Polygenic risk assessment reveals pleiotropy between sarcoidosis and inflammatory disorders in the context of genetic ancestry.

Sarcoidosis is a complex disease of unknown etiology characterized by the presence of granulomatous inflammation. Though various immune system pathways have been implicated in disease, the relationship between the genetic determinants of sarcoidosis and other inflammatory disorders has not been characterized. Herein, we examined the degree of genetic pleiotropy common to sarcoidosis and other inflammatory disorders to identify shared pathways and disease systems pertinent to sarcoidosis onset. To achieve this, we quantify the association of common variant polygenic risk scores from nine complex inflammatory disorders with sarcoidosis risk. Enrichment analyses of genes implicated in pleiotropic associations were further used to elucidate candidate pathways. In European-Americans, we identify significant pleiotropy between risk of sarcoidosis and risk of asthma (R2=2.03%; P=8.89 × 10-9), celiac disease (R2=2.03%; P=8.21 × 10-9), primary biliary cirrhosis (R2=2.43%; P=2.01 × 10-10) and rheumatoid arthritis (R2=4.32%; P=2.50 × 10-17). These associations validate in African Americans only after accounting for the proportion of genome-wide European ancestry, where we demonstrate similar effects of polygenic risk for African-Americans with the highest levels of European ancestry. Variants and genes implicated in European-American pleiotropic associations were enriched for pathways involving interleukin-12, interleukin-27 and cell adhesion molecules, corroborating the hypothesized immunopathogenesis of disease.

Effect of superstimulation protocols on nuclear maturation and distribution of lipid droplets in bovine oocytes.

Our objective was to study the effect of superstimulation protocols on nuclear maturation of the oocyte and the distribution of lipid droplets in the ooplasm. Heifers (n=4 each group) during the luteal phase were either treated with FSH for 4 days (Short FSH), FSH for 4 days followed by 84h of gonadotropin free period (FSH Starvation) or for 7 days (Long FSH) starting from the day of wave emergence. In all groups, LH was given 24h after induced luteolysis (penultimate day of FSH) and cumulus-oocyte complexes were collected 24h later. Oocytes were stained for nuclear maturation (Lamin/chromatin) and lipid droplets (Nile red). The Long FSH group had a greater proportion of mature oocytes (metaphase II) compared with heifers in the Short FSH and FSH Starvation groups (59/100 vs 5/23 and 2/25, respectively; P<0.01). On average across all groups, oocytes contained 22pL of lipids (3.3% of ooplasm volume) distributed as 3000 droplets. Average volume of individual lipid droplets was higher in the FSH Starvation (11.5±1.5 10(-3) pL, P=0.03) compared with the Short and Long FSH groups (7.2±0.6 10(-3) and 8.0±0.8 10(-3) pL, respectively). In conclusion, both FSH Starvation and Short FSH treatments yielded a lower proportion of mature oocytes compared with the Long FSH treatment. Furthermore, FSH starvation led to an accumulation of larger lipid droplets in the ooplasm, indicating atresia. Our results indicate that a longer superstimulation period in beef cattle yields higher numbers and better-quality oocytes.

Role of MYH9 and APOL1 in African and non-African populations with lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by autoantibody production and organ damage. Lupus nephritis (LN) is one of the most severe manifestations of SLE. Multiple studies reported associations between renal diseases and variants in the non-muscle myosin heavy chain 9 (MYH9) and the neighboring apolipoprotein L 1 (APOL1) genes. We evaluated 167 variants spanning MYH9 for association with LN in a multiethnic sample. The two previously identified risk variants in APOL1 were also tested for association with LN in European-Americans (EAs) (N = 579) and African-Americans (AAs) (N = 407). Multiple peaks of association exceeding a Bonferroni corrected P-value of P < 2.03 × 10(-3) were observed between LN and MYH9 in EAs (N = 4620), with the most pronounced association at rs2157257 (P = 4.7 × 10(-4), odds ratio (OR) = 1.205). A modest effect with MYH9 was also detected in Gullah (rs8136069, P = 0.0019, OR = 2.304). No association between LN and MYH9 was found in AAs, Asians, Amerindians or Hispanics. This study provides the first investigation of MYH9 in LN in non-Africans and of APOL1 in LN in any population, and presents novel insight into the potential role of MYH9 in LN in EAs.

