Poly(I:C) source, molecular weight and endotoxin contamination affect dam and prenatal outcomes, implications for models of maternal immune activation.

The viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) is increasingly used to induce maternal immune activation (mIA) to model neurodevelopmental disorders (NDDs). Robust and reproducible phenotypes across studies are essential for the generation of models that will enhance our understanding of NDDs and enable the development of improved therapeutic strategies. However, differences in mIA-induced phenotypes using poly(I:C) have been widely observed, and this has prompted the reporting of useful and much needed methodological guidelines. Here, we perform a detailed investigation of molecular weight and endotoxin variations in poly(I:C) procured from two of the most commonly used suppliers, Sigma and InvivoGen. We demonstrate that endotoxin contamination and molecular weight differences in poly(I:C) composition lead to considerable variability in maternal IL-6 response in rats treated on gestational day (GD)15 and impact on fetal outcomes. Specifically, both endotoxin contamination and molecular weight predicted reductions in litter size on GD21. Further, molecular weight predicted a reduction in placental weight at GD21. While fetal body weight at GD21 was not affected by poly(I:C) treatment, male fetal brain weight was significantly reduced by poly(I:C), dependent on supplier. Our data are in agreement with recent reports of the importance of poly(I:C) molecular weight, and extend this work to demonstrate a key role of endotoxin on relevant phenotypic outcomes. We recommend that the source and batch numbers of poly(I:C) used should always be stated and that molecular weight variability and endotoxin contamination should be minimised for more robust mIA modelling.

Immunolesion of melanopsin neurons causes gonadal regression in Pekin drakes (Anas platyrhynchos domesticus).

Several light sensitive receptors have been described in the avian brain that are thought to regulate the reproductive axis independently from the eyes and pineal gland. Recently, our lab has described the presence of three of these photoneuroendocrine systems in the Pekin duck: opsin, opsin 5, & melanopsin. We set out to test the hypothesis that melanopsin receptive neurons are necessary to maintain seasonal reproductive status along with growth and development in the Pekin drake. To accomplish these goals we first investigated 50-week-old Pekin drakes that were housed in the aviary at Hope College under long day length (18h lights on) conditions in floor pens. To specifically lesion melanopsin-receptive neurons, 3µl of an anti-melanopsin-saporin conjugate (MSAP, 100ng/ul) was injected into the lateral ventricle (n=10). Control drakes were injected with 3µl of equimolar unconjugated anti-melanopsin and saporin (SAP, n=10). Reproductive behaviors were analyzed weekly in a test pen with adult hens and MSAP drakes showed a significant (p<0.01) reduction in reproductive behaviors after week 2. After 5weeks, drakes were euthanized and body weights were measured, and brains, pituitaries, and testes collected and stored for analyses. Mature MSAP-treated drakes had significantly (p<0.001) reduced relative teste weights compared to SAP controls. qRT-PCR analyses of hypothalamus showed a significant reduction (p<0.001) in GnRH and melanopsin mRNA levels, but not opsin 5, vertebrate ancient opsin, or opsin 2 (rhodopsin). Immunocytochemical analyses showed a significant reduction (p<0.01) in tyrosine hydroxylase-immunoreactivity in the PMM. These data suggest that although blue light alone is not able to maintain testicular function, the blue-light sensitive melanopsin activity is critical to maintain gonadal function.

OARSI Clinical Trials Recommendations: Hip imaging in clinical trials in osteoarthritis.

Imaging of hip in osteoarthritis (OA) has seen considerable progress in the past decade, with the introduction of new techniques that may be more sensitive to structural disease changes. The purpose of this expert opinion, consensus driven recommendation is to provide detail on how to apply hip imaging in disease modifying clinical trials. It includes information on acquisition methods/techniques (including guidance on positioning for radiography, sequence/protocol recommendations/hardware for magnetic resonance imaging (MRI)); commonly encountered problems (including positioning, hardware and coil failures, artifacts associated with various MRI sequences); quality assurance/control procedures; measurement methods; measurement performance (reliability, responsiveness, and validity); recommendations for trials; and research recommendations.

