Cytosolic free calcium-ion concentration in cleaving embryonic cells of Oryzias latipes measured with calcium-selective microelectrodes.

Calcium-selective microelectrodes were used to measure the free calcium-ion concentration ([Ca2+]i) in early-cleaving embryonic cells of the golden medaka, Oryzias latipes, a fresh water teleost fish. Embryos could be dechorionated as early as the four-cell stage using a three-step technique consisting of removal of some yolk to enlarge the perivitelline space, partial digestion of the chorion with pancreatin, and removal of the weakened chorion with forceps. Dechorionated embryos underwent cleavage at a normal rate. Intracellular cytosolic [Ca2+]i was monitored by impaling blastomeres first with a microelectrode filled with 5 M potassium acetate to measure membrane potential, and a few minutes later with a calcium-selective microelectrode. During nine rounds of cytokinesis from a total of six different embryos, cytosolic [Ca2+]i remained constant (with apparently random fluctuations of less than +/- 0.1 microM). During two successive cleavages in one embryo, however, [Ca2+]i rose transiently fourfold above the original resting level to 1.32 and 1.20 microM in synchrony with each period of cytokinesis and returned after each rise to submicromolar levels. Because a calcium-selective microelectrode can detect [Ca2+]i changes only in the immediate vicinity of its 2-microns tip, we interpreted these data to suggest that, although [Ca2+]i in most areas of the cytosol remains between 0.01 and 0.40 microM (mean of 0.14 microM), there may be small regions of the cell in which [Ca2+]i undergoes a substantial increase at the time of cleavage. Evidence also is presented to suggest that the membrane potential in these blastomeres undergoes a slow net hyperpolarization during early cleavage stages.

Association of creatine phosphokinase with the cytoskeleton of cultured mammalian cells.

Using an antibody specific for creatine phosphokinase (CPK), we have discovered an association between that enzyme and the cytoskeleton. Immunofluorescence observations show that CPK is associated with intermediate filaments in PTK cells and BALB/3T3 cells. The CPK distribution also follows intermediate filaments when cells are treated with colchicine.

Isolation of human anti-idiotypic antibodies by phage display for clinical immune response assays.

The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody. We generated anti-ids to two human antibodies by antibody phage display using the MorphoSys HuCAL GOLD Fab library. To selectively target the CDR regions, serum and a framework-matched mAb were included as competitors during the phage selection process. Panels of CDR-specific Fabs, with low to sub-nM affinities, were isolated against both targets. The CDR specificity of these Fabs was shown by their lack of binding to a framework-matched control mAb and by competition of this binding with the soluble antigens of the respective therapeutic mAb targets. The candidate anti-id Fabs were able to detect both immobilized and soluble target Ab without being affected by serum, a requirement for both PK assay and the IR bridging assay format. Combinations of the Fabs for PK detection assays were identified by pairwise binding studies, although the pair for one target mAb lacks the desired sensitivity for PK assays. To evaluate their potential as anti-drug antibodies (ADAs), the best Fabs for one of the targets were converted and produced as the required bivalent human mAbs. In comparison to rodent mAbs and primate polyclonal serum, the phage display derived human mAbs were equally effective as reference standards. Our results demonstrate that competition-based phage selection can be an effective method for the isolation of anti-idiotypic antibodies for PK and IR assay development, and in this latter case, overcome limitations of current methods using rodent derived anti-ids.

Membrane Protein Insertion into and Compatibility with Biomimetic Membranes.

