GWAS identifies novel SLE susceptibility genes and explains the association of the HLA region.

In a genome-wide association study (GWAS) of individuals of European ancestry afflicted with systemic lupus erythematosus (SLE) the extensive utilization of imputation, step-wise multiple regression, lasso regularization and increasing study power by utilizing false discovery rate instead of a Bonferroni multiple test correction enabled us to identify 13 novel non-human leukocyte antigen (HLA) genes and confirmed the association of four genes previously reported to be associated. Novel genes associated with SLE susceptibility included two transcription factors (EHF and MED1), two components of the NF-κB pathway (RASSF2 and RNF114), one gene involved in adhesion and endothelial migration (CNTN6) and two genes involved in antigen presentation (BIN1 and SEC61G). In addition, the strongly significant association of multiple single-nucleotide polymorphisms (SNPs) in the HLA region was assigned to HLA alleles and serotypes and deconvoluted into four primary signals. The novel SLE-associated genes point to new directions for both the diagnosis and treatment of this debilitating autoimmune disease.

Effect of anaerobic digestion temperature on odour, coliforms and chlortetracycline in swine manure or monensin in cattle manure.

AIMS: This study evaluated the effect of anaerobic digestion at 22, 38 and 55°C on odour, coliforms and chlortetracycline (CTC) in swine manure or monensin (MON) in cattle manure. METHODS AND RESULTS: Swine or cattle were fed the respective growth promotant, manure was collected, and 2-l laboratory methane digesters were established at the various temperatures and sampled over 25 or 28 days. After 21 days, the concentration of CTC in the 22, 38 and 55°C swine digester slurries decreased 7, 80 and 98%, respectively. Coliforms in the 22°C digester slurries were still viable after 25 days; however, they were not detectable in the 38 and 55°C slurries after 3 and 1 days, respectively. After 28 days, the concentration of MON in the 22, 38 and 55°C cattle digester slurries decreased 3, 8 and 27%, respectively. Coliforms in the 22°C cattle digester slurries were still viable after 28 days; however, they were not detectable in the 38 and 55°C slurries after 14 and 1 days, respectively. CONCLUSIONS: These studies indicate that anaerobic digestion at 38 or 55°C may be an effective treatment to reduce coliforms and CTC; however, it is not an effective treatment to reduce MON. SIGNIFICANCE AND IMPACT OF THE STUDY: More studies are needed to determine which pharmaceuticals are susceptible to degradation by a specific manure treatment to prevent negative environmental consequences.

Identification of new SLE-associated genes with a two-step Bayesian study design.

In our earlier study, we utilized a Bayesian design to probe the association of approximately 1000 genes (approximately 10,000 single-nucleotide polymorphisms (SNPs)) with systemic lupus erythematosus (SLE) on a moderate number of trios of parents and children with SLE. Two genes associated with SLE, with a multitest-corrected false discovery rate (FDR) of <0.05, were identified, and a number of noteworthy genes with FDR of <0.8 were also found, pointing out a future direction for the study. In this report, using a large population of controls and adult- or childhood-onset SLE cases, we have extended the earlier investigation to explore the SLE association of 10 of these noteworthy genes (109 SNPs). We have found that seven of these genes exhibit a significant (FDR<0.05) association with SLE, both confirming some genes that have earlier been found to be associated with SLE (PTPN22 and IRF5) and presenting novel findings of genes (KLRG1, interleukin-16, protein tyrosine phosphatase receptor type T, toll-like receptor (TLR)8 and CASP10), which have not been reported earlier. The results signify that the two-step candidate pathway design is an efficient way to study the genetic foundations of complex diseases. Furthermore, the novel genes identified in this study point to new directions in both the diagnosis and the eventual treatment of this debilitating disease.

Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin.

