[Thin layer chromatography for identification of 9 species of traditional Chinese medicine in baihe gujin tang granules]. Baihe Gujin Tang granules are mainly composed of nine species of traditional Chinese medicine and other admixtures, so the composition is very complex. In order to confirm the presence of each traditional Chinese medicine in the preparation, a thin layer chromatographic method was developed successfully for analysis of Scrophulariae, Platycodon, Paeoniae and Glycyrrhizae.


[Granule-making technology for preparing luohanguo chrysanthemum granule medicine with orthogonal design]. L9 (3(4)) orthogonal design was adopted to inspect the consumption of white sugar, water and ethanol and the duration of raw material mixing in relation to the granule-attaining rate in preparing Luohanguo-Chrysanthemum granule medicine. The result shows that with a consumption of 170 kg of white sugar, 6000 ml of water and 800 ml of ethanol and a duration of 8 minutes, the granule attaining rate reaches about 80%.


[Chemical constituents of Actinidia kolomikta (Rupr. et Maxim.) Planch]. Two crystalline components I and II were isolated from the roots of Actinidia kolomikta. I was identified as delta 7-stigmasterol and II was named alpha-kolomiktriose and elucidated as 2, 3-di-0-beta-D-galactopyranosyl-alpha-D-galactopyranose on the basis of spectral data (13C NMR, 1H NMR, IR and EI-MS).


[Chemical components of the leaves of Pistacia Chinensis Bge]. Six compounds have been isolated from the leaves of Pistacia chinensis, a species of Anacardiaceae family. Their structures were identified on the basis of UV, IR, NMR, and MS as gallic acid, m-digallic acid, quercetin, 6-0-galloyl arbutin-quercitrin and quercetin-3-0(6''-galloyl)-beta-D-glucosides.


[Pharmacokinetics of sinomenine by HPLC]. Pharmacokinetics of sinomenine in rabbit was studied by reverse-phase HPLC method. After i.v. injection of 20 mg/kg of sinomenine to rabbits, the pharmacokinetic characteristics were found to fit a two-compartment open model.


[The effects of dachengqi decoction, shaogan decoction, emodin and sennoside on the histamine level of intestinal mucosa in intestinally obstructed rats]. The experimental intestinal obstruction model was made by partly ligating the ileum in rat. The histamine levels of obstruction groups (8.14 +/- 2.28 micrograms/g) were obviously lower than those of control groups (P < 0.01). After administration of Dachengqi decoction, Shaogan decoction, emodin and sennoside, the levels of histamine were significantly higher than those of obstruction groups, but there were no significant differences as compared with control groups.


[Hypoglycemic effect of fructus Ligustri Lucidi]. The decoction of Fructus Ligustri Lucidi (FLL) 15.30 g/kg ig for 10 days significantly decreased the blood glucose level in normal mice. FLL 30 g/kg before or after the treatment of alloxan also decreased the blood glucose level in alloxan diabetic mice. The elevation of blood glucose level induced by adrenaline or glucose was antagonized by FLL.


A new role for the cerebellum in cognitive operations. Over the last 2 centuries, the predominant view of the cerebellum has been that it is part of a motor control system. Evidence is now presented that the neocerebellum, the evolutionarily newest region of the cerebellum, may also be involved in a key mental operation: the voluntary shift of selective attention between sensory modalities. It is theorized that this newly recognized function may operate via previously described sensory modulation properties of the cerebellum and its many connections with areas known to be important for selective attention, such as the pulvinar, the superior colliculus, and the parietal and frontal cortices.


Cerebellar stimulation as an unconditioned stimulus in classical conditioning. Rabbits were implanted with chronic stimulating electrodes in white matter underlying lobule HVI of the cerebellar cortex. Stimulation elicited movements of the face or neck and, when paired with a tone conditioned stimulus (CS), produced learning comparable to that seen with peripheral unconditioned stimuli (USs). CS-alone trials produced extinction. Reinstatement of paired trials produced reacquisition with savings. Additional groups received either explicitly or randomly unpaired CS-US trials before paired conditioning. Low-frequency responding during these sessions indicated that the paired training results were associative and not due to pseudoconditioning or sensitization. Explicitly unpaired sessions retarded learning on subsequent paired trials compared with groups that received either randomly unpaired or no CS-US preexposure. These results are interpreted in terms of the role of the cerebellum and associated pathways in classical conditioning of motor responses.


Hippocampus, fimbria-fornix, amygdala, and memory: object discriminations in rats. Rats with lesions of the fimbria-fornix, hippocampus, or hippocampus and amygdala were tested in object discriminations commonly used with monkeys. Two 1-pair object discriminations were learned preoperatively and tested postoperatively. Additional postoperative testing included acquisition of a third 1-pair object discrimination, an 8-pair concurrent object discrimination, and spatial alternation. All lesions impaired performance in the 8-pair object discrimination and in spatial alternation but not in the 1-pair object discriminations. Data from this study and from previous studies indicate that the hippocampus in both rats and monkeys has an important role in the mnemonic processes required for concurrent object discriminations and that variations of the procedure for concurrent object discriminations can be an effective tool for investigating hippocampal function.


Complementary roles of the orbital prefrontal cortex and the perirhinal-entorhinal cortices in an odor-guided delayed-nonmatching-to-sample task. Continuing efforts toward designing odor-guided tasks for rats that are similar in memory demands to tasks used typically with primates have resulted in the development of a continuous delayed-nonmatching-to-sample (cDNM) task that is guided by olfactory stimuli. The results indicate that normal subjects acquire the cDNM task rapidly and that subsequent performance deteriorates with increases in memory delay or interitem interference. Moreover, different aspects of cDNM performance were shown to be differentially sensitive to selective lesions of the orbitofrontal and parahippocampal areas. Orbitofrontal cortex lesions disproportionately impaired cDNM acquisition; delay performance was impaired only under conditions of elevated levels of interitem interference. Combined perirhinal and entorhinal cortical lesions had no effect on cDNM acquisition but impaired cDNM performance at longer delays across all levels of interference. Fornix lesions did not impair either acquisition of cDNM or subsequent performance across long delays and increased interference. This pattern of impaired and spared capacities is similar to that observed in monkeys after lesions of analogous areas and is consistent with the notion that the prefrontal cortical system contributes preferentially to learning general task "rules" such as the nonmatching rule that is inherent in cDNM, whereas the perirhinal and entorhinal cortical areas are involved in the intermediate-term maintenance of memories for specific information.


Increased training in an aversively motivated task attenuates the memory-impairing effects of posttraining N-methyl-D-aspartate-induced amygdala lesions. This study was designed to examine the effect of variations in the amount of preoperative training on the retention deficit produced by posttraining lesions of the amygdaloid complex (AC). Rats received 1, 10, or 20 training trials in a footshock-motivated escape task 7 days before receiving N-methyl-D-aspartate lesions of the AC. Inhibitory avoidance retention performance, which was measured 4 days postoperatively, indicated that increased training improved retention in AC-lesioned animals as well as in control animals. The retention performance of AC-lesioned animals was impaired when compared with that of controls; however, the impairment was partially attenuated by increased preoperative training. The finding that AC-lesioned animals displayed greater locomotor activity on the retention test compared with nonlesioned controls suggests that the increased activity may have contributed to the impaired inhibitory avoidance retention performance. Two days after the retention test, some of the AC-lesioned animals were subsequently trained on a continuous multiple-trial inhibitory avoidance response in the same apparatus. AC lesions did not block acquisition or retention of the task. These findings suggest that the amygdala may not be a critical site for the permanent changes mediating stimulus-affect associations based on extensive training.


Effects of unilateral parietal lesions on spatial localization in the rat. In the first of two experiments, rats with left or right parietal lesions and controls were tested in place and landmark navigation in the water maize. Right parietal lesions resulted in deficits in both tasks, but especially landmark navigation. Lateralized effects appeared mainly in latency to find the platform. Experiment 2 investigated the role of the corpus callosum. Split-brain rats with unilateral parietal lesions were tested on the same two tasks. Place and landmark deficits were particularly severe, but lateralization was weaker. Callosum section had its own effect, impairing the learning of both tasks. There appear to be additive effects of unilateral cortical lesions and bisection of the hemispheres. The impairment from left lesions equaled the right-lesion deficit because of the interruption of compensatory information from the intact right hemisphere and the effect of callosum section itself.


Effects of prenatal exposure to cocaine on conditional discrimination learning in adult rats. Adult male rats that were gestationally exposed to cocaine and control offspring were trained on an instrumental conditioning task for assessment of the acquisition and reversal of an appetitive conditional discrimination based on olfactory cues. Offspring were derived from Sprague-Dawley dams that had received subcutaneous injections of 40 mg/kg/3 cc cocaine hydrochloride (C40) daily on Gestational Days 8-20, pair-fed (PF) dams that were injected with saline, nutritional control dams (NC) that had received saline injections, and nontreated control dams (LC). There were no differences among the prenatal treatment groups in acquisition of the barpress response or response rate throughout all phases of training. All prenatal treatment groups required approximately the same number of sessions to criterion on the initial odor discrimination. In contrast, adult C40 offspring required more sessions to acquire the reversal of the conditional discrimination than did animals from the other treatment groups (PF, NC, and LC). In addition, even at criterion performance for acquisition of the reversal discrimination, C40 animals exhibited lower accuracy on the first 10 responses and made significantly more errors before the first reward. Taken together with previous results, these findings suggest that gestational exposure to cocaine results in long-lasting alterations in performance on conditioning tasks that are evident early in life and that persist into adulthood.


Circadian modulation of the rat acoustic startle response. The acoustic startle response (ASR) of male rats was measured during several sessions over a 24-hr period in both a light-dark cycle and a constant-dark condition. Each session consisted of 10 trials each at 80, 90, 100, 110, and 120 dB white noise. The results indicate robust daily and circadian modulation of ASR amplitude that consist of an approximately twofold nocturnal increase at eliciting-stimuli intensities above 80 dB. Similar results were observed in female rats in constant-dark conditions. To determine whether daily changes in auditory thresholds were responsible for the observed modulation, ASR reflex modification procedures were used. These procedures were designed to measure auditory thresholds at frequencies of 10 and 40 kHz at several times of day. The results suggest a lack of significant circadian differences in auditory thresholds at these frequencies. This study demonstrates a novel role of the rat circadian system in the modulation of ASR amplitude.


The habenular complex mediates hormonal stimulation of maternal behavior in rats. The role of the habenular complex (Hbc) in the hormonal onset and nonhormonal maintenance of maternal behavior in rats was examined. In Experiment 1, bilateral lesions were produced in the Hbc on Gestational Day (GD) 12. On GD 16, animals were hysterectomized-ovariectomized and given estradiol benzoate (EB); they were then tested for maternal behavior 48 hr later. Hbc lesions delayed the appearance of all components of maternal behavior for several days. In Experiment 2, large Hbc lesions that were produced on Postpartum Day 4 caused only 1- or 2-day deficits in maternal behavior. These data suggest that the Hbc mediates the hormonal onset of maternal behavior. During the postpartum period, however, the importance of the Hbc for maternal behavior diminishes as the hormones of pregnancy become less important.