Membrane protein and membrane protein-mimic functionalized materials are rapidly gaining interest across a wide range of applications, including drug screening, DNA sequencing, drug delivery, sensors, water desalination, and bioelectronics. In these applications, material performance is highly dependent on activity-per-protein and protein packing density in bilayer and bilayer-like structures collectively known as biomimetic membranes. However, a clear understanding of, and accurate tools to study these properties of biomimetic membranes does not exist. This paper presents methods to evaluate membrane protein compatibility with biomimetic membrane materials. The methods utilized provide average single protein activity, and for the first time, provide experimentally quantifiable measures of the chemical and physical compatibility between proteins (and their mimics) and membrane materials. Water transport proteins, rhodopsins, and artificial water channels are reconstituted into the full range of current biomimetic membrane matrices to evaluate the proposed platform. Compatibility measurement results show that both biological and artificial water channels tested largely preserve their single protein water transport rates in biomimetic membranes, while their reconstitution density is variable, leading to different overall membrane permeabilities. It is also shown that membrane protein insertion efficiency inversely correlates with both chemical and physical hydrophobicity mismatch between membrane protein and the membrane matrix.

Abciximab readministration: results of the ReoPro Readministration Registry.

BACKGROUND: Platelet glycoprotein IIb/IIIa blockade with abciximab (ReoPro) improves the clinical outcomes of percutaneous coronary intervention. This registry was conducted to characterize the effects of repeated administration of abciximab during intervention. METHODS AND RESULTS: We recruited 500 consecutive patients at 22 centers in the United States who were receiving abciximab for at least a second time during percutaneous coronary intervention. Safety was measured as the incidence of hypersensitivity reactions, major bleeding, and thrombocytopenia. Efficacy was assessed as event-free clinical success. Human antichimeric antibody (HACA) responses were also characterized. There were no cases of hypersensitivity (95% upper confidence bound, 0.3%), major bleeding, or death. Clinical success was 94.4%. Thrombocytopenia occurred in 23 patients (4.6%; 95% CI, 2.8% to 6.4%), including 12 (2.4%; 95% CI, 1.1% to 3.7%) who developed profound thrombocytopenia (<20x10(9) cells/L). In 2 patients (0.4%), profound thrombocytopenia did not develop until after hospital discharge; in 4 (0.8%), profound thrombocytopenia recurred despite platelet transfusion. Before a first readministration, a positive HACA titer was present in 22 of 454 patients (4.8%); after a first readministration, an additional 82 of 432 (19.0%) became HACA-positive. HACA did not neutralize the in vitro inhibition of platelet aggregation by abciximab or correlate with clinical events. CONCLUSIONS: The results, including overall rates of thrombocytopenia, were consistent with randomized clinical trials of first abciximab treatment. However, there was a shift from mild to profound thrombocytopenia, and cases of delayed presentation and of recurrent thrombocytopenia were seen. These findings suggest that indications and guidelines for first-time use apply to retreatment, particularly the systematic monitoring for thrombocytopenia.

Mechanisms of polar lobe formation in fertilized eggs of molluscs.

Polar lobe formation in the marine mudsnail, Ilyanassa obsoleta, involves formation of a microtubule-, microfilament-dependent furrow that constricts at two rates, then stops and relaxes. Some artificial seawater mixtures allow relatively normal development, facilitate insertion of microelectrode tips, and prevent artifactual bleb formation during such punctures. Membrane events may affect formation of polar lobe constrictions: (1) Brief treatment with digitonin prevents constrictions, but not cytokinesis per se, and the suppression of constrictions is permanent. Tomatine (but not tomatidine) and filipin act similarly, although filipin often also stops cytokinesis as well. (2) Responses to digitonin, tomatine, and filipin occur with little change in membrane potential. (3) Adhesion to substrata in response to brief treatment at low pH prevents both constrictions and cytokinesis. (4) Adhesion to substrata via polylysine allows both constrictions and cytokinesis, but embryos are smaller in volume and develop abnormally. Formation of lobe constrictions may be sensitive to perturbations of the plasma membrane.

Testicular microlithiasis with sterility.

A case report of male infertility associated with testicular microlithiasis is presented. This rare condition has not previously been associated with infertility. A variable maturation arrest was observed in the spermatogenic epithelium. The question of whether the microliths are the cause of the infertility or whether both conditions are the result of some unknown agent must await further elucidation.

