse strain and was imported from Japan to The Joslin Diabetes Center in 1984. NOD mice are now the most widely used preclinical model of T1D, in part due to the availability of genetic analysis and manipulation as well as the wide array of reagents available for mechanistic studies. The most commonly cited source for NOD mice is The Jackson Laboratory (Bar Harbor, ME), where female NOD mice develop disease at a frequency of 65% to 100% by 30 wk of age, whereas male NOD mice develop disease at a frequency of 35% to 85% (inbred for more than 83 generations). The incidence can vary from year to year³⁴ and from facility to facility depending on several factors, the most important being housing conditions.^15,26 The incidence of T1D in female NOD mice at our VAF barrier facility has been 65% to 80% over the past 3 y; this frequency can be far lower in nonVAF facilities. Diabetic NOD mice exhibit mild ketoacidosis, which allows them to survive for as long as several weeks after the onset of hyperglycemia without supportive insulin treatment. NOD mice also present with insulin resistance and a distinct stage of insulitis, referred to as peri-insulitis, that is not found in either human T1D or in diabetic BBDP rats.^5,18 Although both NOD mouse and BBDP rat models of T1D have particular advantages and disadvantages, a prudent path of drug development would include the examination of the therapeutic efficacy of novel agents in both models.^2,31To standardize and improve current testing protocols, we developed insulin treatment regimens that maintain blood glucose levels near normal levels throughout day and night activities over prolonged periods, as would be expected to occur in interventional clinical trials. We show here that whereas 2 daily injections of insulin to diabetic BBDP rats were sufficient to achieve our goal, diabetic NOD mice required continuous delivery of insulin through the implantation of osmotic pumps.     

Short and long-term safety of the 2009 AS03-adjuvanted pandemic vaccine

Background

This study assessed the short and the long term safety of the 2009 AS03 adjuvanted monovalent pandemic vaccine through an active web-based electronic surveillance. We compared its safety profile to that of the seasonal trivalent inactivated influenza vaccine (TIV) for 2010–2011.

Methodology/Principal Findings

Health care workers (HCW) vaccinated in 2009 with the pandemic vaccine (Arepanrix ® from GSK) or HCW vaccinated in 2010 with the 2010–2011 TIV were invited to participate in a web-based active surveillance of vaccine safety. They completed two surveys the day-8 survey covered the first 7 days post-vaccination and the day-29 survey covered events occurring 8 to 28 days after vaccination. Those who reported a problem were called by a nurse to obtain details. The main outcome was the occurrence of a new health problem or the worsening of an existing health condition that resulted in a medical consultation or work absenteeism. For the pandemic vaccine, a six-month follow-up for the occurrence of serious adverse events (SAE) was conducted. Among the 6242 HCW who received the pandemic vaccine, 440 (7%) reported 468 events compared to 328 of the 7645 HCW (4.3%) who reported 339 events after the seasonal vaccine. The 2009 pandemic vaccine was associated with significantly more local reactions than the 2010–2011 seasonal vaccine (1% vs. 0.03%, p<0.001). Paresthesia was reported by 7 HCW (0.1%) after the pandemic vaccine but by none after the seasonal vaccine. For the pandemic vaccine, no clustering of SAE was found in the 6 month follow-up.

Conclusion

The 2009 pandemic vaccine seems to have a good safety profile, similar to the 2010–2011 TIV, with the exception of local reactions. This surveillance was adequately powered to identify AE associated with an excess risk ≥1 per 1000 vaccinations but is insufficient to detect rare AE.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01289418","term_id":"NCT01289418"}}NCT01289418, {"type":"clinical-trial","attrs":{"text":"NCT01318876","term_id":"NCT01318876"}}NCT01318876     

Multiple SNP Markers Reveal Fine-Scale Population and Deep Phylogeographic Structure in European Anchovy (Engraulis encrasicolus L.)

Geographic surveys of allozymes, microsatellites, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have detected several genetic subdivisions among European anchovy populations. However, these studies have been limited in their power to detect some aspects of population structure by the use of a single or a few molecular markers, or by limited geographic sampling. We use a multi-marker approach, 47 nDNA and 15 mtDNA single nucleotide polymorphisms (SNPs), to analyze 626 European anchovies from the whole range of the species to resolve shallow and deep levels of population structure. Nuclear SNPs define 10 genetic entities within two larger genetically distinctive groups associated with oceanic variables and different life-history traits. MtDNA SNPs define two deep phylogroups that reflect ancient dispersals and colonizations. These markers define two ecological groups. One major group of Iberian-Atlantic populations is associated with upwelling areas on narrow continental shelves and includes populations spawning and overwintering in coastal areas. A second major group includes northern populations in the North East (NE) Atlantic (including the Bay of Biscay) and the Mediterranean and is associated with wide continental shelves with local larval retention currents. This group tends to spawn and overwinter in oceanic areas. These two groups encompass ten populations that differ from previously defined management stocks in the Alboran Sea, Iberian-Atlantic and Bay of Biscay regions. In addition, a new North Sea-English Channel stock is defined. SNPs indicate that some populations in the Bay of Biscay are genetically closer to North Western (NW) Mediterranean populations than to other populations in the NE Atlantic, likely due to colonizations of the Bay of Biscay and NW Mediterranean by migrants from a common ancestral population. Northern NE Atlantic populations were subsequently established by migrants from the Bay of Biscay. Populations along the Iberian-Atlantic coast appear to have been founded by secondary waves of migrants from a southern refuge.     