Opioid control of the fetal stretch response: implications for the first suckling episode. Upon their first experience with milk, fetal rats express a stretch response that is similar to the postnatal behavior exhibited by infant rats at the nipple. Fetuses also possess a functional opioid system that is activated by prenatal milk exposure. The opioid receptor antagonist naloxone and the specific kappa antagonist nor-binaltorphimine blocked the stretch response and prevented the increase in rearlimb activity that is typically induced by milk. The mu antagonist beta-funaltrexamine blocked the stretch while permitting the expression of rearlimb activity. The kappa agonist U50,488 promoted rearlimb activity in the absence of milk, whereas the mu agonist [D-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) exerted little influence on fetal behavior. Fetuses pretreated with U50,488 stretched to nonmilk stimuli (saline or lemon), but fetuses pretreated with DAMGO did not. Opioid activation is part of a chain of events that culminates in the fetal stretch response and may be important in promoting milk ingestion during the newborn's first suckling episode.


Phase I cancer trials: limitations and implications. The goal of Phase I clinical trials is to establish a maximum tolerated dose (MTD) and a tolerable dose range for future efficacy testing. Various issues include schedule of drug delivery, starting dose, number of patients to be tested, pitfalls in the selection of MTD, risk:benefit ethical issues, and the problems introduced by patient heterogeneity. Statistical evaluation of various Phase I strategies illustrate the limitations of various approaches in terms of patient expectations regarding efficacy and the scientific goals of such trials. Patients should be given the opportunity to receive the drug at more than one single dose level.


A gelonin-containing immunotoxin directed against human breast carcinoma. Toxins may be specifically directed to tumor cells and the toxins' potency greatly increased by covalent conjugation to monoclonal antibodies recognizing tumor-associated antigens. Antibody 15A8, an immunoglobulin G1 (IgG1) subclass anti-human breast carcinoma murine monoclonal antibody and gelonin, a plant toxin, were covalently modified with N-succimindyl 3-(2-pyridyldithio) proprionate and iminothiolane, respectively, and allowed to cross-link. 15A8-gelonin conjugates were purified from unreacted antibody and free gelonin by gel filtration and blue sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the final product contained two bands corresponding to antibody:gelonin conjugates of 1:1 (predominant) and 1:2. There were no contaminating amounts of free antibody or free toxin in the preparation. The yield of the final purified 15A8-gelonin conjugate was approximately 20% based on the amount of starting antibody. The protein synthesis inhibitory activity of the immunoconjugate was assessed by in vitro rabbit reticulocyte translation assay. This functional activity was normalized to that of unmodified gelonin for use in in vitro antiproliferative assays against antigen-negative (Hs294t human melanoma) and antigen-positive (ME-180 human cervical carcinoma) cell lines. Antigen-negative Hs294t cells incubated for 72 hours with 15A8-gelonin immunotoxin showed no increased cytotoxicity compared with HS294t cells exposed to free gelonin alone. However, the immunotoxin was preferentially toxic to antigen-positive ME-180 cells; over 5 logs greater cell kill was observed after 72 hours exposure to 15A8-gelonin than after the same exposure to gelonin alone. Various lysosomotropic agents augmented 15A8-gelonin cytotoxicity; the most effective potentiating agent appeared to be monensin. In addition, the chemotherapeutic agents L-phenylalanine mustard (L-PAM), 5-fluorouracil, vincristine, and bleomycin, and the biological response modifiers interferon-alpha and tumor necrosis factor-alpha were shown to augment 15A8-gelonin cytotoxicity. Should in vivo pharmacology and therapeutic studies confirm these in vitro findings, 15A8-gelonin conjugate may be a potent agent for therapy of cancer in man.


Our experience with interferon alpha: renal cell carcinoma. Our 3-year clinical experience using recombinant interferon (rIFN) alpha-C in patients with metastatic renal cell carcinoma (RCC) is summarized. This type of IFN is a new subspecies of the IFN-alpha protein family. Its specific activity is 1-2 x 10(9) U/mg protein, the highest among IFN-alpha species presently available. Pharmacokinetic study indicated good bioavailability of the preparation from the intramuscular injection. A phase II study was performed to assess the response rate related to rIFN-alpha C at a low dosage. A dose of 3 x 10(6) U daily was administered, followed by 3 x 10(6) U/m2 every other day to avoid severe toxicity. Among 33 treated patients, a partial remission rate of 9.7% and stable disease rate of 25.8% were achieved. Side effects were usually mild and the treatment was well tolerated by the patients. However, mental deterioration and behavioral changes were observed in five patients with RCC treated by rIFN-alpha C and were related to neurotoxicity of IFN. The role of vinblastine in addition to IFN in the treatment of RCC was assessed in nine patients who had failed on IFN alone. No response was observed. It appeared that vinblastine had little if any effect in being added to IFN as second-line therapy. We conclude that rIFN-alpha C has moderate activity in the treatment of RCC. Familiarity with the possible toxicity of this agent will lead to more careful management of patients.


Our experience with interferon alpha: metastatic malignant melanoma. Interferon-alpha and dacarbazine combination is a milestone in the treatment of metastatic malignant melanoma. Objective response rate ranged from 3% to 25%. Our phase II study included 34 patients; the overall response was 29.4%. Median time of survival of the responders was significantly longer than that of the nonresponders. Nine of the 34 patients had previously progressed on interleukin-2 (IL-2) and dacarbazine treatment, or had been withdrawn because of unacceptable toxicity. Two patients (22.2%) achieved partial responses. There seemed to be no cross-resistance between the two biologic response modifiers. Successful treatment of melanoma patients by interferon resulted in complete disappearance of all extracerebral lesions, but left the brain vulnerable to involvement by metastases, and was frequently a site of relapse. Brain irradiation is suggested by several investigators to prevent cerebral involvement. Ongoing protocols are an adjuvant treatment for high-risk patients and combination of interferon-alpha, IL-2, dacarbazine and cisplatinum for metastatic melanoma after failure of interferon-dacarbazine regimen.


Interferon-alpha induced autoimmune hepatitis in a patient with Philadelphia chromosome-positive chronic myeloid leukemia with cytogenetically normal T lymphocytes. We report the occurrence of autoimmune hepatitis after treatment with interferon-alpha in a patient with Philadelphia chromosome-positive chronic myeloid leukemia. Cytogenetic and molecular genetic studies of the T lymphocytes in this patient demonstrated that the T lymphocytes were not part of the leukemic clone. The role of interferon in the production of autoimmune abnormalities is reviewed.


Phytochemicals potentiate interleukin-2 generated lymphokine-activated killer cell cytotoxicity against murine renal cell carcinoma. The success of adoptive immunotherapy using recombinant interleukin-2 (rIL-2) and lymphokine-activated killer (LAK) cells in several cancers has been hampered by severe toxicity associated with high doses of rIL-2. Methods that reduce the dosage of rIL-2 without loss of clinical efficacy are needed. In this study we determined the in vitro effect of a phytochemical immune modulator, Astragalus membranaceus (AM), and two fractions isolated by high-performance liquid chromatography on the cytotoxicity of rIL-2-generated LAK cells against a murine renal cell carcinoma. Our results indicated a 10-fold potentiation of rIL-2-generated LAK cell cytotoxicity manifested by tumor cell lysis of 88% in the group with 100 U/ml of rIL-2 plus AM versus 86% in the group with 1,000 U/ml of rIL-2 alone. Potentiation was obtained with the purified fractions as well. A significantly reduced number of LAK cells was required to achieve the tumor cytotoxicity after LAK cell generation with rIL-2 plus the phytochemicals as compared with rIL-2 alone. Our data indicate that AM is an effective immune modulator, capable of potentiating in vitro the antitumor activity of rIL-2-generated LAK cells.


Application of Corynebacterium cutis lysate as an immune stimulant in cattle. An ultrasonicated lysate of Corynebacterium cutis (Ultracorn, Virbac, France) was administered to 10-day-old calves, 5-month-old calves, and pregnant dams kept under Egyptian environmental conditions. Ninety-five calves and 50 dams were used in the study. All animals were treated with 2 ml/100 kg body weight of killed C cutis. Its effects on body weight gain and on calf mortality and morbidity were recorded. The results obtained showed that treated calves had greater weight gains, reduced susceptibility to common viral pathogens, and lower mortality. When given simultaneously with rinderpest vaccine, an immunopotentiating or adjuvant effect was seen. Thus, treated calves had higher neutralizing antibody titers to rinderpest as compared with untreated calves. When administered to pregnant cows in the last month of pregnancy, the offspring of these animals had higher birth weight, better weight gain, and reduced morbidity.


Recombinant interleukin-2 significantly increases the survival of mice with peritonitis, but not acute Staphylococcus aureus peritoneal infection. The effect of recombinant interleukin 2 (rIL-2) on survival of mice with peritonitis and acute Staphylococcus aureus strain 5/2 infection was studied. rIL-2 was ineffective in the case of acute infection when administered simultaneously with LD95 dose of bacteria. The antibiotics (gentamycin or a combination of penicillin and streptomycin) administered in the same fashion cured 100% of animals. rIL-2 proved to be a potent healing agent in the two of three models of S aureus peritonitis. In this case animals received bacteria at days 0 and 2, 4, or 6. rIL-2 was injected at day 0 (group 1), days 0 and 2 (group 2), and days 0, 2, and 4 (group 3). Treatment with rIL-2 was ineffective in group 1; however, in groups 2 and 3 rIL-2 increased the survival up to 90% (in comparison with 30% in the untreated animals of group 2 and 64% in group 3). On the contrary, administration of antibiotics instead of rIL-2 in the group 3 decreased survival to 25%. The perspectives of rIL-2 use in the treatment of bacterial peritonitis, including purous ones, and the cases complicated by immunodepression, are discussed.


Structure of sp-9-hydroxy-9-pivaloylfluorene, product of base-catalyzed autoxidation of ap-9-pivaloylfluorene. 1-(9-Hydroxyfluoren-9-yl)-2,2-dimethyl-1-propanone, C18H18O2, M(r) = 266.34, orthorhombic, Pbcn, a = 18.917 (10), b = 11.843 (8), c = 13.177 (7) A, V = 2952 (5) A3, Z = 8, Dx = 1.198 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.72 cm-1, F(000) = 1136, T = 296 K, R = 0.044 for 1042 unique observed reflections. Conversion of ap-9-pivaloylfluorene, ap-(I), into lithiated (I)-9-anion followed by the addition of MeOH, then H2O, led to unexpected hydroxylation to provide 20-40% of sp-9-hydroxy-9-pivaloyfluorene, sp-(II), and 60-80% recovery of ap-(I). The singular sp conformation of (II) in solution suggested by NMR was confirmed in the crystalline state by X-ray diffraction which showed the O(1)-C(9)-C(10)-O(2) torsion angle approximately 0 degrees and the C(10) = O(2)...H(9)-O(1) non-bonding distance 1.95 (4) A, suggesting strong intramolecular hydrogen bonding in this conformation.


