The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.


The mouse peripheral blood micronucleus test with 2-acetylaminofluorene using the acridine orange supravital staining method.

The peripheral blood micronucleus test using the acridine orange (AO) supravital staining method was validated with the potent bone marrow clastogen 2-acetylaminofluorene (2-AAF). 2-AAF induced micronuclei in peripheral blood reticulocytes dose-dependently as well as in bone marrow polychromatic erythrocytes. The incidence of micronucleated reticulocytes (MNRETs) peaked 48 h after a single treatment in both CD-1 and BDF1 mice, and the incidence of micronucleated polychromatic erythrocytes (MNPCEs) peaked 24 or 48 h after treatment. The maximum incidences of MNRETs were always higher than those of MNPCEs in both mouse strains treated once. In the double-treatment regime, the maximum incidence of MNRETs was observed at 24 h after the second treatment in each strain. The incidences of MNRETs in BDF1 mice were higher than in CD-1 mice after a single treatment but were comparable after double treatment. These results indicate that the peripheral blood micronucleus test using AO supravital staining is as sensitive as the conventional bone marrow assay. The new staining method can be performed more easily than the original smear method using either bone marrow or peripheral blood cells. Thus, the peripheral blood method using AO supravital staining is a possible alternative to the conventional bone marrow assay.


The micronucleus test with peripheral reticulocytes from phenacetin-treated mice.

The in vivo micronucleus test is conventionally performed using mouse bone marrow cells (BM assay). Using phenacetin as a test chemical, an alternative method using reticulocytes (RET assay) was examined to determine if this could be substituted for the BM assay. Single doses of 400, 600, and 800 mg/kg gave negative results 24 h after i.p. administration, but positive results were obtained with 600 and 800 mg/kg after 48 h. Responses were weak at 72 h. Double treatment enhanced the responses; 400 mg/kg gave a positive result. Maximum responses were generally reached 24 h after the second treatment, 48 h if doses were highly toxic. When the BM and RET assays were compared, the BM assay seemed to be slightly more sensitive than the RET assay; double treatment was superior to a single treatment in both BM and RET assays. Both assays can be used routinely but in the RET assay, sequential samples can be obtained from the same individuals without killing them, providing a firm basis to substitute it for the BM assay. Taking advantage of this characteristic of the RET assay, a regimen of double treatments and double sampling at 24 and 48 h is recommended for a wide range of doses. These data were obtained with CD-1 mice; MS/Ae mice gave a higher incidence of micronuclei than did the CD-1 strain.


The micronucleus test of benzo[a]pyrene with mouse and rat peripheral blood reticulocytes.

The micronucleus test using peripheral blood reticulocytes (RETs) was evaluated in CD-1 and BDF1 mice and Sprague-Dawley rats treated with benzo[a]pyrene at two independent laboratories. The maximum incidence of micronucleated reticulocytes (MNRETs) appeared in both strains of mice 48 h after the treatment; interlaboratory differences were small. The incidence of MNRETs in BDF1 mice was higher than in CD-1 mice. In rats, significant increases of MNRETs with the maximum response at 72 h were detected when B[a]P was administered i.p.; slight but significant increases were observed at 24 h or later, with the maximum at 24-48 h, when it was administered p.o. These results suggest that the new method for the micronucleus test using circulating RETs will be useful in the detection of the clastogenicity of chemicals.


Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene.

Micronucleus assays using mouse peripheral blood stained vitally on acridine orange (AO)-coated slides were evaluated at two laboratories with 7,12-dimethylbenz[a]anthracene (DMBA) and compared with the standard bone marrow assay. DMBA was administered by single intraperitoneal injection to CD-1 mice at doses ranging from 5 to 80 mg/kg, then 5 microliters of peripheral blood was sampled from a tail vein at 24, 48, 72, 96, and 120 h after treatment. Similar incidences of micronucleated young erythrocytes were observed in peripheral blood reticulocytes and bone marrow polychromatic erythrocytes. The dose response of micronucleated reticulocytes was delayed compared to that of micronucleated polychromatic erythrocytes. The dose-response curves after treatment with DMBA differed depending on the sampling times, which revealed the difficulty of obtaining accurate dose-response relations in the micronucleus assay. The present result demonstrated that the simple and rapid AO supravital staining method is a valuable and easier method for obtaining dose- and time-response data for quantification of micronucleus induction by chemicals.


Micronucleus test with vincristine sulfate and colchicine in peripheral blood reticulocytes of mice using acridine orange supravital staining.

The induction of micronuclei in mouse peripheral blood reticulocytes (RETs) was studied with the spindle poisons vincristine sulfate (VINC) and colchicine (COL) using acridine orange (AO) supravital staining. Each chemical was studied independently in two laboratories using the same protocol. Blood samples were prepared at 0, 24, 48, and 72 h after a single intraperitoneal treatment with VINC (0.0625, 0.125, and 0.25 mg/kg) or COL (0.25, 0.5, 1.0, and 2.0 mg/kg). Both VINC and COL induced micronucleated RETs (MNRETs) significantly and dose-dependently with a peak at 48 h after treatment. Maximum frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment with VINC; thus, the transition time from MNPCEs to MNRETs was about 24 h. Both spindle poisons gave comparable results in the paired laboratories, indicating that the present AO supravital staining method is highly reproducible.


Effects of benzene in a micronucleus test on peripheral blood utilizing acridine orange-coated slides.

A micronucleus test was conducted on the peripheral blood of mice and rats utilizing acridine orange-coated slides (AO-coated method) after oral administration of benzene. Blood was sampled at 0, 24, 48, and 72 h after administration of benzene at doses of 500, 1000, and 2000 mg/kg in both mice and rats. The highest occurrence of micronucleated reticulocytes (MNRETs) was observed at 48 h after administration in both species. Species differences was found in the frequency of MNRETs, with the number being lower in rats than in mice. The present results indicate that the micronucleus test can easily detect chromosome aberrations in peripheral blood induced by benzene administration in both mice and rats. Furthermore, the AO-coated method used in this study was simpler to perform and allowed for easier detection of the effect than the conventional method.


Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.


Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane.

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF1 mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.


Detection of micronuclei in peripheral blood of mitomycin C-treated mice using supravital staining with acridine orange.

The induction of micronuclei in peripheral blood from mitomycin C (MMC)-treated mice was examined using a supravital acridine orange staining method. Male ICR mice were intraperitoneally given MMC at a single dose of 0.25, 0.5, 1, or 2 mg/kg. Blood was sampled from the tail 24, 48, 72, and 96 h after treatment, and the frequency of micronucleated reticulocytes (MNRETs) was examined. The induction of MNRETs peaked at 48 h after treatment with MMC; there was a clear, dose-related increase in MNRETs. In a multiple-treatment study, mice were treated with 4 consecutive daily injections of MMC at a dose of 0.13, 0.25, 0.5, or 1 mg/kg. The frequency of MNRETs increased markedly 24 h after the second treatment as compared with the first treatment, and did not change significantly until 24 h after the fourth treatment. The frequency of MNRETs decreased to approximately control values 96 h after the last treatment. In addition, a slight but statistically significant increase in the number of micronucleated normochromatic erythrocytes in peripheral blood was detected by means of Giemsa staining 7 days after the last treatment. These results confirm the usefulness of the supravital acridine orange staining method to evaluate micronucleus induction in mouse peripheral blood.


The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.


Induction of micronucleated reticulocytes by potassium bromate and potassium chromate in CD-1 male mice.

Micronucleus tests of potassium bromate (KBrO3) and potassium chromate (K2CrO4) were conducted with peripheral blood reticulocytes (PB-RETs) of CD-1 male mice dose intraperitoneally. Peripheral blood cells collected from the tail were stained supravitally with acridine orange (AO) using AO-coated glass slides. Both KBrO3 and K2CrO4 induced micronuclei in PB-RETs in the same manner as in polychromatic erythrocytes of bone marrow.


A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.

In the hereditary blistering condition epidermolysis bullosa simplex, the skin blisters on trauma following rupture of epidermal basal cells. Clinical variations range from severely incapacitating, especially in early childhood, to mild forms that may not even present clinically. Dowling-Meara epidermolysis bullosa simplex is characterized by clusters of epidermal blisters and keratin clumping in the cytoplasm; recent reports describe potentially causal mutations in keratin 14 (refs 2, 3). Here we describe a 'complementary' mutation at the other end of the other keratin expressed by these cells (K5, coexpressed with K14), a change from a Glu to a Gly in the helix termination peptide, detected by altered antibody binding and confirmed by sequencing using the polymerase chain reaction. The two conserved helix boundary peptides are predicted to be essential for filament assembly, and the requirement for two complementary (type I and type II) keratins is absolute. Epidermolysis bullosa simplex diseases demonstrate the function of the keratin cytoskeleton in resisting compaction stresses which otherwise lead to cell lysis.


Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: the summary report of the 5th collaborative study by CSGMT/JEMS.MMS. The Collaborative Study Group for the Micronucleus Test.

The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced by Hayashi et al. (1990), using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-1 mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0, 24, 48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytes per mouse. All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences were observed with some chemicals, there were no discrepancies in qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (greatest response, 24-48 h). These results suggest that the peripheral blood assay using the AO supravital staining technique generates reproducible and reliable data to evaluate the clastogenicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.


Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin.

Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi Cl- channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying Cl- channels were clustered in discrete loci. They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for Cl- over monovalent cations (Na+ and K+) and gluconate. They were blocked by 20-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and were insensitive to changes in the Ca2+ concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion.