A functional haplotype of UBE2L3 confers risk for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations characterized by the development of pathogenic autoantibodies manifesting in inflammation of target organs such as the kidneys, skin and joints. Genome-wide association studies have identified genetic variants in the UBE2L3 region that are associated with SLE in subjects of European and Asian ancestry. UBE2L3 encodes an ubiquitin-conjugating enzyme, UBCH7, involved in cell proliferation and immune function. In this study, we sought to further characterize the genetic association in the region of UBE2L3 and use molecular methods to determine the functional effect of the risk haplotype. We identified significant associations between variants in the region of UBE2L3 and SLE in individuals of European and Asian ancestry that exceeded a Bonferroni-corrected threshold (P<1 × 10(-4)). A single risk haplotype was observed in all associated populations. Individuals harboring the risk haplotype display a significant increase in both UBE2L3 mRNA expression (P=0.0004) and UBCH7 protein expression (P=0.0068). The results suggest that variants carried on the SLE-associated UBE2L3 risk haplotype influence autoimmunity by modulating UBCH7 expression.

Recent insights into the genetic basis of systemic lupus erythematosus.

Genetic variation was first shown to be important in systemic lupus erythematosus (SLE or lupus) in the 1970s with associations in the human leukocyte antigen region. Almost four decades later, and with the help of increasingly powerful genetic approaches, more than 25 genes are now known to contribute to the mechanisms that predispose individuals to lupus. Over half of these loci have been discovered in the past 2 years, underscoring the extraordinary success of genome-wide association approaches in SLE. Well-established risk factors include alleles in the major histocompatibility complex region (multiple genes), IRF5, ITGAM, STAT4, BLK, BANK1, PDCD1, PTPN22, TNFSF4, TNFAIP3, SPP1, some of the Fcgamma receptors, and deficiencies in several complement components, including C1q, C4 and C2. As reviewed here, many susceptibility genes fall into key pathways that are consistent with previous studies implicating immune complexes, host immune signal transduction and interferon pathways in the pathogenesis of SLE. Other loci have no known function or apparent immunological role and have the potential to reveal novel disease mechanisms. Certainly, as our understanding of the genetic etiology of SLE continues to mature, important new opportunities will emerge for developing more effective diagnostic and clinical management tools for this complex autoimmune disease.

Peripheral blood gene expression profiling in Sjögren's syndrome.

Sjögren's syndrome (SS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands. The affected cases commonly present with oral and ocular dryness, which is thought to be the result of inflammatory cell-mediated gland dysfunction. To identify important molecular pathways involved in SS, we used high-density microarrays to define global gene expression profiles in the peripheral blood. We first analyzed 21 SS cases and 23 controls, and identified a prominent pattern of overexpressed genes that are inducible by interferons (IFNs). These results were confirmed by evaluation of a second independent data set of 17 SS cases and 22 controls. Additional inflammatory and immune-related pathways with altered expression patterns in SS cases included B- and T-cell receptor, insulin-like growth factor-1, granulocyte macrophage-colony stimulating factor, peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha and PI3/AKT signaling. Exploration of these data for relationships to clinical features of disease showed that expression levels for most interferon-inducible genes were positively correlated with titers of anti-Ro/SSA (P<0.001) and anti-La/SSB (P<0.001) autoantibodies. Diagnostic and therapeutic approaches targeting interferon-signaling pathway may prove most effective in the subset of SS cases that produce anti-Ro/SSA and anti-La/SSB autoantibodies. Our results strongly support innate and adaptive immune processes in the pathogenesis of SS, and provide numerous candidate disease markers for further study.

Meta-analysis and imputation identifies a 109 kb risk haplotype spanning TNFAIP3 associated with lupus nephritis and hematologic manifestations.