Witnessing Phenotypic and Molecular Evolution in the Fruit Fly

This multi-day exercise is designed for a college genetics and evolution laboratory to demonstrate concepts of inheritance and phenotypic and molecular evolution using a live model organism, Drosophila simulans. Students set up an experimental fruit fly population consisting of ten white-eyed flies and one red-eyed fly. Having red eyes is advantageous compared to having white eyes, allowing students to track the spread of this advantageous trait over several generations. Ultimately, the students perform polymerase chain reaction and gel electrophoresis at two neutral markers, one located in close proximity to the eye color locus and one located at the other end of the chromosome. Students observe that most flies have red eyes, and these red-eyed flies have lost variation at the near marker but maintained variation at the far marker hence observing a ??selective sweep?? and the ??hitchhiking?? of a nearby neutral variant. Students literally observe phenotypic and molecular evolution in their classroom!     

Common garden experiments reveal uncommon responses across temperatures,locations, and species of ants

Population changes and shifts in geographic range boundaries induced by climate change have been documented for many insect species. On the basis of such studies, ecological forecasting models predict that, in the absence of dispersal and resource barriers, many species will exhibit large shifts in abundance and geographic range in response to warming. However, species are composed of individual populations, which may be subject to different selection pressures and therefore may be differentially responsive to environmental change. Asystematic responses across populations and species to warming will alter ecological communities differently across space. Common garden experiments can provide a more mechanistic understanding of the causes of compositional and spatial variation in responses to warming. Such experiments are useful for determining if geographically separated populations and co‐occurring species respond differently to warming, and they provide the opportunity to compare effects of warming on fitness (survivorship and reproduction). We exposed colonies of two common ant species in the eastern United States, Aphaenogaster rudis and Temnothorax curvispinosus, collected along a latitudinal gradient from Massachusetts to North Carolina, to growth chamber treatments that simulated current and projected temperatures in central Massachusetts and central North Carolina within the next century. Regardless of source location, colonies of A. rudis, a keystone seed disperser, experienced high mortality and low brood production in the warmest temperature treatment. Colonies of T. curvispinosus from cooler locations experienced increased mortality in the warmest rearing temperatures, but colonies from the warmest locales did not. Our results suggest that populations of some common species may exhibit uniform declines in response to warming across their geographic ranges, whereas other species will respond differently to warming in different parts of their geographic ranges. Our results suggest that differential responses of populations and species must be incorporated into projections of range shifts in a changing climate.     

Mobilizing the genome of Lepidoptera through novel sequence gains and end creation by non-autonomous Lep1 Helitrons

Transposable elements (TEs) can affect the structure of genomes through their acquisition and transposition of novel DNA sequences. The 134-bp repetitive elements, Lep1, are conserved non-autonomous Helitrons in lepidopteran genomes that have characteristic 5'-CT and 3'-CTAY nucleotide termini, a 3'-terminal hairpin structure, a 5'- and 3'-subterminal inverted repeat (SIR), and integrations that occur between AT or TT nucleotides. Lep1 Helitrons have acquired and propagated sequences downstream of their 3'-CTAY termini that are 57-344-bp in length and have termini composed of a 3'-CTRR preceded by a 3'-hairpin structure and a region complementary to the 5'-SIR (3'-SIRb). Features of both the Lep1 Helitron and multiple acquired sequences indicate that secondary structures at the 3'-terminus may have a role in rolling circle replication or genome integration mechanisms, and are a prerequisite for novel end creation by Helitron-like TEs. The preferential integration of Lep1 Helitrons in proximity to gene-coding regions results in the creation of genetic novelty that is shown to impact gene structure and function through the introduction of novel exon sequence (exon shuffling). These findings are important in understanding the structural requirements of genomic DNA sequences that are acquired and transposed by Helitron-like TEs.     

