A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa) but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia, because both PCa and benign prostatic hyperplasia release PSA into the serum. We have identified previously a truncated form of precursor PSA (pPSA) in prostate tumor extracts consisting of PSA with a serine-arginine pro leader peptide ([-2]pPSA) instead of the normally expressed 7 amino acid pro leader peptide. In the current study we developed monoclonal antibodies to detect [-2]pPSA and other isoforms of pPSA for Western blot analysis. PSA was immunoaffinity purified from 100 to 200 ml of serum from each of five men with biopsy-proven cancer and three biopsy-negative men, all with total PSA levels in the diagnostically relevant range near 10 ng/ml. The truncated [-2]pPSA was estimated to range from 25 to 95% of the free PSA in the five PCa samples but only 6-19% of the free PSA in the biopsy-negative men. Immunohistochemical studies showed positive staining for [-2]pPSA in PCa epithelium and that [-2]pPSA was enriched in cancer cell secretions. In vitro activation studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a stable but inactive isoform of PSA. Truncated [-2]pPSA may represent an important new diagnostic marker for the early detection of PCa.

Occipital brain perfusion deficits in children with major depressive disorder.

UNLABELLED: Occipital lobe perfusion defects have been identified on regional cerebral blood flow (rCBF) SPECT scans of adolescent children and young adults with major depressive disorder (MDD). We reinvestigated a series of rCBF SPECT scans obtained several years ago on drug-naive children with a clinical diagnosis of MDD and on healthy children. METHODS: To test whether visually apparent abnormalities in rCBF constitute statistically significant differences between patients, given the relatively small sample sizes, we applied the technique of statistical parametric mapping (SPM). RESULTS: Two groups of patients were identified: 8 with significant posterior flow deficits in the occipital cortex (Brodmann's areas 18 and 19), usually symmetric, and best visualized on paramedian sagittal sections, and 13 without obvious occipital perfusion deficits but with anterior rCBF deficits in a pattern often described in the literature, attaining statistical significance in the right frontal region. Other localizations in the left frontal and bilateral prefrontal regions did not attain significance, but each localization contained statistically significant maxima (z scores). The scan findings of all 18 healthy children were normal. CONCLUSION: With the aid of SPM, 2 groups of children with significantly different rCBF behavior were identified. The reason for this difference is not known but should be investigated to determine its possible significance to patients with MDD.

Large-scale propagation of recombinant adherent cells that secrete a stable form of human glandular kallikrein, hK2.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease that is expressed predominantly in the prostate epithelium and has 78% aa identity with prostate-specific antigen (PSA). hK2 has been recognized as a potential prostate cancer marker and has been demonstrated to be highly expressed in prostate cancer compared to benign prostatic tissue. Purification and characterization of hK2 have been impeded due to its lower expression in bodily fluids and tissues compared to PSA and its ability to autodegrade. Therefore, to study biochemical and biological characteristics of hK2, a stable and enzymatically inactive mutant form of hK2, hK2(A217V), was expressed in a hamster cell line, AV12-664 (AV12-hK2(A217V)). AV12-hK2(A217V) cells secreted prohK2(A217V) (phK2(A217V)) in the spent medium at approximately 2.5 microgram/ml. Since AV12-hK2(A217V) are adherent cells, it was necessary to develop an efficient system to propagate large numbers of cells to obtain significant quantities of phK2(A217V). In this paper, we compared ceramic core bioreactor and microcarrier beads as alternatives to static culture to propagate adherent cells. Considering production levels, ease of operation, cost effectiveness, and labor, microcarrier beads were found to be a better alternative. Our findings led to the development of a general protocol for large-scale propagation of adherent cells on microcarrier beads eliminating the need for propagating AV12-hK2(A217V) in culture flasks or bioreactors. Microcarrier beads coated with AV12-hK2(A217V) cells could be propagated in 1- or 3-liter spinner flasks and were passed from one spinner to the next in a manner analogous to static culture or could be frozen and later used as inoculum for subsequent spinners. Using this protocol, >40 liters of spent medium was harvested within 30 days, which in turn was used to purify phK2(A217V). phK2(A217V) purified from spent medium of cells grown either on microcarrier beads or in culture flasks were biochemically similar as indicated by HIC-HPLC profile followed by sequencing of relevant peaks.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES: Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS: We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS: The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS: Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

Comparison of technetium-99m-HMPAO and xenon-133 measurements of regional cerebral blood flow by SPECT.