Micronucleus test with cyclophosphamide using mouse peripheral blood reticulocytes.

The usefulness of the micronucleus test using supravital staining of peripheral blood reticulocytes with acridine orange was evaluated in two laboratories after administering cyclophosphamide (CYP) as a model chemical by intraperitoneal injection (i.p.) to CD-1 mice. The frequencies of micronucleated peripheral reticulocytes (MNRETs) increased dose-dependently at each sampling time. There were no significant differences in the results obtained with this new method by the two laboratories. Although the induction of MNRETs was delayed by about 24 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optical sampling time, 24 h for MNPCEs and 48 h for MNRETs. Therefore, it is concluded that this method is a suitable alternative to that using femoral marrow cells.


Pathways of NH3/NH4+ permeation across Xenopus laevis oocyte cell membrane.

While acid loading with extracellular NH4Cl solutions usually first alkalinizes the cells through NH3 influx, and acidifies only when NH4Cl is removed, Xenopus oocytes became immediately acidic upon NH4Cl addition and the cells did not acidify further upon its removal. Since NH4Cl solutions also collapsed the membrane potential (Vm) and resistance (Rm), we conclude that primarily NH4+ entered the cells where it liberated H+, with NH3 being trapped in intracellular lipid stores. To identify the NH4+ permeation pathway we have used K+ channel blockers (Ba2+, Cs+, tetraethylammonium, quinidine), various cation transport inhibitors (ouabain, bumetanide, amiloride) and other inhibitors, some of which block non-selective cation channels (La3+, diphenylamine-2-carboxylate, and p-chloromercuribenzoate). However, only the latter substances partially prevented the collapse of Vm and Rm. This suggests, that NH4+ passes through non-selective cation channels. In accordance with the voltage dependence and/or stretch activation of such channels NH4+ fluxes appeared to be asymmetric. NH4+ influx, which depolarized and swelled the cells, was large and acidified rapidly, while the efflux, which repolarized and shrank the cells, was slow and alkalinized only slowly.


The safety and efficacy of chronic ventricular pacing at 1.6 volts using a steroid eluting lead.

Steroid eluting leads may allow for lower chronic pacing thresholds and therefore lower pacing outputs. Twenty-two patients (15 presenting with syncope) were implanted with VVI or VVIR pacemakers and transvenous steroid eluting leads and followed for a mean of 20.6 months while being paced at 1.6 V and 0.6 msec. Mean acute voltage pacing thresholds were 0.40 V at 0.5 msec and chronic pulse width thresholds were 0.21 msec at 0.8 V. Pacemaker function was documented with one to three 24-hour Holter monitors, attached during the 2-6 week postimplant period, bimonthly transtelephonic monitoring, and monthly pacemaker clinic visits. No patient developed recurrent symptoms and consistent capture was verified in all patients on every 24-hour Holter recording and transtelephonic monitor. Chronic ventricular pacing at an output of 1.6 V at 0.6 msec is safe and effective when using a steroid eluting lead and potentially has implications for pacemaker longevity.


Prospective evaluation of heart block complicating early Lyme disease.

Lyme disease is a recognized cause of heart block/carditis. The incidence of heart block complicating early Lyme disease has not been prospectively evaluated. In this study, 61 patients with early Lyme disease documented by the rash of erythema migrans were prospectively evaluated for carditis. Fifty five of 61 patients had a repeat examination 3 to 4 weeks after initiation of antibiotic therapy. Only one of 61 patients (1.6%) presented with heart block, which resolved with antibiotics. None of the 54 patients without heart block on initial presentation had a change in any measured electrocardiographic parameter or progressed to heart block after antibiotics. Therefore, early Lyme disease appears to be infrequently complicated by heart block. Early administration of antibiotics may prevent the development of heart block/carditis.


Late development of conduction block over the Mahaim fibers after electrical atrioventricular junction ablation for Mahaim fiber tachycardia.

A patient suffering from so called "Mahaim fiber" tachycardia is presented who developed complete heart block over Mahaim fibers after 18 months of AV junction ablation. Antegrade conduction along the Mahaim fiber was intact immediately after the procedure. This finding has not been previously described and suggests that permanent pacemaker implantation is mandatory in such patients.


Safe introducer technique for pacemaker lead implantation.

Over the last several years, an introducer approach for pacemaker lead insertion has evolved that eliminates most introducer-related complications. The approach consists of defining a safe region for intrathoracic cannulation of the subclavian vein. If specific conditions cannot be met for entering the "safe" region or if the vein cannot be found, the subclavian vein is cannulated extrathoracically. Recently, this technique was used in 263 consecutive patients undergoing pacemaker implantation. The intrathoracic portion of the subclavian vein was used in 239 (90.9%) cases and the extrathoracic portion in 24 (9.1%). One hundred and ninety-eight (75.3%) cases were right-sided and 65 (24.7%) were left-sided. On the right side, 177 (89.4%) used the intrathoracic portion of the subclavian vein and 21 (10.6%) used the extrathoracic portion. On the left side, 62 (95.4%) used the intrathoracic portion and three (4.6%) used the extrathoracic. The introducer technique was successful in all cases and there were no introducer-related complications.


Correlations between the localization of accessory atrioventricular pathway and Doppler indices of left ventricular output and function in patients with Wolff-Parkinson-White syndrome.

Left ventricular (LV) output and function was investigated, using pulsed Doppler echocardiography in 52 patients with various localizations of accessory AV pathway (AP) in sinus rhythm and during paroxysm of AV reentrant tachycardia. In patients with sinus rhythm and ventricular preexcitation the most marked decrease in LV output and function (reduced aortic flow peak velocity [PV], mean acceleration [MA], stroke distance [SD], minute distance [MD], and lengthened time to peak velocity [TTP]) was noted in the presence of right parietal AP and less marked changes (decreased MA, lengthened TTP)--in the presence of posteroseptal AP, in comparison with the controls (P less than 0.005). During antidromic tachycardia the pronounced decrease in PV, MA, SD, and MD was noted, especially in patients with left parietal and posteroseptal AP, while in the case of right parietal AP changes in MA and MD were insignificant. During orthodromic tachycardia the decrease in LV function was less marked and no significant differences in the magnitude of LV output and function changes were found in various localizations of AP, except MA, which was more severely decreased in patients with posteroseptal AP. We conclude, that the alterations in Doppler indices of LV output and function are related to the localization of AP during AV reentrant tachycardia and regular sinus rhythm.


Optimized hemodynamics by implantation of a dual chamber pacemaker after heterotopic cardiac transplantation.

A patient who underwent prior heterotopic cardiac transplantation had persistent complaints of dyspnea, palpitations, and fatigue in spite of normal pump function of the donor heart. Repeated Holter monitoring excluded paroxysmal arrhythmias. It was thought that synchronization of both heart rates might alleviate his symptoms. The intrinsic heart rate of the donor heart was 90 beats/min, the recipient heart was 60 beats/min with acceleration up to 130 beats/min on exercise. A DDD pacemaker was implanted, the atrial lead was positioned in the right ventricule of the donor heart and the ventricular lead in the atrium of the recipient heart. Search for an optimal AV interval was evaluated by echo-Doppler and intraarterial pressure recordings. By increasing the AV interval from 125 to 300 msec, the maximum aortic flow velocity of the recipient heart increased from 1.0 to 1.2 m/sec. Left ventricular end-diastolic diameter remained unchanged, left ventricular end-systolic diameter decreased from 52 to 48 mm. Wall motion of the recipient left ventricle improved. At an AV interval of 125 msec there was alternate systolic contraction of both hearts, resulting in arterial pressure waves at a rate of 180/min. This did not relieve his symptoms and he complained further of headaches. At an AV interval of 300 msec contraction of the recipient heart just preceded that of the donor heart, resulting in arterial pressure waves at a rate of 90/min, normalization of the wave form, relief of symptoms, and improvement of exercise tolerance.


Radiofrequency catheter ablation of an automatic atrial tachycardia in an adult.

Automatic atrial tachycardia (AAT) is often refractory to medical management. While surgical ablation and isolation procedures such as atrioventricular node ablation have been useful in the management of AAT, important limitations remain. Reported experience with catheter ablation of AAT is limited. This report describes the successful application of transvenous radiofrequency catheter ablation in an adult with AAT. Potential limitations of catheter ablation in the management of AAT are discussed.


Should a VVIR pacemaker increase the heart rate with standing?

To assess the usefulness of incorporating a posture sensor into a ventricular inhibited rate modulated pacemaker, the hemodynamic effects of increasing the ventricular pacing rate with standing were studied in 15 pacemaker dependent patients aged 55 +/- 3.5 years. In a randomized cross-over design, the pacing rate remained at 70 or was increased to 100 beats/min immediately prior to standing. Blood pressure was monitored continuously and forearm blood flow was measured by venous occlusion plethysmography. There was no difference in supine blood pressure (117 +/- 4/63 +/- 3 compared to 118 +/- 5/64 +/- 4 mmHg) or forearm blood flow (2.88 +/- 0.36 vs 2.94 +/- 0.32 mL/100 mL/min) before the 70 or 100 pacing rate intervention. With standing, blood pressure fell to an equivalent degree at the two pacing rates (fall in mean blood pressure at 70 beats/min 6 +/- 4 and at 100 beats/min 8 +/- 2 mmHg, P = 0.7). After 1 minute of standing differences in blood pressure were similar, but after 2.5 minutes of standing the increase in mean blood pressure was less at 70 than at 100 beats/min (increase from control 28 +/- 2 compared to 36 +/- 3 mmHg, P = 0.002). Forearm blood flow decreased after standing for 1 and 2.5 minutes but there was no difference between the 70 and 100 pacing rates (fall in forearm blood flow at 2.5 minutes 0.50 +/- 0.24 and 0.59 +/- 0.25 mL/100 mL/cm2).(ABSTRACT TRUNCATED AT 250 WORDS)


Reproducibility of successful drug trials in patients with inducible sustained ventricular tachycardia.