The sodium channel blocker, tetrodotoxin (TDT), was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice. Anti-TDT antibodies were detected in serum by ELISA and reached stable levels 4-5 wk after the first immunization. Spleens from immunized mice were fused with NS-1 mouse myeloma cells and approximately 9,329 resultant hybrids were screened by ELISA for reactivity to TDT. Two stable hybrids were isolated, subcloned, and characterized. These hybrids, termed TD13a1 and TD2C5, secreted specific anti-TDT antibodies that recognized TDT but not the related sodium channel blocker, saxitoxin (STX), as determined by competition ELISA. Both antibodies were of the IgG1k subclass with Ka's approaching 10(7) M-1. The inhibitory ability of these antibodies was tested by a competitive displacement assay for [3H]STX on rat brain membranes. Both antibodies strongly inhibited TDT binding to membranes. A nanomole of TD2C5 was able to bind approximately 1.8 nmol of TDT, whereas a comparable amount of TD13a1 bound half as much. Furthermore, TD2C5 was able to protect against TDT-induced reduction of peripheral nerve action potentials in rat tibial nerve when administered in situ. These antibodies thus represent potentially useful reagents for neurobiologic research, detection of toxin contamination and diagnosis of poisoning, and may provide protection against the toxicity of TDT in vivo.

Characterizing pharmaceutical, personal care product, and hormone contamination in a karst aquifer of southwestern Illinois, USA, using water quality and stream flow parameters.

Karst aquifers are drinking water sources for 25% of the global population. However, the unique geology of karst areas facilitates rapid transfer of surficial chemicals to groundwater, potentially contaminating drinking water. Contamination of karst aquifers by nitrate, chloride, and bacteria have been previously observed, but little knowledge is available on the presence of contaminants of emerging concern (CECs), such as pharmaceuticals. Over a 17-month period, 58 water samples were collected from 13 sites in the Salem Plateau, a karst region in southwestern Illinois, United States. Water was analyzed for 12 pharmaceutical and personal care products (PPCPs), 7 natural and synthetic hormones, and 49 typical water quality parameters (e.g., nutrients and bacteria). Hormones were detected in only 23% of samples, with concentrations of 2.2-9.1ng/L. In contrast, PPCPs were quantified in 89% of groundwater samples. The two most commonly detected PPCPs were the antimicrobial triclocarban, in 81% of samples, and the cardiovascular drug gemfibrozil, in 57%. Analytical results were combined with data of local stream flow, weather, and land use to 1) characterize the extent of aquifer contamination by CECs, 2) cluster sites with similar PPCP contamination profiles, and 3) develop models to describe PPCP contamination. Median detection in karst groundwater was 3 PPCPs at a summed concentration of 4.6ng/L. Sites clustered into 3 subsets with unique contamination models. PPCP contamination in Cluster I sites was related to stream height, manganese, boron, and heterotrophic bacteria. Cluster II sites were characterized by groundwater temperature, specific conductivity, sodium, and calcium. Cluster III sites were characterized by dissolved oxygen and barium. Across all sites, no single or small set of water quality factors was significantly predictive of PPCP contamination, although gemfibrozil concentrations were strongly related to the sum of PPCPs in karst groundwater.

Calcium channel regulation by calcineurin, a Ca2+-activated phosphatase in mammalian brain.

The enzymatic addition or removal of phosphate esters on serine and threonine hydroxyls alters the activity of many proteins that contribute to the characteristic structure and function of nerve cells. Recently, calcineurin, a major calmodulin-binding protein in mammalian brain, has been purified and identified as a Ca2+-activated protein phosphatase. Preliminary experiments suggest that calcineurin may limit Ca2+ influx through dihydropyridine-sensitive Ca2+ channels in the plasma membrane by dephosphorylating the channel, or a closely associated protein, and inactivating it.

An enzymatic mechanism for potassium channel stimulation through pertussis-toxin-sensitive G proteins.

Many neurotransmitters inhibit secretion from electrically excitable cells by activating pertussis-toxin-sensitive G proteins that modulate voltage-gated ion channels. Recent electrophysiological studies of metabolically intact cells from mammalian and molluscan neuroendocrine systems have implicated protein phosphatases in this process. In this article David Armstrong and Richard White review these studies and suggest a biochemical pathway that might link one of the G proteins to protein phosphatase activity.

N-myc amplification and neuronal differentiation in human primitive neuroectodermal tumors of the central nervous system.

