Dose-dependent reversal of KCC2 hypofunction and phenobarbital-resistant neonatal seizures by ANA12.

Neonatal seizures have an incidence of 3.5 per 1000 newborns; while hypoxic-ischemic encephalopathy (HIE) accounts for 50-60% of cases, half are resistant to 1st-line anti-seizure drugs such as phenobarbital (PB). Tyrosine receptor kinase B (TrkB) activation following ischemic injury is known to increase neuronal excitability by downregulation of K-Cl co-transporter 2 (KCC2); a neuronal chloride (Cl-) co-transporter. In this study, three graded doses of ANA12, a small-molecule selective TrkB antagonist, were tested in CD1 mice at P7 and P10 following induction of neonatal ischemia by a unilateral carotid ligation. The PB loading dose remained the same in all treatment groups at both ages. Evaluation criteria for the anti-seizure efficacy of ANA12 were: (1) quantitative electroencephalographic (EEG) seizure burden and power, (2) rescue of post-ischemic KCC2 and pKCC2-S940 downregulation and (3) reversal of TrkB pathway activation following ischemia. ANA12 significantly rescued PB resistant seizures in a dose-dependent manner at P7 and improved PB efficacy at P10. Additionally, female pups responded better to lower doses of ANA12 compared to males. ANA12 significantly reversed post-ischemic KCC2 downregulation and TrkB pathway activation at P7 when PB alone was inefficacious. Rescuing KCC2 hypofunction may be critical for preventing emergence of refractory seizures.

When should we test for voltage-gated potassium channel complex antibodies? A retrospective case control study.

Patients with voltage-gated potassium channel (VGKC)-complex antibodies are increasingly recognized as having central, peripheral or combined phenotypes. With increasing awareness, more patients are tested and the clinical spectrum is expanding. Consequently, clinicians may be uncertain as to which patients should or should not be tested. Previous studies have identified common clinical features, but none has looked at the usefulness of these in predicting seropositive disease. We conducted a case-control study of patients tested for VGKC-complex antibodies over 10years at a regional tertiary neurology centre determining which clinical/biochemical features were associated with antibody-positive disease. We found a marked increase in the numbers tested, although the percentage positive remained low. Antibody titre was highest in central disease (p<0.001). Time from presentation to testing was shorter in those with VGKC-disease (p=0.01). Seizures were present in 11 (69%) of those with VGKC-disease versus three (18%) without (odds ratio [OR] 10.3, 95% confidence interval [CI]: 2.0-52.7, p=0.005). There was an inverse correlation between the antibody titre and serum sodium. A multivariate model selected seizures and hyponatraemia as predictive of VGKC disease (sensitivity 75% and specificity 82%); faciobrachial dystonic movements were specific but insensitive. Interestingly serum alkaline phosphatase was higher in those with VGKC-disease (p=0.016) and highest in those with peripheral disease (p=0.015). An ALP>70u/L was strongly associated with antibody positivity (OR 4.11 95% CI: 1.43-11.8, p=0.007) with a sensitivity of 74.2%. The presence of seizures, faciobrachial movements, and hyponatraemia should raise suspicion of VGKC-disease; alkaline phosphatase may represent a novel biomarker, particularly in those with peripheral disease.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T-cells.

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 6.4 years.

BACKGROUND: Prophylactic human papillomavirus (HPV) vaccines have to provide sustained protection. We assessed efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. METHODS: Women aged 15-25 years, with normal cervical cytology, who were HPV-16/18 seronegative and oncogenic HPV DNA-negative (14 types) at screening participated in a double-blind, randomised, placebo-controlled initial study (n=1113; 560 vaccine group vs 553 placebo group) and follow-up study (n=776; 393 vs 383). 27 sites in three countries participated in the follow-up study. Cervical samples were tested every 6 months for HPV DNA. Management of abnormal cytologies was prespecified, and HPV-16/18 antibody titres were assessed. The primary objective was to assess long-term vaccine efficacy in the prevention of incident cervical infection with HPV 16 or HPV 18, or both. We report the analyses up to 6.4 years of this follow-up study and combined with the initial study. For the primary endpoint, the efficacy analysis was done in the according-to-protocol (ATP) cohort; the analysis of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was done in the total vaccinated cohort (TVC). The study is registered with ClinicalTrials.gov, number NCT00120848. FINDINGS: For the combined analysis of the initial and follow-up studies, the ATP efficacy cohort included 465 women in the vaccine group and 454 in the placebo group; the TVC included 560 women in the vaccine group and 553 in the placebo group. Vaccine efficacy against incident infection with HPV 16/18 was 95.3% (95% CI 87.4-98.7) and against 12-month persistent infection was 100% (81.8-100). Vaccine efficacy against CIN2+ was 100% (51.3-100) for lesions associated with HPV-16/18 and 71.9% (20.6-91.9) for lesions independent of HPV DNA. Antibody concentrations by ELISA remained 12-fold or more higher than after natural infection (both antigens). Safety outcomes were similar between groups: during the follow-up study, 30 (8%) participants reported a serious adverse event in the vaccine group versus 37 (10%) in the placebo group. None was judged related or possibly related to vaccination, and no deaths occurred. INTERPRETATION: Our findings show excellent long-term efficacy, high and sustained immunogenicity, and favourable safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. FUNDING: GlaxoSmithKline Biologicals (Belgium).