Patients whose inducible sustained ventricular tachycardia is suppressed during serial electrophysiological testing have a small but gradually increasing actuarial incidence of recurrent arrhythmias despite therapy with the "successful" drug. In an effort to improve the predictive value of a drug response, in 1990 we began to require that our full stimulation protocol be repeated successfully several times before considering a drug to be effective. In 23 consecutive patients who had inducible sustained ventricular tachycardia which was suppressed by at least one drug during invasive serial drug testing using a standard stimulation protocol, the identical stimulation protocol was performed six times during therapy with the initially successful drug (three trials on Day 1 and three trials on Day 2). Repeat trials were completed (i.e., either all six trials were successfully finished or sustained tachycardia was induced) for 29 initially successful drugs in these 23 patients. With 18 of these 29 initially successful drugs (62%), sustained ventricular tachycardia was eventually induced during repeat trials. The eventual drug failures could not be correlated with specific drugs tested, subtherapeutic or falling serum drug levels, marked fluctuations in autonomic tone, or changes in anatomic substrate. The proportion of patients failing each repeat trial was relatively constant: 4/29 (14%) failed Trial 2, 2/25 (8%) failed Trial 3, 7/23 (30%) failed Trial 4, 2/16 (13%) failed Trial 5, and 3/14 (21%) failed Trial 6. The increase in the cumulative incidence of drug failure during repeat trials was nearly linear. Inducibility of ventricular tachycardia appears to be a probability function; a successful drug study should not be regarded as an absolute phenomenon.


Longevity in patients with high degree atrioventricular block paced in the atrial synchronous or the fixed rate ventricular inhibited mode.

Survival in patients paced for high degree AV block has been demonstrated to be influenced by underlying cardiac disease in particular congestive heart failure. One previous study has suggested that dual chamber pacing may improve the vital prognosis for such patients. To investigate this, 74 patients treated with rate adaptive atrial synchronous (VDD) and 74 patients treated with VVI pacemakers for high degree AV block, were retrospectively studied for a mean of 5.4 years by life-table analysis. The two groups had an equal distribution of age, sex, date of pacemaker implantation, and concomitant cardiovascular diseases. Total mortality and estimated survival did not differ between the two groups. The estimated survival in the VDD group at 1, 3, and 5 years for patients without and with congestive heart failure was 94%, 86% and 78%, and 92%, 83% and 72%, respectively. In the VVI group the corresponding values were 95%, 90%, and 83% for patients without congestive heart failure and 82%, 64%, and 47% for those with congestive heart failure (P = 0.008). Compared to the expected survival rate of the general Swedish population, only the VVI group with congestive heart failure, had an excess mortality (P = 0.007). Patients with high degree AV block have a fairly normal vital prognosis irrespective of pacing mode. The prognosis for patients with congestive heart failure was negatively affected by VVI pacing. Thus, for patients with congestive heart failure the choice of pacing mode is of vital importance, whereas for patients without congestive heart failure, other factors such as feeling of well-being and exercise capacity should decide the final choice of pacing mode.


Effect of bilateral stellectomy on electrical instability of the atrium in the dog with hypokalemia.

To investigate the effect of sympathetic nerve activity on electrical instability of the atrium in the presence of hypokalemia, open chest electrophysiological study was performed before and after bilateral stellectomy (BS) in 15 dogs with hypokalemia (hypokalemia group) and in 15 dogs with normokalemia (control group). Hypokalemia was created by infusion of 5.0 g/kg of polystyrene sulfonic acid calcium into the colon. Serum level of potassium was significantly lower in the hypokalemia group (2.94 +/- 0.52 mEq/L) than in the control group (4.86 +/- 0.51 mEq/L, P less than 0.01) before BS. There was no significant change in serum level of potassium in the two groups after BS. Incidence of electrically induced atrial fibrillation (AF) was significantly higher in the hypokalemia group (80%) than in the control group (13%, P less than 0.001) before BS. It was significantly reduced in the hypokalemia group (40%, P less than 0.05), but not in the control group (6%) after BS. Dispersion of effective refractory period of the atrium (delta ERP) was significantly greater in the hypokalemia group (26.1 +/- 2.8 msec) than in the control group (22.0 +/- 3.3 msec, P less than 0.005) before BS. It was significantly decreased to 23.1 +/- 3.2 msec in the hypokalemia group (P less than 0.001) and to 20.6 +/- 2.5 msec in the control group (P less than 0.01) after BS. Maximum conduction delay in the atrium (MaxCD) was 36.1 +/- 3.5 msec before and 36.2 +/- 4.1 msec after BS in the hypokalemia group and 31.1 +/- 4.2 msec before and 32.3 +/- 4.9 msec after BS in the control group. There was a significant difference in MaxCD between the two groups before BS. Atrial fibrillation threshold (AFT) was significantly lower in the hypokalemia group (3.9 +/- 0.7 mA) than in the control group (13.8 +/- 3.1 mA, P less than 0.001) before BS. It was significantly increased both in the hypokalemia group (6.5 +/- 1.3 mA, P less than 0.001) and in the control group (15.0 +/- 2.7 mA, P less than 0.005) after BS. It is concluded that sympathetic nerve activity may play some role in the increase in electrical instability of the atrium in the presence of hypokalemia.


Results of electrophysiological testing and long-term follow-up in patients sustaining cardiac arrest only while receiving type IA antiarrhythmic agents.

Therapeutic management of patients sustaining a cardiac arrest while receiving antiarrhythmic agents can be difficult since the role of the drug in possibly facilitating the arrhythmia is often difficult to define. To determine if the response to programmed stimulation could give insight into which patients may have experienced a drug-induced cardiac arrest, we studied 29 patients (61 +/- 9 years) with no prior history of sustained ventricular tachyarrhythmias (VT) who suffered a cardiac arrest only while receiving type Ia antiarrhythmic agents. Patients with documented myocardial infarction, acute ischemia, electrolyte abnormalities, or torsade de pointes were excluded from the study. Twenty-four patients had coronary artery disease with prior myocardial infarction (ejection fraction 28% +/- 9%) and five patients had idiopathic dilated cardiomyopathy (ejection fraction 31% +/- 6%). During baseline electrophysiological testing, 19 patients (66%) had inducible sustained ventricular arrhythmias: uniform VT, n = 14 (group I), polymorphic VT or ventricular fibrillation, n = 5 (group II). Ten patients (group III) had no inducible sustained ventricular arrhythmias. To determine if rechallenge with a type Ia agent could facilitate induction of a sustained ventricular arrhythmia in group III, eight patients underwent ten electrophysiological studies during therapy with either procainamide or quinidine. Only two patients developed sustained VT in response to programmed stimulation. Patients in groups I and II received therapy guided by electrophysiological testing, including antiarrhythmic agents alone (n = 8), subendocardial resection (n = 4), or an implantable cardioverter defibrillator (n = 7). Patients in group III received antiarrhythmic agents empirically (n = 3), or for treatment of atrial tachyarrhythmias (n = 2) or nonsustained VT (n = 1). In addition, four patients in group III received an implantable cardioverter defibrillator. During a mean follow-up of 28 +/- 27 months (range: 1 day-84 months) 13 patients died suddenly or received a defibrillator shock preceded by syncope or presyncope: group I: n = 5; group II: n = 2; group III: n = 6. (1) most patients sustaining a cardiac arrest only in the presence of type Ia antiarrhythmic agents have inducible sustained VT in the absence of antiarrhythmic agents, and (2) the risk of recurrent VT persists in patients without inducible sustained arrhythmias in the drug-free state, regardless of whether they manifest inducible arrhythmias after rechallenge with a type Ia agent.


Clinical experience with an activity sensing DDDR pacemaker using an accelerometer sensor.

The rate adaptive characteristics and pacemaker mediated tachycardia protection algorithm of an accelerometer based DDDR pacemaker were evaluated in 11 patients with bradycardia (seven atrioventricular block, four sick sinus syndrome). Rate adaptive programming was effected by collecting the acceleration level during a 3-minute moderate exercise ("tailoring" of sensor). In comparison with an externally attached piezoelectric sensor, the accelerometer sensor showed lower rate changes during external tapping of the pacemaker (16 +/- 3 vs 29 +/- 4 ppm, P less than 0.02) and applied direct pressure (1 +/- 1 vs 40 +/- 3 beats/min, P less than 0.001) on the pacemaker. At nominal setting, the accelerometer sensor showed improved rate stability and higher rate response to jogging and standing, although responses to other daily activities and treadmill exercise were similar. Apart from changing the rate responsive slope, rate response could be improved by repeat "tailoring" of the sensor at a lower exercise level, resulting in better overall rate response characteristics. The ability of the rate monitoring software to collect acceleration levels for an activity and profile the projected rate response at different rate responsive settings allowed programming to be effected with the minimum amount of exercise testing. The pacemaker also discriminated atrial tachyarrhythmias from normal sinus response using the sensor to judge the appropriateness of the atrial rate, which correctly identified and prevented rapid ventricular tracking in two patients during atrial flutter/fibrillation.


