Ketamine modulates hippocampal neurochemistry and functional connectivity: a combined magnetic resonance spectroscopy and resting-state fMRI study in healthy volunteers.

A growing body of evidence suggests glutamate excess in schizophrenia and that N-methyl-d-aspartate receptor (NMDAR) hypofunction on γ-aminobutyric acid (GABA) interneurons disinhibiting pyramidal cells may be relevant to this hyperglutamatergic state. To better understand how NMDAR hypofunction affects the brain, we used magnetic resonance spectroscopy and resting-state functional magnetic resonance imaging (MRI) to study the effects of ketamine on hippocampal neurometabolite levels and functional connectivity in 15 healthy human subjects. We observed a ketamine-induced increase in hippocampal Glx (glutamate+glutamine; F=3.76; P=0.04), a decrease in fronto-temporal (t=4.92, PFDR<0.05, kE=2198, x=-30, y=52, z=14) and temporo-parietal functional connectivity (t=5.07, PFDR<0.05, kE=6094, x=-28, y=-36, z=-2), and a possible link between connectivity changes and elevated Glx. Our data empirically support that hippocampal glutamatergic elevation and resting-state network alterations may arise from NMDAR hypofunction and establish a proof of principle whereby experimental modelling of a disorder can help mechanistically integrate distinct neuroimaging abnormalities in schizophrenia.

Lack of detectable bluetongue virus in skin of seropositive cattle: implications for vertebrate overwintering of bluetongue virus.

The overwintering mechanism of bluetongue virus (BTV) has eluded researchers for many years. It was recently proposed that ovine gamma delta T-cells may become persistently infected with BTV, and serve as a reservoir for infection of naive vectors in the next transmission season. Since cattle are more numerous than sheep in the western United States (where BTV is endemic), this hypothesis was tested in bovines. In the winter of 2002-2003, 54 cattle from an endemic site in northern Colorado were age-selected to ensure that possible BTV exposure must have occurred in the summer of 2002. These cattle were tested for the presence of anti-BTV antibody by ELISA; 53 were seropositive, and one was seronegative. Naive Culicoides sonorensis colony insects were fed on skin sites of four seropositive and one seronegative cattle at day 1 (135 days after the first frost), then sequentially on separate sites for three days. Virus isolation and/or reverse transcriptase-nested polymerase chain reaction from engorged insects and 6 mm skin biopsy samples were performed for detection of viable BTV or BTV nucleic acid; all were negative. These data suggest that cattle are not a reservoir host for BTV overwintering in the western United States. The role of sheep in the trans-seasonality of BTV still remains to be determined.

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection.

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2x threshold voltage) or A and C fibres (2 Hz at 20x threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.

Design and expression of polymeric immunoglobulin fusion proteins: a strategy for targeting low-affinity Fcgamma receptors.

We have developed a family of cloning vectors that direct expression of fusion proteins that mimic aggregated immunoglobulin (IgG) (AIG) and immune complex function with respect to their interactions with FcgammaR and that allow for the inclusion and targeting of a second protein domain to cells expressing FcgammaR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG(1) fused to the framework region of human IgG(1). Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. The resulting molecule is tripartite. The carboxyl-IgG(1) framework domain provides stability and permits dimerization, and the intervening polymer provides increased effector function and targeting to FcgammaR while the amino-terminal domain can deliver an additional signal to cells expressing FcgammaR. To demonstrate the utility of the vectors, the extracellular domain of human CD8alpha was expressed as a HCH2 polymer fusion protein. The fusion proteins were secreted in useful amounts from polyclonal cell lines established in Sf9 cells following transfection and selection with G418. The biological activity of the recombinant CD8alpha-HCH2 polymers was determined and compared to those of AIG and an anti-CD16 monoclonal antibody using an in vitro assay. The activity of the fusion proteins positively correlates to the number of HCH2 units. The largest polymer tested was severalfold more potent than AIG at similar concentrations. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcgammaR in autoimmune disorders.

Neurotrophin-3 antisense oligonucleotide attenuates nerve injury-induced Abeta-fibre sprouting.