Correlation of meniscal T2* with multiphoton microscopy, and change of articular cartilage T2 in an ovine model of meniscal repair.

OBJECTIVE: To correlate meniscal T2* relaxation times using ultra-short echo time (UTE) magnetic resonance imaging (MRI) with quantitative microscopic methods, and to determine the effect of meniscal repair on post-operative cartilage T2 values. DESIGN: A medial meniscal tear was created and repaired in the anterior horn of one limb of 28 crossbred mature ewes. MR scans for morphological evaluation, meniscal T2* values, and cartilage T2 values were acquired at 0, 4 and 8 months post-operatively for the Tear and Non-Op limb. Samples of menisci from both limbs were analyzed using multiphoton microscopy (MPM) analysis and biomechanical testing. RESULTS: Significantly prolonged meniscal T2* values were found in repaired limbs than in control limbs, P < 0.0001. No regional differences of T2* were detected for either the repaired or control limbs in the anterior horn. Repaired limbs had prolonged cartilage T2 values, primarily anteriorly, and tended to have lower biomechanical force to failure at 8 months than Non-Op limbs. MPM autofluorescence and second harmonic generation data correlated with T2* values at 8 months (ρ = -0.48, P = 0.06). CONCLUSIONS: T2* mapping is sensitive to detecting temporal and zonal differences of meniscal structure and composition. Meniscal MPM and cartilage T2 values indicate changes in tissue integrity in the presence of meniscal repair.

GM-CSF Promotes Immune Response and Survival in a Mouse Model of COVID-19.

COVID-19 results in increased expression of inflammatory cytokines, but inflammation-targeting clinical trials have yielded poor to mixed results. Our studies of other disorders with an inflammatory component, including Alzheimer's disease, chemobrain, Down syndrome, normal aging, and West Nile Virus infection, showed that treatment with the 'pro-inflammatory' cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in humans or mouse models alleviated clinical, behavioral, and pathological features. We proposed that human recombinant GM-CSF (sargramostim) be repurposed to promote both the innate and adaptive immune responses in COVID-19 to reduce viral load and mortality1. Here, we report the results of a placebo-controlled study of GM-CSF in human ACE2 transgenic mice inoculated intranasally with SARS-CoV2 virus, a model of COVID-19. Infection resulted in high viral titers in lungs and brains and over 85% mortality. GM-CSF treatment beginning one day after infection increased anti-viral antibody titers, lowered mean lung viral titers proportionately (p=0.0020) and increased the odds of long-term survival by up to 5.8-fold (p=0.0358), compared to placebo. These findings suggest that, as an activator of both the innate and adaptive immune systems, GM-CSF/sargramostim may be an effective COVID-19 therapy with the potential to protect from re-infection more effectively than treatment with antiviral drugs or monoclonal antibodies.

Imaging near metal with a MAVRIC-SEMAC hybrid.

The recently developed multi-acquisition with variable resonance image combination (MAVRIC) and slice-encoding metal artifact correction (SEMAC) techniques can significantly reduce image artifacts commonly encountered near embedded metal hardware. These artifact reductions are enabled by applying alternative spectral and spatial-encoding schemes to conventional spin-echo imaging techniques. Here, the MAVRIC and SEMAC concepts are connected and discussed. The development of a hybrid technique that utilizes strengths of both methods is then introduced. The presented technique is shown capable of producing minimal artifact, high-resolution images near total joint replacements in a clinical setting.

Helicobacter species in the human colon.