Effect on ventricular performance of direct current electrical shock for catheter ablation of the atrioventricular junction.

In ten patients undergoing catheter ablation of the atrioventricular junction (CAVJ) because of therapy refractoriness of supraventricular arrhythmias, the effect of repeated high energy direct current (DC) shock on left ventricular function has been investigated. End-systolic pressure-volume relation (ESPVR) and the positive first derivative of ventricular pressure (dP/dt) have been used as indices of left ventricular systolic function, while the time constant of isovolumic pressure decay, the diastolic stiffness, and the negative dP/dt represented the diastolic function parameters, respectively. Each patient received at least two and no more than three DC shocks for successful CAVJ, with an energy of 360 joules. Significant acute reduction of both systolic and diastolic function was noted after each DC shock, with a slow partial recovery of both phases. The recovery process involved the systolic phase earlier and more completely than the diastolic one. The alterations observed could not be predicted from preablation values, but were significantly related to cumulative energy dose index for body weight. In conclusion, repeated high energy DC shocks acutely, but reversibly, impair left ventricular function; in addition, the ventricular function reduction is primarily related to the total ablation energy indexed for body weight.


The influence of sucrose, dextran, and hydroxypropyl-beta-cyclodextrin as lyoprotectants for a freeze-dried mouse IgG2a monoclonal antibody (MN12).

The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-beta-cyclodextrin (HP beta CD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56 degrees C). For comparison, solutions of MN12 were stored under the same conditions. Regardless of the lyoprotectant used, precipitation and a concomitant reduction of the antigen-binding capacity by about 10% were observed upon reconstitution of lyophilized MN12. HP beta CD proved to be the most effective stabilizer to prevent degradation of lyophilized MN12 during storage. Compared with MN12 solutions, HP beta CD-containing lyophilized MN12 cakes were more resistant to heat-induced charge alterations and loss of antigen-binding capacity.


Prenatal identification of small mosaic markers of different chromosomal origins.

In situ hybridization using a series of alphoid DNA probes has demonstrated the origin of two small accessory mosaic marker chromosomes ascertained from 1079 amniocenteses. These markers appeared to be de novo, derived from acrocentric chromosomes, and identical by traditional cytogenetic staining (G, Q, C, AgNOR, Hoechst-distamycin). Molecular characterization showed that one marker had originated from chromosome 14, the other from chromosome 22. Clinical outcome in both cases was normal.


Amniotic fluid levels of human chorionic gonadotropin and its alpha and beta subunits in second-trimester chromosomally abnormal pregnancies.

Amniotic fluid from 135 pregnancies was assayed for human chorionic gonadotropin (hCG) and its free alpha (ahCG) and free beta (bhCG) subunits. Forty-six chromosomally abnormal pregnancies between 14 and 20 weeks' gestation were matched with 89 chromosomally normal samples. Compared with controls, trisomy 21 pregnancies exhibited significantly elevated levels of all three peptides, whereas trisomy 18 gestations gave rise only to significant elevation of ahCG. Female fetuses in both the trisomy 21 and trisomy 18 pregnancies provided significantly elevated levels of hCG and bhCG compared to their male counterparts. On converting the values to multiples of the median, it was determined that 6 of 7 trisomy 18 samples had abnormally elevated alpha/beta ratios, as did 6 of 21 Down's syndrome pregnancies. Further, 11 of 21 trisomy 21 gestations had abnormal amniotic fluid hCG levels. Using only ahCG, bhCG and their ratio, a 61 per cent sensitivity was found for these trisomies, with a 96 per cent specificity.


Characterization of the cytokeratins of human colonic, pancreatic, and gastric adenocarcinoma cell lines.

Cytokeratin-type intermediate filaments are a polygenic family of insoluble proteins that vary according to cell of origin and have been proposed as potentially useful markers of differentiation in epithelial malignancies. Because gastrointestinal malignancies resemble each other in their expression of many soluble antigens, we compared the cytokeratins of seven colonic, three gastric, six pancreatic, and one duodenal carcinoma cell lines, and one colon villous adenoma cell line. Cytokeratins were characterized by one- and two-dimensional gel electrophoresis, immunoblotting, and immunocytochemistry. These cell lines expressed combinations of cytokeratins 7, 8, 18, and 19, which are typical of the "simple" epithelial pattern found in normal ductal and glandular tissues of the gastrointestinal tract. However, pancreatic carcinoma cell lines expressed additional cytokeratins that are normally found in stratified squamous epithelium and epidermoid (squamous cell) carcinomas. These additional cytokeratins consisted of cytokeratin 16 in all six cell lines and cytokeratins 4, 13, and 16 in one cell line. These results suggest that cytokeratin patterns represent stable markers that may aid in distinguishing gastrointestinal malignancies.


Expression of elongation factor-1 gamma-related sequence in human pancreatic cancer.

A cDNA clone designated pPDC-1 was isolated from a cDNA library prepared against poly(A+)RNA isolated from the human pancreatic adenocarcinoma cell line, Capan-2. The cDNA corresponds to a 1.7-kilobase mRNA that is expressed at higher levels in seven of nine pancreatic tumors than in their corresponding normal tissues. It is also expressed in normal human kidney, intestine, pancreas, stomach, placenta, lung, brain, spleen, and liver. A computer search of the Intelligenetics System of all available nucleotide sequences revealed a 60% homology between the nucleotide sequence of the pPDC-1 cDNA and that of elongation factor-1 gamma from Artemia. The deduced amino acid sequence shared 53% identity with the amino acid sequence for the Artemia elongation factor-1 gamma.


Detection of occult metastases in pancreatic adenocarcinoma with anticytokeratin antibody.

Fifty-six lymph nodes excised from 11 patients with pancreatic adenocarcinoma were studied for evidence of metastasis by standard light microscopy and immunohistochemistry using an anticytokeratin monoclonal antibody. Eight of 56 nodes contained metastatic carcinoma as demonstrated by conventional techniques. By immunohistochemistry, positive-staining cells were easily identified. In 10 of 56 nodes, the positive staining was accompanied by cytologic atypia, changes consistent with malignancy. In seven additional nodes, positive-staining cells were present, but cytologic atypia was lacking. The use of immunohistochemistry facilitated recognition of malignant cells and raised questions that single-cell metastasis may be present. Positive staining for cytokeratin should prompt a close examination of cells to determine if morphologic features of malignancy are present.


Changes in peptidergic innervation in chronic pancreatitis.

We sought to identify characteristics of peptidergic innervation that altered in patients with chronic pancreatitis. Pancreatic tissue removed from patients with chronic pancreatitis was analyzed by immunohistochemistry using antisera against neuropeptide Y, tyrosine hydroxylase, vasoactive intestinal polypeptide, peptide histidine isoleucine, calcitonin gene-related peptide, and substance P, respectively. In accordance with recent findings, the number and diameter of intralobular and interlobular nerve bundles were found to be increased as compared with control pancreas from organ donors. The striking change in the peptidergic innervation pattern in chronic pancreatitis concerned these altered nerves. It consisted of an intensification of the immunostaining for calcitonin gene-related peptide and substance P in numerous fibers contained in these nerves. Adjacent sections showed that immunoreactive substance P and immunoreactive calcitonin gene-related peptide coexisted in these fibers. Because both of these peptides are generally regarded as pain transmitter candidates, our findings provide further evidence that changes in pancreatic nerves themselves might be responsible for the long-lasting pain syndrome in chronic pancreatitis.


Effects of FK506 on exocrine pancreas in rats.

The efficacy of FK506 on exocrine pancreas was studied in rats. Male Sprague-Dawley rats (230-250 g) received an i.m. daily injection of FK506 (0.1, 0.5, or 5.0 mg/kg), cyclosporine (CS; 25 mg/kg), or saline for 2 weeks. Isolated dispersed pancreatic acini were prepared from rats, and enzyme content of the cells and secretory response to cholecystokinin (CCK) were determined. Amylase and trypsin contents were increased in a dose-related manner by FK506 (p less than 0.01) and by CS at 25 mg/kg (p less than 0.01). The release of amylase in response to CCK was reduced by FK506 in a dose-related manner (p less than 0.01) and by CS at 25 mg/kg (p less than 0.01). Histologic examination showed that treatment of rats with FK506 at 0.1 mg/kg did not affect morphology of the acinar cells. FK506 at 0.5 mg/kg induced a minimal number of small vacuoles in cytoplasm of acinar cells and FK506 at 5.0 mg/kg, and CS at 25 mg/kg induced numerous cytoplasmic vacuoles and pyknotic nuclei. Increased enzyme storage and suppressed responsiveness of amylase release may have an association with the histologic changes. Therefore, the results of this study suggest that FK506, even when used in a low dose, may have adverse effects on the exocrine pancreas. Understanding of the mechanism of action of FK506 on pancreas will provide essential basic information that will allow transplant practitioners to more fully explore the benefits of this drug.


Reduced pancreatic content of the inhibitory neurotransmitter galanin in genetically obese, hyperinsulinemic mice.