It is proposed that following peripheral nerve injury abnormal sprouting of Abeta-fibre primary afferent neurons in the spinal cord contributes to the allodynia that often occurs with such injury. Allodynia is characterized as pain due to a stimulus which is normally non-noxious. Our recent in vivo experiments show that intrathecal administration of neurotrophin-3 (NT-3), in normal animals, induces allodynia and sprouting of Abeta-fibres. In this study, we examine whether intrathecal administration of NT-3 antisense oligonucleotides (50 microM), via an osmotic pump for 14 days, attenuates nerve injury-induced sprouting and allodynia. The oligonucleotides used in this study were phosphorothioate modified and control experiments, using an ELISA, confirm that intrathecal administration of the antisense induces a significant decrease in NT-3 levels in the spinal cord. All surgery was conducted on anaesthetized Wistar rats (sodium pentobarbitone, i.p. 50 mg/kg). Consistent with previous studies, transganglionic labelling of Abeta-fibres with choleragenoid-horseradish peroxidase (C-HRP) shows that complete transection of the sciatic nerve induces an expansion of C-HRP label into lamina II of the spinal dorsal horn. Using image analysis, we find that intrathecal administration of NT-3 antisense attenuates the density of C-HRP labelling in lamina II in nerve injured animals. A NT-3 sense oligonucleotide (50 microM) has no effect. To test the effect of NT-3 antisense on allodynia, the nociceptive flexion reflex is examined, using an Ugo Basile Analgesymeter, in animals with partial sciatic nerve ligation. Intrathecal administration of 50 microM NT-3 antisense significantly attenuates nerve injury-induced allodynia, whereas the sense oligonucleotide has no effect. These results provide further evidence that endogenous NT-3 contributes to both nerve injury-induced Abeta-fibre sprouting and allodynia and demonstrates the potential of neurotrophin-3 antisense oligonucleotides as therapeutic agents for neuropathic pain.

CREB contributes to the increased neurite outgrowth of sensory neurons induced by vasoactive intestinal polypeptide and activity-dependent neurotrophic factor.

Our recent experiments suggest that vasoactive intestinal polypeptide (VIP) enhances neurite outgrowth of dissociated rat dorsal root ganglion cells, indirectly, via the release of a trophic factor from the spinal cord. In this study, we have examined the possible contribution of activity-dependent neurotrophic factor (ADNF) to the trophic actions of VIP. In addition, as we have shown that the factor mediating the trophic actions of VIP acts via protein kinase A we have also examined the contribution of CREB, which is a transcription factor activated by protein kinase A. As previously shown, supernatant taken from spinal cord incubated with VIP, significantly increased the percentage of sensory neurons with neurites. Antiserum against ADNF attenuated the trophic effect of the VIP-conditioned supernatant. Consistently, the ADNF agonist, ADNF(14) (0.001-0.1 fM), significantly enhanced the percentage of cells with neurite outgrowth. Furthermore, the trophic action of ADNF(14) was attenuated by a protein kinase A inhibitor, Rp-cAMPS, whereas the inactive isomer, Sp-cAMPS, had no effect. Preincubation of cells with 5 mcM CREB antisense oligonucleotides, attenuated the increase in neurite outgrowth induced by either the supernatant or ADNF(14). The sense oligonucleotide had no influence on the enhanced neurite outgrowth. We also found that both the supernatant and ADNF(14) induced an increase in the percentage of cells expressing phosphorylated CREB. The data suggests that VIP induces a release of neurotrophic factors, such as ADNF, which enhance neurite outgrowth. In addition, protein kinase A and CREB appear to contribute to the neurotrophic actions of VIP and ADNF. The mechanisms underlying the neurotrophic action of VIP, may have important implications for sprouting and/or synaptic reorganization of central terminals of sensory neurons, which may contribute to neuropathic pain that commonly occurs following peripheral nerve damage.

Intrinsic connections in the caudal subdivision of the dorsolateral visual area (DL(C)) in squirrel monkeys.