Despite the phenotypic similarities between primitive neuroectodermal tumors of the central nervous system, childhood neuroblastoma, and peripheral neuroepithelioma, a histogenetic relationship among these neoplasms has not been shown. High levels of N-myc expression occur selectively in developing brain and in some embryonic tumors of neural origin. N-myc amplification and high levels of N-myc expression in childhood neuroblastoma have been correlated with disease stage and prognosis. To determine whether the copy number of the N-myc gene in primitive neuroectodermal tumors of the central nervous system is altered, we examined 20 primitive neuroectodermal tumors by Southern and/or slot blot hybridization to a 1-kilobase N-myc genomic DNA sequence and a 492-base pair N-myc-specific subclone as well as to a 1.1-kilobase albumin complementary DNA sequence as a control for gene copy number. Amplification of the N-myc gene was detected in two cerebellar tumors both of which exhibited neuronal differentiation by light microscopy. These tumors had not been treated previously. Of the remaining 18 tumors, eight were undifferentiated, three showed early neuroblastic, and seven focal glial differentiation. These findings suggest a possible relationship between N-myc amplification and neuronal differentiation.

Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells.

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.

Morphology of the developing visual cortex of the human infant: a quantitative and qualitative Golgi study.

A renaissance of the Golgi impregnation method has focused interest on dendritic aspects of neuronal development. The visual cortices of 39 "neurologically normal" infants from 14 weeks' gestation to 6 months of age were prepared at postmortem for rapid Golgi impregnation studies. These were stained and duplicated with camera lucida drawings. The total number of cells in defined columns of the visual cortex was counted on cresyl violet-stained sections, and differentiated neurons were identified. The number of spines on the apical and basal dendrites of selected cells was counted for a given interval along the dendrites. The camera lucida drawings, cell counts, and spine counts were used to illustrate the normal ontogeny of the visual cortex.

Myelination of the human vagus nerve from 24 weeks postconceptional age to adolescence.

Significant changes in respiratory reflexes occur with maturation. The vagus nerve, the pathway for the Hering-Breuer and irritant-receptor reflexes, was studied quantitatively in 33 infants and 5 adolescents. In the infants, total myelinated vagus fibers increased linearly (r m0.682, p less than 0.001) with postconceptional age (PCA), and by 40 weeks after conception, total counts were comparable to those of adolescent group. Counts of total myelinated vagus fibers in 16 term infants (greater than 41 weeks PCA) were comparable to those in the adolescent group (p less than 0.40), whereas 17 preterm infants (less than 38 weeks PCA) showed significantly fewer total myelinated vagus fibers than term or adolescent groups (p less than 0.001). Smaller-diameter (less than 2 micrometer) myelinated vagus fibers depended upon PCA in the preterm group (p less than 0.005), but were independent of PCA in the term group (p less than 0.5). Preterm infants have a higher percentage of small to total myelinated vagus fibers than term infants (p less than 0.1).

Disproportionate loss of CA4 parvalbumin-immunoreactive interneurons in patients with Ammon's horn sclerosis.

We studied differences in the number and morphology of parvalbumin-immunoreactive (PV-IR) interneurons in 43 hippocampal specimens from patients with classical Ammon's horn sclerosis (AHS) who underwent anterior temporal lobectomy, as compared with 14 autopsy and non-AHS surgical control specimens. PV-IR neuronal loss in the AHS specimens varied significantly from that expected based on overall AHS-associated pyramidal and granule neuron loss. Most striking was the loss of PV-IR interneurons in CA4 of the AHS specimens, which was 12 times greater than AHS-associated pyramidal neuron loss, and significantly exceeded the PV-IR interneuron loss observed in the other sectors of the hippocampus. In addition, the PV-IR interneurons in the AHS specimens had markedly smaller and less defined cell bodies and shortened and simplified dendritic arbors compared with the PV-IR interneurons in the control specimens. Other differences noted in the AHS specimens included prominent dendritic varicosities; the loss or interruption of a band formed by PV-IR terminals in the dentate gyrus; and the virtual absence of a small, intensely staining PV-IR interneuron with a short, exuberant dendritic arbor that was readily identified in the autopsy specimens. We discuss these findings in relationship to the development of classical AHS and complex partial seizures (CPS).

Corpora amylacea and heat shock protein 27 in Ammon's horn sclerosis.