UNLABELLED: This study compares 99mTc-HMPAO count ratios and derived regional cerebral blood flow (rCBF) to 133Xe rCBF ratios and true rCBF (ml/min/100 g), respectively. METHODS: Technetium-99m-HMPAO distribution was evaluated in 14 patients and 5 normal control subjects. Immediately after 133Xe SPECT, subjects received 22 +/- 4mCi 99mTc-HMPAO, and images were acquired 15 min after injection. rCBF (ml/min/100 g, 133Xe) or regional count density (99mTc-HMPAO) were extracted from 24 ROI located 6 cm above the cantho-meatal line. These data were also normalized to global cerebral blood flow (gCBF) for 133Xe or to global count density (gCD) for 99mTc-HMPAO. Technetium-99m-HMPAO ROI data also were expressed in units of ml/min/100 g by relating gCD to gCBF. Comparisons between 133Xe and 99mTc-HMPAO were evaluated using a Bonferroni-corrected paired t-test and by linear regression analysis. RESULTS: Profile plots demonstrated agreement in the pattern of relative distribution between rCBF ratios (133Xe) and count density ratios (99mTc-HMPAO). Regression analysis indicated a significant correlation (r = 0.78), with a modest slope (0.52) and a large intercept (0.48). A closer correlation (r = 0.92) was found for the comparison between rCBF (133Xe) and derived 99mTc-HMPAO rCBF. The slope was closer to one (0.82) and the intercept closer to zero. This relationship was also examined during high rCBF after a subset of these subjects (n = 7) was injected intravenously with 1 g acetazolamide. Again, profile plots and regression analysis demonstrated agreement in the pattern of distribution (ratios) between 133Xe and 99mTc-HMPAO (r = 0.66). However, the slope was reduced and the intercept increased relative to resting data. Absolute flow correlations showed some improvement relative to the ratio data (r = 0.77). CONCLUSION: The distribution of 99mTc-HMPAO is linearly related to rCBF measured by 133Xe SPECT, although our data suggest that 99mTc-HMPAO mildly underestimates rCBF above 80 ml/min/100 g. These results are similar to our previous comparison of 99mTc-ECD and 133Xe.

Site-specific prodrug activation by antibody-beta-lactamase conjugates: preclinical investigation of the efficacy and toxicity of doxorubicin delivered by antibody directed catalysis.

Antibody directed catalysis (ADC), the catalytic conversion of prodrugs to drugs by enzymes localized at disease targets by appropriate monoclonal antibodies, has shown promise in the treatment of cancer in nude mouse xenograft models. We investigated this concept using antibody enzyme conjugates constructed from beta-lactamase and Fab's reactive with carcinoembryonic antigen, CEA, and tumor associated glycoprotein, TAG-72, to convert prodrugs that are cephalosporin sulfoxide derivatives into oncolytic drugs. Previous work focused on ADC delivery of the potent vinca alkaloid derivative desacetylvinblastine carboxhydrazide (DAVLBHYD). In the current study the ability of the system to deliver doxorubicin was tested in MCF7 breast carcinoma xenografts and OVCAR3 ovarian carcinoma xenografts, and in T380 and LS174T colon tumor xenografts for comparison with previous DAVLBHYD results. ADC enhanced the delivery of doxorubicin in the model systems investigated. Tumor growth suppression was equivalent to or greater than that observed with free doxorubicin at its maximum tolerated dose (MTD). In contrast to the DAVLBHYD results, ADC delivery of doxorubicin did not regress tumors, but did result in a substantial increase in the MTD.

Comparison of technetium-99m-ECD to Xenon-133 SPECT in normal controls and in patients with mild to moderate regional cerebral blood flow abnormalities.