A community intervention trial to promote judicious antibiotic use and reduce penicillin-resistant Streptococcus pneumoniae carriage in children.

OBJECTIVE: Inappropriate use of antibiotics is common in primary care, and effective interventions are needed to promote judicious antibiotic use and reduce antibiotic resistance. The objective of this study was to assess the impact of parent and clinician education on pediatric antibiotic prescribing and carriage of penicillin-nonsusceptible Streptococcus pneumoniae in child care facilities. METHODS: A nonrandomized, controlled, community intervention trial was conducted in northern Wisconsin Clinicians. Clinic staff received educational materials and small-group presentations; materials were distributed to parents through clinics, child care facilities, and community organizations. Prescribing data were analyzed for 151 clinicians who provided primary pediatric care; nasopharyngeal carriage of penicillin-nonsusceptible S pneumoniae was assessed for 664 children in the baseline period (January-June 1997) and for 472 children in the postintervention period (January-June 1998). RESULTS: The median number of solid antibiotic prescriptions per clinician declined 19% in the intervention region and 8% in the control region. The median number of liquid antibiotic prescriptions per clinician declined 11% in the intervention region, compared with an increase of 12% in the control region. Retail antibiotic sales declined in the intervention region but not in the control region. Among participating children in child care facilities, there were no significant differences in antibiotic use or penicillin-nonsusceptible S pneumoniae colonization between the intervention and control regions. CONCLUSIONS: A multifaceted educational program for clinicians and parents led to community-wide reductions in antibiotic prescribing, but in child care facilities, there was no apparent impact on judicious antibiotic use or colonization with drug-resistant S pneumoniae. Longer follow-up time or greater reductions in antibiotic use may be required to identify changes in the pneumococcal susceptibility.

RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function.

Mice with targeted RelB mutations demonstrated an essential role for RelB in immune responses and in myeloid dendritic cell differentiation. Human studies suggested a more global transcriptional role in antigen presentation. Burkitt lymphoma cell lines were used as a model to examine the role of RelB in antigen presentation. After transient transfection of BJAB with RelB, strong nuclear expression of RelB-p50 heterodimers was associated with increased APC function and expression of CD40 and MHC class I. Antisense RelB in DG75 reduced antigen-presenting capacity and CD40-mediated up-regulation of MHC molecules. The data indicate that RelB transcriptional activity directly affects antigen presentation and CD40 synthesis. Stimulation of RelB transcriptional activity may provide a positive feedback loop for facilitating productive APC/T cell interactions.

Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120.

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.

Profound hypothermia and circulatory arrest with skull base approaches for treatment of complex posterior circulation aneurysms.

OBJECTIVE: Cardiopulmonary bypass with profound hypothermia and circulatory arrest has seen a resurgence as an adjunct technique in neurological surgery. We report our experience with this technique in treating seven complex vertebro-basilar aneurysms. METHODS: Skull base approaches were used in all cases, providing excellent exposure and minimizing brain retraction. There were six basilar artery aneurysms and one giant fusiform vertebro-basilar artery aneurysm. All aneurysms but one had an apparent neck, which could be clipped. The fusiform vertebro-basilar artery aneurysm was trapped, partially resected, and the circulation was reestablished with a saphenous vein graft from the cervical internal carotid artery to the mid-basilar artery. RESULTS: Five patients had an excellent outcome and two had a good outcome at one year or at latest follow up. Two of the patients showed improvement of neurological deficits which were present before the surgical intervention. CONCLUSION: Applying very strict selection criteria in this small series of patients with posterior circulation aneurysms, excellent or good results were achieved using the profound hypothermic circulatory arrest technique.