TNFAIP3 encodes the ubiquitin-modifying enzyme, A20, a key regulator of inflammatory signaling pathways. We previously reported association between TNFAIP3 variants and systemic lupus erythematosus (SLE). To further localize the risk variant(s), we performed a meta-analysis using genetic data available from two Caucasian case-control datasets (1453 total cases, 3381 total control subjects) and 713 SLE trio families. The best result was found at rs5029939 (P=1.67 x 10(-14), odds ratio=2.09, 95% confidence interval 1.68-2.60). We then imputed single nucleotide polymorphisms (SNPs) from the CEU Phase II HapMap using genotypes from 431 SLE cases and 2155 control subjects. Imputation identified 11 SNPs in addition to three observed SNPs, which together, defined a 109 kb SLE risk segment surrounding TNFAIP3. When evaluating whether the rs5029939 risk allele was associated with SLE clinical manifestations, we observed that heterozygous carriers of the TNFAIP3 risk allele at rs5029939 have a twofold increased risk of developing renal or hematologic manifestations compared to homozygous non-risk subjects. In summary, our study strengthens the genetic evidence that variants in the region of TNFAIP3 influence risk for SLE, particularly in patients with renal and hematologic manifestations, and narrows the risk effect to a 109 kb DNA segment that spans the TNFAIP3 gene.

Banking North American buffalo semen.

The purpose of this study was to develop a procedure to collect and preserve semen from wood bison (Bison bison athabascae) and plains bison (Bison bison bison). Semen samples from three wood and three plains bison bulls were collected by electroejaculation from June through October. In addition, sperm was collected from the cauda epididymis of seven plains bison. Semen was cryopreserved using two commercially available cryopreservation media, an egg yolk-based medium (Triladyl), and a medium free of products of animal origin (Andromed). Sperm morphology and motility were recorded on fresh and post-thawed semen samples. Total sperm motility was not different between plains and wood bison for the months of June (50%), July (69%) and October (54%). However, total sperm motility for wood bison was higher (P<0.05) than plains bison for the months of August and September (August: 80% vs 55%; September: 73% vs 40%). Plains and wood bison did not differ in mean total and mean progressive motility (35 and 15%, respectively) of frozen-thawed sperm samples. The post-thaw motility of Triladyl-treated sperm was higher (P<0.05) than Andromed-treated sperm (35% vs 13%, respectively). Interestingly, post-thawed epididymal spermatozoa had higher total motility (P<0.05) than post-thawed electroejaculated sperm when cryopreserved with a medium free of products of animal origin (Andromed; 35% vs 9%, respectively). In conclusion, we used electroejaculation to collect high quality bison semen, and cryopreserved it for future needs.

Encapsulation of foscarnet in liposomes modifies drug intracellular accumulation, in vitro anti-HIV-1 activity, tissue distribution and pharmacokinetics.

OBJECTIVE: To improve the in vitro anti-HIV-1 activity, intracellular accumulation in macrophages and in vivo pharmacokinetics and tissue distribution of foscarnet (trisodium phosphonoformate; PFA) by encapsulation in liposomes. METHODS: The accumulation of free and liposome-encapsulated PFA was determined in monocyte-macrophage RAW 264.7 cells and human premonocytoid U937 cells. The antiviral activity was evaluated in U937 cells infected with HIV-1IIIB. Tissue distribution and pharmacokinetics of free and liposomal PFA were determined in female Sprague-Dawley rats following the administration of an intravenous bolus dose (10 mg PFA/kg). RESULTS: The entrapment of PFA in liposomes resulted in a higher drug accumulation in both U937 and RAW 264.7 cells. A slightly greater efficacy against HIV-1IIIB replication into U937 cells was observed upon encapsulation of PFA into liposomes. Improved pharmacokinetics was observed upon entrapment of PFA in liposomes. Much higher drug levels were found in plasma for the liposomal formulation. The systemic clearance of the liposomal drug was 77 times lower than that of free drug. The encapsulation of PFA in liposomes greatly enhanced the drug accumulation in organs of the reticuloendothelial system. CONCLUSION: The encapsulation of PFA in liposomes modified the tissue distribution and plasma pharmacokinetics of the antiviral agent, resulting in a marked improvement of drug accumulation in organs involved in HIV immunopathogenesis and in a greater PFA bioavailability. The antiviral activity of liposomal PFA was slightly greater than that of free drug in HIV-1IIIB-infected U937 cells.

