A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest.

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at 99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was

The green tea catechins, (-)-Epigallocatechin-3-gallate (EGCG) and (-)-Epicatechin-3-gallate (ECG), inhibit HGF/Met signaling in immortalized and tumorigenic breast epithelial cells.

The hepatocyte growth factor (HGF) receptor, Met, is a strong prognostic indicator of breast cancer patient outcome and survival, suggesting that therapies targeting Met may have beneficial outcomes in the clinic. (-)-Epigallocatechin-3-gallate (EGCG), a catechin found in green tea, has been recognized as a potential therapeutic agent. We assessed the ability of EGCG to inhibit HGF signaling in the immortalized, nontumorigenic breast cell line, MCF10A, and the invasive breast carcinoma cell line, MDA-MB-231. HGF treatment in both cell lines induced rapid, sustained activation of Met, ERK and AKT. Pretreatment of cells with concentrations of EGCG as low as 0.3 microM inhibited HGF-induced Met phosphorylation and downstream activation of AKT and ERK. Treatment with 5.0 microM EGCG blocked the ability of HGF to induce cell motility and invasion. We assessed the ability of alternative green tea catechins to inhibit HGF-induced signaling and motility. (-)-Epicatechin-3-gallate (ECG) functioned similar to EGCG by completely blocking HGF-induced signaling as low as 0.6 microM and motility at 5 microM in MCF10A cells; whereas, (-)-epicatechin (EC) was unable to inhibit HGF-induced events at any concentration tested. (-)-Epigallocatechin (EGC), however, completely repressed HGF-induced AKT and ERK phosphorylation at concentrations of 10 and 20 microM, but was incapable of blocking Met activation. Despite these observations, EGC did inhibit HGF-induced motility in MCF10A cells at 10 microM. These observations suggest that the R1 galloyl and the R2 hydroxyl groups are important in mediating the green tea catechins' inhibitory effect towards HGF/Met signaling. These combined in vitro studies reveal the possible benefits of green tea polyphenols as cancer therapeutic agents to inhibit Met signaling and potentially block invasive cancer growth.

ELISA for thyroglobulin in serum: recovery studies to evaluate autoantibody interference and reliability of thyroglobulin values.

An ELISA for measuring thyroglobulin (Tg) in serum was developed with polyclonal antibodies to Tg on the solid phase and two monoclonal antibodies to Tg as the second antibodies. The assay has a detection limit of 1 microgram/L, a within-run imprecision (CV) of <7%, and a between-run CV of < 10%. Parallelism of the assay was shown in dilution studies, in which the percent observed/expected values for n = 5 autoantibody-containing samples gave a mean of 99% (SD 13.1 %); for n = 5 samples with undetectable autoantibody concentrations, the mean was 103% (SD 11.8%). The correlation of the ELISA with an RIA for Tg in 46 normal samples was ELISA = 1.11(RIA) + 0.52, S y/x = 2.23, SD intercept = 0.54, SD slope = 0.03, range = 0 to 53 micrograms/L, r = 0.980. Comparison of the ELISA with a reference laboratory RIA for 29 clinical samples gave a correlation of: ELISA = 1.53(RIA) - 0.48, S y/x = 9.00, SD intercept = 2.19, SD slope = 0.10, range = 0 to 98 micrograms/L, r = 0.950. To provide additional information concerning the reliability of the Tg measurement in samples containing autoantibodies to Tg, we developed a procedure for determining the percent recovery. A percent recovery greater than or equal to 80% indicates minimal interference by autoantibodies in this assay. The assay is straightforward to perform, results can be posted within 8 h, and the routinely good recovery of Tg in the presence of Tg autoantibodies indicates minimal autoantibody interference in this assay.

99mTc-bicisate (neurolite) SPECT brain imaging and cognitive impairment in dementia of the Alzheimer type: a blinded read of image sets from a multicenter SPECT trial.