Technetium-99m-1,1-ethyl cysteinate dimer (ECD) has been proposed as a "chemical microsphere" for SPECT measurement of regional cerebral blood flow (rCBF). However, its distribution has not yet been compared in humans to an established rCBF measure. Therefore, we compared the uptake and distribution of ECD with rCBF measured by 133Xe SPECT in subjects with mild to moderate flow abnormalities and in normal volunteers. Blood and urine chemistries and vital signs were unchanged from pre-ECD values up to seven days postinjection. Profile plots demonstrated pattern agreement between rCBF ratios (133Xe) and ECD count density ratios. A significant correlation of rCBF ratios to ECD count density ratios was observed (r = 0.77), with a slope of 0.64 and intercept of 0.36. To explore whether or not the relationship between rCBF and ECD was dependent on absolute flow, ECD region of interest data were expressed in units of ml/min/100 g by equating global CBF (133Xe) and ECD global count density. A closer correlation (r = 0.88) was found for these data than for the count ratio data. The slope was closer to one (m = 0.83) and the intercept was closer to zero (b = 8.2). Also, a significant correlation was observed between ECD-derived rCBF and 133Xe rCBF in the lesion area (r = 0.92) for patients with well-demarcated rCBF lesions. The slope (0.80) suggested a slight underestimation of lesion flow by ECD. Finally, ECD clearance from cortical gray matter ROIs derived from high-resolution scans from 1 to 4 hr postinjection was slow (2.4%/hr). In summary, ECD is a safe and effective marker of regional cerebral perfusion. The distribution of ECD is linearly related to rCBF measured by 133Xe SPECT, although our data suggest a mild underestimation of flow at the high end of the normal range.

Dual-isotope brain SPECT imaging with technetium-99m and iodine-123: clinical validation using xenon-133 SPECT.

Our phantom studies indicate that the energy resolution (9.7% FWHM) of a new three-headed single-photon tomograph (PRISM-3000) separates the distribution of 99mTc from 123I for 10% asymmetric or 15% or 10% centered 99mTc windows when combined with a 10% asymmetric 123I window. This technique is now applied to the simultaneous measurement of resting rCBF and changes induced by vasodilation (1 g acetazolamide) in 10 subjects with cerebrovascular disease. Resting and vasodilated 133Xe SPECT images were obtained first. Within 48 hr, 99mTc HMPAO was given at rest, acetazolamide injected, and after 20 min either [123I] IMP or [123I] HIPDM was administered. Subjects were scanned for 99mTc and 123I simultaneously using 10% asymmetric windows. Regression analyses demonstrated a linear relationship between 133Xe SPECT and dual-isotope SPECT measurements of lesion-to-cerebellum ratios in baseline (r = 0.92), vasodilated (r = 0.86) and rest-minus-vasodilated data (r = 0.85). Technetium-99m and 123I images obtained through dual-isotope imaging are by definition in perfect anatomic registration.

Dual-isotope brain SPECT imaging with technetium-99m and iodine-123: validation by phantom studies.

Phantom studies were employed to determine whether the enhanced energy resolution (9.7% FWHM) of a new high-resolution, three-headed single-photon emission computed tomograph might permit the simultaneous acquisition of 99mTc and (123)I. Various window widths (15% and 10%) and positions (centered and asymmetric to the photopeak) were used to examine cross-contamination between these two isotopes. Brain phantom experiments using a 15% centered 99mTc window in conjunction with a 10% asymmetric (123)I window (upper half of the (123)I photopeak) demonstrated that approximately 95% of observed counts were derived from the isotope of interest. Shifting the (123)I window from asymmetric to centered resulted in a significant increase in contamination of the (123)I window. Shifting the 99mTc window from centered to asymmetric did not significantly alter image quality for 99mTc. Separate experiments employing vials with varying isotope concentrations demonstrated that quantitative recovery from mixed 99mTc and (123)I sources was equivalent to that from matched single-isotope sources (r2 > or = to 0.90).

Long-term consequences of exposure to an imprinting stimulus on 'spontaneous' impulse activity in the chick brain.

Young chicks were exposed in a running wheel to an imprinting object, a rotating flashing red box (n = 36 chicks) or a rotating stuffed jungle fowl (n = 18). The net number of revolutions of the wheel toward the imprinting object provided a measure of approach activity during training. The chicks were subsequently anaesthetized and the 'spontaneous' firing rates of units were recorded simultaneously in two regions of the left forebrain hemisphere. One microelectrode penetration was made through a region, the intermediate and medial part of the hyperstriatum ventrale (IMHV) known to be critical for imprinting; simultaneously another microelectrode penetration was made through the visual Wulst. The electrodes were advanced in steps of 250 micron and the spontaneous discharge of multiple units at each site recorded. The mean firing rate from greater than or equal to 3 sites was calculated for IMHV and for the hyperstriatum accessorium of the Wulst. There was a negative correlation between mean firing rate of units in IMHV and approach activity for chicks trained on the box. The effect was: stimulus specific since it was not found in chicks trained on the jungle fowl; and regionally specific since approach activity, depending on conditions which are described, was either not correlated or positively correlated with mean firing rate for neurons in the hyperstriatum accessorium. The positive correlation was not dependent on the training stimulus but on the presence of a visually responsive lamina deep to the hyperstriatum accessorium.