OBJECTIVE: To determine the prevalence, frequency and colonization patterns of Helicobacter species throughout the colon. METHOD: Patients having initial colonoscopy for nonspecific gastrointestinal disturbance had colonic biopsies taken from up to four sites during colonoscopy and examined for evidence of the Helicobacteraceae family using a group-specific PCR. Serum was also collected and examined for IgG reactivity to Helicobacter pylori. RESULTS: 100 patients had colonoscopy of whom 35 were found to have DNA evidence of Helicobacter species throughout the colon, with 22 having H. pylori. Fifteen patients had a demonstrable serum IgG response to H. pylori that was not always associated with molecular evidence of H. pylori DNA in colon biopsies and vice versa. No specific association with colon disease was found in patients with H. pylori infection. CONCLUSION: We found evidence of Helicobacter infection in a significant number of patients presenting for colonoscopy but no specific association between the presence of these bacteria and colon disease. Our finding of disparity between molecular and serological techniques to detect Helicobacter species suggests that future studies should not rely on serology alone to detect these bacteria in the human colon.

Quantitative magnetic resonance imaging and histological hoof wall assessment of 3-year-old Quarter Horses.

BACKGROUND: Few noninvasive methods are available for equine hoof wall evaluation. The highly organised wall structures and composition of proteoglycans and collagens may make this region amenable to quantitative MRI (qMRI) techniques of T1ρ and T2 mapping to identify pathology related to proteoglycan content and collagen organisation respectively. OBJECTIVE: To establish normative T1ρ and T2 values of the equine hoof wall of 3-year-old Quarter Horses with histological comparison. STUDY DESIGN: Cadaveric anatomical study. METHODS: Six cadaveric left thoracic feet from 3-year-old racing Quarter Horses with no reported lameness were evaluated using T1ρ and T2 mapping. Mapping was performed at six regions of interest at the toe of each hoof including proximal and distal regions of the inner epidermis, stratum lamellatum and corium. Histology was evaluated for standard hoof morphology and proteoglycan staining. RESULTS: T2 values of the stratum lamellatum and corium were similar (42.9 [95% CI: 41.6-44.2] ms and 44 [95% CI: 42.7-45.3] ms respectively), but both were significantly different to the inner epidermis (35.8 [95% CI: 34.5-37.1] ms, P<0.001). T1ρ values for the inner epidermis, stratum lamellatum and corium were significantly different (25.1 [95% CI: 23.1-27.1] ms, 44.4 [95% CI: 42.4-46.4] ms and 50.1 [95% CI: 48.1-52.1] ms, respectively, P<0.001). Histology demonstrated normal organised morphology. Proteoglycan staining was only visible in the stratum lamellatum and corium. MAIN LIMITATIONS: Cadaveric study with frozen samples used. CONCLUSIONS: Variation of qMRI metrics through the depth of the equine hoof wall was found. Although the highly ordered environment of collagen may contribute to T2 values, there was lack of evidence to support proteoglycan content as a major contributor of T1ρ values. It is possible T1ρ values had a greater dependence on total water content as the lowest values were seen in the epidermis. Additional research using qMRI is needed to determine mapping values in different disease states.

Local cytokine profiles in knee osteoarthritis: elevated synovial fluid interleukin-15 differentiates early from end-stage disease.

OBJECTIVE: Much of what is known about the inflammatory response in the synovial membrane (SM) of patients with osteoarthritis (OA) comes from studies of synovial tissues from end-stage disease. In this study, we sought to better characterize the inflammatory infiltrate in symptomatic patients with early signs of knee OA, and to determine how inflammatory cell populations relate to the pattern of cytokine and degradative enzyme production. METHODS: Study populations comprised patients with degenerative meniscal tears and early cartilage thinning undergoing arthroscopic procedures (early OA) and patients undergoing total knee replacement for end-stage OA. Quantitative real-time polymerase chain reaction (PCR) was used to measure expression of SM cytokines and enzymes implicated in the pathogenesis of inflammatory arthritis and OA, as well as cell lineage-specific markers. We quantified synovial fluid (SF) cytokines and enzymes by enzyme-linked immunosorbent assay (ELISA) and SM cell populations by immunohistochemistry. RESULTS: We found increased levels of SF interleukin-15 (IL-15) protein in the early knee OA patients when compared to end-stage OA. Both SF IL-15 protein and numbers of CD8 cells within SM correlated with matrix metalloproteinase-1 (MMP-1) and three levels. TNF-alpha, IL-6 and IL-21 were also detectable in the SF of the majority of patients, and IL-15 levels were associated with IL-6 levels. CONCLUSION: IL-15 is elevated in early knee OA, suggesting activation of an innate immune response in the SM. The association of IL-15 expression with CD8 transcripts and MMPs implicates this cytokine in OA pathogenesis and as a candidate therapeutic target.