Patterns of intrinsic connections and features of individual intrinsic axons in the caudal subdivision of the dorsolateral visual area (DL(C)) were investigated in four squirrel monkeys (Saimiri) following extracellular injections of the tracers biocytin, biotinylated dextran amine, and wheat germ agglutinin conjugated to horseradish peroxidase. Injections were defined in DL(C) by reference to architectonic borders and patterns of connections with other cortical areas. Intrinsic connections extended up to 6 mm from an injection and were usually anisotropic, extending farther dorsoventrally than anteroposteriorly. Injections that involved the supragranular layers produced up to 20 mainly supragranular patches of projections that had a width of 285 +/- 8 microm (mean +/- standard error) and area of 0.125 +/- 0.016 mm(2). Seventy-four intrinsic axon segments with a total of 3,290 boutons were drawn and their bouton spacing measured. The sample included axons in layers 1, 2-3, 5, and multiple (>2) layers; horizontally and vertically oriented axons; and axons in an injection halo, patch, or nonpatch region of projections. There were no differences in bouton spacing for axons in halo, patch, or nonpatch regions. Layer 1 axons (n = 7) had a significantly sparser distribution of boutons (median interbouton interval of 45.2 +/- 17.8 microm) than the layers 2-3 (n = 35) and layer 5 axons (n = 26), which did not differ in bouton spacing (median interbouton intervals of 8.1 +/- 0.4 microm and 8.4 +/- 0.8 microm, respectively). Patterns of intrinsic connections in DL(C) are related to neural organization and properties reported for DL or visual area V4, and are compared to intrinsic connections of other areas.

An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity.

Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT) cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites), and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (beta site) and all four insertions cause premature translation terminations, whereas the other deletion (alpha site) is 36 bp and lies within reverse transcriptase (RT) motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the alpha, beta or both alpha and beta deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the alpha site deletion variant (hTERT alpha-) construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT alpha- inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

The prevalence of polycystic ovaries in women with infertility.

Polycystic ovaries (PCO) are highly prevalent in women presenting with hirsutism or recurrent miscarriage but the functional significance of PCO in ovulatory women presenting with infertility remains unclear. We examined the prevalence of PCO, on ultrasonography, among women presenting with infertility. Among 289 couples classified in four main diagnostic categories, PCO were found in 81 (83%) of 98 anovulatory patients, 40 (53%) of 76 patients whose partners had sperm dysfunction, 26 (50%) of 52 patients with tubal disease and in 28 (44%) of 63 patients with unexplained infertility. By comparison, in a control group of 67 parous volunteers, 19 (28%) were found to have PCO. PCO patients with unexplained infertility had higher midfollicular luteinizing hormone and testosterone compared with the group with normal ovaries. The prevalence of PCO was significantly higher in each of the infertility groups than in controls, and a similar tendency (not significant) was observed among women with unexplained infertility. Ovulatory PCO women with infertility had higher testosterone concentrations in comparison with PCO controls. In summary, the prevalence of PCO among ovulatory women with infertility is higher than that in the normal population, suggesting that PCO may, perhaps by virtue of an effect of hyperandrogenaemia, contribute to the causes of subfertility in women with regular menses.

The long-term effect of epidural administration of butamben suspension on nerve injury-induced allodynia in rats.

UNLABELLED: Although local anesthetics can, in some situations, alleviate neuropathic pain, currently available preparations are short-acting and nonselective, producing, for example, motor dysfunction. Clinical studies report that a novel suspension preparation of butamben has the advantage of a prolonged duration of action, and it can be used epidurally, without impairment of motor function. In this behavioral study, we investigated the effect of the epidural administration of a 5% butamben suspension on nerve injury-induced allodynia. Behavioral studies were performed using an established animal model of neuropathic pain, which involves a partial ligation of the sciatic nerve. Nociceptive thresholds to mechanical stimulation were determined by the paw withdrawal method. The allodynia to mechanical stimulation induced by partial nerve ligation was significantly attenuated by daily injections, for 5 days, of 10 microL of butamben suspension. The analgesia lasted at least 7 days after the final injection. Daily injections of 10 microL of vehicle, for 5 days, had no significant effect on allodynia. During the period of daily injections, both the butamben and vehicle treated rats had temporary impairment of motor coordination compared with untreated controls. Motor function recovered after the final injection. Neither daily injections of butamben for 2 or 3 days, nor smaller volumes for 5 days (2.5-5 microL), had a long-lasting effect. We conclude that repeated epidural administration of butamben suspension for several days provides long-lasting analgesia in rats with nerve injury-induced allodynia to mechanical stimulation. IMPLICATIONS: In this animal behavioral study, using rats with nerve injury-induced pain, we examined the possible long-term analgesic effects of epidural administration of a suspension of the local anesthetic, butamben. We found that multiple doses for several days were required to provide a prolonged analgesia.

Effect of subcutaneous administration of calcium channel blockers on nerve injury-induced hyperalgesia.

