Update to the Hong Kong Epilepsy Guideline: evidence-based recommendations for clinical management of women with epilepsy throughout the reproductive cycle.

Since the publication of the Hong Kong Epilepsy Guideline in 2009, there has been significant progress in antiepileptic drug development. New AEDs have emerged, and data about their uses have been published. Women require special attention in epilepsy care. Drug teratogenicity, pregnancy, breastfeeding, contraception, reproduction technology, menopause, and catamenial epilepsy are major topics. Antiepileptic drugs should be chosen individually for patients who are pregnant or may become pregnant with consideration of their teratogenicity and seizure control properties. Folate is commonly prescribed for women of childbearing age who are taking antiepileptic drugs. Spontaneous vaginal delivery and breastfeeding are not contra-indicated in most cases but need to be considered individually based on the patient's medical condition and wishes. Serum drug level monitoring of certain antiepileptic drugs during pregnancy and puerperium can guide dosage adjustment. For catamenial epilepsy, intermittent benzodiazepines such as clobazam during the susceptible phase of the menstrual cycle could be a treatment option.

Ron tyrosine kinase receptor synergises with EGFR to confer adverse features in head and neck squamous cell carcinoma.

BACKGROUND: Although EGFR inhibitors have shown some success in the treatment of head and neck squamous cell carcinomas (HNSCCs), the results are not dramatic. Additional molecular targets are urgently needed. We previously showed that the loss of Ron receptor activity significantly slowed squamous tumour growth and progression in a murine model. Based on these data, we hypothesised that Ron expression confers an aggressive phenotype in HNSCCs. We prospectively collected and evaluated 154 snap-frozen, primary HNSCCs for Ron and EGFR expression/phosphorylation. Biomarker correlation with clinical, pathological and outcome data was performed. The biological responses of HNSCC cell lines to Ron knockdown, its activation and the biochemical interaction between Ron and EGFR were examined. RESULTS: We discovered that 64.3% (99 out of 154) HNSCCs expressed Ron. The carcinomas expressed exclusively mature functional Ron, whereas the adjacent nonmalignant epithelium expressed predominantly nonfunctional Ron precursor. There was no significant association between Ron and sex, tumour differentiation, perineural/vascular invasion or staging. However, patients with Ron+HNSCC were significantly older and more likely to have oropharyngeal tumours. Ron+HNSCC also had significantly higher EGFR expression and correlated strongly with phosphorylated EGFR (pEGFR). Newly diagnosed HNSCC with either Ron/pEGFR or both had lower disease-free survival than those without Ron and pEGFR. Knocking down Ron in SCC9 cells significantly blunted their migratory response to not only the Ron ligand, MSP, but also EGF. Stimulation of Ron in SCC9 cells significantly augmented the growth effect of EGF; the synergistic effect of both growth factors in SCC9 cells was dependent on Ron expression. Activated Ron also interacted with and transactivated EGFR. CONCLUSION: Ron synergises with EGFR to confer certain adverse features in HNSCCs.

Active suppression of immunoglobulin allotype synthesis. 3. Identification of T cells as responsible for suppression by cells from spleen, thymus, lymph node, and bone marrow.

Thymus-derived cells (T cells) that actively suppress production of IgG2a immunoglobulins carrying the Ig-1b allotype have been found in adult (SJL x BALB/c)F(1) mice exposed to anti-Ig-1b early in life. The suppression is specific for Ig-1b. The allelic product, Ig-1a, is unaffected. Spleen, lymph node, bone marrow, or thymus cells from suppressed mice suppress production of Ig-1b by syngeneic spleen cells from normal F(1) mice. When a mixture of suppressed and normal cells is transferred into lethally irradiated BALB/c mice, there is a short burst of Ig-1b production after which Ig-1b levels in the recipient fall rapidly below detectability. Pretreatment of the cells from the suppressed mice with antiserum specific for T cells (anti-Thy-1b) plus complement before mixture destroys the suppressing activity. Similar results with suppressor cells were obtained in vitro using Mishell-Dutton cultures. Mixture of spleen cells from suppressed animals with sheep erythrocyte (SRBC)-primed syngeneic normal spleen before culture suppresses Ig-1b plaque-forming cell (PFC) formation while leaving Ig-1a PFC unaffected. Treatment of the suppressed spleen with anti-Thy-1b before transfer removes the suppressing activity.