Absolute configuration of (-)-alpha-acetylmethadol hydrochloride. beta-[2-(Dimethylamino)propyl]-alpha-ethyl-beta-phenylbenzeneethanol+ ++ acetate ester hydrochloride, C23H32NO2+. Cl-.H2O, M(r) = 407.9, monoclinic, P2(1), a = 15.608 (4), b = 8.637 (2), c = 17.273 (5) A, beta = 97.71 (2) degrees, V = 2307.4 (1) A3, Z = 4 (two methadol hydrochlorides and two water molecules per asymmetric unit), Dx = 1.17 Mg m-3, lambda (Cu K alpha) = 1.54184 A, mu = 1.64 mm-1, F(000) = 880, T = 295 K, final R = 0.069, wR = 0.064 for 2559 independent observed reflections. Both asymmetric C atoms in the two independent methadol molecules are 'S'. Of the two N-H moieties one acts as a hydrogen-bond donor to a Cl atom (N-H = 1.21, H...Cl = 1.85, N...Cl = 3.03 A, N-H...Cl = 162.3 degrees) and the other is a donor to a water molecule (N-H = 0.90, H...O = 2.06, N...O = 2.80 A, N-H...O = 138.2 degrees). In addition, both water molecules hydrogen bond to both Cl atoms with O...Cl distances in the range 3.06-3.36 A. A comparison of torsion angles for the two independent methadol molecules indicates that there is very little stereochemical similarity between them.


Structure of cis,cis-4,6-diphenyl-2-(2-propenyl)-1,3-dioxa-2-phosphorinane 2-oxide. C18H19O3P, M(r) = 314.32, monoclinic, P2(1)/n, a = 5.726 (6), b = 14.234 (7), c = 20.08 (1) A, beta = 92.54 (7) degrees, V = 1635 (4) A3, Z = 4, Dx = 1.277 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.71 cm-1, F(000) = 664, T = 296 K, R = 0.042, 1813 unique observed reflections. The X-ray structure determination of the title compound shows that the dioxaphosphorinane ring has a chair conformation in which the phosphoryl O atom (P=O) is equatorial, which explains the absence of substantial NMR deshielding by its P=O group on H(4) and H(6), which are axial.


Structure of a chiral intermediate in the synthesis of (+)-19-epiajmalicine. [2S*-(2 beta,3 alpha,6 alpha,12b beta)]-Methyl 3-acetyl-1,2,3,4,6,7,12,12b-octahydro-6-methoxycarbonyl-indolo+ ++[2,3-a] quinolizine-3-ethanoate, C22H26N2O5, M(r) = 398.46, orthorhombic, P2(1)2(1)2(1), a = 9.463 (2), b = 11.251 (3), c = 18.871 (6) A, V = 2009.2 (9) A3, Z = 4, Dx = 1.32 g cm-3 (178 K), lambda(Mo K alpha) = 0.7107 A, mu = 0.8762 cm-1, F(000) = 848, T = 178 K, R = 0.0536 for 1673 reflections [Fo > or = 6 sigma (Fo)]. Molecules are hydrogen bonded along the 2(1)-screw axis parallel to a. The hydrogen-bond geometric parameters for N12-H12...O19 (related by 0.5 + x, 1.5 - y, 1 - z) are N...O 2.986 (6), H...O 2.30 (5) A, N-H...O 161 (5) degrees. The C and D rings are trans fused with ring-junction torsion angles of -39.6 (5) and 63.8 (5) degrees for C12a-C12b-N5-C6 and C1-C12b-N5-C4, respectively. The conformation of the C ring is half chair with N5 and C6 -0.168 (4) and 0.552 (5) A, respectively, out of the plane defined by the remaining four atoms of the ring. The D ring is in the chair conformation.


(2S-[2 alpha,3 alpha,3a beta,6 beta(R*),7 alpha, 7a alpha])-6-(3-benzyloxy-2-propyl)-2-hydroxy-2,3,7-trimethylhexahydro-4H- furo [3,2-c]pyran-4-one, a rearrangement product of pyranone derivatives in the tirandamycin A series. The six- and five-membered heterocyclic rings are cis-fused and adopt twist-boat and half-chair conformations, respectively. The average bond distances are: Csp3-Csp3 1.520 (6), Csp3-Csp2 1.494 (6), C-Cbenz 1.360 (10) and Csp3-O 1.425 (5) A; the C = O and Csp2-O bond lengths are 1.214 (5) and 1.324 (5) A, respectively.


Structure of pentaamminechlororuthenium(III) bisulfate tetrahydrate. [RuCl(NH3)5](HSO4)2.4H2O, M(r) = 487.88, triclinic, P1, a = 10.0422 (5), b = 14.1044 (7), c = 6.3273 (6) A, alpha = 100.369 (5), beta = 98.655 (6), gamma = 81.354 (4) degree, V = 864.5 (3) A3, Z = 2, Dm = 1.86 (1), D chi = 1.874 g cm-3, lambda(MO K alpha) = 0.71073 A, mu = 13.3 cm-1, F(000) = 498, T = 296 (1) K, R = 0.025 for 4622 unique reflections with I greater than 3 sigma(I). The structure contains (NH3)5RuCl2+ cations, bisulfate anions and solvate water molecules all linked by an extensive hydrogen-bonding network. Each Ru ion is coordinated in a distorted octahedral fashion by five ammonia molecules and a chloride ion. The Ru--Cl distance [2.3742 (5) A] is typical for RuIII--Cl linkages. The Ru--NH3 distances span a narrow range [2.096 (2)-2.119 (2) A] and provide no evidence for a trans effect.


A chiral N-crotonyloxazolidinone Diels-Alder adduct. (4S)-4-Benzyl-3-[(4S,5S)-(1-methoxy-5-methylcyclohexen-4- yl)carbonyl]-2-oxazolidinone, C19H23NO4, M(r) = 329.40, monoclinic, P2(1), a = 11.453 (3), b = 7.163 (4), c = 11.929 (2) A, beta = 111.86 (2) degree, V = 908.3 (5) A3, Z = 2, D chi = 1.20 g cm-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.79 cm-1, F(000) = 352, T = 297 K, R = 0.034 for 885 reflections with Fo2 greater than 0. The molecule is extended in the crystal; there is a small twist, -13.1 (2) degree, about the amide-like C--N bond joining the oxazolidinone ring to the carbonyl group. The configurations at the two optical centers in the cyclohexene ring confirm the anticipated stereospecificity of the Diels-Alder cycloaddition synthesis.


Structure of 2,5:3,4-dianhydro-D-altritol. 3,6-Dioxabicyclo[3.1.0]hexane-2,4-dimethanol, C6H10O4, M(r) = 146.1, orthorhombic, P2(1)2(1)2(1), a = 7.6209 (2), b = 9.1292 (3), c = 9.6135 (5) A, V = 668.8 (1) A3, Z = 4, Dx = 1.451 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 1.15 cm-1, F(000) = 312, T = 298 K, R = 0.029 for 1280 observations with I greater than 3 sigma(I) (of 1695 unique data). The tetrahydrofuran ring has the envelope conformation, OE, with P of 94.3 degrees and tau m = 24.0 degrees. C atoms deviate from their best plane by +/- 0.0006 (1) to 0.010 (1) A, and the O atom lies 0.331 (1) A from that plane. The epoxide O atom is syn to the tetrahydrofuran O atom. Each hydroxy group is involved in intermolecular hydrogen bonding both as donor and acceptor. The two hydrogen bonds have O...O distances of 2.743 (1) and 2.729 (1) A, and angles about H of 166.3 (12) and 172 (2) degrees, respectively.


Structure of a tricyclic subunit of manzamine A. (4a alpha,7a beta,10a beta)-2-Benzyl-1,2,3,4,4a,7,7a,8,9,10-decahydro-8,8- dimethylpyrrolo[2,3-i]isoquinolinium iodide, C20H29N2+.I-, M(r) = 424.37, monoclinic, P2(1)/n, a = 9.441 (4), b = 10.378 (4), c = 20.023 (9) A, beta = 91.56 (3) degrees, V = 1961 (1) A3, Z = 4, Dx = 1.44 g cm-3 (188 K), lambda(Mo K alpha) = 0.7107 A, mu = 16.16 cm-1, F(000) = 864, T = 188 K, R = 0.0288 for 3070 reflections [Fo greater than or equal to 4 sigma(Fo)]. The crystal structure determination was undertaken in order to establish the configuration around C10a. The rings of the isoquinoline group are trans, with the pyrrole moiety cis fused. The A ring is in the chair conformation, while the cyclohexene ring, B, is in the boat conformation owing to the cis fusion of the five-membered pyrrole ring. The pyrrole ring, C, assumes the half-chair conformation. The C-N bonds of the quaternary N atom, N8, are longer than those of the tertiary N atom, N2 [1.517 (2) for N8 and 1.463 (2) A for N2].


3D structure determination from electron-microscope images: electron crystallography of staurolite. Resolution of better than 2 A has been obtained in many crystals by high-resolution electron microscopy. Although this resolution is sufficient to resolve interatomic spacings, structures are traditionally interpreted by comparing experimental images with contrast calculations. A drawback of this method is that images are 2D projections in which information is invariably obscured by overlap of atoms. 3D electron crystallography, developed by biophysicists to study proteins, has been used to investigate the crystal structure of staurolite. Amplitudes and phases of structure factors are obtained experimentally from high-resolution images (JEOL ARM 1000 at the National Center for Electron Microscopy at LBL), taken in different directions from thin regions where dynamic scattering is minimal. From images in five orientations (containing 59 independent reflections to a resolution of 1.38 A), a 3D electron potential map is constructed which resolves clearly all cations (Al, Si, Fe, including those with partial occupancy) and all O atoms. This method has great potential in crystal structure determinations of small domains in heterogeneous crystals which are inaccessible to X-ray analysis. It is estimated that 3D structure determinations should be possible on regions only about ten unit cells wide and should resolve not only atom positions but also site occupancies. The method is also applicable to space-group determination.


Polarized dispersion, glide-rule-forbidden reflections and phase determination in barium bromate monohydrate. Reflections forbidden by a glide-plane rule are observed in diffraction experiments with a crystal of barium bromate monohydrate using linearly polarized synchrotron radiation with wavelength near the bromine K-absorption edge. Their intensities change with azimuth in agreement with equations derived using a tensor model of the anomalous scattering of the bromate ion and are consistent in scale with earlier measurements of that tensor in sodium bromate. The intensity of each forbidden h0l reflection gives the magnitude and phase of the bromine part of the structure factor of the allowed 2h,0,2l reflection. The x and z coordinates of the Br atom determined from such data for 11 reflections are within 0.02 A of those from two crystal structure determinations.