Increased numbers of corpora amylacea have been observed in the resected mesial temporal lobe of many patients with complex partial seizures (CPS) and Ammon's horn sclerosis (AHS). Several heat shock proteins (HSPs) are induced by seizures and have been suggested as an etiologic factor in the formation corpora amylacea. We quantified corpora amylacea and HSP27-immunoreactive astrocytes in temporal lobe specimens from patients with CPS (28 AHS; 10 non-AHS) and in 5 autopsy controls. Corpora amylacea were increased in each sector of Ammon's horn in the AHS group, significantly so in CA1 and CA3 (p < 0.0001 and p = 0.0097, respectively), compared with the non-AHS group, although there was considerable variability among the specimens. We found HSP27 to be significantly but nonspecifically increased in the resected temporal lobe specimens from all patients with CPS, regardless of the underlying pathology. HSP27 was not, however, expressed within the corpora amylacea, and did not correlate with the number of corpora amylacea in any of the 9 mesial and lateral temporal lobe areas examined.

Glial cell nuclear hypertrophy in complex partial seizures.

The white matter of resected temporal lobes from patients with intractable complex partial seizures shows increased cellularity which appears to be related to glia and neurons. This study, using quantitative methods, defines an increase in glial cell numbers and a significant increase in glial nuclear size within a defined area of white matter in the lateral temporal lobe. Evaluation was made on specimens from ten patients with complex partial seizures compared with two patients with non-epileptic brain lesions and five autopsy patients with no neurologic disease. The importance of recognizing these alterations in glia and the possible relevance to the pathoetiology of epilepsy are discussed.

The clinical significance of extracellular matrix in gangliogliomas.

Based on in vitro studies which demonstrate that collagen IV and laminin inhibit the proliferation and invasiveness of glioma cells, we investigated the clinical significance of these extracellular matrix proteins (ECM) in patients with gangliogliomas, tumors in which ECM is often a prominent feature. Our study compared the relative presence and deposition pattern of collagen IV and laminin in 19 gangliogliomas and in 18 gliomas without ganglion cell differentiation (8 low-grade astrocytomas, 7 anaplastic astrocytomas, and 3 anaplastic mixed gliomas). We also examined whether the presence of collagen IV and laminin correlated with other features often observed in gangliogliomas, including perivascular lymphocytic inflammation, granular bodies, microcalcification, and subarachnoid extension, and whether any of these features were associated with the patient's clinical course. Significant deposition of collagen IV and laminin was found in 9 gangliogliomas (47%), but in none of the other gliomas. The presence of these extracellular proteins in gangliogliomas correlated with both perivascular inflammation (P = 0.003), and involvement of the leptomeninges by tumor (P = 0.008). The duration of symptoms prior to surgical resection was significantly longer for patients whose tumors showed extracellular deposition of collagen IV and laminin than for patients whose tumors lacked deposition of these proteins (mean 13.7 vs 5.1 years; P = 0.02). In addition, the duration of symptoms was significantly longer for patients whose tumors exhibited perivascular inflammation than for patients whose tumors displayed little or no perivascular inflammation (mean 14.8 vs 4.8 years; P = 0.01). These findings suggests that collagen IV and laminin and perivascular inflammation are related to the indolent behavior of gangliogliomas.

The hippocampal tissue slice in animal models of CNS disorders.

The use of in vitro brain slice preparations has increased in the past ten years, particularly the hippocampal slice because of the dissection ease, well-defined cell layers, and self-contained afferent systems. Developing the slice as a model for screening neurotoxins is of particular interest because of the sensitivity of hippocampal neurons to chemical or metabolic insult and the need to keep animal usage to a minimum. Experiments where the slice has been used to study the neurotoxin trimethyltin are discussed, along with its use as a model for investigating the neurodegenerative problems associated with hypoxia, ischemia, and epilepsy.

Subcortical leukomalacia. Relationship to development of the cerebral sulcus and its vascular supply.

The development of the microvascular architecture of the cerebral cortex and white matter was studied in the preterm, term, and older infant, using postmortem cerebral angiography and metal impregnation studies. During the first few months after birth, a relatively avascular triangle forms in the white matter at the depth of the sulcus. Subcortical leukomalacia occurs in this triangle as well as in border zones between the major cerebral arteries.

Somatostatin stimulates BKCa channels in rat pituitary tumor cells through lipoxygenase metabolites of arachidonic acid.