Because abnormalities of the autonomic nervous system have been described in several animal models of obesity, and because galanin has been proposed to be a sympathetic neurotransmitter in the endocrine pancreas, we hypothesized that the hyperinsulinemia observed in genetically obese (ob/ob) mice may result either from defective ability of galanin to inhibit insulin release or from a reduced degree of pancreatic galaninergic innervation. To address these possibilities, we examined the effect of exogenous galanin on immunoreactive insulin (IRI) levels in ob/ob mice and compared the pancreatic content of galaninlike immunoreactivity (GLIR) in ob/ob mice with that in lean littermates. Intravenous administration of synthetic porcine galanin significantly reduced basal IRI levels in ob/ob mice, suggesting that a defect in galanin action is unlikely to account for the hyperinsulinemia in this model. In contrast, reduced pancreatic galaninergic innervation was supported by findings that pancreatic content of GLIR in ob/ob mice was less than 10% of that in age- and sex-matched lean littermates. The reduction of pancreatic GLIR in ob/ob mice appeared organ specific; no such reduction was observed in adrenal GLIR content when comparing obese and lean mice. In addition, the relationship between pancreatic GLIR content and plasma IRI levels was examined in groups of obese and lean mice. It was found in young females, young males, and older mice of mixed sex that there was a significant negative correlation between pancreatic GLIR and plasma IRI in lean mice, whereas no such correlation was observed in obese mice.(ABSTRACT TRUNCATED AT 250 WORDS)


On the helix sense of gramicidin A single channels.

In order to resolve whether gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A- and M- channels are structurally equivalent, while gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.


Renal argininosuccinate synthetase: purification, immunohistochemical localization, and elastin-binding property.

Several proteins interact with elastin, an essential extracellular matrix element. We describe here an additional elastin-binding protein from the porcine kidney. This protein was extractable with an isotonic buffer, and was purified by a rapid and simple procedure employing its characteristic properties: high affinity for red A-agarose and spontaneous formation of cryoprecipitate. Immunohistochemical studies using antibody against the purified protein demonstrated its localization in the cytoplasm of proximal tubules of the normal rat kidney. On Western blotting using the antibody, an immunoreactive protein was detected in the liver as well as in the kidney. On the basis of the partial amino acid sequences of the purified protein, it was identified as argininosuccinate synthetase. Some biochemical properties of the protein were different from the data reporting a hepatic form of argininosuccinate synthetase, a key enzyme in the urea cycle in the liver. These results suggest that the elastin-binding property of a renal form of the enzyme may be related to a tissue-specific function in the kidney.


Characterization of renal Na-K-ATPase gene expression by in situ hybridization.

Na-K-ATPase is a heterodimer of alpha- and beta-subunits often referred to as the 'sodium pump' responsible for the maintenance of cell volume and electric potential across cell membranes. In the present study we have used antisense RNA probes to localize these subunits in the rat kidney by in situ hybridization. alpha 1-Subunit and beta-subunit gene expression are highest in cortical distal convoluted tubules, and the thick ascending limb of the loops of Henle; high in some proximal convoluted tubules, and low in the collecting ducts and blood vessels. Expression of the alpha 11 and alpha 111 isoforms is very low or absent throughout the kidney. These results confirm and extend studies of Na-K-ATPase enzyme activity levels and immunocytochemical localization in the kidney.


Local immune response in the chicken Harderian gland to antigen given by different ocular routes.

The effectiveness of three ocular routes of antigen administration to produce a local immune response in the Harderian gland was studied. The routes were by eyedrop, injection into the ocular conjunctiva and injection into the nictitating membrane. The antigen was observed in the cytoplasm of macrophages located within the lymphoid tissue only after the injection into the nictitating membrane. The numbers of germinal centres and plaque forming cells found in the gland after injection into the nictitating membrane was higher than the numbers observed following the other two ocular applications. These findings indicate that the injection of the antigen into the nictitating membrane is the most effective ocular route for producing a local immune response in the Harderian gland.


Zonal changes of guanidine 3', 5'-cyclic monophosphate related to endothelium-derived relaxing factor in dog renal medulla.

This study was undertaken to determine the basal and the stimulated profiles of endothelium-derived relaxing factor (EDRF) production along the cortical medullary axis of the dog kidney. To this end, slices (0.5 mm thick) were obtained from six zones equally spaced along the cortical medullary axis. Zone 1 included the medullary crest, while zones 2 and 3 included the inner medulla, zone 4 the outer medulla, zones 5 and 6 the the middle and superficial cortex, respectively. The guanidine 3', 5'-cyclic monophosphate (cGMP) content (an index of EDRF production) was determined by radioimmunoassay under basal conditions and after acetylcholine (10(-5) M), bradykinin (10(-5) M) and SIN-1 (10(-4) M) stimulation. Under basal and stimulated conditions, the cGMP concentrations were highest in the midinner medulla and decreased progressively to lowest concentration in the cortex. These responses were inhibited by NG-monomethyl-L-arginine (LNMMA), a specific antagonist of EDRF synthesis. In contrast, LNMMA did not alter the stimulation of cGMP produced by SIN-1 (10(-4) M) an endothelium-independent vasodilator. This particular localization of EDRF-mediated stimulation on the midinner medulla may have a specific role in the regulation of sodium tubular reabsorption.


Immunoglobulin classes synthesised by the chicken Harderian gland after local immunisation.

The immunoglobulin (Ig) levels in tears and sera were compared after antigen administration (salmonella O antigen) by eyedrop and injection into the nictitating membrane, to determine the Ig classes synthesised by the plasma cells in the chicken Harderian gland. Samples of tears and sera were collected from immunised and control birds between 24 hours and 24 days after the antigen or sterile saline was administered. Samples were assayed for IgA, IgG and IgM concentrations using radial immunodiffusion. It is suggested that most of the IgG found in tears after local immunisation has an extraglandular origin.


Antigenic changes of the glomerular basement membrane after incorporation of hydroxyproline isomers.

We investigated antigenic changes of murine glomerular basement membrane (GBM) collagen type IV after oral feedings of 3-cis- or 4-cis-hydroxyproline. As shown by thin layer chromatography and gas chromatographic-mass spectrometric studies, 3-cis- and 4-cis-hydroxyproline were incorporated into the GBM collagen instead of the normal trans isomer and led to a considerable reduction of the collagen antibody reactivity on indirect immunofluorescence, passive hemagglutination and radioimmunoassay when compared to GBM collagen of control animals. We speculate that antigen modification due to the incorporation of stereoisomers could serve as a model for basement membrane immunopathology.


Hypoxic contraction of pre-stretched human pulmonary artery.

To clarify the mechanism of hypoxic pulmonary vasoconstriction in man, human pulmonary artery segments (2 mm O.D.) were suspended and changes in isometric force were measured. The arteries were contracted by hypoxia (PO2 43 +/- 2 Torr) developing a tension of 127 +/- 36 mg over the course of 15 min. This contraction was completely blocked by 10(-6) M L-isoproterenol, 10(-6) M nitroglycerin, partially blocked by 10(-8)-10(-6) M verapamil, unchanged by 10(-6) M phentolamine, 10(-6) M L-propranolol, 10(-6) M diphenhydramine, 10(-6) M guanethidine, 10(-7) M FPL 55712 and enhanced by 10(-6) M BAY K 8644, 10(-3) M procaine, 3 x 10(-6) M quinacrine, 10(-6) M indomethacin or 10(-6) M methylene blue. Removal of the endothelium significantly enhanced the magnitude of hypoxia-induced contraction. These results suggest that the human pulmonary artery constricts in response to hypoxia, at least in part, through activation of the voltage-dependent Ca2+ channels and that neither alpha, beta, H1 receptors, the lipoxygenase pathway nor neural reflexes are involved. They also show that the endothelium is not required for hypoxic contraction and that its presence reduces sensitivity to hypoxia.


Specific binding site for pro atrial natriuretic factors 1-30, 31-67, and 99-126 on distal nephrons, proximal tubules, renal cortical and medullary membranes.

Two peptides with natriuretic and diuretic properties consisting of amino acids (a.a.) 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF), which normally circulates in humans and animals, were investigated to determine if they have specific binding sites on distal nephrons, proximal tubules, renal cortical and medullary membranes. Competitive binding experiments revealed that proANFs 1-30, 31-67, and 99-126 (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants (Kd) for proANFs 1-30, 31-67, and 99-126 binding to renal cortical membranes were similar at 9.8, 5.1, and 4.7 nM, respectively. In renal medullary membranes, on the other hand, proANF1-30 (Kd = 3.7 nM) and proANF31-67 (Kd = 2.9 nM) had a 10-fold higher binding affinity than ANF (Kd = 56 nM). All three peptides had very weak binding to proximal tubules with the respective Kds for proANF1-30, proANF31-67, and ANF of 892, 789, and 550 nM indicating a 50- to 100-fold less affinity for their binding sites in proximal tubules than in distal nephrons (Kds of 4.9, 5.3, and 11 nM, respectively). Each peptide bound to the respective kidney membranes or tubules between 10(-8) and 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7) M. Neither insulin, growth hormone, nor adrenocorticotrophic hormone competitively inhibited the binding of proANF 1-30, proANF 31-67 or ANF. These results suggest that proANF 1-30 and proANF31-67 do not work through the ANF receptor but rather have their own separate and distinct receptors to mediate their diuretic and natriuretic effects in the kidney.


Brain-gut peptides and the renal hemodynamic response to an oral protein load: a study of gastrin, bombesin, and glucagon in man.