Improving multiple isomorphous replacement phasing by heavy-atom refinement using solvent-flattened phases. Solvent flattening of macromolecular MIR electron density maps is frequently used to improve the quality of the phases and the interpretability of resultant electron density maps. A new method is presented by which the heavy-atom parameters of isomorphous derivatives are refined against these same solvent-flattened phases and is shown to enhance convergence of the parameters by decoupling heavy-atom-parameter adjustment from parent-phase calculation. This approach is described here in the first example of its application in the solution of the glutaminyl-tRNA synthetase-tRNA(Gln)-ATP co-crystal structure.


Heavy-atom refinement against solvent-flattened phases. A new algorithm for refinement of heavy-atom parameters is defined by an iterative procedure where external phases are provided by density modification. This algorithm is applied to two cases, tRNA(Asp) and the complex between tRNA(Asp) and aspartyl-tRNA synthetase. In the first case, where the structure was solved by multiple isomorphous replacement (MIR) methods, it was found that the new method gives accurate values for the native-derivative scale and four occupancy of heavy-atom sites. Position refinement was more delicate and it needed to be handled in a restricted resolution range. In the second case, where a similar method was used in the early stages of solving the phase problem, it slightly decreased the phase error. It was followed by an improvement of the density-modification masks, which led to better maps at higher resolution.


A gas-chromatographic determination of the ppb levels of volatile halogenated hydrocarbons in water for injections. Some volatile halogenated hydrocarbons have been found in commercial large volume parenterals (LVPs) prepared from untreated or treated (disinfected) water. To monitor the presence of volatile halogenated hydrocarbons in the source water and also in the water for injections, a low cost and sufficiently simple procedure has been developed, specifically for the following components: 1,1,1 trichloroethane, 1,1,2 trichloroethylene, 1,1,2,2 tetrachloroethylene, carbon tetrachloride, chloroform, dichlorobromomethane and dibromochloromethane. A Head-space technique coupled with ECD-gaschromatography was used. The procedure is thoroughly discussed in the article, including the results of a ring test for a preliminary validation of this method.


New oral system for timing-release of drugs. Polymeric barriers applied by compression have already been used to control drug release rate from matrix tablets. In this paper, polymeric barrier layers, used to prepare and develop a new device able to release the drug after a programmable period of time, are described. Some matrix core formulations, containing Trapidil or Sodium Diclofenac as a model drug, were dry-coated using either a swellable or an erodible shell. This coating prevents drug release from the core until the polymeric layer is not completely eroded or swollen. The time-lag can be modified by changing the barrier formulation and/or the coating thickness. Also drug release profiles (release rate and kinetics), can be widely modified changing the barrier layer characteristics.


[A new inclusion complex of silibinin and beta-cyclodextrins: in vitro dissolution kinetics and in vivo absorption in comparison with traditional formulations]. The very low bioavailability of silybinin, the main constituent of silymarin, so far prevented the development of an oral pharmaceutical specialty based on this active ingredient. To overcome this difficulty, an inclusion complex between Silybinin and beta-Cyclodextrin was prepared. The new complex was compared in vitro tests (dissolution rate) and in a in vivo test (rat bile elimination) with silybinin, silymarin and one traditional formulation based on silybinin. The results show a dramatic increase in the dissolution rate of the complex (> 90% within 5 min) respect to the silybinin that confirm to be practically insoluble (< 5%). The in vivo results agree with the dissolution rates; after administration of the silybinin complex p.o., the silybinin concentration in the rat bile was near 20 times more than after administration of silybinin as is or in a traditional formulation. In the last two cases, the silybinin concentration was even 6 times less than after administration of the same amount of silymarin. These data show that the beta-CD complex solved the problem of the bioavailability of silybinin which, in the traditional formulation utilised as reference, proved to be not bioavailable.


Stability of phenobarbital sodium in liquid pharmaceutical preparations. Shelf-lives of liquid pharmaceutical preparations (injectable and oral solution) containing phenobarbital sodium, commercially available in Brazil, were predicted by accelerated stability test using isothermal method (at 37 degrees C, 50 degrees C and 75 degrees C). Data obtained in high temperature studies using Arrhenius relation were applied to predict shelf-lives at room temperature. The shelf-lives were calculated by linear regression analysis. UV difference spectrophotometry was used for phenobarbital determination. Thin-layer chromatography was used for separation and identification of phenylethylacethylurea, the main degradation product of phenobarbital.


Standardization of process parameters for salbutamol sulphate microcapsules coated with cellulose acetate phthalate and computation of in-vitro release kinetics. Salbutamol sulphate, a bronchodilatatory drug for asthma, is encapsulated by Emulsion-Solvent-Evaporation technique using cellulose acetatephthalate as coating polymeric material. Different process parameters like Stirring Speed, Drug-Polymer Ratio, Solvent-Polymer Ratio, Internal Phase-External Phase Ratio, Effects of Temperature, and of Surfactants have been studied to standardise the process. At optimal condition of process parameters maximum encapsulation efficiency is obtained and microcapsules produced are free flowing and spherical. The dissolution of the microcapsules is carried out and the data obtained from in-vitro dissolution profile are computed in the light of different kinetic models. Diffusion coefficient (Da) and diffusivity rate constant (KBL) are evaluated with the help of Baker-Lonsdale model.


The microbial controls of antiseptics and disinfectants by membrane filtration: experimental evaluation of the interferences. Microbiological investigations made by membrane filtration method on antiseptics and disinfectants demonstrated that the filtering membranes present very frequently a remarkable antimicrobial activity, even after washing with 300 ml of peptone water according to the guidelines of the Pharmacopoeia. The observed phenomena (appearance of inhibition zones, disappearance or decrease of the number of colonies forming units, release of antimicrobial properties to the cultural broths) can seriously diminish the validity of microbiological controls of antiseptics and disinfectants.


Oncogene activation in rheumatoid synovium. Rheumatoid arthritis (RA) is a chronic systemic disorder that is dominated by the debilitating sequelae associated with the progressive destruction of articular joints. The molecular and cellular basis of rheumatoid joint destruction is characterized by an abnormal expression of oncogenes modulating cellular proliferation and the induction of lysosomal and metalloproteinases. Based on the observation that the synovial hyperplasia in RA is associated with the proliferation of transformed-appearing synovial lining cells and an overexpression of such oncogenes, the possibility that a hitherto unknown HTLV related retrovirus is involved in the etiopathogenesis of RA is discussed.


Factors affecting quantitative assessment of Pseudomonas aeruginosa adherence to buccal epithelial cells. The influence of experimental protocol on estimation of adherence of P. aeruginosa to buccal epithelial cells is highlighted. Use of membrane filtration to remove non-adherent bacteria was significantly affected by membrane pore size and rinse volume, but was not affected by inoculum size or test isolate. Even with optimum filtration conditions, over 58% of the non-adherent bacterial population were retained on the filters. Separation of non-adherent bacteria was greatly improved when density centrifugation was employed, with less than 30% of non-adherent bacteria present in the buccal epithelial suspension. Adherence values measured by a radiological and fluorometric method were significantly higher than those determined by microscopic counting. Radiological assays had the lowest variance. Gradient centrifugation was used to prepare a standard suspension of buccal epithelial cells. However, cells collected on different days showed significantly different adherence measurements. Adherence of bacteria to trypsinized buccal epithelial cells and to buccal epithelial cells from cystic fibrosis patients was similar, whereas adherence to normal buccal epithelial cells was significantly lower.


Descriptive epidemiology of infections due to thermotolerant Campylobacter spp. in Norway, 1979-1988. This report reviews the first ten years of Campylobacter surveillance in Norway. During 1979-1988, a total of 3,545 isolates of thermotolerant Campylobacter spp. were reported. The isolation rate increased from 1.8 per 100,000 persons per year in 1979 to 13.1 in 1988. The highest isolation rate for both sexes occurred during the first five years of life (31.0 per 100,000). A smaller second peak was detected in the age group 15-24 years (11.1 per 100,000). The male-to-female ratio was 1.52:1 for infants less than five years of age, compared with a ratio of 1.35:1 for all ages combined. Thirty-eight percent of the infections had most probably been acquired abroad. The isolation rate in urban areas (12.4 per 100,000) was over twice that observed in rural agricultural municipalities (5.5 per 100,000). However, this difference was largely due to a higher proportion of imported cases in urban areas, only small variations in isolation rate were observed when imported cases were excluded. The seasonal distribution of Campylobacter isolates showed a peak during the warm months of the year. Travel activity during summer holidays did not account for this trend, since the summer peak became even more pronounced when imported cases were excluded. A north-south gradient in the seasonality was observed; when domestic cases were considered, the summer peak became more accentuated with increasing latitude reaching a maximum in subarctic areas. This might be explained by corresponding variations in occurrence of campylobacters in surface water sources.


Capsular polysaccharide is linked to the outer surface of type 6A pneumococcal cell walls. The in situ attachment of capsular polysaccharide of type 6A pneumococci was examined by immunoelectron microscopy using anti-type 6A monoclonal antibody. The result discloses an asymmetrical cross-section of pneumococcal cell walls because capsular polysaccharides are located on the outer surface of the walls only, in contrast to the cell wall polysaccharide, which has been shown to be located on both surfaces.


Serum levels of fetal antigen 2 in hyperthyroidism and primary hyperparathyroidism. Serum concentration of fetal antigen 2 (FA2) in patients with hyperthyroidism (n = 18) (median: 12.9 mAU/l; range: 3.2-22.4 mAU/l) was significantly (p < 0.002) higher than in age- and sex-matched healthy controls (median: 4.1 mAU FA2/l; range: 2.4-10.0 mAU FA2/l). Serum FA2 was positively correlated with thyroxine (T4) (Rs = 0.51; p < 0.05), triiodothyronine (T3) (Rs = 0.64; p < 0.01), bone-Gla protein (BGP) (Rs = 0.70; p < 0.01), total alkaline phosphatase (total-AP) (Rs = 0.62; p < 0.01), bone isoenzyme alkaline phosphatase (bone-AP) (Rs = 0.63; p < 0.01), N-terminal procollagen type III (PIIINP) (Rs = 0.65; p < 0.01) and urine OH-proline (OHP) (Rs = 0.79; p < 0.01). In patients with hyperparathyroidism the pretreatment levels of FA2 (n = 8) (median: 17.6 mAU/l; range: 5.2-35.0 mAU/l) were significantly (p < 0.001) higher than those of age- and sex-matched controls (median: 3.7 mAU FA2/l; range: 3.4-9.0 mAU/l). The pretreatment level of FA2 was positively correlated with the parathyroid hormone (PTH) (Rs = 0.80; p < 0.05). Following surgical treatment the serum concentrations of FA2, PTH, and BGP decreased compared to pretreatment levels and the fall in these three parameters revealed parallelism. These data indicate that serum FA2 can be used as a marker in the evaluation of metabolic bone diseases.