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.

Comparison of cellular accumulation, tissue distribution, and anti-HIV activity of free and liposomal 2',3'-dideoxycytidine.

We have investigated the cellular accumulation, tissue distribution, and antihuman immunodeficiency virus activity of free dideoxycytidine (ddC) and liposomal ddC (L-ddC). We have found that L-ddC was more efficiently taken up than its free form by RAW 264.7 cells (a monocyte-macrophage cell line) (p < 0.01) while a comparable uptake was seen in U937 cells (a promonocytic cell line). In the rat, L-ddC accumulated preferentially in liver and spleen when injected intravenously (p < 0.01), and mostly in spleen when given intraperitoneally (p < 0.01). In contrast, free ddC was rapidly eliminated out of the body. Liposomal ddC showed a similar anti-HIV activity in comparison with free ddC in U937 cells. Given the fact that encapsulation of ddC in liposomes does not affect its anti-HIV activity but enhances its in vitro cellular accumulation and its in vivo distribution in reticuloendothelial system (RES) tissues, we conclude that ddC in liposomal formulation is a promising anti-HIV agent with a targeted action on the RES, which is considered a reservoir for dissemination of virus to other cells, tissues, and organs.

Cryopreservation alters the levels of the bull sperm surface protein P25b.

Fertility of frozen-thawed bull sperm is reduced by cryopreservation. Freezing-thawing procedures can result in as much as a sevenfold fertility decrease. Sperm mortality and loss of motility do not fully explain the reduced fertility of cryopreserved semen; they may be partially explained by the loss of sperm surface proteins, which are necessary for fertilization. We have previously identified P25b, a sperm surface protein, which is associated with the fertility index of bulls used for artificial insemination. Using Western blotting techniques, we have evaluated P25b levels before and after cryopreservation of bull spermatozoa in extenders based on either egg yolk or milk. Long storage periods (28 days) in liquid nitrogen results in a threefold decrease of P25b levels associated with cryopreserved versus fresh spermatozoa. Over a short storage period (3-7 days), a stable P25b level was observed on spermatozoa cryopreserved in extender containing either egg yolk or milk. A decrease in P25b levels associated with spermatozoa was observed after 5 days of storage in egg yolk extender, whereas a significant decrease was observed after 14 days of sperm storage in milk extender (P < .05). Therefore, the loss of P25b may be responsible, at least in part, for the decrease in fertility following the freezing-thawing procedure of bull semen. Moreover, the cryopreservation extender used may have different effects on the loss of sperm surface proteins after even brief storage periods in liquid nitrogen. Considering that a sperm protein similar to P25b exists in humans (P34H), these results may have significant clinical applications in which frozen semen is used.

Application of phonocardiography for detecting hypoxia-induced cardiovascular adaptation in the chicken.

Phonocardiography was evaluated as a noninvasive technique for diagnosis of cardiovascular adaptation and disease in broiler chickens. Heart sounds (HSs) were compared in a fast-growing (FG) commercial broiler line that is highly susceptible to chronic right heart failure resulting from pulmonary hypertension syndrome (PHS) and in a non-selected slow-growing (SG) broiler line that is resistant to PHS. HSs were analyzed in broilers reared in hypobaric hypoxia (HYP) and normobaric (CON) conditions. PHS was induced by a combination of embryonic hypoxia and HYP exposure. HSs were recorded with a microphone placed at the thoracic inlet of each chicken. Electrocardiograms were used to mark the sampling interval for the first, second, and total HS. Digitized HS signals were analyzed for peak frequency, mean peak frequency, and band width. Birds were examined for PHS lesions when 6 wk of age, at the end of each experiment. HSs were compared by line and treatment (Experiment 1) or by treatment and week (Experiment 2). In addition, HS frequencies were analyzed within the HYP treatment group for differences between birds with severe or no gross PHS lesions. HS frequencies generally decreased with age and were also lower in the FG than the SG line. Hypobaric exposure decreased all HS frequencies in the SG line and components of the first HSs in the FG line. The SG line did not develop gross lesions of PHS. In the FG line, significant differences in HS frequencies were observed between HYP and CON groups but not between PHS- and PHS+ broilers. Frequency changes described in humans with PHS were not observed. Further development to maximize the resolution of the HS waveforms and improved matching of the sampling interval to the electrical or hemodynamic output of the chicken heart may allow its use as a diagnostic tool for differentiating broilers with normal cardiac function or physiologic adaptation from those with cardiovascular disease.