With the aim of disclosing a possibility for gastrin and bombesin to participate in the postprandial regulation of the renal hemodynamic response, 10 healthy males were studied before and after a meat meal (2 g/kg BW of proteins as cooked red meat). We evaluated the time course changes of glomerular filtration rate (GFR) renal plasma flow (RPF), and the plasma concentrations of gastrin, bombesin, glucagon, and total amino acids. After the meat meal a significant increase in GFR and RPF was seen, within 30 min, along with an increase in plasma gastrin and glucagon. Bombesin and amino acid concentrations increased at a later time. The data suggest but cannot demonstrate a causal role for gastrin and glucagon in the genesis of the hyperfiltration response to acute protein administration.


A single amino acid that determines the sensitivity of progesterone receptors to RU486.

The progesterone analog RU486, an abortifacient, inhibits the action of progestins in humans but not in chickens or hamsters. Substitution of cysteine at position 575 by glycine in the hormone binding domain (HBD) of the chicken progesterone receptor (cPR) generated a cPR that binds RU486 and whose activity is antagonized by that compound. In fact, all receptors that bind RU486 have a glycine at the corresponding position. The hamster PR, like cPR, has a cysteine. Only glycine--not methionine or leucine--at position 575 allowed binding of RU486 to cPR. Substitution of this glycine by cysteine in the human PR (hPR) abrogated binding of RU486 but not that of an agonist. The corresponding mutation in the human glucocorticoid receptor resulted in a loss of binding of both dexamethasone and RU486. Examination of a series of 11 beta-substituted steroids showed that antagonism is not an intrinsic property of an antihormone, because one hPR antagonist acted as an agonist for a mutated hPR. The positioning of an aromatic 11 beta-substitution in the PR HBD appears to be critical for generating agonistic or antagonistic activity.


Renal handling of sodium after an oral protein load in adult humans.

This investigation was designed to study (1) renal sodium handling after an oral protein load and (2) its relationship to some known determinants of the hemodynamic response (glucagon, insulin, growth hormone, renin, aldosterone, and plasma amino acid concentration). To this end of group of 8 adult subjects was studied before (three 30-min clearances) and after a meat meal (MM; five 30-min clearances at 30, 60, 90, 120 and 180 min). The MM provided 2 g/kg BW of protein. Within 30 min from the MM an hyperfiltration response was seen, which was paralleled by a 2-fold increase in plasma alanine concentration while total plasma amino acid concentration was not different from the baseline values. The hemodynamic response was associated with a normally operating tubuloglomerular feedback mechanism independent of renin-aldosterone activity, but possibly associated with an early increase in plasma glucagon concentration and later on with a modest increase in postmeal plasma insuling concentration.


Selection of intrinsic horizontal connections in the visual cortex by correlated neuronal activity.

In the visual cortex of the brain, long-ranging tangentially oriented axon collaterals interconnect regularly spaced clusters of cells. These connections develop after birth and attain their specificity by pruning. To test whether there is selective stabilization of connections between those cells that exhibit correlated activity, kittens were raised with artificially induced strabismus (eye deviation) to eliminate the correlation between signals from the two eyes. In area 17, cell clusters were driven almost exclusively from either the right or the left eye and tangential intracortical fibers preferentially connected cell groups activated by the same eye. Thus, circuit selection depends on visual experience, and the selection criterion is the correlation of activity.


Inhibition of myeloid differentiation by the helix-loop-helix protein Id.

Id is a helix-loop-helix (HLH) protein that represses activity of several basic helix-loop-helix (bHLH) proteins involved in cell type--specific transcription and cell lineage commitment. The myeloid precursor cell line 32DC13(G) expressed Id messenger RNA, which was transiently decreased when cells were induced to terminally differentiate with granulocyte--colony-stimulating factor. Concomitant with the decrease of Id messenger RNA was the appearance in nuclear extracts of DNA binding proteins that recognized a canonical E-box motif, a DNA binding site for some bHLH proteins. Constitutive expression of an Id complementary DNA in 32DC13(G) cells blocked their ability to differentiate and to induce E-box-binding activity. These results suggest that Id and, hence, bHLH proteins function in the process of myeloid differentiation.


Salvage therapy in recurrent testicular cancer.

Patients with testicular cancer who relapse after primary chemotherapy are still curable and should be treated aggressively with this goal in mind. These patients should receive a cisplatin-based regimen, usually including ifosfamide and either vinblastine or etoposide (depending upon prior exposure to drugs). As with first-line therapy, chemotherapy should not be delayed because of blood counts alone. Because this patient population has a higher incidence of significant myelosuppression, the use of hematopoietic growth factors may be of value and is currently being investigated. Patients who achieve a CR after salvage chemotherapy can be followed as usual with monthly serum markers and chest x-rays. For those patients who have a marker-negative partial remission, resection of all residual disease (if possible) is appropriate. If the surgical specimen contains only necrotic material or teratoma, then they should also be followed without further therapy. The appropriate approach for patients with residual carcinoma at the time of surgical resection is uncertain. The standard approach at Indiana University has been to give two more cycles of chemotherapy post-operatively, however most of these patients will eventually relapse and require further treatment. Patients are currently being treated with oral etoposide for 3 months after surgery in an attempt to improve long-term survival. Whether this approach will be of benefit remains to be seen. Patients who fail second-line chemotherapy should be considered for high-dose chemotherapy with autologous bone marrow rescue. Patients who do not normalize their serologic markers with first-line chemotherapy or who relapse within 1 month after chemotherapy are truly platinum refractory and do poorly with standard-dose second line cisplatin-based chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS)


Current perspectives on the role of adjunctive surgery in combined modality treatment for patients with germ cell tumors.

The role of surgery in patients with advanced GCT after chemotherapy has evolved substantially in the era of combined modality therapy. In evaluating patients for surgery after chemotherapy, the clinician must consider carefully the histology (seminoma v NSGCT) of the primary as well as the extent of the residual disease. In patients with seminoma, the size of residual disease (greater than or equal to 3 cm) permits selection of patients with a high incidence of residual malignancy. In contrast, criteria designed to select NSGCT patients in whom surgical intervention after chemotherapy can be avoided are associated with substantial error. A normal radiographic evaluation in patients with NSGCT does not indicate a negative pathology and the treating physician must consider the approximately 20% risk of residual teratoma or carcinoma despite evidence of a radiographic CR. Continued research is needed to improve the sensitivity and specificity of case selection for patients requiring surgery after chemotherapy in order to limit the toxicity of curative therapy in this patient population.


Hematopoietins in combination with 1-beta-D-arabinofuranosylcytosine: a possible strategy for improved treatment of myeloid disorders.

Inhibitors of DNA synthesis, such as 1-beta-D-arabinofuranosylcytosine, have been proven useful in the management of acute myelogenous leukemia. Despite significant improvement of response rates, long-term disease-free survival can be presently achieved only in a small percentage of patients. As S-phase--specific drugs, such as the inhibitors of DNA synthesis, only reach the cycling part of their target populations, dormant cells are left mainly unaffected. Their survival, however, is largely responsible for later disease relapse. This paper reports experiments that show that cell kinetic manipulations by hematopoietic growth factors may help to overcome the problem of cytokinetic and pharmacologic resistance. Hematopoietic growth factors synergize with inhibitors of DNA synthesis given at low doses to induce a more mature phenotype or to enhance leukemia cell kill when combined with high doses of DNA inhibitors. In addition, we present data to unravel several aspects of the molecular mechanism underlying this synergism.


Antineoplastic activity of the combination of 5-fluorouracil and interferon: preclinical and clinical results.

Interferon (IFN) is capable of modulating the cytotoxic effects and clinical activity of the fluorinated pyrimidine, 5-fluorouracil (5-FU). This occurs in the absence of a demonstrable effect on immune function, suggesting that these effects occur at the biochemical level. Pharmacokinetic studies have also demonstrated that IFN can increase serum levels of 5-FU. The relative importance of these effects on enhancement of 5-FU activity remains to be investigated.


Combined treatment of metastatic melanoma with interferons and cytotoxic drugs.

The treatment of disseminated melanoma with monotherapy and combined chemotherapy is still associated with rather low objective response rates and significant toxicity. Therefore, more effective substances and regimens with less toxicity are desired in the pharmacologic treatment of metastatic melanoma. The initial high expectations placed in systemic cancer treatment with interferons (IFNs) were not fulfilled, but IFN-alpha as a single substance revealed an objective response rate of 10% to 15% in melanoma patients. Clinical and experimental results suggest that the antitumoral activity of IFNs is mainly related to their antiproliferative effect, whereas immunomodulatory effects were not substantiated in clinical investigations. Results of in vitro trials showed that type-I IFNs may produce antagonistic effects combined with some agents (eg, cisplatin) and synergistic effects combined with others (eg, vindesine and BCNU). Clinical trials with combined IFN and cytostatic drug therapy were started a few years ago and have yielded promising initial results. Several studies with an entire number of more than 200 patients have already been performed to evaluate the combination of IFN-alpha and dacarbazine. This regimen was effective in over 50% of the patients, leading to complete or partial remissions in 27% and stabilization of the disease in an additional 28%. Toxicity is significant but still manageable, especially with a new generation of antiemetic drugs (serotonin receptor blockers). Several clinical trials performed so far did not find an improved efficacy by adding IFN-alpha to cisplatin or to vinblastine. The combination of IFN-alpha and vindesine seems to be more favorable and superior to the response rates of single agents and was well tolerated in an ongoing trial in our clinic. Recently, a new generation of multidrug combinations including IFN-alpha has been initiated and several reports with overall response rates greater than 50% have been published. In conclusion, combined regimens with IFN-alpha and different cytostatic drugs seem to be superior to treatments with cytostatic drugs alone and rather safe in disseminated melanoma. Additional combinations of IFNs and cytostatic drugs should be evaluated in future in vitro studies and in clinical trials.