Haemagglutination patterns of Helicobacter pylori. Frequency of sialic acid-specific and non-sialic acid-specific haemagglutinins. Thirty-two Helicobacter pylori strains were screened for haemagglutination (HA) activity with erythrocytes of 11 different animal species. Twenty-three strains (72%) that agglutinated human erythrocytes exhibited a broad-spectrum HA profile. Human, guinea pig and bovine erythrocytes high in sialoglycoconjugates were strongly agglutinated by most strains. Except for two, seven strains (22%) that did not agglutinate human erythrocytes exhibited a narrow-spectrum HA profile, commonly not inhibitable by sialoglycoconjugates or N-acetylneuraminlactose (NANLac). Strains were classified into three major HA classes. HA of 10 strains (31%) in class I was inhibited by different combinations of NANLac, orosomucoid or fetuin, but not by asialofetuin, suggesting the presence of sialic acid-specific HAs probably recognizing NeuAc alpha-(2-3)- Gal isomer. Twelve strains (38%) in class II exhibited a different receptor specificity binding to different combinations of NANLac, orosomucoid and fetuin, as well as asialofetuin. No inhibition was observed with 10 strains (31%) in class III; thus, this receptor seems different from both the other classes. Of 21 strains (66%) in classes I and II, HA of 11 strains (34%) was inhibited with NANLac, 14 strains (44%) with orosomucoid and 15 strains (47%) with fetuin. The great heterogeneity observed in HA patterns indicates that the HAs of different strains may recognize a heterogeneous class of sialoglycoconjugates on the erythrocyte membrane.


Ultrastructural morphometric analysis of human blood platelets exposed to minimal handling procedures. A detailed morphometric study of normal human blood platelets is described. The purpose has been to evaluate the morphological characteristics of platelets exposed to minimal handling procedures in order to obtain an optimal basis for the appraisal of platelets in disease. Blood from 10 healthy volunteers was collected directly into buffered glutaraldehyde and processed for electron microscopy. This platelet fixation procedure resulted in excellent preservation of resting platelet ultrastructure with one exception: the dense bodies. Compared to platelets fixed following washing procedures, our directly fixed platelets comprised fewer pseudopods and contained more glycogen. An unexpected feature of the open canalicular system was the apparent release of blisters interpreted as microvesicles. Employing a computerized image analyzer, 300 of the platelets were examined morphologically. The morphometric data thus obtained were analyzed statistically, resulting in a set of standard values for morphological characteristics of human platelets which we have found useful in subsequent evaluations of platelet morphology in disease. Significant inter-individual variance was, however, detected in two instances, in the section area of the alpha granules, as well as the area fraction of platelet sections occupied by channels of the open canalicular system (OCS). This should be taken into consideration when appraising platelet ultrastructure in health and disease.


Tumours in Iceland. 16. Malignant tumours of the stomach. Histological classification and description of epidemiological changes in a high-risk population during 30 years. Iceland is one of the high-risk countries for stomach cancer. During the period 1955-84 the incidence declined from 76 to 28 per 10(5) p.a. for males and from 30 to 12 for females. Tissue material from the primary site in 978 males and 448 females was available for histological typing. By the WHO classification tubular carcinoma was most frequent in both sexes, 66% in males and 63% in females, and signet-ring carcinoma second, 13% in males and 16% in females. By the Laurén classification in males 78.1% were intestinal and 16.5% diffuse carcinomas, and in females 73.1% were intestinal and 20.7% diffuse carcinomas. The decline in stomach cancer in Icelanders has mostly affected the intestinal type of tumour (Laurén) and the tubular type of tumour (WHO). Diffuse type tumours (Laurén) have declined slightly. This supports the theory that intestinal carcinomas are more influenced by environmental and especially dietary factors, and that diffuse carcinomas are more influenced by other as yet unknown factors. For epidemiological studies both histological classifications have their value, the WHO especially in that it is based on standard histopathological criteria and the Laurén especially in that it only includes two tumour types. The WHO classification can roughly be transcribed to the Laurén classification as tubular, mucinous and papillary carcinomas fall into the group of intestinal tumours, and signet-ring and more than half of undifferentiated carcinomas into the group of diffuse tumours.


Tumours in Iceland. 17. Malignant tumours of the oesophagus. Histological classification and epidemiological considerations. We studied all primary malignancies of the oesophagus diagnosed in Iceland between 1955 and 1984 and reclassified tumours where histological material was available using the WHO classification system. Of a total of 329 tumours diagnosed in the time period, seven were excluded for various reasons. Of the remaining 322 tumours, 178 were in males and 144 in females. The age standardized incidence was 5.3/10(5) for males and 3.1/10(5) for females. The incidence of oesophageal tumours decreased for both sexes during the time period under investigation. Of 250 reclassified tumours (142 in males and 108 in females), squamous cell carcinomas comprised 81.6%. If the undifferentiated tumours are excluded, the squamous group accounted for 89.1% of the remaining tumours. Small cell carcinomas comprised 3.2% of all cases, which was higher than expected. Most of the tumours appear to be located in the middle part of the oesophagus. The vast majority of resected tumours extended through the wall of the oesophagus. A relatively higher proportion of tumours was confined to the submucosal or muscular layers in the latter half of the period. In conclusion, the epidemiological data in our study appear to resemble what is observed in the other Nordic countries for oesophageal tumours, except for slightly higher overall incidence in Iceland, especially for women.


Detection of gastrin mRNA by in situ hybridization using radioactive- and digoxigenin-labelled probes: a comparative study. The hormone gastrin is mainly produced by the G cells of the antral mucosa and plays a major role in the regulation of digestive mucosal growth. Since it permits identification of cell types containing mRNA, in situ hybridization (ISH) appears to be an interesting method for studying gastrin-producing tissues. In this study, in situ detection of gastrin mRNA has been carried out on frozen sections of four human normal antral mucosa samples and of six colonic carcinomas removed from patients with high levels of plasma gastrin, using a gastrin oligonucleotidic DNA probe. We have compared the results provided respectively by the [35s] labelling and the digoxigenin labelling of the synthetic probe. Positive cells were found in each normal sample analysed with radioactive- as well as digoxigenin-labelled antisense probes. The total number of cells expressing gastrin mRNA appeared slightly higher with the [35s]-labelled probe, while the digoxigenin-labelled probe gave a better definition of positive signals. In contrast, neither radioactive nor cold probes gave positive signals in the six colonic carcinoma samples, although gastrin expression had been demonstrated in these tumours using a reverse transcriptase-PCR method. These results show that, although ISH does not seem sensitive enough to allow the detection of very low levels of gastrin expression, it would appear to be a reliable method for visualizing gastrin mRNA in human antral mucosa.


beta-lactamases in Shigella.

beta-lactamases in Shigella. The occurrence of resistance and production of beta-lactamases was investigated in 60 Shigella strains. Ampicillin resistance was found in 28 (47%) of the isolates, the resistance being more frequent in Sh. flexneri than in Sh. sonnei. All strains were susceptible to cefotaxime, mecillinam, and ciprofloxacin. The beta-lactamases produced by Shigella were similar to TEM-1, OXA-1, or the low-level chromosomally mediated cephalosporinase produced by Escherichia coli. The beta-lactamases produced by Sh. flexneri were most often the OXA-1-like enzymes.


Factors to be considered in the evaluation of bioavailability and bioequivalence of topical formulations. In this paper, an attempt is made to find functional definitions of bioavailability and bioequivalence for topical products and to examine critical factors that influence topical bioavailability and bioequivalence. A physical model approach for quantifying the problem and increasing our understanding is presented here. The key assumptions are (1) that the target site is in the lower epidermis (basal layer) or in the dermis, and (2) that it is the thermodynamic activity (i.e., the free drug concentration, C*, of the active drug species) at the target site that is the true correlate of drug effectiveness. Studies initiated to implement the physical model approach involved first validating a 'three-tiered' model for finite dose drug uptake/transport in skin with experimentally determined input parameters (partition coefficient, K, and steady-state permeability coefficients, P, for the stratum corneum, viable epidermis, and dermis). Hydrocortisone was used as the model drug with hairless guinea pig skin as the model membrane. The physical model is used to show, via the C* concept, how formulation factors may influence bioavailability and bioequivalence. Finally, a method is presented for predicting the efficacy of topical formulations employing appropriate in vitro data and physical model calculations.


Absorption and metabolism of topically applied testosterone in an organotypic skin culture. The Living Skin Equivalent (LSE) is an organotypic coculture of human dermal fibroblasts in a collagen-containing matrix and a stratified epidermis composed of human epidermal keratinocytes. In order to establish the feasibility of using this in vitro system as a model for cutaneous biotransformation, the metabolic fate of topically applied testosterone (T) was monitored in the LSE. After a 24-hour exposure period (37 degrees C) to radiolabelled T, LSE extracts analyzed by high-performance thin-layer chromatography showed that approximately 50% of the applied T had been metabolized. Identified metabolites included bands which comigrated with polar metabolites and products of T 5 alpha-reductase. The general distribution of the observed metabolites was similar to that obtained using biopsied human skin. The rates of T penetration (32 degrees C) through the LSE were monitored after application of T in two vehicles (water and petrolatum) and yielded permeability constants (Kps) of 29 and 1.6 x 10(-3) cm/h, respectively. These Kp values were 4- to 6-fold higher than those reported for human abdominal skin, and reflect the vehicle-related shift in penetration seen in human skin. The Kp values for two additional steroids, estradiol and hydrocortisone, and for T were also determined at 22 degrees C and compared to published Kp values. These Kp values in the LSE were, respectively, 63-, 187- and 35-fold higher than those reported for human skin. The data suggest that compared to human skin the LSE has only a partial barrier function to the passage of test chemicals.(ABSTRACT TRUNCATED AT 250 WORDS)


Topical and systemic absorption of sodium pyrithione following topical application to the nails of the rhesus monkey. A study was performed to investigate the local penetration into the nail, the systemic absorption into the rest of the body, and the routes of excretion of sodium pyrithione following topical application to the nail. Approximately 20 microliters of a film-forming 3% sodium 14C-pyrithione solution was applied once daily to 5 fingernails and 5 toenails of 4 rhesus monkeys for 6 or 7 days. Following dose removal on study day 7, 2 animals were sacrificed, and the treated nails were analyzed for radioactivity. The other 2 monkeys received the topical dose for 1 more day and were monitored during the postdosing period. Sodium 14C-pyrithione was absorbed slowly into and across the nail following topical application, with the nails serving as reservoirs for the drug. Further evidence of the slow movement of sodium pyrithione across the nail was provided by peak plasma 14C equivalents obtained on day 9, 1 day after the last dose had been removed from the nails. Only slight drug concentrations were measurable in plasma, with no radioactivity observed beyond day 12. The urinary excretion data exhibited a delay in peak urinary excretion (days 8 and 9), and an elimination half-life of 2 days, so that approximately 90% of the absorbed drug was eliminated within 1 week following treatment. Including a minor excretion pathway through the feces, total excretion as a percent of dosage was 8.5%, indicating that less than 10% of the applied topical dose of sodium pyrithione was absorbed systemically.(ABSTRACT TRUNCATED AT 250 WORDS)


The possibility of lidocaine ion pair absorption through excised hairless mouse skin. The purpose of the present research was to test the ion pair absorption hypothesis with respect to the topical route of drug delivery. The experiment consisted of preparing various lidocaine-n-alkanoate ion pairs, then characterizing them by proton magnetic resonance spectroscopy, elemental analysis and conductivity. Percutaneous absorption studies through excised hairless mouse skin were carried out using ethanolic solution of radiolabeled 14C-lidocaine-octanoate, 14C-lidocaine-decanoate and 14C-lidocaine-dodecanoate. Studies were conducted under steady-state conditions using Bronaugh's flow-through apparatus and normal saline as the receptor fluid. Ethanolic solution of a lidocaine base served as a control. The apparent differences in flux between lidocaine and the various ion pairs were statistically significant (p less than 0.05). The differences among the fluxes of the various ion pairs were not statistically significant (p greater than 0.05), nor were the differences in lag times (p greater than 0.05). The difference between the flux values of lidocaine-1-14C-dodecanoate and 14C-lidocaine-dodecanoate infers that lidocaine-dodecanoate did not cross the excised, full-thickness, hairless mouse skin as an intact 1:1 ion pair. The formation of weakly associated ion pairs was suggested by the apparent low-association constants (Ka = 15-17 liters/mol) obtained at 25 degrees C in methanol by conductometric analysis.