Immunological memory in mice. 3. Memory to heterologous erythrocytes in both T cell and B cell populations and requirement for T cells in expression of B cell memory. Evidence using immunoglobulin allotype and mouse alloantigen theta markers with congenic mice.

Using anti-allotype sera and AKR anti thetaC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-theta serum and guinea pig serum (Anti theta + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti theta + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti theta + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.

Cell interaction in an immune response in vitro: requirement for theta-carrying cells.

Cytotoxic antiserum to theta antigen reduces the capacity of mouse spleen cells to generate direct and indirect plaque-forming cells to sheep erythrocytes in vitro but does not affect plaque-forming cells, their precursors, or hemopoietic stem cells. The response of spleen cells treated with antiserum to theta antigen is restored by thymus cells incubated in vivo with sheep erythrocytes.

Effect of different amounts of sodium intake for 4 months on calcium metabolism in normal and oophorectomized rats.

The effects of different amounts of salt intake on urinary calcium and hydroxyproline (OHP) excretion were studied in oophorectomized (O) and sham-operated (S) rats on normal calcium diet. Groups of O and S rats were given either tap water or 2, 6, or 18 g/liter of NaCl to drink. Urinary excretion of sodium, calcium, and OHP was monitored at 0, 2, and 4 months. Urinary excretion of calcium and OHP increased with increasing salt intake (P < 0.001 by ANOVA) in S and O groups. Oophorectomy did not have any additional effect on calcium or OHP excretion. We conclude that increased salt intake causes increased excretion of calcium and OHP and oophorectomy does not further aggravate the loss.

Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein.

A Myelin Basic Protein (MBP) epitope encephalitogenic for the Lewis rat (amino acid residues 68-86) was synthesized and acylated by the attachment of a palmitoyl residue. Lewis rats treated intravenously (i.v.) with the palmitoylated peptide alone were better protected against clinical manifestations of experimental allergic encephalomyelitis (EAE) than rats treated with the peptide inserted into liposomes or with the native peptide at similar doses. The administration of the acylated peptide (PAL68 86) conferred excellent protection against a challenge with the encephalitogenic peptide (p68-86) or with the intact MBP molecule, both before and after induction of active disease, and also when administered to recipients after the transfer of lymphocytes from MBP-challenged donors. Histological manifestations were also reduced to a statistically significant degree. Treatment with a palmitoylated peptide from a non-encephalitogenic region of the MBP molecule (PAL44-62) or with a palmitoylated unrelated peptide were ineffective. In vitro Ag-specific proliferative responses as well as the ability to transfer disease to syngeneic recipients, by lymph node lymphocytes from PAL68-86-treated donors, were considerably reduced. Addition of IL-2 to these cultures failed to restore either Ag-specific responsiveness or the ability of the cells to transfer disease. The results suggest that the administration of acylated peptides induces a profound state of unresponsiveness, and thus may provide an effective means for treating T cell-mediated autoimmune inflammatory disorders.

Human coronary endothelial cell activation by endotoxin is characterized by NF-kappa B activation and TNF-alpha synthesis.

Tumor necrosis factor-alpha (TNF-alpha), an early inflammatory mediator typically regulated by nuclear factor kappa B (NF-kappa B), plays a critical role in the development of cardiovascular dysfunction in sepsis. While several myocardial cell types synthesize TNF-alpha, the importance of the myocardial endothelium in sepsis-related cardiac cytokine production is unclear. To determine the role of the human coronary artery endothelial cell (HCA-EC) in the cytokine response to endotoxin we measured in vitro TNF-alpha synthesis, TNF-alpha mRNA, and the associated NF-kappa B response to LPS. To determine the magnitude of the HCA-EC response we assessed the TNF-alpha and NF-kappa B response to LPS in a human monocytic cell line (THP-1) as well. We observed an increase in supernatant TNF-alpha from LPS-stimulated HCA-EC (12 h) that was ablated by the proteosome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal). Similarly, ALLN-sensitive TNF-alpha was produced by monocytes following LPS, although at concentrations 100-fold higher than HCA-EC. TNF-alpha mRNA from HCA-EC was detected at 60 min in LPS-stimulated cells, but not in unstimulated cells or cells pretreated with ALLN. NF-kappa B p50/p65 subunits were detectable in endothelial nuclear protein 60 min following LPS. In contrast, NF-kappa B subunits from monocytes were detected at 15 min. Also, while ALLN only attenuated endothelial NF-kappa B translocation, monocyte NF-kappa B translocation was completely inhibited. These data suggest endotoxin-stimulated human coronary endothelial cells express TNF-alpha, which is regulated in part by NF-kappa B activation, in a manner and degree distinct from human monocytes.