Treatment of myelodysplastic syndromes with cytokines and cytotoxic drugs.

Clinical trials with hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF], interleukin-3, erythropoietin] have been done in patients with myelodysplastic syndromes. Treatment with GM-CSF or G-CSF has resulted in an increase of neutrophil counts into the normal range in the vast majority of patients. Progression to acute leukemia does not appear to occur more frequently in the patients receiving GM-CSF or G-CSF. Increases in platelet counts and hemoglobin levels have been reported after treatment with interleukin-3 and erythropoietin, respectively, although the response is only seen in a minority of treated patients. Combination therapy with GM-CSF and low-dose cytosine arabinoside has been studied, but present data do not indicate an advantage over other treatment strategies. Cytogenetic and molecular genetic analyses demonstrate that both normal and malignant precursor cells are stimulated by cytokine therapy.


Neural elements in human cervical intervertebral discs.

This study attempted to characterize neural elements within the human cervical intervertebral disc. Cervical intervertebral discs were obtained from four adult human subjects at autopsy. Discs were stained in bulk with gold chloride, sectioned, and viewed with the light microscope. Nerve fibers appeared to enter the disc in the posterolateral direction and course both parallel and perpendicular to the bundles of the anulus fibrosus. Nerves were seen throughout the anulus but were most numerous in the middle third of the disc. Receptors resembling Pacinian corpuscles and Golgi tendon organs were seen in the posterolateral region of the upper third of the disc. These results provide further evidence that human cervical intervertebral discs are supplied with both nerve fibers and mechanoreceptors.


[The percutaneous-endocavitary irradiation of esophageal carcinomas].

In a prospective, nonrandomized study 43 patients with inoperable oesophageal carcinoma were treated with a combined therapy of external and intracavitary irradiation according to the Heidelberg protocol adjusted to tumor stage, general condition and age. The proportion of external beam to afterloading doses was 2/3:1/3. The reference doses were between 50 and 75 Gy. Intracavitary radiotherapy was carried out with a HDR-afterloading device in single doses of 5 Gy. In a median follow-up of 23 months 46% had a complete remission and 42% had a partial remission. Within ten months 17 patients (39.5%) showed local tumor progression or recurrence. Presently the estimated median survival time of the whole collective is eleven months. The median survival was significantly influenced by achievement of complete remission (17.7 months in comparison to 8.7 months by missing complete remission). After completion of therapy 90% had sufficient oral nutrition. During long-term follow-up in 44% of the cases repeated measures had to be taken to eliminate initial or recurrent dysphagia. Almost all postradiogenic stenoses were caused by tumor progression. Radiogenic side-effects caused by HDR-afterloading boosts, exceeding the acceptance, were not found. The combined therapy reduces the period of hospitalisation and has the same palliative effects as an exclusively external radiotherapy.


Soluble and nuclear type I and II androgen-binding sites in benign hyperplasia and cancer of the human prostate.

This paper presents an approach for the assessment of the androgen receptor (AR) status in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) tissues. Evaluation of AR was carried out in both soluble and nuclear fractions by a standard competition method, using tritiated mibolerone as radioligand. Based on our experience with breast and endometrial cancer, this approach focused on both type I (high affinity, low capacity) and type II (reduced affinity, higher capacity) binding sites, aiming mainly at establishing a putative "functional" receptor mechanism, i.e., the presence of type I AR in both cytosol and nucleus. Ancillary studies were carried out to exclude a potential overestimation of the AR content by interference with other steroid receptors, namely, progesterone (PgR) or glucocorticoid (GcR) receptors. Results showed that the interaction by PgR or GcR upon AR measurement was not relevant. The distribution of AR, namely the percent of positivity either in a single or in both cell compartments, was not significantly different in BPH (N = 32) or PCa (N = 24) tissues. For type I binding, the percent of positivity in both soluble and nuclear fractions (i.e., the "functional" AR status) was very close to that observed for other endocrine-related tumors, like breast cancer. Concentrations of type I AR appeared significantly higher in PCa than in BPH tissues; this was true for both soluble and nuclear fractions. In contrast, no significant difference was found in type II AR concentrations in either cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)


Partial purification and characterization of a growth factor from human hyperplastic prostatic tissues.

A growth factor capable of stimulating DNA synthesis of Balb/c 3T3 cells was purified by heparin-Sepharose column chromatography about 1900-fold from the cytosol of human prostatic tissues obtained at autopsy or open prostatectomy. This growth factor bound to heparin-Sepharose in the presence of 0.5 mol/l NaCl and was eluted by 1.0-1.55 mol/l NaCl. Its molecular weight was estimated to be 68,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition was determined and compared with the data of other growth factors, which revealed no striking conformity. Distribution of growth factor activity was investigated in mechanically separated prostatic tissues of benign prostatic hyperplasia. The separation scheme provided two fractions: the stromal fraction consisting mainly of fibroblasts, fibers and smooth muscle, and the epithelial fraction consisting of epithelial cells. The specific growth-stimulating activity in the stromal fraction was about 2-fold that in the epithelial fraction. Referred to the total activity of whole tissue, about 74% of the activity could be detected in the stromal fraction, while only about 5% was detectable in the epithelial fraction. This study demonstrates the existence of a growth factor in human benign hyperplastic prostatic tissues, showing a remarkable distribution of growth factor activity, which may play a role in the pathogenesis of benign prostatic hyperplasia.


Estrogenic action of commonly used fragrant agent citral induces prostatic hyperplasia.

A rat model for benign prostatic hyperplasia in man (BPH) was investigated. Citral treatment of male Copenhagen rats for 4 months via the transdermal route resulted in a marked hyperplasia of glandular epithelium and interglandular stroma in the ventral prostate. Despite the cellular hyperplasia there was not a significant increase in prostate weight. Investigations of the mechanism of action of citral showed that application of citral directly to the vagina in female, ovariectomized rats resulted in an increased proliferation of vaginal epithelium and a significant increase in the BrdUrd incorporation in vaginal epithelial cells, in short a similar effect to that of estrogen application. In an in vitro assay citral proved to inhibit estrogen binding to estrogen receptors, while no such inhibition was observed with testosterone for androgen receptors. These observations together with the estrogen implication in the BPH and the reported incidence of gynecomastia following exposure to geraniol, a precursor of citral, strongly suggest that the prostatic hyperplasia-inducing capacity of citral may be due to its estrogenic action.


[Effect of TUR and transvesical prostatectomy on symptomatology and quality of life].

The influence of transurethral resection and transvesical prostatectomy on symptoms and quality of life in patients with benign prostatic hyperplasia was evaluated and compared in a prospective study. Two groups of 20 patients each were matched for preoperative symptoms, flow and residual urine. A detailed questionnaire was completed by the patients preoperatively and at various intervals postoperatively. In the patients' judgement both operations brought about a fast and significant improvement of their symptoms. Sexuality, erectile function and libido were not reduced. During the 6-month follow-up no significant differences was found between the two operative procedures in their effects on the parameters examined. Thus, the choice of operation can be based entirely on other parameters, such as prostate weight.


[Incidental prostatic cancer--"wait and see" or radical prostatectomy?].

In a comparative and retrospective study, outcome was examined in two groups of patients with incidental carcinoma of the prostate (stage T1a). In the 47 patients in the first group the carcinoma was kept under observation for 2-10 years without the administration of any treatment. During the mean follow-up of 5 years, the disease progressed in 11 (23.4%) of these patients, and 4 of them died. The other group was made up of 20 patients who underwent radical prostatectomy. The clinical and pathological staging were quite different in this group than in the other: more advanced stages and higher grades were seen in 10 patients, and the specimens taken from 6 were found not to contain any tumour. After a mean follow-up period of 4.5 years, all these 20 patients are alive with no evidence of disease. The management of patients with T1a tumour is still a problem. Observation only will be followed by progression in 23% of cases, while radical prostatectomy actually constitutes overtreatment in 30%. In conclusion, we prefer to perform prostatectomy in all patients with T1a carcinoma who are young and have a good life expectancy.


[Solitary osseous metastasis in a patient with germ cell tumor--successful chemotherapy].

A 25-year-old patient with testicular teratoma (pathological stage I) relapsed with a solitary, symptomatic metastasis in the right humerus 8 months after unilateral orchiectomy and lymphadenectomy. Treatment consisted in three cycles of standard chemotherapy with etoposide, bleomycin and cisplatin (the last replaced by carboplatin in courses 2 and 3). No supplementary treatment proved necessary to achieve complete remission and complete physical rehabilitation. Only four cases of solitary bone metastasis of testicular germ cell tumors have been reported previously. The present case illustrates that, contrary to previous reports, symptomatic skeletal metastases of germ cell tumors can safely be treated with standard chemotherapy alone.


[New conservative therapeutic approaches in benign prostatic hyperplasia].