In vivo microdialysis estimation of histamine in human skin. Microdialysis, a new bioanalytical sampling technique, enables the measurement of substances in the extracellular space. This study investigates the use of the technique in the in vivo measurement of histamine levels in human skin. Microdialysis probes are equipped at the tip with a semipermeable polycarbonate membrane which permits the passive diffusion of substances. 16 probes were inserted, via a guide, into the skin of the ventral forearm of 8 patients or volunteers. The probe was perfused at a flow of 5 microliters/min, with samples being collected at intervals of 10 min and analysed by RIA technique. The mean histamine level in the first 10-min sample following probe insertion was 39.4 nM. The mean histamine value fell with successive 10-min samples (8.8, 4.6, 2.3 nM). An equilibration period of 40 min following probe insertion is suggested for histamine studies, where provocation of the skin is to be performed. Microdialysis appears to be a promising new tool for quantitative and chronological studies of cutaneous inflammatory mediators.


Repeated application of dinitrochlorobenzene to the ears of sensitized guinea pigs: a preliminary characterization of a potential new animal model for contact eczema in humans. We have evaluated a subchronic model of contact hypersensitivity in the guinea pig to mimic human chronic/recurrent eczema. Repeated challenges of the ears of previously sensitized guinea pigs with 0.1% dinitrochlorobenzene (once a week for 4 weeks) induced a typical oedema response, which increased during the first 48 h after each challenge. Crusts were detectable (48 h after challenge) and histological observations (72 h after challenge) revealed hyperplasia, papillomatosis, hyperkeratosis and some mononuclear cell infiltrates in the dermis. In agreement with clinical observations in humans, topical treatment of challenged animals with corticosteroid (1% hydrocortisone) reduced the oedema, hyperplasia, papillomatosis, and leucocyte infiltrates, while application of 5% bufexamac (a non-steroidal drug) was associated with a slight enhancement of the inflammatory response. Thus, this model presents clinical and histological similarities with human eczema. Its pharmacological relevance is also suggested, although further investigations are required to better define its selectivity.


[The results of drug holiday in Parkinson's disease]. The transitory suppression of L-dopa in patients with advanced Parkinson's disease improves motor response upon reintroduction of the drug although this improvement is not constant. The degree and duration of clinical improvement obtained following L-dopa drug holiday were studied and the factors which may intervene in the result of 32 patients with Parkinson's disease were analyzed. Improvement was found in 100% (24.9 +/- 6.7%) with significant differences being observed in the degree of disability (p < 0.05) and the final decrease of the doses of L-dopa (p < 0.05). Improvement was maintained during 10.3 +/- 7.9 weeks. Linear regression studies did not show any relation between the length of the drug holiday, longer than 4 days, and the degree and maintenance of improvement or final decrease of the doses of L-dopa. The authors consider this procedure useful in patients with important fluctuations in treatment response or with important refractory side effects to other measures.


[Treatment with ubiquinone in Kearns-Sayre syndrome. Improvement in ocular motility and visual evoked potentials]. In 1985 Ogasahara observed that treatment with ubiquinone produced improvement in the cardiac conduction and the metabolism of the lactic and pyruvic acids in the Kearns-Sayre syndrome. The results of the administration of 150 mg/day of ubiquinone for 3 years in a patient diagnosed with the Kearns-Sayre syndrome is described. The patient improved notably in strength, ocular movement, visual evoked potentials and in the metabolism of lactic and pyruvic acids. Other beneficial effects reported in the literature have been improvement of ataxia and the somato-sensitive evoked potentials. No side effects have been described.


[Rhombencephalitis caused by Listeria monocytogenes]. The observation of an abscess in the brain stem and cerebellum due to Listeria monocytogenes is presented. The patient was a 55 year old diabetic, alcoholic, gastrectomized male in whom a febrile and meningeal syndrome developed. Three days later he had a progressive unilateral dysfunction of the cranial nerves. The cerebrospinal fluid showed pleocytosis of the polymorphonuclear cells, an increase in proteins and low glucose in relation with the glycemia. No parenchymatous lesion was observed upon emergency computerized tomography. Magnetic resonance imaging showed a hyper-signal lesion in the brain stem and cerebellum with a contrast enhancement in ring. Following antibiotic treatment the clinical evolution of the patient was favorable with slight focal sequelae persistent at discharge. A review of the epidemiology, clinical manifestations and treatment of this infection is carried out.


Amino acid substitution of proteins coded for in mitochondrial DNA during mammalian evolution. Three Markov models (Dayhoff, Proportional and Poisson models; Hasegawa et al., 1992a) for amino acid substitution during evolution were used for maximum likelihood analyses of proteins coded for in mitochondrial DNA in estimating a phylogenetic tree among human, bovine and murids (mouse and rat) with chicken as an outgroup. It turned out that Dayhoff model is the most appropriate model among the alternatives in approximating the amino acid substitutions of proteins coded for in mitochondrial DNA. In spite of the presence of the complete sequence data of mitochondrial genomes, we could not resolve the trichotomy among human, bovine and murids, probably because the time length separating two branching events among these three lines was short and because chicken is too distant from mammals to be used as an outgroup. It was suggested that the average substitution rate of amino acids coded for in mitochondrial DNA is lower along the bovine line than those along the human or murid lines. Advantages of amino acid sequence analysis over nucleotide sequence analysis in phylogenetic study were discussed.


Compensatory changes in silver-stainability of nucleolar organizer regions in mice. Silver-stainability of nucleolar organizer regions (NORs) that contain genes for ribosomal RNA (rDNA) was investigated using two mouse strains, BALB/cCrSlc and MOA, and their hybrid progeny. The patterns of segregation of the rDNA clusters were analyzed in terms of chromosomal C-banding and by use of a polymorphic probe for the variable region in backcrossed N2 and N3 individuals. The results indicate that the intensity of Ag-NOR staining is stably inherited in most of the rDNA clusters, irrespective of different genetic backgrounds. In some clusters, such as those on chromosome 12 of BALB/cCrSlc, a modulation of the intensity is observed. This modulation seems to be due to compensatory activation via a change in the number of actively transcribed genes. The change from silver-negative to silver-positive staining of the NOR of chromosome 12 of BALB/cCrSlc was correlated with demethylation of the genes.


Cytological mapping of Om mutants of Drosophila ananassae. Semidominant, optic morphology (Om) mutants in Drosophila ananassae have been genetically mapped to at least 25 loci throughout the genome (Hinton, 1984; 1988). Among them, four X-linked Om mutants were proved to be associated with the insertion of a transposable element, tom (Shrimpton et al., 1986; Tanda et al., 1988). In the present study, cytological mapping of autosomal Om mutants was carried out by in situ hybridization to polytene chromosomes using a cloned tom element as a probe. The cytological site for each autosomal Om mutant has been determined to a single band of the salivary gland chromosomes.


Trends in mortality from malignant cutaneous melanoma in The Netherlands, 1950-1988. This paper presents an analysis of trends in mortality from malignant melanoma of the skin in The Netherlands, 1950-1988. Statistical analyses show that time period effects are needed to describe the mortality trends in The Netherlands. Because this contrasts with reports from other countries, in which the trends were ascribed to a cohort effect only, log-linear models including the three factors age, time period and birth cohort, were fitted to the data. To be able to separate time period effects from birth cohort effects we assumed a mathematical function for the mortality rates in relation to age. The results obtained in this way indicate that time period effects increased up to 1970. An increase of birth cohort effects is seen for cohorts born between 1900 and 1955. For cohorts born after 1955 the mortality from melanoma seems to decrease. The most plausible explanation for the time period effect probably is improvement in death certification.


Diet and colorectal cancer: results of two case-control studies in Russia. Two case-control studies of diet and colorectal cancer were conducted in Moscow and Khabarovsk. The Moscow study comprised 100 cases of colorectal cancer and 100 neighbourhood controls. The Khabarovsk study consisted of 117 cases of colorectal cancer and 117 population controls. A history of the usual dietary intake one year prior to interview was taken using a food frequency questionnaire. Effects were adjusted in analysis for energy intake and education. Significantly reduced risks were observed with high intakes of cellulose (P = 0.001), beta-carotene (P = 0.002), vitamin C (P = 0.007), polyunsaturated fatty acids (PUFA) (P = 0.004), cholesterol (P = 0.04), and with a high ratio of PUFA to saturated fatty acids (SFA) (P = 0.002). Significant increases in risk were observed in association with high ratios of intakes of protein to cellulose (P = 0.002) and of fat to cellulose (P = 0.008). High intake of total fat was associated with non-significant decrease in the risk (P = 0.12), while high intake of SFA resulted in statistically non-significant increase in risk (P = 0.40). Significant reductions in risk were associated with high frequencies of consumption of vegetables (P = 0.001) and fruit (P = 0.009). There were results suggestive of a decreased risk with a high frequency of milk consumption (P = 0.06) and an increased risk in association with a ratio of meat to vegetable frequencies (P = 0.09). After adjustment among factors effecting risk of colorectal cancer statistically significant increase in the risk was seen only for protein/cellulose ratio and significant protective effect for PUFA/SFA ratio, beta-carotene and vegetable consumption.


Metabolic response of AH13r rat tumours to cyclophosphamide as monitored by pO2 and pH semi-microelectrodes. The composition of the microenvironment has an important influence on the cellular response to cytotoxic agents. Using pH and pO2 semi-microelectrodes, we have monitored metabolic changes in AH13r rat tumours as a function of time after subcurative chemotherapy. Prior to therapy, tumours contained large areas considered hypoxic (mean pO2 approximately 4 mmHg) and are characterised by a marked accumulation of acidic metabolites (mean pH 6.65). Administration of cyclophosphamide (40 mg/kg body weight) resulted in tumour regression to 15% of pretreatment volumes and a growth delay of 12 days. Concomitant with volume reduction, tumours became reoxygenated (mean pO2 approximately 7 mmHg), with maximum values being reached within 2-4 days, paralleled by a shift of pH to more alkaline values (0.17 U on average). These changes coincided with the development of subtotal necrosis. During early tumour regrowth, the pH and pO2 histograms returned to control values. These data corroborate and extend the results of previous studies in which noninvasive techniques had been applied for the monitoring of treatment-induced metabolic changes in malignant tumours in vivo. In addition, these results support the notion that the effectiveness of anticancer therapy might be improved by selecting and scheduling therapeutic agents in consideration of physiological changes caused by preceding courses of treatment.