Prediction of ovulation.

The purpose of this investigation was to evaluate all available ovulatory diagnostics with respect to sensitivity, specificity, diagnostic specificity (predictive value of a positive test, PVP) and diagnostic sensitivity (predictive value of a negative test, PVN). Twenty-one ovulatory women with more than 3 years of infertility problems were included in the study. PVP and PVN were highest for detection of urinary luteinizing hormone (LH) peak at ovulation (PVP = 90%, PVN = 95%) and for serum-estradiol peak 1 day before ovulation (PVP = 83%, PVN = 97%). The predictive values were lower for all other tests. The PVP (54%) and PVN (90%) were rather low for detection of ovulation with vaginal electric impedance. However, all ovulations were predicted when urinary LH peak and vaginal impedance were combined. Two women were stimulated with human chorionic gonadotropin to investigate a possible connection between the LH peak and the preovulatory vaginal electric impedance. No close connection between them could be demonstrated. Basal body temperature should not be used for the prediction of ovulation (PVP = 25%). We suggest that ovulation should primarily be predicted from the identification of the urinary LH peak and that other methods be supplementary.

The microscopical pathology of primary parathyroid hyperplasia.

The microscopical pathology of the two types of primary parathyroid hyperplasia is reviewed. The importance of the differential diagnosis between chief cell hyperplasia and adenoma is stressed. The problems of the surgical pathologist in masking this differential diagnosis of a biopsy, especially on a frozen section sample, are discussed.

Consequences of immunogenicity to the therapeutic monoclonal antibodies ReoPro and Remicade.

The clinical consequences of immune antibodies generated to abciximab (ReoPro) and infliximab (Remicade) are described. Abciximab, a chimaeric Fab fragment that binds to the beta3 integrin of the GPIIb/IIIa and alphavbeta3 receptors on human platelets, is approved in the US and Europe for use in percutaneous coronary intervention (PCI) to prevent cardiac ischaemic complications. The effects of induced antibodies upon the safety and efficacy of repeat administration of abciximab have been evaluated in the ReoPro Re-administration Registry Study, in which 5.7% of patients were HACA positive before re-treatment. An interim evaluation of 1000 patients has indicated that re-administration of abciximab can be accomplished in the setting of PCI with an acceptable safety and efficacy profile. Infliximab is a chimaeric IgG1 antibody specific for human TNFalpha, and is approved in the US and Europe for the acute treatment of the signs and symptoms of Crohn's disease and for the chronic treatment of rheumatoid arthritis (RA). The incidence of antibodies to infliximab is reported to be approximately 10%; however, an inverse dose-immunogenicity relationship was observed, indicating that higher doses of infliximab (> or = 3 to 10 mg/kg) could reduce the incidence of immune antibodies. The induction of immune antibodies could also be reduced by concomitant administration of low-dose methotrexate and other immunosuppressant agents. Although antibodies to infliximab appeared to be associated with lower serum infliximab concentrations and a slightly higher incidence of infusion reactions, these immune antibodies were generally not associated with a reduction in clinical efficacy. In addition, the antibodies induced to infliximab are specific for infliximab, and do not cross-react with other currently available therapeutic antibodies.

[Long-term follow-up of patients after a single-freezing treatment of ectocervical intraepithelial neoplasms]. / Langtidsopfølgning af enkeltfrysebehandling af ektocervikale intraepiteliale neoplasier.

Fifty-five women aged 21-51 years were treated in 1977 and 1978 with cryocoagulation (single-freezing technique) for cervical intraepithelial neoplasia (CIN I-III). 92% of these patients were followed up 7-8 years after this treatment. The primary success rate i.e. the proportion between the number of women in whom dysplastic changes were not re-encountered within the first 15 months after treatment and the total number of women treated was 94.5%. After the period of observation of 7-8 years, the success rate was 100% for CIN I + II and 79.5% for CIN III.