The efficacy of treatment for benign prostatic hyperplasia (BPH) is presently under critical consideration. In addition, various new therapeutic modalities are currently being evaluated. When medicamentous treatment is planned, in particular, the natural history of the disease must be carefully considered. Summarized data from several studies indicate that spontaneous improvement of symptoms may occur within 3-6 months, while in most cases deterioration takes a longer period of time. As intraprostatic urethral pressure depends on prostatic volume as well as on tone of the prostate smooth muscle, different medical treatment modalities seem reasonable. The dynamic component of the smooth muscle cells may be influenced by alpha-blockers. Administration of selective alpha 1-blockers will be advantageous as these have fewer side effects. Prostate volume represents the static component, which can be influenced by hormone treatment. Androgen deprivation via surgical castration must now be regarded as of historical interest only. Antiandrogens or LH-RH analogues have undesirable side effects and are expensive, making such treatment unacceptable for routine use. 5 alpha-Reductase inhibitors may emerge as a new treatment form allowing androgen suppression with a low rate of side effects. As it has been proposed that estrogens play an important role in the regulation of prostatic growth, aromatase inhibitors, which inhibit metabolization from androgens to estrogens, may receive special attention in the near future. Based on the theory that androgens may be of special importance for the epithelium, while estrogen action may be concentrated on the stroma, a combined treatment with inhibitors of 5 alpha-reductase plus aromatase may be even more effective.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential diagnostic patterns of lung neuroendocrine tumours. A clinico-pathological and immunohistochemical study of 122 cases.

A series of 3 tumourlets (TLs), 81 typical carcinoids (TCs), 14 atypical carcinoids (ACs) (well-differentiated neuroendocrine carcinomas, WDNCs) and 24 small cell-intermediate cell carcinomas (SCC-ICCs) of the lung were studied. Histopathological features were correlated with amine and peptide hormone immunoreactivity and with clinical data. All types of tumours expressed general neuroendocrine (NE) markers: Grimelius positivity and chromogranins were detected more frequently in well-differentiated (TLs, TCs) than in less well differentiated tumours [ACs (WDNCs) and SCC-ICCs] whereas neuron specific enolase (NSE) was prominent in the latter tumours. TLs and peripheral TCs were benign, often showing a paraganglioid pattern and frequently expressing gastrin-releasing peptide (GRP), which is present in the peripheral airways of normal lung. Central TCs were associated with lymph node metastases in 8.5% of the cases, frequently had a trabecular architecture, often associated with human milk fat globule 2 (HMFG2)-positive acinar and rosette-like structures, and were mainly immunostained for the alpha-subunit of human chorionic gonadotrophin (alpha-hCG) and serotonin. ACs (WDNCs) were associated with intrathoracic and/or extrathoracic metastases in 57.1% of the cases with a mortality rate of 35.7%. Their histological and cytological features were intermediate between those of TCs and SCC-ICCs. ACs (WDNCs) expressed serotonin and alpha-hCG less frequently than TCs. All SCC-ICCs were surgically treated and displayed a mortality rate of 91.6% with a mean survival of 10.2 months after operation. These tumours were characterized by high expression of HMFG2 and NSE, while the expression of both orthotopic (serotonin, GRP) and ectopic (ACTH) specific NE substances was very low. Since all TCs (either central or peripheral) had a favourable outcome, while about 36% of ACs (WDNCs) were fatal, the latter seem more appropriately designated "well-differentiated NE carcinomas". The differential diagnosis between different NE tumours of the lung is important and is mainly based on morphology. Both panendocrine and specific immunohistochemical markers are helpful in distinguishing the less aggressive, mostly benign varieties from the more malignant varieties.


Granular cell tumour of the breast.

Eight cases of benign granular cell tumour of the breast are reported. Seven patients were women and one was male. The age at the time of the excision ranged from 17 to 73 (average 40.1) years. All tumours were positive for S-100 protein and negative for keratin, myoglobin and gross cystic disease fluid protein. In two cases ultrastructural studies revealed findings identical to those in the previously reported cases of granular cell tumours. None of these cases were diagnosed preoperatively. In six cases the clinical and mammographic findings, and in one case the frozen section, led to an erroneous diagnosis of malignancy. The clinico-pathological features of the cases are delineated in order to draw attention to a benign condition which closely simulates malignancy.


Histological reactivity of a monoclonal antibody against rat colon cancer cells on human and rat normal gut and colonic tumours.

A monoclonal antibody, F11C, was raised against rat colon cancer cells. Its immunoreactivity on normal human and rat gut as well as human and rat colonic tumours was studied by the avidin-biotin-peroxidase complex technique. In both normal rat and human gastrointestinal tract, F11C stained surface epithelial cells from the fundus to distal colon, mainly as supranuclear vesicles. These vesicles appeared to be part of the Golgi apparatus on electron microscopy with immunogold labelling. Twenty primary rat colon tumours and 28 of 43 human colon tumours were also stained, with a heterogeneous pattern but much more strongly than the normal colonic mucosa. Biochemical purification suggested that in rat tumours F11C epitope was carried by a high molecular weight glycoprotein. Absorption experiments with synthetic oligosaccharides showed that F11C monoclonal antibody reacted with blood group A-related oligosaccharides. Nevertheless, F11C reactivity on human tissues was not related to the individual ABO or Lewis phenotype.


[Itching following therapy with hydroxyethyl starch (HES) in otoneurological diseases].

It is generally assumed, that a disturbance of microcirculation is the common pathogenetic end factor in various cochleovestibular disorders of different etiology. Therefore improvement of microcirculation is an important therapeutic goal. Several studies demonstrated, that hydroxyethylstarch (HES) has better haemorheological effects than Dextran and less side effects. For this reason we have changed the therapy with Dextran since 1987 to hydroxyethylstarch in several oto-neurological disorders (as sudden hearing loss, neuronopathia vestibularis, idiopathic facial palsy). As after the therapy with HES--generally after dismissal from the ENT-department--some patients complained of general pruritus, so we performed a retrospective study with a standardized interview-protocol. Of 481 treated patients we investigated 237 (49%): of 149 patients treated with HES 200/0.5, 43 patients (28.8%) complained of pruritus; from 88 patients treated with Dextran 40, only 5 patients (5.7%) reported pruritus. The difference is significant (p less than 0.0001). In nearly half of the patients (more than 40%) the pruritus started in normal skin 1 to 3 weeks after the HES-therapy and lasted for 6 weeks to 6 months; the itching was very resistant to therapy (f.e. with antihistaminics). We want to draw the attention to this possible, in the literature until now quite neglected, for some patients extremely uncomfortable and socially embarrassing side effects after HES-therapy when given in relatively high doses. It is therefore suggested therapeutic recommendations should be developed to prevent this undesired side effect.


[The fresh weight and the concentration of DNA, RNA and protein in different tissues of swine of different ages].

Female pigs aged between one day (body weight 1.39 +/- 0.20 kg) and 1123 days (b. w. 233 +/- 32 kg) were allotted to 10 groups of various age and analyses of the concentration of DNA, RNA and protein in 9 different tissues were performed. The wet weight: DNA-, the protein: DNA- and the RNA: DNA-ratio were determined. With the exception of the liver the content in DNA, RNA and protein increased to the age of 1123 days. The highest fresh weight: DNA ratio of 2041 was reached on the 1123rd day in the M.longissimus; the smallest (111) existed at this time in the spleen. In the liver the highest content in fresh weight, in DNA and RNA was found on the 180th day. The smallest increases in the content of DNA and RNA in the mentioned period were in the brain, the highest in the spleen, the liver and the M.longissimus dorsi. The values can be used as a basis for studies on the influence of feeding and of diseases (infections) on growth.


[Demonstration of glycosaminoglycans (GAGs) in fetal human trabecular tissue].

The ultrastructural localization of glycosaminoglycans (GAGs) in the developing human outflow apparatus was investigated. The aqueous outflow system from human eyes at 26th and 36th fetal week and 2 years of age was stained with ruthenium red to identify GAGs with the transmission electron microscope. Luminal surface of the inner wall of the Schlemm's canal, basal lamina of the endothelial cells, basal lamina-like material, amorphous substances and collagen fibrils in juxta-canalicular tissue were associated with ruthenium red-stainable material. The basal lamina of the endothelial cells of Schlemm's canal was stained less obviously in 2-year-old trabecular tissue. The composition of the ruthenium red-stainable material was determined by treatment of each tissue with streptomyces hyaluronidase, chondroitinase AC, and chondroitinase ABC respectively. Hyaluronic acid was identified in each ruthenium red-stainable extracellular component. Chondroitin sulfate was identified in all ruthenium red-stainable components except luminal surface of the canal. The presence of dermatan sulfate was confirmed in the amorphous components and collagen fibrils of juxta-canalicular tissue. The results suggest that GAGs in fetal trabecular tissue already contribute to the outflow resistance and that alterations of the pattern of GAGs may take place as development proceeds.


[Lymphocyte subsets of peripheral blood in patients with sarcoidosis].

Cytofluorometric analysis of lymphocyte subsets was performed in the peripheral blood samples from fifteen sarcoidosis patients with uveitis as well as normal controls. The percentage of T cells (CD3+) was significantly decreased, whereas that of B cells (CD19+) was markedly increased in patients with sarcoidosis compared to controls. Natural killer cells (CD8+CD57+) as well as activated T cells (CD3+HLA-DR+) were significantly increased in patients with sarcoidosis. The percentage of the Leu 1-B cells and the ratio of Leu 1-B/B were significantly higher in sarcoidosis patients with respect to controls. Moreover, the ratio of Leu 1-B/B was significantly correlated with the serum level of angiotensin converting enzyme (ACE) in sarcoidosis. Therefore, it is considered that the Leu 1-B/B value could be a useful indicator not only for the activity but also for the diagnosis of sarcoidosis. No significant difference was found in any subset between the patients with active uveitis and patients with inactive uveitis.