Survival trends for neuroblastoma patients in Finland: negative reflections on screening. Based on 257 neuroblastoma patients in the age group 0-14 years and reported to the Finnish Cancer Registry, the 5-year cumulative survival rates have improved from 15% in the 1950s to 57% in 1980-1986. The potential benefit of screening for neuroblastoma was assessed on the basis of these nationwide survival trends. It is likely that any decrease in the overall neuroblastoma mortality due to screening would be small, because the survival rates of the Finnish neuroblastoma patients are already, even without screening, similar to those in Japan, which has a nationwide public health policy to screen for neuroblastoma.


Effect of dequalinium on K1735-M2 melanoma cell growth, directional migration and invasion in vitro. Cationic lipophilic compounds have an antiproliferative effect on certain tumour systems in vitro and in vivo. We have investigated whether the cationic lipophilic compound dequalinium affects not only proliferation but also motility and invasion of the highly metastatic and highly invasive melanoma cell line K1735-M2. Proliferation was assessed in monolayer cultures and in multicellular spheroids, motility was estimated in the assay of directional migration, and invasiveness was tested through confrontation cultures of tumour multicellular spheroids with embryonic chick heart tissue evaluated by computerized image analysis. 2 mumol/l dequalinium impaired melanoma cell proliferation, reduced directional migration and significantly blocked invasion in vitro. On the ultrastructural level, dequalinium caused obvious changes in mitochondria of both melanoma and embryonic chick heart cells. The mechanisms of the antiproliferative, antimigrating and antiinvasive effects remain to be determined. Inhibition of protein kinase C, calmodulin antagonism, DNA intercalation and/or direct effects on mitochondrial functions may be considered.


Long-term stability of 5-fluorouracil and folinic acid admixtures. 5-Fluorouracil (5-FU) and d,1-folinic acid (FA) are used in association to treat a wide variety of malignancies. The stability and the compatibility of 5-FU and FA in combination in intravenous admixtures were studied under various storage conditions and with drug concentrations matching their clinical use (0.9% sodium chloride, 5% dextrose, protected from light or not). 5-FU and FA concentrations (mg/ml) were 6.5 or 50 and 4.0 or 30.8, respectively. Successive aliquots of the drugs mixtures were withdrawn during 60 h from 500 ml glass bottles and 500 ml polyvinyl chloride (PVC) bags (at room temperature) and during 120 h from cassettes (at 32 degrees C). Drug concentrations were measured by high performance liquid chromatography. For all conditions tested, the changes in 5-FU and FA relative to the initial concentrations remained within the assay reproducibility (10%). In complement, infrared Fourier transformation spectrophotometry has not shown a significant fixation of FA or 5-FU on the PVC bags, in all tested conditions. Under the conditions examined above 5-FU and FA can be mixed in the same container for their use in cancer chemotherapy. This can have practical consequences by simplifying the widely used treatment protocols associating 5-FU and FA.


Serum osteocalcin in the management of myeloma. We have measured serum osteocalcin, a vitamin K-dependent glycoprotein synthesised by osteoblasts in 62 patients, 49 with myeloma, 26 at presentation and 23 previously treated, 7 with Waldenstrom's macroglobulinaemia (WM), and 6 with monoclonal gammopathy of uncertain significance (MGUS). Osteocalcin levels were normal in WM and MGUS. High values were found in 5/26 (19%) patients with myeloma at presentation. There was no relationship between serum osteocalcin and stage of disease. Osteocalcin was normal in all patients in plateau phase, falling to low levels in relapsed patients who failed to respond to further treatment. Serum osteocalcin may be a useful indicator of bone metabolism in myeloma.


CA 15.3 determination in patients with breast cancer: clinical utility for the detection of distant metastases. In 81 healthy women, 26 pregnant women, 25 patients with fibrocystic disease and 144 breast cancer patients, the overall diagnostic sensitivity and specificity of the CA 15.3 test was 27 and 97%, respectively. The positive and negative predictive values were 93 and 43%. In 150 node-negative patients taking part in a chemoprevention trial CA 15.3 was assayed at baseline and every 4 months for a median follow-up of 24 months (range 4-48). In these patients, 5 had local recurrences, 1 had a regional recurrence, 9 had distant metastases and 3 developed cancer in the contralateral breast. Among the patients with recurrences, those with distant metastases showed the highest ratio of CA 15.3 increase (8/9); in local and regional recurrences, this ratio was lower (2/6). The patients with contralateral breast cancer had no significant increase in CA 15.3. Patients in whom metastases were detected showed an increase in CA 15.3 4-48 months before clinical or instrumental detection of the metastases.


Diethylstilboestrol: II, pharmacology, toxicology and carcinogenicity in experimental animals. Diethylstilboestrol (DES) exerts several toxic effects in experimental animals, by mechanisms which are still unclear. The genotoxicity of the drug has been attributed to a quinone metabolite and is mainly clastogenic, including sister chromatid exchange, unscheduled DNA synthesis, chromosomal aberrations, disruption of mitotic spindle and aneuploidy. There is evidence that genotoxic effects may occur also transplacentally. Intrauterine and early postnatal exposure to DES can cause a variety of dysplasias. In the offspring of female mice exposed to DES during pregnancy, histological changes are observed in the vaginal and cervical epithelium, the endometrium, the ovary, the testis and the epididymis. Prenatal exposure of rats to DES led to decreased litter size and to urethrovaginal cloaca, penile and testicular hypoplasia, and cryptorchidism. Vaginal ridging, vaginal adenosis, testicular hypoplasia and cryptorchidism have been observed in rhesus monkeys following prenatal exposure. There is sufficient evidence that diethylstilboestrol is carcinogenic in experimental animals, after either prenatal or postnatal exposure. Mice show a similar type of carcinogenicity to that observed in humans, target organs being vagina, cervix, uterus, ovary, mammary gland and testis. In rats, prenatal exposure to DES produces mostly mammary and pituitary tumours, but also some tumours of the vagina. Hamsters develop tumours of vagina, cervix, endometrium, epididymis, testis, liver and kidney. DES induces ovarian papillary carcinomas in dogs, and malignant uterine mesotheliomas in squirrel monkeys. Some experimental evidence points to the possibility of a transgenerational carcinogenic effect, since prenatal treatment of mice with DES is followed by an increased incidence of uterine and ovarian carcinomas in the second-generation descendants. Experimental results could have been used to predict the adverse effects of DES observed in humans in the early 1970s: DES had been reported to be carcinogenic in mice in the 1930s, while experiments in the 1960s had provided evidence that exposure during pregnancy could result in an increased cancer risk in the progeny.


Long term effects of tamoxifen. Biological effects of Tamoxifen Working Party. A total of 153 breast cancer patients who participated in two trials of adjuvant tamoxifen and who had not recurred were recruited into a study of the long term effects of tamoxifen. There were 60 controls (no tamoxifen), 73 ex-users (mostly for 2 years) and 20 current users (median treatment duration 72 months) and the median follow-up time was 7 years. A wide ranging study of lipids, hormones, bone density and haemostasis was undertaken. When compared with controls, current users had lower cholesterol levels (especially low density cholesterol), and increased triglyceride levels. Thyroid hormones were higher and sex hormone binding globulin was almost doubled. Bone density was non-significantly higher, clotting times were slightly shorter and fibrinogen and antithrombin III levels were reduced. However few of these changes persisted in ex-users, suggesting that most of the biological effects of treatment are reversible on cessation of treatment. This is reassuring for potentially negative side-effects, but also indicates that potentially positive 'side-effects' such as cholesterol lowering only occur while on active treatment.


Chemotherapy induced amenorrhoea in a randomised trial of adjuvant chemotherapy duration in breast cancer. We have previously reported the results of a clinical trial in patients with stage II breast cancer which compared a 12 week chemohormonal regimen with a 36 week chemotherapy regimen. Both pre and post menopausal women were entered. The 12 week regimen was inferior both in terms of disease-free survival and overall survival. The effect of chemotherapy on menstrual function was prospectively documented in 95 of 114 premenopausal women at 3 of the 4 participating centres. 67 of the 95 women (70.5%) developed permanent amenorrhoea. There was a statistically significant difference in the rate of induced amenorrhea between the 12 week and the 36 week groups; 23/42 vs. 44/53, respectively (P = 0.003). Recurrence and mortality rates were lower in the patients who became amenorrheic; 38% vs. 57% (P = 0.03) and 18% vs. 32% (P = 0.17), respectively. Similar trends were observed within treatment groups. The effect of induced amenorrhoea on outcome was seen predominantly in patients under 40 years old. These results suggest that the induction of ovarian failure is a potential mechanism for the observed effect of adjuvant chemotherapy in these patients. The difference in the ovarian failure rates between groups may be a possible explanation for the inferiority of the 12 week regimen.


Early stage Hodgkin's disease: ten-year results of a non-randomised study with radiotherapy alone or combined with MOPP. From September 1976 to June 1982, 201 consecutive patients with stage I (A and B)-IIA Hodgkin's disease were stratified in two groups according to prognostic factors. The F group included 116 patients with favourable presentation: they were staged with laparotomy and treated with subtotal or total nodal radiotherapy alone. The U group included 85 cases with unfavourable presentation who were staged by laparoscopy and treated with 3MOPP (mechlorethamine, vincristine, procarbazine, prednisone)-radiotherapy-3MOPP. At 10 years the F group showed a freedom from progression (FFP) of 71% with significant difference between stage I and II (85% vs. 59%; P = 0.003) and an overall survival of 84%. The results of the U group were: FFP 83%, overall survival 74%, and the findings were not influenced by stage. FFP in patients with bulky vs. not bulky lymphoma was 70% vs. 87% (P = 0.04). No secondary acute non-lymphocytic leukaemia developed among patients treated with radiotherapy and in continuous complete remission, while acute leukaemia occurred in the F group patients who received salvage chemotherapy (4 of 31 cases) and in the U group (3 of 85 cases). Present results confirm the usefulness of radiotherapy alone in favourable pathological stage IA. All other disease stages will require a different strategy that should consist of radiotherapy combined with short-term effective regimens, such as ABVD (doxorubicin, bleomycin, vinblastine and decarbazine) or VBM (vinblastine, bleomycin and methotrexate) to reduce the incidence of MOPP-associated gonadal dysfunction and leukaemogenesis.