The stimulation of large-conductance, calcium-activated (BK) potassium channels by somatostatin through protein dephosphorylation in rat pituitary tumor cells (White et al., Nature 351, 570-573, 1991) is blocked by drugs that interfere with arachidonic acid release by phospholipase A2 and metabolism by 5-lip-oxygenase. In contrast, higher concentrations of the same drugs had no effect on BK channel gating in cell-free patches, on the inhibition of adenylyl cyclase by somatostatin, or on the stimulation of BK channels by protein dephosphorylation through a cGMP-dependent pathway (White et al., Nature 361, 263-266, 1993). Exogenous arachidonic acid (1-20 muM) stimulated BK channel activity through protein dephosphorylation as effectively as somatostatin and was also blocked by inhibitors of lipoxygenases but not by inhibitors of phospholipase A2. These results support the hypothesis that lipoxygenase metabolites of arachidonic acid are second messengers linking pertussis toxin sensitive G-proteins to protein phosphatases regulating potassium channel activity (Armstrong and White, Trends Neurosci. 15, 403-408, 1992).

A chronic focal epilepsy with mossy fiber sprouting follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infant rats.

Studies were conducted to characterize a chronic epileptic condition that follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infancy. Wistar rat pups received a single injection of tetanus toxin in the right CA3 region on postnatal day 10. Animals were monitored for epileptiform activity by video electroencephalographic or visual observation during the following three to five days. Repeat evaluation six months later demonstrated interictal discharges in 79% (11 of 14) and electrographic seizures in 42% (six of 14) of adult rats with tetanus toxin-induced seizures in infancy. Five of the animals had interictal activity which occurred focally in either the left (n = 2) or right (n = 3) hippocampus. One animal had focal interictal activity independently in these regions and in the left and right cortical regions. The remaining five animals had interictal activity in the hippocampus and synchronously in the ipsilateral cortex or the contralateral hippocampus. Electrographic seizures were focal (nine of 14) or bilateral (five of 14) in onset. The behaviors that accompanied these seizures were quite variable. Clonic face and forelimb movements were observed in some animals. However, a significant portion of rats had electrographic seizures with no associated behavioral change. Timm staining was performed on hippocampal sections from experimental and control animals. There was a significantly greater Timm score (aberrant Timm granules) in the inner molecular layer of the dentate gyrus in tetanus toxin-treated rats than in control rats. Our findings suggest that intrahippocampal tetanus toxin injection in infant rats results in a chronic focal epilepsy that persists for at least six months and is associated with aberrant mossy fiber sprouting in the dentate gyrus. The model described here contributes significantly to the evidence for chronic effects of recurrent seizures in early life, and provides a model for investigation of the molecular and cellular events that contribute to the development of chronic epilepsy.

Reduction in incidence of periventricular, intraventricular hemorrhages in hypertensive newborn beagles pretreated with phenobarbital.

Periventricular and intraventricular intracerebral hemorrhages occur frequently in premature newborns and are markers for, or contribute significantly to, neurologic morbidity in survivors. Hemorrhages are hypothesized to result from rapid fluctuations in cerebral perfusion pressure and/or cerebral blood flow. Phenobarbital sodium has been given to premature infants in anticonvulsant dosages in attempts to prevent hemorrhages, but its efficacy in clinical studies has been disputed. In this study, in the 24 to 72-hour-old newborn beagle, an animal model for periventricular and intraventricular intracerebral hemorrhage, phenobarbital sodium was administered to obtain anticonvulsant serum levels prior to phenylephrine-induced hypertensive insult. None of the animals was hypoxic or hypercarbic. Resulting hypertensive systemic mean arterial BPs were 99 +/- 8 mm Hg in the 15 control pups and 93 +/- 15 mm Hg in the 15 phenobarbital sodium-treated pups (not statistically significantly different). The duration of the hypertension was the same in both groups. Phenobarbital caused a significant decrease in mean arterial BP in the treated group just after its administration (P less than .05). Six of the 15 control pups (40%) and one of the 15 treated pups (7%) demonstrated macroscopic and microscopic periventricular and intraventricular hemorrhage (P less than .05, chi 2, Yates correction). Thus, in nonasphyxiated newborn beagles, phenobarbital sodium significantly reduced the incidence of hemorrhage after hypertensive insult.