A blinded read of images obtained with 99mTc-bicisate and single photon emission computed tomography (SPECT) was conducted to determine if a relationship exists between the severity of abnormalities on SPECT brain images and the severity of cognitive impairment in patients with dementia of the Alzheimer type (DAT) and to examine the interreader agreement for visual reading of images in a multicenter SPECT study. Images for a total of 86 subjects were available for the blinded read. The images for 28 subjects were rated as noninterpretable due to technical inadequacies. Images for 58 subjects (45 DAT patients and 13 normal volunteers) from 10 SPECT centers were selected for further analyses. The severity of abnormality was rated as mild, moderate, or severe by three readers. In DAT patients, a significant negative correlation (p < 0.05) of Mini-Mental State Examination (MMSE) score with global severity of abnormality was noted for two of the three readers. A significant correlation (p < 0.05) between MMSE score and severity of abnormality was observed for all three readers for the posterior temporoparietal region. The blinded readers rated a median of 92.3% of normal volunteers' images as normal and a median of 82.2% of DAT patients' images as abnormal. For the regional severity of abnormality, the median percentage interrater agreement across all regions ranged from 95 to 100% in normal volunteers and from 81 to 98% in DAT patients. These results suggest that SPECT brain imaging with 99mTc-bicisate provides functional information about the severity of cognitive impairment in DAT patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Is sudden reperfusion more arrhythmogenic during thrombolysis for myocardial infarction?]. / Une reperfusion plus soudaine est-elle plus arythmogène au cours de la thrombolyse pour infarctus du myocarde?

The object of this study was to analyse ventricular arrhythmias occurring during intravenous thrombolysis for acute myocardial infarction with respect to ST segment changes on 24 hour Holter ECG monitoring initiated at the same time as thrombolytic therapy and on repeated 12 lead electrocardiogrammes. Forty-one patients in whom the infarct-related artery was patent at coronary angiography carried out 30.5 +/- 3.1 hours (< or = 24 hours in 59% of cases) after the onset of chest pain were included. The time to normalisation of the ST segment was defined as the interval between maximum ST elevation to a steady state and helped identify rapid (< or = 60 minutes, n = 13) from intermediate (60-180 minutes, n = 15) and slow (> 180 minutes, n = 13) reperfusion. The incidence of ventricular arrhythmias was the same in all three groups, except for prolonged ventricular tachycardias (> 15 complexes): 69%, 13% and 15% respectively (p = 0.002). The number of arrhythmias was greater when the ST segment changes were rapid than when they were intermediate or slow. This was true for ventricular extrasystoles (p < 0.05), accelerated idioventricular rhythms (p < 0.05), early (< or = 6 hours from onset of thrombolysis) accelerated idioventricular rhythms (p < 0.01) and ventricular tachycardias (p < 0.05). Therefore, the number of ventricular arrhythmias seems to be related to the speed of ST segment change, suggesting that more sudden reperfusion is more arrhythmogenic.

Is arrhythmogenicity related to the speed of reperfusion during thrombolysis for myocardial infarction?

The objective of this study was to relate the number of ventricular arrhythmias (VA) to the normalization time of the ST segment during thrombolysis for acute myocardial infarction. The 24 h Holter recordings, begun on start of intravenous thrombolytic therapy, and the 12-lead electrocardiograms of 41 patients with a patent infarct-related artery according to coronary angiography were analysed. The mean time from onset of chest pain to angiography was 30.5 +/- 3.1 h, < or = 24 h in 59%. The normalization time of the ST segment, assessed by the time of decrease of ST segment elevation from start of Holter recording to normal or steady state was < or = 60 min in 13 patients (group 1), 60 to 180 min in 15 patients (group 2) and > 180 min in 13 patients (group 3). The incidence of VA was similar in all groups, except for ventricular tachycardias (VT) > 15 beats (group 1:69%, group 2:13%, group 3:15%, P = 0.002). The frequency of accelerated idioventricular rhythms (AIVR), early AIVR (< or = 6 h) and of VT was significantly higher in group 1 than in group 3 with a 8-, 30- and 6-fold increase, respectively (back transformed mean). We conclude that the number of VAs is related to the normalization time of the ST segment during reperfusion. This may suggest that faster reflow is more arrhythmogenic.

Intracranial hemorrhage in term or near-term newborns with persistent pulmonary hypertension.

To substantiate the clinical impression of an increased incidence of intracranial hemorrhage (ICH) in term and near-term infants with persistent pulmonary hypertension (PPH), a retrospective chart review of 35 affected patients was performed. ICH was diagnosed in 40 percent of the patients. Multiple regions of the brain were affected; in many patients, at more than one location. In addition, 43 percent of ICH victims had hemorrhages of the type associated with periventricular bleeding in preterm newborns. Of multiple obstetric and neonatal factors analyzed, many of which are identified risk factors for periventricular hemorrhage, only thrombocytopenia (p = 0.02) was significantly associated with ICH. We conclude that the risk of ICH in newborns with PPH is significant and warrants consideration by clinicians caring for this population. Risk factors (except thrombocytopenia) previously implicated in other types of neonatal ICH, particularly periventricular hemorrhage, do not significantly correlate with ICH in infants with PPH.