Prognosis of lymphoma from a fine-needle aspirate. The prognostic value of S-phase fraction (SPF) determined by flow cytometry from a fine-needle aspirate was investigated in a prospective series of 52 non-Hodgkin lymphomas. The aspirates were drawn either at diagnosis (n = 16) or at lymphoma recurrence (n = 36). Patients with lymphoma with a large SPF (> 10%, n = 24) had only a 21% 3-year survival rate corrected for intercurrent deaths as calculated from the date of aspiration, whereas a smaller SPF was associated with a 71% 3-year survival rate (n = 28, P = 0.0009). SPF size also correlated with Working Formulation grading (P = 0.002). In a multivariate analysis the relative risk of death from lymphomas with a large SPF was 4.01 (1.60-10.1), whereas histological grading, age, and sex had no additional independent prognostic value. SPF determined from a fine needle aspirate had unexpectedly good prognostic value, and the result suggests that the method is of clinical importance.


A pilot study of intensive cyclophosphamide, epirubicin and fluorouracil in patients with axillary node positive or locally advanced breast cancer. A multicentre pilot study has been conducted to determine an intensive regimen of cyclophosphamide, epirubicin, and fluorouracil which was tolerable and acceptable to patients with node positive breast cancer. Consecutive patients with operable axillary node positive breast cancer (T1-3, N1-2, M0), 266 patients, or locally advanced breast cancer (T4), 22 patients, were treated with cyclophosphamide post-operatively for 14 days and epirubicin and fluorouracil, both intravenously on days 1 and 8. Each cycle was repeated monthly for 6 months. Dosages were increased according to predetermined guidelines. Outcome measures were admission to hospital for febrile neutropenia and change in cardiac function as assessed by radionuclide angiography. The first 46 patients were treated at the doses of cyclophosphamide = 75 mg/m2, epirubicin = 50 mg/m2, fluorouracil = 375 mg/m2 (level 1), then 42 patients at cyclophosphamide = 75 mg/m2, epirubicin = 50 mg/m2 and fluorouracil = 500 mg/m2 (level 2), 69 patients at cyclophosphamide = 75 mg/m2, epirubicin = 60 mg/m2, and fluorouracil = 500 mg/m2 (level 3), and 42 patients at cyclophosphamide = 75 mg/m2, epirubicin = 70 mg/m2, and fluorouracil = 500 mg/m2 with concurrent antibiotics (level 4). The rates of febrile neutropenia were 8.7% (level 1), 7.1% (level 2), 18.8% (level 3), and 31% (level 4), respectively, P = 0.002. Accrual to level 4 was discontinued according to study guidelines and a further 89 patients were recruited at level 3 dosages with antibiotic prophylaxis (level 3a), resulting in a 5.6% rate of febrile neutropenia. The difference in febrile neutropenia rates between levels 3 and 3a was statistically significant. There were no toxic deaths and 2 cases of heart failure. In conclusion, through a careful dose-finding study in patients with operable or locally advanced breast cancer, an intensive epirubicin-containing adjuvant regimen has been established which is presently being compared with standard CMF (cyclophosphamide, methotrexate, 5-fluorouracil) chemotherapy in a randomised trial. In addition, this study suggests that antibiotic prophylaxis reduces the risk of febrile neutropenia in breast cancer patients receiving intensive chemotherapy.


Prognostic importance of various clinicopathological features in papillary thyroid carcinoma. The influence of various pathological features on tumour recurrences and cancer deaths has been studied in 173 consecutive cases of surgically treated papillary thyroid carcinoma recorded in 1971-1985. During the follow-up (median 7.3 years), 18.6% of the 161 radically treated patients had recurrent disease, and 8.7% died of thyroid cancer. In the univariate life-table analysis, recurrence-free survival was significantly related to age, pTNM category, tumour size, presence of certain growth patterns, tumour necrosis, tumour infiltration in surrounding thyroid tissue and thyroid gland capsule, lymph node metastases, presence of extra-nodal tumour growth and number of positive lymph nodes, whereas only tumour diameter, thyroid gland capsular infiltration and presence of extra-nodal tumour growth remained as significant prognostic factors in the multivariate analysis. Regarding thyroid cancer deaths, sex, age, pTNM category, radicality of surgical treatment, tumour diameter, macroscopic appearance, cellular atypia, tumour necrosis, thyroid gland capsular infiltration, vascular invasion, extra-thyroidal extension and lymph node metastases were all significant variables in the univariate analysis. However, only sex, age, radicality of surgical treatment and vascular invasion were found to be significant predictors of thyroid cancer deaths in the final multivariate Cox model, whereas cellular atypia and necrosis showed a borderline significance. Our study thus documents the independent importance of certain histological features for morbidity and mortality in surgically treated cases of papillary thyroid cancer.


Treatment of melphalan-resistant multiple myeloma with vincristine, BCNU, doxorubicin, and high-dose dexamethasone (VBAD). A total of 65 patients (35 male/30 female) with multiple myeloma primarily (33) or secondarily (32) resistant to melphalan and prednisone were treated with vincristine, carmustine (BCNU), doxorubicin, and high-dose dexamethasone (VBAD) at 4-week intervals. Among 60 evaluable patients the overall response was 36.6% (21.6% objective response plus 15% improvements). The response rate was significantly higher in primarily resistant patients than in those becoming resistant after a prior response (48.4 vs. 24.1%, P < 0.05). The median duration of response was 17.5 months. When survival of responders and non-responders were compared by the conventional method, a highly significant difference was observed (P < 0.001). However, using the Mantel and Byar procedure and the landmark method, only a trend for longer survival in the responders was registered. These results indicate that although VBAD is effective in at least one third of patients with advanced multiple myeloma resistant to melphalan, its impact on survival is limited.


Prediction of superficial bladder cancer by nuclear image analysis. A cohort of 270 superficial transitional cell bladder tumours (Ta-T1) was followed-up for over 8 years. WHO grade, papillary status and six nuclear factors were related to progression, recurrence-free survival (RFS) and bladder cancer-related survival (BS) during the follow-up period. Mean nuclear area (NA), standard deviation of nuclear area (SDNA), nuclear perimetry (PE), standard deviation of nuclear perimetry (SDPE), shortest nuclear axis (Dmin) and longest nuclear axis (Dmax) were significantly related to WHO grade and papillary status (P < 0.0001). All the nuclear factors were related significantly to progression in univariate analysis (P < 0.01) whereas in a multivariate analysis WHO grade (P < 0.0001) and papillary status (P = 0.048) included independent prognostic information. RFS was related to PE (P = 0.009), SDPE (P = 0.013), Dmin (P = 0.021), Dmax (P = 0.028) and SDNA (P = 0.029). In papillary tumours SDPE (P = 0.007) and Dmin (P = 0.024) predicted RFS. BS was related to WHO grade, papillary status, NA, SDNA, PE, Dmax, Dmin (all P < 0.0001) and to SDPE (P = 0.003). In papillary tumours PE (P < 0.0001), Dmax (P = 0.0022), Dmin (P = 0.0027), WHO grade (P = 0.0036), NA (P = 0.0005), SDNA (P = 0.0355) and SDPE (P = 0.0718) predicted BS. In multivariate analysis SDPE (P = 0.029) predicted RFS and survival was related to WHO grade (P < 0.001) and PE (P = 0.014) independently. In papillary tumours only Dmax (P = 0.001) predicted survival independently. The results show that superficial papillary transitional cell bladder tumours can be efficiently categorised into prognostic groups by nuclear image analysis and the results provide a new classification system for superficial papillary bladder tumours. Tumours with high nuclear factor values should be considered for radical primary therapy and adjuvant therapy after transurethral resections.


Tissue polypeptide antigen in breast cancer cytosol: a new effective prognostic indicator. Since 1982 we have been evaluating oestrogen and progesterone receptors (PgR), cathepsin D and the cytosolic levels of the tumour marker, tissue polypeptide antigen (TPA), in 257 patients radically resected for breast cancer (follow-up 24-81 months). TPA was measured by an immunoradiometric assay previously validated for cytosol. No significant associations were found between cytosolic TPA and age, tumour size, lymph-node status, receptor status and cathepsin D. TPA+ cases showed a significantly longer disease-free survival (DFS) and overall survival (OS) than TPA-patients (log-rank P < 0.0001). The prognostic value of cytosolic TPA was also demonstrated after stratification by nodal status, PgR and cathepsin D. The prognostic value of TPA was independent of the other prognostic indicators, being the most powerful among the evaluated indices (Cox multivariate analysis: chi 2 15.5 for DFS, 11.4 for OS). We conclude that cytosolic TPA is a powerful additional prognostic factor in primary breast cancer. Its prognostic role should therefore be extensively evaluated.


Tumour infiltrating lymphocytes as an independent prognostic factor in transitional cell bladder cancer. The prognostic value of tumour infiltrating lymphocytes (TIL) was assessed in a cohort of 514 patients with a transitional cell bladder cancer (TCC) during a follow up period of over 9 years. The density of TIL were positively correlated to WHO grade (P < 0.0001), non-papillary growth architecture (P < 0.0001), morphometric nuclear factors (P < 0.007) and volume corrected mitotic index (M/V index) (P < 0.0001). Dense TIL predicted progression in Ta-T1 tumours (P < 0.0006) whereas in a multivariate analysis they had no independent predictive value. Dense TIL were related to short recurrence-free survival in Ta-T1 tumours in a univariate analysis (P = 0.06) as well as in a multivariate analysis (P = 0.005). Dense TIL predicted unfavourable prognosis in the entire cohort (P = 0.0316) and in papillary tumours (P = 0.062) whereas in nodular tumours TIL were a sign of good prognosis (P = 0.0141). Also in T3-T4 tumours TIL were related to less aggressive behaviour of TCC (P = 0.0259). In a multivariate analysis including clinical stage (T-category), WHO grade, papillary status, six morphometric nuclear factors and M/V index dense TIL were a highly significant indicator of a favourable prognosis (P = 0.007). Particularly TIL categorized rapidly proliferating TCC into prognostic groups (P = 0.001). The results show that TIL are a sign of efficient host defence mechanisms in TCC and TIL predict a favourable prognosis in invasive TCC.


Relations between epidermal growth factor receptor and oestrogen and progesterone receptors in breast cancers of premenopausal and postmenopausal patients in Kuwait. The levels of cell membrane epidermal growth factor receptor EGFR and cytosol (c) and nuclear (n), oestrogen (E) and progesterone (P) receptors (R) were determined in 132 specimens of primary breast cancers. In the tumours of postmenopausal women an inverse significant correlation was demonstrated between the concentrations of EGFR vs. ERc, ERn, and PRc while no such correlation was noted in the tumours of premenopausal women. Premenopausal and postmenopausal EGFR positive tumours (> or = 10 fmol/mg membrane protein) could be regarded as homogenous with respect to the concentration of ER and PR whose mean values were low and without being significantly different. EGFR negative tumours were heterogeneous with respect to the ER and PR concentrations. Postmenopausal EGFR negative (< 10 fmol/mg membrane protein) tumours had evidently higher mean values of ER and PR than premenopausal EGFR negative tumours, but these differences were statistically significant for oestrogen receptors only. The levels of ER and PR of premenopausal EGFR negative tumours were approximated to the corresponding levels of EGFR positive tumours.