Predicting patch occupancy in fragmented landscapes at the rangewide scale for an endangered species: an example of an American warbler

AIM: Our objective was to identify the distribution of the endangered golden-cheeked warbler (Setophaga chrysoparia) in fragmented oak-juniper woodlands by applying a geoadditive semiparametric occupancy model to better assist decision-makers in identifying suitable habitat across the species breeding range on which conservation or mitigation activities can be focused and thus prioritize management and conservation planning. LOCATION: Texas, USA. METHODS: We used repeated double-observer detection/non-detection surveys of randomly selected (n = 287) patches of potential habitat to evaluate warbler patch-scale presence across the species breeding range. We used a geoadditive semiparametric occupancy model with remotely sensed habitat metrics (patch size and landscape composition) to predict patch-scale occupancy of golden-cheeked warblers in the fragmented oak-juniper woodlands of central Texas, USA. RESULTS: Our spatially explicit model indicated that golden-cheeked warbler patch occupancy declined from south to north within the breeding range concomitant with reductions in the availability of large habitat patches. We found that 59% of woodland patches, primarily in the northern and central portions of the warbler's range, were predicted to have occupancy probabilities ≤0.10 with only 3% of patches predicted to have occupancy probabilities >0.90. Our model exhibited high prediction accuracy (area under curve = 0.91) when validated using independently collected warbler occurrence data. MAIN CONCLUSIONS: We have identified a distinct spatial occurrence gradient for golden-cheeked warblers as well as a relationship between two measurable landscape characteristics. Because habitat-occupancy relationships were key drivers of our model, our results can be used to identify potential areas where conservation actions supporting habitat mitigation can occur and identify areas where conservation of future potential habitat is possible. Additionally, our results can be used to focus resources on maintenance and creation of patches that are more likely to harbour viable local warbler populations.     

<Emphasis Type="Italic">Acer rubrum</Emphasis> (red maple) growth is negatively affected by soil from forest stands dominated by its invasive congener (<Emphasis Type="Italic">Acer platanoides</Emphasis>, Norway maple)

Invasive species continue to alter the plant communities of the eastern United States. To better understand the mechanisms and characteristics associated with invasive success, we studied competition between two Acer species. In a greenhouse, we tested (1) the effect of forest soil type (beneath an invasive and native stand) on seedling growth of the invasive Acer platanoides (Norway maple) and native A. rubrum (red maple), and the (2) effects of full (above- and below-ground) and partial inter-specific competition on species growth. We found A. rubrum growth was negatively affected by soil from the invaded stand, as it had lower above-ground (32%) and below-ground (26%) biomass, and number of leaves (20%) than in the native soil. The root:shoot resource allocations of A. platanoides depended on soil type, as it had 14% greater root:shoot mass allocation in the native soil; this ability to change root:shoot allocation may be contributing to the ecological success of the species. Widely published as having a large ecological amplitude, A. rubrum may be a useful species for ecological restoration where A. platanoides has been present, but the impacts of A. platanoides on soil functioning and subsequent plant interactions must be addressed before protocols for native reintroductions are improved and implemented.     

Foliar production and decomposition rates in urban forests invaded by the exotic invasive shrub, <Emphasis Type="Italic">Lonicera maackii</Emphasis>

Exotic invasive shrubs can form dense monocultures in forest understories, which can have cascading effects on ecosystem structure and function. Amur honeysuckle, an exotic shrub that forms dense canopies in eastern forests, has the potential to alter plant community structure and ecosystem functions, such as primary production and decomposition. The goal of this study was to examine foliar productivity and leaf litter decomposition in forests invaded by Amur honeysuckle (Lonicera maackii) and to determine the extent to which the presence of this dominant exotic species may alter ecosystem function in these forests. We found that forests invaded by Amur honeysuckle had 16 times greater honeysuckle foliar biomass and 1.5 times lower total foliar biomass than forests of equivalent tree basal area, but having few honeysuckle shrubs. This suggests that productivity of native tree and shrub species may be reduced where honeysuckle density is high. Additionally, honeysuckle litter decayed four times faster and released nitrogen more rapidly than sugar maple litter, and sugar maple litter decayed 19% faster in forests invaded by Amur honeysuckle. These findings suggest that forests invaded by Amur honeysuckle may exhibit lower rates of organic matter accrual and less nitrogen retention in the forest floor. Since honeysuckle leaves develop in early spring before those of other shrubs or trees in the area, the rapid release of nitrogen from honeysuckle litter that we measured in early spring is timed to benefit this invasive species. The temporally coincident phenologies of nitrogen release during decomposition with the foliar growth needs of this shrub indicates that a potential positive feedback loop may exist between these processes that promotes continued growth and dominance of honeysuckle shrubs in these forested systems.