Age-related changes in transmitter glutamate and NMDA receptor/channels in the brain of senescence-accelerated mouse.

The senescence-accelerated mouse (SAM-P/8) is known as a murine model of aging and memory dysfunction. In the hippocampus and cerebral cortex of P/8, the contents of glutamic acid and glutamine were significantly higher than those of normal strain R/1 during 2 and 14 months. High K(+)-evoked endogenous glutamic acid release from the slices of P/8 was increased in comparison with R/1 at 9 and 11 months. In addition, the Bmax of [3H]dizocilpine (MK-801, channel blocker for N-methyl-D-aspartic acid receptor/channel) binding in the cerebral cortex was age-dependently decreased in P/8 but not in R/1. These results suggest that synaptic dysfunctions in the glutamatergic system occur in the CNS of SAM-P/8.


Inhibition of cisplatin-induced emesis in ferrets by the non-NMDA receptor antagonists NBQX and CNQX.

The excitatory amino acid (EAA) receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(f)quinoxaline (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), which preferentially block non-N-methyl-D-aspartate (non-NMDA) subtypes of EAA receptors, effectively inhibit cisplatin-induced emesis in ferrets. A high dose of cisplatin (10 mg/kg i.v.) was used which induced emesis in all saline-treated control ferrets. At 10 mg/kg i.v., NBQX totally prevented cisplatin-induced emesis in 5 of 6 ferrets and CNQX totally prevented emesis in 3 of 5 ferrets. By comparison, each of the 5-HT3 inhibitors, zacopride and ondansetron, at 1.0 mg/kg i.v. (a dose considered in the high therapeutic range for controlling emesis by these compounds), totally prevented emesis in 2 of 5 ferrets. It is concluded that non-NMDA antagonists effectively inhibit cisplatin-induced emesis. They are potential antiemetic compounds, alone or in combination with 5-HT3 antagonists or other more conventional drugs of choice.


Low concentrations of N-methyl-D-aspartate inhibit the induction of long-term potentiation in rat hippocampal slices.

We have examined the effects of excitatory amino acids on tetanus-induced long-term potentiation (LTP) in the CA1 region of rat hippocampal slices. Agonists acting at N-methyl-D-aspartate (NMDA) receptors, including NMDA, aspartate and glutamate, completely block the development of LTP when administered for 5 min prior to tetanic stimulation. Additionally, and unlike the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV), NMDA blocks LTP when administered for 5 min immediately following the electrical tetanus. The effective concentrations of NMDA are subthreshold for depolarizing CA1 neurons. These results indicate that under certain conditions NMDA receptor activation prevents CA1 LTP.


Depletion of dopamine binding sites and changes in dopamine and dihydroxyphenylacetic acid levels in 17- and 90-day-old rat striatum after irreversible receptor antagonism.

Irreversible antagonism of dopamine (DA) receptors by the alkylating compound N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) results in a depression of DA agonist mediated behaviors in adult, but not preweanling rats. DA D1 and D2 receptors, dihydroxyphenylacetic acid (DOPAC) and DA levels were assessed in 17- and 90-day-old rats in order to identify a neurochemical mechanism for this difference. EEDQ caused a depletion in D1 and D2 receptors in both young and adult rats, with the depletion in adults being relatively greater. In both age groups, EEDQ caused an unexpected decrease in DA levels which returned to normal levels by 30 days post-injection. In addition, DOPAC levels of adult rats, but not rat pups, were elevated after EEDQ treatment. The depletion of endogenous DA levels must be considered when interpreting the effects of EEDQ. Age-dependent differences in DA metabolism may be important for understanding the ontogenetic differences in EEDQ's behavioral effects.


Perspectives on nursing knowledge.

On May 19, 1991, in Tokyo, Japan, four nurse theorists participated in a panel discussion at Discovery International, Inc.'s Biennial Nurse Theorist Conference. The participants were Imogene M. King, Hildegard E. Peplau, Rosemarie Rizzo Parse, and Martha E. Rogers. The goal of the conference was to present the latest views on nursing knowledge of these nurse leaders. The panel discussion provided the nurse theorists with an opportunity to engage in dialogue regarding issues of concern to the audience. The panel moderator was Hiroko Minami of St. Luke's College of Nursing in Tokyo.


Basic concepts of molecular biology as applied to pediatric and adolescent gynecology.