Self-organising discovery, recognition and prediction of haemodynamic patterns in the intensive care unit.

To care properly for critically ill patients in the intensive care unit (ICU), clinicians must be aware of haemodynamic patterns. In a typical ICU, a variety of physiological measurements are made continuously and intermittently in an attempt to provide clinicians with the most accurate and precise data needed for recognising such patterns. However, the data are disjointed, yielding little information beyond that provided by instantaneous high/low limit checking. Although instantaneous limit checking is useful for determining immediate dangers, it does not provide much information about temporal patterns. As a result, the clinician is left to sift manually through an excess of data in the interest of generating information. In the study, an arrangement of self-organising artificial neural networks is used to automate the discovery, recognition and prediction of haemodynamic patterns in real time. It is shown that the network is capable of recognising the same haemodynamic patterns recognised by an expert system, DYNASCENE, without being explicitly programmed to do so. Consequently, the system is also capable of discovering new haemodynamic patterns. Results from real clinical data are presented.

Analysis of nystagmus response to pseudorandom velocity input.

This report describes a method to analyse the vestibular system when subjected to a pseudorandom angular velocity input. A sum of sinusoids (pseudorandom) input lends itself to analysis by linear frequency methods. Resultant horizontal ocular movements were digitized, filtered and transformed into the frequency domain. Programs were developed and evaluated to obtain the auto spectra of input stimulus and resultant ocular response, cross spectra, the estimated vestibular-ocular system transfer function gain and phase, and coherence function between stimulus and response functions.

Localization of current dipole within a sphere by magnetic measurements.

The principal objective of this study is the development of computer programs to determine the location and strength of neural electric activity within the brain from noninvasive magnetic field measurements at the surface of the head. This report presents theoretical calculations and computer programs derived from the method described by Williamson and Kaufman to determine the depth and strength of a current dipole in a sphere. From the location of the magnetic field radial component extremes, Br maximum and Br minimum, the orientation and location of the current dipole can be determined. The accuracy of the solution is dependent on precise location of of the magnetic field extremes as measured from the surface of a sphere, e.g. the head. To validate the program for locating the dipole, theoretical calculations and computer programs related to the total magnetic field vector resulting from a hypothetical current source within a homogeneous sphere were generated. The errors in calculations of the current dipole depth and strength are presented.

Vestibular response to pseudorandom angular velocity input: progress report.

Space motion sickness was not reported during the first Apollo missions; however, since Apollo 8 through the current Shuttle and Skylab missions, approximately 50% of the crewmembers have experienced instances of space motion sickness. One of NASA's efforts to resolve the space adaptation syndrome is to model the vestibular response for both basic knowledge and as a possible predictor of an individual's susceptibility to the disorder. This report describes a method to analyze the vestibular system when subjected to a pseudorandom angular velocity input.

Effects of proposed preflight adaptation training on eye movements, self-motion perception, and motion sickness: a progress report.

A program has been initiated to develop apparatus and procedures to preadapt astronauts to the sensory rearrangement associated with weightlessness in spaceflight. If space motion sickness is a consequence of adaptation to that sensory rearrangement, preflight training could afford astronauts significant relief from the motion sickness. The preflight adaptation trainer (PAT) was designed to produce rearranged relationships between visual and otolith signals analogous to those experienced in space. Investigations have been undertaken with three prototype trainers. The results indicated that exposure to the PAT sensory rearrangement altered self-motion perception, induced motion sickness, and changed the amplitude and phase of the horizontal eye movements evoked by roll stimulation. However, the changes were inconsistent. Appropriate measures of adaptation and protocols for producing the adaptation efficiently remain to be determined.