A potential noninvasive neurobiological test for Alzheimer's disease.

Currently Alzheimer's disease, which affects more than 20 million people worldwide, can only be definitely diagnosed by histological examination of brain tissue obtained at autopsy or biopsy. There is a great need for an early, noninvasive, sensitive, and easily administered diagnostic test of Alzheimer's disease. Here it is reported that patients diagnosed with probable Alzheimer's disease by standard clinical criteria exhibited a marked hypersensitivity in their pupil dilation response to a cholinergic antagonist, tropicamide, placed in their eyes. It was possible to distinguish 18 of 19 individuals (95%) either clinically diagnosed with Alzheimer's disease or classified as suspect Alzheimer's individuals by neuropsychological screening from 30 of 32 normal elderly controls (94%).

Translocations among antibody genes in human cancer.

The characteristic chromosomal translocations that occur in certain human malignancies offer opportunities to understand how two gene systems can affect one another when they are accidentally juxtaposed. In the case of Burkitt lymphoma, such a translocation joins the cellular oncogene, c-myc, to a region encoding one of the immunoglobulin genes. In at least one example, the coding sequence of the rearranged c-myc gene is identical to that of the normal gene, implying that the gene must be quantitatively, rather than qualitatively, altered in its expression if it is to play a role in transformation. One might expect to find the rearranged c-myc gene in a configuration that would allow it to take advantage of one of the known immunoglobulin promoters or enhancer elements. However, the rearranged c-myc gene is often placed so that it can utilize neither of these structures. Since the level of c-myc messenger RNA is often elevated in Burkitt cells, the translocation may lead to a deregulation of the c-myc gene. Further, since the normal allele in a Burkitt cell is often transcriptionally silent in the presence of a rearranged allele, a model for c-myc regulation is suggested that involves a trans-acting negative control element that might use as its target a highly conserved portion of the c-myc gene encoding two discrete transcriptional promoters.

Expression of the Alzheimer amyloid-promoting factor antichymotrypsin is induced in human astrocytes by IL-1.

The amyloid deposits of Alzheimer's disease contain, in addition to the beta protein (A beta), lesser amounts of other proteins including the protease inhibitor alpha 1-antichymotrypsin (ACT). We have recently shown that ACT acts as a pathological chaperone, binding to the beta protein and strongly promoting its polymerization into amyloid filaments in vitro. The data of this paper show that ACT synthesis is induced in cultured human astrocytes by IL-1, a lymphokine whose expression is strongly up-regulated in microglial cells in affected areas of Alzheimer's disease brain. Furthermore, unfractionated glial cultures containing both astrocytes and microglia from human cortex (which develops amyloid in Alzheimer's disease) spontaneously express IL-1 and ACT as they reach confluence. In contrast, confluent mixed glial cultures similarly prepared from human cerebellum or brain stem, or from rat brain-tissues not prone to amyloid formation-do not express ACT unless supplemented with exogenous IL-1. The same regional difference in IL-1 expression by microglia is seen in vivo in Alzheimer's disease. These results indicate that the IL-1-induced expression of ACT may help direct the region-specific production of mature amyloid filaments in the Alzheimer brain.

Three cases of congenital adrenal hypoplasia with novel mutations in the (NROB1) DAX-1 gene.