Influence of menstrual cycle variations on natural killer cytotoxicity and antibody-dependent cellular cytotoxicity to cells infected with herpes simplex virus.

Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) represent the ability of human leukocytes to destroy target cells. Those systems have been shown to influence herpes simplex virus (HSV) infections. Prostaglandins are known to inhibit these nonspecific immunologic defenses. Since prostaglandin production varies with the menstrual cycle, this study was undertaken to test for variations in cytotoxic activity against HSV-infected cells during the proliferative, secretory and menstrual phases in 13 normal volunteers. NKC and ADCC activity was not statistically different between the three menstrual phases when testing was done for both mononuclear and polymorphonuclear cells from donor sera. This study suggested that NKC and ADCC cannot be used to explain anecdotal experiences suggesting menstrual variations as a triggering mechanism for HSV recurrences.

Needle localization of nonpalpable breast masses.

A series of 146 women underwent 150 preoperative localizations of mammographically suspicious but nonpalpable breast lesions. The lesions were localized using the hook-wire method of Frank in 133 of these patients. Carcinoma was discovered in 24 (16%) of the women; 18 (75%) of these women had invasive and six women (25%) had noninvasive carcinomas. Sixty-seven patients demonstrated calcification, and of these, 16 patients (24%) turned out to have malignancies. Eighty percent of the cancers were less than 1 cm in diameter, and 38% met the criteria of minimal carcinoma as described by Gallagher and Martin in 1969. Fourteen percent of the patients with carcinoma had lymph node metastases. We conclude that this is a safe, rapid, and accurate method for localizing small, potentially highly curable breast cancers with minimal sacrifice of breast tissue.

Prophylactic diazepam in prevention of contrast media-associated seizures in glioma patients undergoing cerebral computed tomography.

Five milligrams of intravenous diazepam given prior to contrast media injection statistically significantly reduced the incidence of contrast media-associated seizures from 16% to 2% in a prospective, randomized series of glioma patients. Factors related to increased risk of contrast media-associated seizures are (1) prior seizure history due to glioma and/or prior contrast media and (2) prior or concurrent brain antineoplastic therapy. Factors not related to an increased risk of contrast media-associated seizures are (1) contrast media dosage, (2) type or grade of glioma, and (3) computerized tomographic appearance of the glioma. Theories regarding the etiology of contrast media-associated seizures are discussed.

Ontogeny of protection of neonatal mice from lethal herpes simplex virus infection by human leukocytes, antiviral antibody, and recombinant alpha-interferon.

We have studied the peripheral blood leukocytes from human infants in an assay involving the protection of neonatal mice from herpes simplex virus (HSV) infection by human antibody, interferon, and leukocytes. Recombinant DNA alpha-interferon (IFLrA), antibody, and Ficoll-Hypaque-purified mononuclear cells (MC) from human adults administered intraperitoneally protected neonatal mice from a lethal HSV challenge 1 day later (73.6% survival). MC obtained from human infants less than 130 days old in combination with IFLrA and antibody afforded no protection (15.2% survival; p less than 0.0005 compared to survival with adults' MC). MC from infants over 130 days protected the neonatal mice [60% survival; not significantly different from survival using adult cells, but significantly (p less than 0.0005) different than survival using MC from younger infants]. The ontogeny of MC protection parallels the clinical development of resistance of infants to HSV infection.

Influence of naproxen therapy on natural killer cytotoxicity and antibody-dependent cellular cytotoxicity against cells infected with herpes simplex virus.

Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) represent one of the body's primary lines of defense against viral infections, including herpes simplex (HSV). This immune defense system is negatively influenced by prostaglandins. A project was undertaken to evaluate the influence of an antiprostaglandin agent in vivo on these cytotoxic effects against cells infected with HSV. Thirteen subjects without previous histories of clinical herpes simplex infection were studied during menses with and without naproxen therapy. A statistically significant augmentation (p = 0.05) of natural killer-cell function was identified in mononuclear cells during therapy. Subjects with baseline cytotoxicity of less than 45% demonstrated consistent elevations during naproxen therapy. No other significant differences could be found for mononuclear or polymorphonuclear cells with regard to NKC and ADCC. There appears to be a subset of patients who may benefit from immunologic augmentation with antiprostaglandin agents when experiencing herpes simplex virus infection.