The use of a confirmatory assay to increase the sensitivity and specificity of the Chlamydiazyme test.

The blocking assay was used to reexamine 15,662 patient specimens (2,565 male specimens, 13,097 female specimens) that were submitted for Chlamydia detection using the Chlamydiazyme EIA assay (Abbott Laboratories, North Chicago, IL). Specimens that gave optical density (OD) readings between 1.99 to cutoff and between cutoff to 3 times the negative control in the Chlamydiazyme EIA assay were analyzed further by the blocking assay during the phase 1 study. In the phase 2 study, another 1,120 specimens (473 male specimens and 647 female specimens) that had the above mentioned OD range in the Chlamydiazyme assay were tested with the blocking assay and the direct fluorescent antibody test using the cytospin method. Significant finding from phase 1 study demonstrated that 42.3% of the male specimens with optical density between cutoff to three times the negative control can be blocked by blocking assay (confirmed positive), whereas only 50% of the female specimens with OD range between cutoff to .5 were blocked by the blocking assay. In the phase 2 study, similar results were obtained with the blocking assay. The direct fluorescent antibody test showed excellent correlation with the blocking assay. These data showed that both blocking or direct fluorescent antibody tests can be used for confirmation purposes to increase the sensitivity and specificity of the assay. However, for specimens with OD values below the cutoff to 3 times the negative control, it was necessary to reassess the specimens by either of the methods. This was true especially with the male specimens.

Determination of synergy by two methods with eight antimicrobial combinations against tobramycin-susceptible and tobramycin-resistant strains of Pseudomonas.

Eleven strains of tobramycin-susceptible Pseudomonas aeruginosa, 12 strains of tobramycin-resistant P. aeruginosa, and 11 strains of P. maltophilia were tested against tobramycin in combination with azlocillin, piperacillin, ceftazidime, and imipenem and against colistin in combination with azlocillin, piperacillin, ceftazidime, and imipenem by microdilution checkerboard and time-kill curve techniques. Synergistic or additive effects were demonstrated in the tobramycin-ceftazidime combination against tobramycin-resistant strains of P. aeruginosa and P. maltophilia, and with all tobramycin combinations against tobramycin-susceptible strains of P. aeruginosa using the checkerboard technique. Synergistic and additive effects were seen with the time-kill curve technique with colistin-ceftazidime, colistin-imipenem, and tobramycin-piperacillin combinations against tobramycin-susceptible P. aeruginosa and in the colistin-ceftazidime combination against tobramycin-resistant P. aeruginosa. Fractional inhibitory concentrations (FIC) and fractional bactericidal concentrations (FBC) from checkerboard studies were compared to the time-kill curve results and vice versa. The overall agreement with FIC was 39.4% and 40.9% and agreement with FBC was 40.9% and 43.9%. This limited study illustrates the lack of agreement between these techniques in demonstrating antimicrobial synergy against pseudomonads.

The comparative in vitro susceptibility of cefazolin-resistant organisms to six cephalosporins, four penicillins, and three aminoglycosides.

One thousand thirty-seven cefazolin-resistant, gram-negative clinical isolates including members of the family Enterobacteriaceae, pseudomonads, and other nonfermenters were tested against a variety of newer antimicrobial agents by microdilution. Most of the Enterobacteriaceae were resistant to the second-generation cephalosporins, but highly susceptible to the third-generation agents and the broad-spectrum penicillins, 90% of the strains being inhibited at attainable serum concentrations. Cefoperazone and the penicillins had good activity against the Pseudomonas species, but the aminoglycosides remained the most active agents against all the gram-negative bacilli tested except Pseudomonas maltophilia.

A comparison of the Genie and western blot assays in confirmatory testing for HIV-1 antibody.