Abnormalities in the DAX-1 gene (dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome) are a well-recognized cause of congenital adrenal hypoplasia. DAX-1 is expressed in the adrenal cortex, gonads, hypothalamus and anterior pituitary, which gives rise to the clinical features of this deletion. Presentations are varied but salt-wasting and/or hypoglycaemia are the most common in an infant, with late onset of hypogonadotrophic hypogonadism. Over 80 different mutations in this gene have been identified. We present three unrelated cases with variable clinical presentations, all with novel mutations in the DAX-1 gene.

Pseudo-hypertriglyceridaemia: a measurement artefact due to glycerol kinase deficiency.

A man presented with elevated plasma triglycerides and was commenced on fibrate treatment. The triglycerides did not fall and compliance was questioned. The triglyceride elevation was inconsistent with the observed lack of turbidity in the plasma sample. Triglyceride elevation was not confirmed by a different analytical method and lipoprotein electrophoresis showed a normal very low density lipoprotein (VLDL) band pattern. Glycerol kinase deficiency was suspected and was supported by elevated urine glycerol, and confirmed by reduced leucocyte enzyme activity and mutational analysis of the GK gene which showed a novel three base pair deletion. Demonstration of a point mutation also excludes a contiguous gene deletion syndrome.

Quantifying the importance of diffuse minewater pollution in a historically heavily coal mined catchment.

There has been considerable progress in developing treatment systems for point sources of minewater pollution in recent years; however, there remains a knowledge gap in the characterisation and remediation of diffuse minewater sources. Data are presented from the River Gaunless catchment, a historically heavily coal mined catchment in the northeast of England. Instream iron (Fe) loadings were monitored alongside loadings arising from point minewater discharges over a 12-month period to assess the dynamic importance of diffuse sources of minewater pollution. In low flow, diffuse sources account for around 50% of instream loading, a proportion which increases to 98% in high flow conditions. The low flow sources appear to be dominated by direct discharge of contaminated groundwater to surface waters in lower reaches of the catchment. In high flow, resuspended Fe-rich sediments, which are both naturally occurring and derived from historic mining, become the dominant diffuse source of Fe in the water column.

Low light intensity in Pekin duck breeder barns has a greater impact on the fertility of drakes than hens.

The Pekin duck is an excellent model for the study of seasonal reproduction. To more completely understand the lighting requirements for maximal fertility in duck breeder houses, we housed adult (45 week old) drakes and hens in the Hope College aviary as 5 drakes and 25 hens. Light conditions in each floor pen were normalized based upon quantal energy and divided into the following categories: (1) to simulate summer, 14.5 h 65 lux with 9.5 h 1 lux; (2) to simulate winter, 8 h 65 lux with 16 h 1 lux; (3) winter augmented, 8 h 65 lux with 16 h at 15 lux. The experiment was repeated with rotation of light treatments among 3 pens until a final N = 6 was obtained. Daily, total number of eggs laid was tallied, and a daily average of eggs laid was calculated throughout the study. Weekly, eggs were weighed and the perivitilline membrane was assayed for the number of sperm holes as an indirect measure of drake fertility. As expected, winter conditions caused a reduction in the percent of eggs laid and a reduction in the number of fertilized eggs compared to the summer light conditions. The augmented winter light conditions prevented the loss in the percent eggs laid and fertilized eggs. Surprisingly, even after 4 wk of the study, the winter conditions did not cause a complete loss of fertility in the Pekin ducks. At the end of the study, no differences in the relative expression in brain deep brain photoreceptors or gonadotropin inhibitory hormone mRNAs were observed among any light treatment. Although a minimum (1 lux) of light can support some fertility, our findings suggest commercial Pekin duck barns may benefit from increasing the augmented light to 15 lux to maintain optimal fertility during winter months. Furthermore, our data suggest that drakes may be more sensitive to environmental light conditions than hens.