Comparison of optokinetic scene effects on the somatogyral illusion.

The objective of this study was to determine during rotation the relative importance of a scene in achieving "visual dominance" over non-visual vestibular orientation inputs, e.g., otolith and semicircular canals. Five visual scenes were presented, while rotating the subject (at three angular acceleration rates), to obtain the vestibular and optokinetic nystagmus and perception of rotation. The onset time and direction of rotation, as well as EOG and chair velocity were recorded. Adaptation times during rotation at constant angular velocity and during post-rotation were also recorded. Analysis of perceived times (onset, adaptation, and post-rotatory adaptation) with the EOG slow-phase velocity at respectively perceived times were analyzed using the SAS procedures Analysis of Variance (ANOVA) and General Linear Models (GLM). Basic hypotheses for the study were: "There are no differences in latencies for either onset time (L1), adaptation to rotation (L2), or adaptation to post rotatory motion (L4), between treatment conditions, or between Groups of subjects, or between acceleration rates, between constant rotation velocities, or between direction of rotation." From the results, it was concluded that pilots are highly visually dependent; additionally, if there is a sensory conflict, a larger percentage of individuals (pilots) will follow visual perceptions even if the demands of the aerial environment and our perception is incorrect. However, in sensory conflict conditions, with degraded visual scene individuals including pilots will revert to vestibular perception rather than visual perception (less visually coupled), approaching percentages as noted with a dark environment.

A strategy for improvement of postthaw quality of bison sperm.

The objective was to improve the postthaw quality of bison semen using zwitterion (ZI)-based extenders, glycerol addition at a lower temperature (4 °C), adding reduced glutathione (GSH) in extender, or treating bison sperm with cholesterol-loaded cyclodextrin (CLC) before freezing. Postthaw sperm motility and structural characteristics were analyzed using a computer-assisted sperm analyzer and flow cytometer respectively, at 0 and 3 hours postthaw incubation at 37 °C. In experiment 1, each ejaculate (N = 11) was diluted in Triladyl extender (control) or in ZI extenders (Tes-Tris or HEPES-Tris). In addition, glycerol in semen was added either at 37 °C or 4 °C before cryopreservation. Extenders had no significant effect on postthaw sperm motilities at 0 hour. However, sperm velocity parameters were higher (P < 0.05) in ZI extenders than in Triladyl. Sperm population with intact plasma membrane (IPM) and acrosomes (IACR) were higher in Triladyl than in ZI extenders (P < 0.05). Postthaw sperm total and progressive motilities, average path velocity, straight-line velocity, IPM, and IPM-IACR did not improve with the addition of glycerol at 4 °C. In experiment 2, semen was diluted (50 × 10(6) sperm per mL) in Triladyl extender containing 0 (control), 0.5, 1.0, or 2.0 mM GSH (an antioxidant) at 37 °C. Postthaw sperm motility and structural characteristics at 0 hour and percentage declined after 3 hour incubation, but did not differ because of GSH in the extender (P > 0.05). In experiment 3, fresh bison sperm (100 × 10(6) sperm in 1 mL) were pretreated with 0, 1, 2, or 3 mg/mL of CLC at 22 °C for 15 minutes and diluted to 50 × 10(6) sperm per mL in Tris-citric acid-egg yolk-glycerol extender before cryopreservation. The CLC pretreated sperm had higher (P < 0.05) postthaw total and progressive motilities, IPM, and IACR at 0 hour and less percentage of decline in these characteristics after 3 hour postthaw incubation. In conclusion, zwitterion extenders (Tes-Tris and HEPES-Tris), temperatures of glycerol addition, and GSH in extender did not significantly improve postthaw quality of bison sperm. However, pretreatment with CLC significantly improved postthaw quality of bison sperm, which should enhance its use in assisted reproductive technologies.