The significance of carotid stenosis or ulceration.

Data from the medical treatment group of the Aspirin in TIA study were reviewed, and prospective analysis of patients with asymptomatic bruits was performed to see whether carotid stenosis (0 to 49% or 50 to 99%) or ulceration produced an increased risk of ipsilateral TIA or infarct. In symptomatic arteries, greater than 50% stenosis without ulceration implied a higher risk of subsequent symptoms. Ulceration was associated with an increased risk only in nonstenotic vessels. Lesion anatomy was not related to outcome in asymptomatic arteries, and the incidence of cerebral infarct was low. Factors other than anatomy must play a large role in determining subsequent risk.

The Bacon Chow study: maternal supplementation and infant growth.

The effect of maternal supplementation during both gestation and lactation on infant growth from birth to 12 mth was investigated in the double-blind, randomly allocated control trial conducted in Taiwan by the late Bacon F. Chow. The supplement was a milk-based formula providing 800 kcal and 40 g protein daily. The placebo provided less than 40 kcal per diem but resembled the supplement in appearance. Supplementation was limited to mothers and began after birth of one infant and continued without interruption until weaning of a second infant. Supplement effects were tested by comparing both supplement and placebo groups and first- and second-infant groups with respect to weight, length and head circumference. Both comparisons of growth curves and analyses of variance were carried out. Both strategies agreed in failing to detect differences in growth between supplement and placebo groups. However, differences were found between first and second infants in the supplement group.

Concentration of intrathecal 3H-labeled metrizamide in normal dog brain: age-related differences.

Responses to intrathecal metrizamide in dogs were found to be age-related: Adult dogs had seizures; 7-week-old dogs appeared stuporous; 6-day-old dogs were clinically unaffected. The brain metrizamide concentrations 4, 6, and 20 hr after intrathecal injection correlated directly with the occurrence and severity of neurotoxic symptoms. Age-related differences in brain metrizamide concentration may be explained by two factors. The first is the failure of current clinical guidelines to adjust the recommended dosage of metrizamide to reflect differences among age groups in brain weight rather than body weight. This error resulted in lower doses/gram of brain weight being given to the puppies. However, the large differences in brain metrizamide concentrations among the three groups of dogs could not be explained solely by differences in the dose. A second factor, physiologic age-related differences in brain penetration, is believed to be operative. The precise nature of these differences is unclear.

Diazepam prophylaxis of contrast media-induced seizures during computed tomography of patients with brain metastases.

The effect of 5 mg of intravenous diazepam (Valium) on contrast media-associated seizure incidence was studied in a randomized controlled trial involving 284 patients with known or suspected brain metastases undergoing cerebral computed tomography. Of these patients, 188 were found to have brain metastases, and it is estimated that for this subgroup prophylactic diazepam reduces the risk of contrast-associated seizure by a factor of 0.26. Seizures occurred in three of 96 patients with metastases on diazepam and in 14 of 92 patients with metastases but without diazepam. Factors related to increased risk of contrast media-associated seizures are: (1) prior seizure history due to brain metastases and/or prior contrast, (2) progressive cerebral metastases, and (3) prior or concurrent brain antineoplastic therapy. Factors not related to an increased risk of these seizures are: (1) contrast media dosage, chemical composition, or osmolarity, (2) computed tomographic appearance of metastases, and (3) type of primary malignancy. Concomitant therapeutic levels of diphenylhydantoin (Dilantin) do not protect completely against contrast media-associated seizures. Pathophysiology of contrast media-associated seizures is discussed in view of the risk factors determined by this study.

Maximizing the harvest of contrast enhancing lesions in multiple sclerosis.

Transient contrast enhancing lesions can be seen with CT in patients with MS, particularly during acute exacerbations. Conventional 40 gm iodine enhancement technique followed by immediate postinfusion scanning was compared with high dose (80 gm iodine) enhancement technique followed by a 1-hour delayed CT scan; delayed high dose technique (DHD) in nine exacerbations. New lesions, totaling 36 in all, were observed in each case with the DHD technique. With time, the borders expanded in 98% of lesions, thus confirming in vivo that a defective blood-brain barrier (BBB) underlies this phenomenon.