A diet high in omega-3 fatty acids does not improve or protect cognitive performance in Alzheimer's transgenic mice.

Although a number of epidemiologic studies reported that higher intake of omega-3 fatty acids (largely associated with fish consumption) is protective against Alzheimer's disease (AD), other human studies reported no such effect. Because retrospective human studies are problematic and controlled longitudinal studies over decades are impractical, the present study utilized Alzheimer's transgenic mice (Tg) in a highly controlled study to determine whether a diet high in omega-3 fatty acid, equivalent to the 13% omega-3 fatty acid diet of Greenland Eskimos, can improve cognitive performance or protect against cognitive impairment. Amyloid precursor protein (APP)-sw+PS1 double transgenic mice, as well as nontransgenic (NT) normal littermates, were given a high omega-3 supplemented diet or a standard diet from 2 through 9 months of age, with a comprehensive behavioral test battery administered during the final 6 weeks. For both Tg and NT mice, long-term n-3 supplementation resulted in cognitive performance that was no better than that of mice fed a standard diet. In NT mice, the high omega-3 diet increased cortical levels of omega-3 fatty acids while decreasing omega-6 levels. However, the high omega-3 diet had no effect on cortical fatty acid levels in Tg mice. Irrespective of diet, no correlations existed between brain omega-3 levels and cognitive performance for individual NT or Tg mice. In contrast, brain levels of omega-6 fatty acids were strongly correlated with cognitive impairment for both genotypes. Thus, elevated brain levels of omega-3 fatty acids were not relevant to cognitive function, whereas high brain levels of omega-6 were associated with impaired cognitive function. In Tg mice, the omega-3 supplemental diet did not induce significant changes in soluble/insoluble Abeta within the hippocampus, although strong correlations were evident between hippocampal Abeta(1-40) levels and cognitive impairment. While these studies involved a genetically manipulated mouse model of AD, our results suggest that diets high in omega-3 fatty acids, or use of fish oil supplements (DHA+EPA), will not protect against AD, at least in high-risk individuals. However, normal individuals conceivably could derive cognitive benefits from high omega-3 intake if it corrects an elevation in the brain level of n-6 fatty acids as a result. Alternatively, dietary fish may contain nutrients, other than DHA and EPA, that could provide some protection against AD.

Cell-specific expression of the human parathyroid hormone gene in rat pituitary cells.

The promoter region of the human parathyroid hormone gene was fused to the Escherichia coli neo gene and introduced into GH4C1 rat pituitary and human HeLa cells. Both TATA boxes of the human parathyroid hormone gene accurately directed transcription in GH4C1 cells; the parathyroid hormone promoter was inactive in HeLa cells.

A general RNA-binding protein complex that includes the cytoskeleton-associated protein MAP 1A.

Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a approximately 160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a approximately 1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.

Thyroid hormone resistance: the role of mutational analysis.

The finding of increased thyroxine (T4) and tri-iodothyronine (T3) levels in a patient with normal or increased thyroid-stimulating hormone is unexpected and presents a differential diagnosis between a thyroid-stimulating hormone-secreting pituitary adenoma, generalized resistance to thyroid hormone (RTH) and laboratory artefact. Without careful clinical and biochemical evaluation, errors may occur in patient diagnosis and treatment. In the case of RTH, mutation of the thyroid hormone receptor beta gene results in generalized tissue resistance to thyroid hormone. As the pituitary gland shares in this tissue resistance, euthyroidism with a normal thyroid-stimulating hormone is usually maintained by increased thyroid hormones. To date, we have identified eight pedigrees in New Zealand with mutations in the thyroid hormone receptor beta gene, including two novel mutations. Mutational analysis of the thyroid hormone receptor beta gene allows definitive diagnosis of RTH, potentially avoiding the need for protracted and expensive pituitary function testing and imaging. Mutational analysis also enables family screening and may help to avoid potential misdiagnosis and inappropriate treatment.