Effect of promoters and signal sequences on the production of secreted HIV-1 gp120 protein in the baculovirus system.

We compared insect cell production levels of secreted HIV-1 gp120 glycoprotein encoded by five different baculovirus expression constructs. Combinations consisting of one of two baculovirus promoters (very late or hybrid late/very late) and one of three different signal sequences [human tissue plasminogen activator (tpa), human placental alkaline phosphatase (pap), or baculovirus envelope glycoprotein (gp67)] were constructed. Production of secreted gp120 from these constructs was analyzed in two enzyme-linked immunosorbent assay formats, one detecting the total amount of secreted gp120 protein and the other measuring the level of "active" gp120 (as defined by the ability to bind to CD4). We found that for all of the constructs, approximately 50 to 90% of the secreted gp120 protein was active. Furthermore, our results indicated that expression from either promoter yielded comparable production of secreted protein, despite the fact that transcription from the hybrid promoter begins at an earlier time. By contrast, the signal sequence had a much greater effect on the levels of secreted gp120: the tpa leader yielded the highest level of secreted protein, followed by the gp67 and pap sequences. This result suggests that transcription is not a limiting factor in the production of secreted gp120, but rather that downstream processing of the protein is more critical. Furthermore, these results confirm the notion that the "optimal" signal sequence is protein dependent and that an insect-derived signal sequence is not optimal in all cases.

Formal pathology rotation during obstetrics and gynecology residency: is it beneficial?

BACKGROUND: Our objective was to assess the educational benefits of a formal pathology rotation during an obstetrics and gynecology residency program and to determine the utility of this information in clinical practice. METHODS: In this descriptive study, the benefits of a 2-month rotation in pathology for obstetrics and gynecology residents were analyzed. A computerized listing of surgical cases processed by each resident was sent to the obstetrics and gynecology program director. RESULTS: Our resident accessioned 5.4% of the total pathology cases processed each month. Reports from previous residents (over a 17-year period) and from program directors at the annual educational retreat indicate that such information was not relevant to our graduates in their clinical practice. CONCLUSIONS: A formal pathology rotation for obstetric residents can improve knowledge base, but the usefulness of this knowledge in clinical practice is dubious.

Cerebral vasospasm after tumor resection. A case report.

OBJECTIVE AND IMPORTANCE: Symptomatic cerebral vasospasm can occur after resection of tumors in or adjacent to the basal cisterns, causing delayed neurological deterioration. This potentially treatable condition may go unrecognized. Delay in its recognition will adversely affect the outcome of the patients. There has been a few cases of vasospasm after tumoral resection reported in the literature, mostly in adults. We report a case of vasospasm after resection of a third nerve schwannoma in a pediatric patient. This is the youngest patient reported to date with vasospasm after resection of a brain tumor. CLINICAL PRESENTATION: A six years old girl presented with sudden onset diplopia. Radiological work-up revealed a third nerve mass. She underwent a craniotomy for resection of her mass. Pathological findings were consistent with a third nerve schwannoma. One week postoperatively, her mental status deteriorated. A CT scan revealed a diffuse hypodense area involving the right frontal and temporal lobes in the middle cerebral artery distribution as well as the midbrain. The absence of these findings on the MRI imaging performed on the first postoperative day made us evoke a vascular etiology. A cerebral angiogram was performed and revealed vasospasm in the right internal carotid artery and in the right middle and posterior cerebral arteries. Hyperdynamic hypervolemic hemodilutional therapy was instituted. CONCLUSION: Delayed clinical deterioration from vasospasm is a potentially reversible condition, if recognized early. A high index of suspicion should be maintained in case delayed clinical deterioration occurs after surgery of tumors in the basal cisterns. Cerebral angiography will confirm the diagnosis. Early institution of hyperdynamic hypervolemic hemodilutional therapy and angioplasty may reverse the deficit and improve outcome.

Construction and characterization of a radio-iodinatable mutant of recombinant human CD4.