Recent studies suggest that calcium contributes to peripheral neural mechanisms of hyperalgesia associated with nerve damage. In this animal behavioural study, we examined further the contribution of calcium in neuropathic pain by testing whether subcutaneous administration of either a calcium chelating agent or voltage-dependent calcium channel blockers attenuate nerve injury-induced hyperalgesia to mechanical stimulation. Studies were carried out in animals with partially ligated sciatic nerves, an established animal model of neuropathic pain. The nociceptive flexion reflex was quantified using an Ugo Basile Analgesymeter. Partial nerve injury induced a significant decrease in mechanical threshold compared to the sham operated controls. Daily subcutaneous injections of the calcium chelating agent, Quin 2 (20 microgram/2.5 microliter), significantly attenuated the nerve injury-induced hyperalgesia. Similarly, SNX-111, a N-type channel blocker, also significantly attenuated the nerve injury-induced hyperalgesia. SNX-230, a P and/or Q-type channel blocker, and nifedipine, a L-type channel blocker, had no effect on the hyperalgesia to mechanical stimulation. In control experiments, SNX-111 had no effect on mechanical thresholds when administered subcutaneously in either the hindpaw of normal animals or the back of the neck in nerve injury animals. This study shows that neuropathic pain involves a local calcium-dependent mechanism in the receptive field of intact neurons of an injured nerve, since it can be alleviated by subcutaneous injections of either a calcium chelating agent or SNX-111, a N-type calcium channel blocker. These agents may be effective, peripherally acting therapeutic agents for neuropathic pain.

Contribution of neurotrophin-3 to the neuropeptide Y-induced increase in neurite outgrowth of rat dorsal root ganglion cells.

Recent studies show that neuropeptide Y acts indirectly, via release of a neurotrophic factor(s) from the spinal cord, to increase the neurite outgrowth of dissociated adult rat dorsal root ganglion cells. This study examines further the neuropeptide Y-induced increase in neurite outgrowth. To characterize the factor(s) mediating the neuropeptide Y-induced increase in neurite outgrowth, we have examined whether antisera to either nerve growth factor or neurotrophin-3 influence the neuropeptide Y-induced increase in neurite outgrowth. Spinal cord slices were incubated with media alone or in combination with 10 nM neuropeptide Y for 2 h at 37 degrees C. The supernatant of spinal cord incubated with neuropeptide Y significantly enhanced the neurite outgrowth of normal dorsal root ganglion cells. Antiserum against nerve growth factor had no effect on the trophic actions of the supernatant. Antiserum against neurotrophin-3, however, significantly attenuated the increase in neurite outgrowth. Consistent with this finding, neurotrophin-3 also increased the percentage of cells with neurites. Transganglionic labelling of A-fibres with choleragenoid-horseradish peroxidase in animals treated intrathecally with neurotrophin-3 for 14 days via an osmotic pump showed that the area of choleragenoid-horseradish peroxidase label expanded into lamina II. In comparison, saline-treated animals had no label in lamina II. In addition, neurotrophin-3-treated animals also had a significant decrease in mechanical nociceptive threshold. The results suggest that neuropeptide Y acts via neurotrophin-3 to mediate an increase in neurite outgrowth of dorsal root ganglion cells. These results have important implications for the mechanisms underlying neuropathic pain.

Extracorporeal shock-wave lithotripsy for bile duct calculi.

BACKGROUND: Bile duct calculi (BDC) can be cleared or treated with modern endoscopic techniques in most patients. However, large stones, bile duct strictures, or unusual anatomy may make endoscopic clearance difficult. The purpose of the present study was to determine the efficacy of extracorporeal shock-wave lithotripsy (ESWL) in treating patients with complicated BDC. METHODS: Between 1989 and January 1995, 16 patients with BDC were treated at our institution with ESWL using a Dornier HM-3 lithotropter. The average age of patients was 62 years (range 32 to 88). Endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy and attempted stone extraction (100%), nasobiliary drainage (83%), and biliary stents (6%) were used prior to ESWL. Eleven patients (61 %) had solitary stones, ranging in diameter from 0.5 to 2.6 cm, whereas 7 patients had multiple stones, ranging in diameter from 0.5 to 5.0 cm. The indications for ESWL were stone impaction (56%), stone size (38%), and bile duct stricture (6%). RESULTS: The 16 patients received 27 ESWL treatments (mean = 2101 shock at 21 kV); with 4 patients (22%) requiring multiple treatments. Stone fragmentation was achieved in 94% of patients. All patients had ERCP performed post-ESWL, and only 2 (13%) patients required immediate operations. At discharge, 94% of patients were stone-free. Minor complications (eg, pain, hematuria) were common. With an average follow-up of 3 years, only 1 patient (6%) has required retreatment for BDC. Hepatic transplantation was required in an additional patient. CONCLUSIONS: In this cohort of patients with both major medical comorbidities and/or technical contraindications to standard methods of endoscopic and surgical clearance of BDC, we found that ESWL facilitated stone clearance in 94% of patients with minimal morbidity and no mortality. In our opinion, ESWL should be used more frequently in the treatment of these complex patients.