Modifiability of responsiveness in a visual projection area of the chick brain: visual experience is only one of several factors involved.

The effects of visual experience on neuronal responsiveness in the hyperstriatum accessorium, a visual projection area, were investigated in 4 groups of domestic chicks, each comprising dark-reared (total = 44) and visually experienced (total = 34) birds. Visually experienced birds were placed singly in running wheels facing a flashing red light for 3 h; wheel revolutions were used as a measure of the chicks' locomotor activity. At approximately equal to 48 h after hatching each chick was anaesthetized and a microelectrode advanced in 250 micron steps through the left Wulst. After each step the responsiveness of units to diffuse retinal illumination (light flashes) was tested. Each recording site at which responses greater than or equal to 5 successive flashes could be evoked was classified as a response site. In some chicks at least one site responding briskly to greater than or equal to 15 successive flashes was found deep to the hyperstriatum accessorium. The zone of markedly increased responsiveness is referred to as the visually responsive lamina. The effect of visual experience on the response of units in the hyperstriatum accessorium varied between the groups of chicks. Visual experience did not significantly effect neuronal responsiveness in this region for chicks without a visually responsive lamina. For chicks with a lamina there was an effect of visual experience, but the effect again varied between groups. When data from inactive, visually experienced chicks were excluded, the group-to-group variation ceased to be significant. Thus visual experience alone was not adequate consistently to bring about long-term changes in the responsiveness of neurones in the hyperstriatum accessorium. Such experience was likely to increase neuronal responsiveness provided the chicks: were behaviourally active; and possessed a visually responsive lamina.

Differential effects of exposure to an imprinting stimulus on 'spontaneous' neuronal activity in two regions of the chick brain.

Young chicks were placed in running wheels and exposed for a total of 3 h to an imprinting stimulus: a rotating flashing red light. Revolutions made towards the stimulus minus revolutions made away from it were totalled and are referred to as approach counts. After exposure, chicks were anaesthetized and two vertical electrode penetrations were made, one through the visual projection area, hyperstriatum accessorium (HA), and one through the intermediate and medial part of hyperstriatum ventrale (IMHV), a region which has been implicated in the imprinting process. 'Spontaneous' impulse activity was recorded from neurones in these areas. In some of the chicks, after penetrating through HA, discharges evoked by diffuse retinal illumination were monitored and the zona hyperstriatum intercalatus/hyperstriatum dorsale (HI/HD) below HA was classified as being a visually responsive lamina (VRL) if responses were obtained to greater than or equal to 20 consecutive light flashes. In HA there was a positive correlation between spontaneous activity and approach counts in those chicks with a VRL; there was no significant correlation in those chicks without a VRL. In contrast, for IMHV the correlation between spontaneous activity and approach counts was negative and appeared to be independent of the presence or absence of a VRL.

A new method for laryngeal electromyography.

A new method of surface electromyography (EMG) is presented, for sampling the posterior crico-arytenoid, interarytenoid and crico-pharyngeal sphincter. The results obtained in normal volunteers are discussed and the indications for laryngeal electromyography (LEMG) outlined. It has the advantage of being non-invasive and safe, and can be done as an out-patient procedure with minimal discomfort to the patient, and unassociated complications. This has provided a new and fascinating field of investigation in neuromuscular disorders of the larynx. The same principles of surface EMG may also be applied to the muscles of the tongue, palate, pharynx . . . and surface EMG in the upper respiratory tract may become a valuable method of clinical investigation.

Clinical electromyography in ear, nose and throat practice.

A new method of surface 'integrated' electromyography is described for use in ENT practice. It is particularly suitable for the relatively delicate muscles of the mouth and throat. A special bipolar surface electrode has been designed to sample the interarytenoid, posterior crico-arytenoid and crico-pharyngeal sphincter. Very important EMG abnormalities have been found in a variety of neurolaryngological disorders, and it is expected that surface EMG will very soon become the method of choice in otolaryngology.

Laryngeal electromyography in multiple system atrophy with autonomic failure.

In five patients with multiple system atrophy, electromyographic studies using a new surface electrode technique, showed unequivocal evidence of denervation of the posterior crico-arytenoid muscle, and in three of them, of the interarytenoid muscle or crico-pharyngeal sphincter. By comparison, only minor electromyographic abnormalities were found in one-third of patients with other extra-pyramidal syndromes, though in two cases, the abnormalities were of a different kind from those found in multiple system atrophy.