Recombinant soluble human CD4 (rsCD4) has been used in iodinated form to study the interaction of CD4 with its ligands. However, the utility of [125I]-rsCD4 is limited because rsCD4 is inefficiently iodinated and the iodinated protein is poorly active. The iodination properties of rsCD4 most likely reflect the poor accessibility of the tyrosine residues, apparent from the available X-ray structures. We have generated an iodinatable mutant of rsCD4 by substituting Tyr for Phe(179) in the flexible, solvent-exposed C-terminal region of rsCD4(183), a truncated form of CD4 that consists of the first 183 residues of CD4 and includes the binding sites for HIV-1 gp120 and MHC class II molecules. When F179Y rsCD4(183) is iodinated under trace-labeling conditions, the efficiency of 125I incorporation and the percentage of iodinated molecules that are active are much enhanced compared with WT rsCD4. Moreover, trace-labeled [125I]-F179Y rsCD4(183) has the same affinity for HIV-1 rgp120 as unlabeled WT rsCD4. The improved activity of trace-labeled [125I]-F179Y rsCD4(183) appears to be due to effective competition by Y179 for reactive iodine species that, in WT rsCD4, react with traces of denatured protein and/or with residues critical for activity or conformational integrity. The incorporation of accessible tyrosine residues may improve the iodinatibility of a protein both by introducing a readily iodinatable residue and by protecting sensitive proteins from adverse reactions.

Chromosomal structure of the human TYRP1 and TYRP2 loci and comparison of the tyrosinase-related protein gene family.

The structures of the human tyrosinase-related protein genes TYRP1 and TYRP2 have been determined and compared with that of the tyrosinase gene (TYR). The TYRP1 protein is encoded in 7 exons spread over 24 kb of genomic DNA. Characterization of a 55-kb contig encompassing the human TYRP2 locus reveals that the protein coding region is divided into 8 exons. All three members of the TYRP gene family share a common C-terminal membrane spanning exon. Examination of the position of other intron junctions suggests that TYRP1 was derived from a TYR duplication and then was itself duplicated to give rise to the TYRP2 gene. The evidence also suggests that at least some of the introns within the TYR, TYRP1, and TYRP2 coding regions were gained after duplication and that intron slippage is unlikely to have occurred.

Duplication (12p) syndrome--a family.

We report the following cases of Duplication (12p) Syndrome. This is a rare entity which may present with developmental delay, dysmorphic features and malformations.

Expression studies of pigmentation and POU-domain genes in human melanoma cells.

Human melanoma cell lines have been used to examine the regulation of the tyrosinase (TYR) and tyrosinase-related protein genes TRP-1 and TRP-2 in response to differentiating chemicals and UV irradiation. TRP-1 mRNA levels can be repressed by treatment with the differentiating chemicals DMSO and HMBA. There is little effect of UV irradiation on pigment synthesis by human melanoma cell lines or tyrosinase activity, with variable effects on the levels of the TYR, TRP-1, and TRP-2 gene transcripts. The human TRP-1 gene promoter has been isolated and its activity tested by transient cell transfection to begin an examination of signal transduction mechanisms operating in response to pigmenting and differentiating agents. To identify transcription factors that may be involved in melanocytic gene expression, we studied the N-Oct-3 and N-Oct-5 octamer-binding activities normally expressed in the neuroectodermal cell lineage and which are expressed at high levels in melanoma cells. POU-domain-containing cDNA have been isolated from the A2058 human melanoma cell line that are homologous to the brn-2 gene that encodes N-Oct-3 and N-Oct-5.

Coincident bit counting-a new criterion for image registration.

A similarity measure based on the number of coincident bits in multichannel images is presented. The similarity criterion incorporated in the image registration algorithm uses a coincident bit counting (CBC) method to obtain the number of matching bits between the frames of interest. The CBC method not only performs favorably compared with traditional techniques, but also renders simpler implementation in conventional computing machines. An image registration algorithm that incorporates the CBC criterion is proposed to determine the translation motion among sequences of images. The errors caused by noise, misregistration, and a combination of these two are analyzed. Some experimental studies using low-contrast coronary images from a digital angiographic sequence are discussed. The results compare favorably with those obtained by using other nonparametric methods.

Application of the EM algorithm to radiographic images.

The expectation maximization (EM) algorithm has received considerable attention in the area of positron emitted tomography (PET) as a restoration and reconstruction technique. In this paper, the restoration capabilities of the EM algorithm when applied to radiographic images is investigated. This application does not involve reconstruction. The performance of the EM algorithm is quantitatively evaluated using a "perceived" signal-to-noise ratio (SNR) as the image quality metric. This perceived SNR is based on statistical decision theory and includes both the observer's visual response function and a noise component internal to the eye-brain system. For a variety of processing parameters, the relative SNR (ratio of the processed SNR to the original SNR) is calculated and used as a metric to compare quantitatively the effects of the EM algorithm with two other image enhancement techniques: global contrast enhancement (windowing) and unsharp mask filtering. The results suggest that the EM algorithm's performance is superior when compared to unsharp mask filtering and global contrast enhancement for radiographic images which contain objects smaller than 4 mm.