Treating the family with multiple sclerosis.

It is difficult to treat a family, particularly when clinicians only have the opportunity to see one or two members in the medical setting. Still, one can accomplish a great deal by viewing the patient within a family system. Advantages of this systemic approach are similar to those accrued when one views the individual patient's problems as being inter-connected. For example, we know that pain stemming from MS can exacerbate depression that, in turn, increases pain ... and so on. By the same token, if the clinician treats either pain or depression, it will likely reduce the other one. One doesn't need to treat all aspects of the family to show a fundamental improvement in the system. If the well partner attends a support group, his or her ability to care for the patient may increase, which could reduce his or her own depression. If children learn to explain the patient's MS to their friends, then they feel more comfortable with closeness to the parent. Treating the family helps it stay healthy, and it is the family who ultimately cares for the patient. To treat the family is to provide the patient with essential care.

Modern use of clomiphene citrate in induction of ovulation.

Clomiphene citrate is the treatment of first choice in the management of infertility in normally oestrogenized, anovulatory women (WHO group II). The majority of women with 'pure' anovulatory infertility respond to treatment with clomiphene citrate. The rates of pregnancy and miscarriage are close to those expected in a normal fertile population. Basal hormone concentrations do not predict outcome. An increased body mass index is the only factor which is consistently associated with a decreased response to clomiphene citrate; it follows therefore, that weight reduction should be an important part of therapy in anovulatory women. According to our data, only an increased luteinizing hormone value immediately post clomiphene citrate predicted an adverse pregnancy outcome in women who conceived. Clomiphene citrate, along with other ovulation induction therapies, can cause multiple follicular development, with a risk of ovarian hyperstimulation and multiple pregnancy. Ultrasound monitoring of treatment is important in order to choose the appropriate dose of clomiphene citrate in subsequent cycles and to minimize the risks of hyperstimulation and multiple pregnancy. When couples with other factors contributing to subfertility are excluded, the cumulative conception rate continues to rise after 6 months of treatment with clomiphene citrate, reaches a plateau by treatment cycle 12 and approaches that of the normal population. It has been reported that prolonged use of clomiphene citrate may be associated with an increased risk of a borderline or invasive ovarian tumour. Taking into consideration these observations, we recommend that anovulatory women responsive to clomiphene citrate should be treated for at least 6 cycles before considering more complex or invasive methods of ovulation induction, and that treatment should probably be limited to a maximum of 12 cycles.

Beta-trace gene expression is regulated by a core promoter and a distal thyroid hormone response element.

We isolated and characterized the human beta-Trace protein (betaTP) gene promoter. betaTP, also known as prostaglandin D2 synthase, is a lipocalin secreted from the choroid plexus and meninges into cerebrospinal fluid. Basal transcription of the betaTP gene is directed from a core promoter found within the first 325 bases of the 5'-flanking sequence. The betaTP gene promoter is responsive to thyroid hormone (3,3',5-triiodothyronine, T3) and efficiently repressed by unliganded human thyroid hormone receptor beta (TRbeta). Functional analysis of the betaTP promoter in TE671 cells revealed that responsiveness to T3 occurs in sequences 2.5 kilobase pairs 5' of the start site. Within the hormone-responsive region we identified a thyroid hormone response element (TRE) located from -2576 to -2562 base pairs relative to the transcription start site. The betaTP TRE is composed of two directly repeated consensus half-sites separated by a 3-base pair space (DR3). The betaTP TRE forms specific complexes with TRbeta. We have shown that a gene active in the choroid plexus and meninges is responsive to T3. T3 may play a role in the regulated transport of substances into the cerebrospinal fluid and ultimately the brain.

A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket.

The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.