Statin therapy is associated with reduced neuropathologic changes of Alzheimer disease.

BACKGROUND: Treatment with 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitors ("statins") has been associated in some epidemiologic studies with reduced risk of Alzheimer disease (AD). However, direct evidence of statin effects on neuropathologic markers of AD is lacking. We investigated whether antecedent statin exposure is associated with neuritic plaque (NP) or neurofibrillary tangle (NFT) burden in a population-based sample of human subjects. METHODS: Brain autopsies were performed on 110 subjects, ages 65 to 79 years, who were cognitively normal at enrollment into the Adult Changes in Thought Study. Neuropathologic findings were compared between statin users with > or =3 prescriptions of > or =15 pills of simvastatin, pravastatin, lovastatin, or atorvastatin vs nonusers, based on pharmacy dispensing records. RESULTS: After controlling for age at death, gender, cognitive function at study entry, brain weight, and presence of cerebral microvascular lesions, the odds ratio (OR) for each unit increase in Braak NFT stage in statin users vs nonusers was 0.44 (95% CI: 0.20 to 0.95). The OR for each unit increase in Consortium to Establish a Registry for Alzheimer's Disease (CERAD) staging of NPs did not deviate significantly from unity (OR 0.69; 95% CI: 0.32 to 1.52). However, the risk for typical AD pathology (Braak stage > or = IV and CERAD rating > or = moderate) was reduced in statin users (OR 0.20; 95% CI: 0.05 to 0.86). CONCLUSIONS: These findings demonstrate an association between antecedent statin use and neurofibrillary tangle burden at autopsy. Additional study is needed to examine whether statin use may be causally related to decreased development of Alzheimer disease-related neuropathologic changes.

Dendritic degeneration in neostriatal medium spiny neurons in Parkinson disease.

Dysfunction of neostriatal medium spiny neurons (MSNs) is hypothesized to underlie late-stage motor complications of Parkinson disease (PD). The authors demonstrate shortened dendrite length of MSNs that was similar in four regions of neostriatum in late-stage PD. In contrast, MSN dendrite spine degeneration was unevenly distributed with the greatest loss in caudal putamen. The authors propose that these structural changes in MSN may contribute to late-stage motor complications of PD.

A minor complication of cryosurgery--occlusion of the cervical canal.

In a series of 145 patients who underwent cryosurgery of the cervix uteri, six cases (4 per cent) were found with occlusion of the cervical canal. The patients complained of constant pains resembling labor. Inspection of the cervix uteri showed necrosis covering the orificium externum uteri. On removal of the necrotic area there was a discharge of watery fluid. The patients were then free of pain.

Cryosurgery for dysplasia of the uterine ectocervix. A randomized study of the efficacy of the single- and double-freeze techniques.

This prospective, randomized study of 142 women with mild or moderate dysplasia of the uterine ectocervix shows that the double-freeze technique is significantly more effective than the single-freeze technique, the recurrence rates being, respectively, 6.2% and 16.3%. The highest recurrence rate was found in the group treated with a single freeze and having the most extensive portio involvement. All patients were examined colposcopically before cryosurgery in order to exclude patients with lesions extending into the cervical canal and in order to evaluate the size of the lesion. Recurrent lesions were usually diagnosed within the first year (75%) and in only one patient more than 2 years after treatment. None of the patients developed invasive cancer. The mean follow-up period was 27 months (24-42 months). We consider cryosurgery to be an acceptable treatment for selected patients. Our criteria for entry were mild or moderate dysplasia of the uterine ectocervix, and colposcopic visualization of the entire transformation zone to ensure that the lesion does not extend into the cervical canal. Our experience from the present study is that the following criteria should be added: the portio involvement must not be too extensive and the patient must agree to follow-up examination. When these conditions are fulfilled, future treatment in our department will consist of a double freeze extending 3 mm beyond the lesion, with a thaw interval of 4 min.