The Genie HIV-1/2 kit (Sanofi Diagnostics Pasteur, Montreal, Quebec), a synthetic-peptide solid-phase enzyme immunoassay, was evaluated as a confirmatory assay for HIV-1 antibodies in comparison with Western blot (BioRad, Hercules, CA, USA) on 50 stored HIV-1 antibody-positive sera and the 137 sera yielding repeated positive results in the conventional EIA screen out of 13405 fresh patient sera from Saskatchewan in 1993. The stored HIV-1-positive sera were uniformly positive in the Genie test. Of the 137 EIA screen-positive sera, 33 were uniformly positive and 64 were uniformly negative in Genie and Western blot; 36 were Genie-negative and indeterminate by Western blot; and four were Genie indeterminate, of which one was negative and three were indeterminate by Western blot. All HIV-1 Western blot-indeterminate and Genie-interdeterminate sera were negative in radio-immunoprecipitation assay (RIPA) and Western blot for HIV-1 and HIV-2 antibodies performed by a reference laboratory. Genie gave an accurate definitive result for 97% of EIA positive sera compared with 71% for Western blot. There was excellent correlation between Genie, Western blot and RIPA results. However, the Genie assay was faster, less costly and yielded fewer indeterminate results than Western blot in confirmatory testing for HIV-1 antibodies.

Comparison of the Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay with the original standard assay and cell culture.

The new Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay was evaluated by comparison with the original standard assay and cell culture. A total of 853 paired male and female genital tract specimens was tested with both Sanofi Chlamydia Microplate EIA shortened and standard assays and the results were compared with those of cell culture. For confirmation, a blocking assay run in the shortened format was used. Discrepancies between the three methods were resolved by a direct fluorescent antibody (DFA) test on the EIA samples or the culture retentate, or both. After resolution of discrepant results, the standard assay had a sensitivity, specificity, positive predictive value and negative predictive value of 98.5%, 100%, 100% and 99.9%, respectively. The shortened assay results were 100%, 100%, 100% and 100%, respectively. The shortened assay takes approximately 1.5 h less time than the standard assay and this study demonstrated that they have equivalent sensitivity and specificity. The improvement in turnaround time enables results to be reported on the same day.

The effect of antibiotics on the cell morphology of Legionella pneumophila.

Legionella pneumophila, in Buffered Yeast Extract broth, was treated for 5 h at 37 degrees C with rosaramicin, erythromycin, cefotaxime, dibekacin, penicillin, methicillin, cefoxitin, cephalothin, ticarcillin, carbenicillin or polymyxin B at near-MIC levels and above. Electronmicroscopy demonstrated morphological changes to the bacteria in some, but not all, of the antibiotic-treated suspensions. Penicillin, at 1000 micrograms/ml (40 X MIC) but not less, produced smooth bubble-like structures on cell surfaces; methicillin produced rough bubble-like structures at 100 micrograms/ml (MIC) but not at 1000 micrograms/ml. In each case, these structures resembled spheroplasts. Polymyxin B induced small-bleb formation on the bacterial cell surfaces at all concentrations tested (MIC-10 X MIC). The other eight antibiotics did not induce any morphological changes at any concentration tested.

Minimal inhibitory effect of male urine on detection of Chlamydia trachomatis by Roche Amplicor PCR.

A total of 1120 specimens of fresh urine from male patients was tested for Chlamydia trachomatis with the Roche Amplicor PCR Kit and an in-house PCR assay. The in-house PCR had an internal control to monitor inhibitory effects of clinical specimens on the PCR assay. All urine samples were processed within 24-48 h of collection and DNA was extracted on the same day that the assays were performed. Specimens that gave discrepant PCR results were tested by a reference laboratory with both the Roche Amplicor kit and their in-house PCR assay. Of the 1120 samples, 174 gave positive results in both assays and 942 gave negative results in both assays. Only one specimen showed an inhibitory effect on the in-house PCR assays, as indicated by failure to produce the internal control PCR product. This specimen gave negative results by both assays. There were four discrepant results in the two PCR assays. One was a false negative result obtained with the Roche Amplicor kit and the remaining three discrepant results could not be resolved because there was an insufficient quantity of specimen. This study demonstrated that the Roche Amplicor kit could effectively detect C. trachomatis in urine specimens from this population of male patients with negligible inhibition of PCR.

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.

A rapid assay for the measurement of Na+,K(+)-ATPase inhibitors.

A rapid and automated assay for inhibitors of Na+, K(+)-ATPase was developed by determining the amount of inorganic phosphorus (Pi) released by Na+,K(+)-ATPase in a centrifugal analyzer. This method avoids long incubation, strong acids and centrifugation as in the conventional manual method. The coefficients of variation of intra- and inter-assay at ouabain concentration 0.5 mumol/L were 1.0 and 1.4%, respectively. The method is quick, reproducible and easy compared with current techniques.

Comparison of the effectiveness of polymerase chain reaction and enzyme immunoassay in detecting Chlamydia trachomatis in different female genitourinary specimens.

BACKGROUND: In high-volume laboratories, enzyme immunoassay (EIA) is the most commonly used method of detecting Chlamydia trachomatis. The optimal specimen for detecting C trachomatis is a combined urethral and cervical swab. OBJECTIVE: To compare EIA with the combined urethral and cervical swab with polymerase chain reaction (PCR) on urine alone and urine mixed with cervical cells. PATIENTS AND METHODS: Phase 1 of the study included 752 sets of specimens used for comparison. In phase 2, another 212 samples of urine and urine plus cervical cells were added to the study for comparison of the 2 specimen types using PCR. RESULTS: In phase 1, 648 samples were negative and 76 were positive by all 3 methods and specimen combinations. Enzyme immunoassay was able to detect 81 positive samples (10.8%), whereas PCR on urine alone detected 97 positive samples (12.9%) and PCR on urine plus cervical cells detected 102 positive samples (13.6%), giving a sensitivity of 75%, 93.3%, and 98. 1% respectively. In phase 2, PCR on urine alone detected 119 positive samples (12.3%) and PCR on urine plus cervical cells detected 127 positive samples (13.1%), with a sensitivity of 92.2% and 98.5%, respectively. CONCLUSION: Polymerase chain reaction on urine alone or urine plus cervical cells is superior to EIA on combined cervical and urethral swabs. There is a slight advantage of adding cervical cells to the urine compared with the urine specimen alone when PCR is used as the assay for detection. The total inhibition rate in our female population is only 3.1% when PCR is used.

Performance characteristics of the Becton Dickinson ProbeTec System for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in male and female urine specimens in comparison with the Roche Cobas System.

OBJECTIVE: The Becton Dickinson BDProbeTec ET System is a new semiautomated system using strand displacement amplification technology that simultaneously amplifies and detects Chlamydia trachomatis and Neisseria gonorrhoeae DNA. The strand displacement amplification products are hybridized with a fluorescent detector probe and are captured by a chemiluminescent assay in a microwell format. An amplification control is also included to monitor assay inhibition. This study evaluated the performance of the BDProbTec ET system in detecting C trachomatis and N gonorrhoeae in male and female urine specimens, calculated its ability to process large volumes of specimens, and determined the inhibition rate. MATERIALS AND METHODS: Eight hundred twenty-five male and 399 female urine specimens were tested for both C trachomatis and N gonorrhoeae with the BDProbeTec ET system, and results were compared with those of the Roche Amplicor Cobas system. All urine specimens were processed on both assays on the same day they were received, according to the manufacturers' instructions. Discrepant results were resolved by in-house polymerase chain reaction assays. Internal or amplification controls were also used in each specimen assay to monitor inhibition. The throughput of the BDProbTec ET system was further tested with 150 urine specimens on an 8-hour shift for 2 days. RESULTS: The overall sensitivity, specificity, positive predicative value, and negative predicative value for for detection of chlamydia were 95.3%, 99.3%, 95.9%, and 99.2% for strand displacement amplification and 95.9%, 98.3%, 90.6%, and 99.3% for the Roche Amplicor system. For detection of gonorrhea, these values were 100%, 99.7%, 88.2%, and 100% and 96.7%, 98.9%, 69%, and 99.9%, respectively. The overall inhibition rates for both strand displacement amplification and Roche Amplicor were less than 3.5%. The BDProbTec ET system was able to produce 150 results each for chlamydia and gonorrhea and the internal control within the 8-hour shift. CONCLUSIONS: The performance characteristics of the BDProbeTec ET assay are similar to those of the Roche Amplicor polymerase chain reaction for detection of chlamydia and gonorrhea in male and female urine specimens. The system was able to produce 300 results in an 8-hour shift.