Recombinant DNA techniques have increased our understanding of genetic disorders and facilitated their diagnosis and may eventually lead to therapy. Expression may be assessed and quantified by Northern blot analysis, whereas in situ hybridization may localize the transcript. Southern blotting has been the standard method to identify gene mutations and RFLPs, which are useful in the diagnosis of many different disorders. Special multiallelic RFLPs produce a DNA fingerprint particularly useful in paternity testing and forensic medicine. PCR has greatly reduced the time and expense to diagnose genetic disorders, and has an enormous number of applications both clinically and at the research level. These techniques along with DNA sequencing are currently being used in the human genome project, which may some day allow "DNA typing" of individuals for any gene of interest. There is no doubt that new techniques in molecular biology will continue to evolve so that the goal of gene therapy for many disorders may be possible in the future.


A new transthyretin mutation associated with amyloidotic vitreous opacities. Asparagine for isoleucine at position 84.

An inherited type of amyloidosis was suspected in an individual of Italian descent who presented with vitreous opacities. Although no family history of amyloidosis was apparent, the patient's transthyretin gene was examined and found not to possess any of the known transthyretin mutations. Complete DNA sequencing revealed a substitution of adenine for thymine in the second base of codon 84 causing an amino acid change of asparagine for isoleucine. The mutation was confirmed by demonstrating the loss of an Sfa N1 restriction endonuclease site. Allele-specific DNA amplification by polymerase chain reaction also was used to confirm the mutation. Either of these tests can be used for diagnosis. Asparagine 84 represents the second mutation associated with amyloidosis to occur at codon 84.


Ethanol ingestion combined with lowered carbohydrate intake enhances the initiation of diethylnitrosamine liver carcinogenesis in rats.

The effect of ethanol on the initiation of diethylnitrosamine- (DEN) induced liver carcinogenesis was investigated in rats. In the first experiment, eight-week-old male Wistar rats were maintained on four liquid diets: a basal diet (Group 1), a low-carbohydrate (low-CHO) diet (Group 2), a basal diet+ethanol (Group 3), or a low-CHO diet+ethanol (Group 4). After three weeks on these diets, 50 mg/kg of DEN was injected intraperitoneally. The plasma glutamic-oxaloacetic transaminase activity in Group 4 was higher 24 hours after DEN administration than in Groups 1 and 3. The plasma glutamic-pyruvic transaminase activity in Groups 3 and 4 was higher than in Groups 1 and 2. The number of gamma-glutamyltranspeptidase-positive foci per unit liver area 41 weeks after DEN administration was higher in Group 4 than in Group 1. The area of gamma-glutamyltranspeptidase-positive foci was greater in Groups 2 and 4 than in Group 1. In the second experiment, Groups 1 and 4 were given DEN orally (25 or 75 mg/kg). Plasma glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities 24 hours after DEN administration were significantly higher in Group 4 than in Group 1, but only when the dose of DEN was 75 mg/kg. In contrast, the number and area of placental glutathione S-transferase-positive foci per unit liver area were greater in Group 4 than in Group 1 only after 25 mg/kg of DEN. Thus the severity of hepatotoxicity and the incidence of precancerous liver lesions were not necessarily correlated. These findings together indicate that a combination of ethanol and a low-CHO diet enhances DEN-induced liver carcinogenesis in rats by increasing the bioactivation of DEN in the liver.


[Multiple cholesterol embolism mimicking periarteritis nodosa].

Ten men aged 56 to 84 were hospitalized with a diagnosis of periarteritis nodosa, whereas they had multiple cholesterol embolism. The diagnosis was corrected post mortem in the first 3 patients and subsequently in live patients. The particularly misleading clinical manifestations were neurological (polyneuritis in 5 cases, mononeuritis in 1, central nervous system disorders in 3), pulmonary (alveolar haemorrhage in 2 cases, respiratory failure of unknown mechanism in 4) and pericardial (2 cases). Five patients had eosinophilia (more than 500 eosinophils/mm3). The elements that led to the correct diagnosis were the presence of vascular risk factors in all 10 patients (but hyperlipidaemia in only one), severe complications of the atheromatous disease in all cases, a precipitating or aggravating factor in 8 patients (anticoagulant therapy in 7, arteriography in 6) and the finding of purple or necrotic toes (6 cases). Histological (5 cases) and/or ophthalmological (2 cases) evidence was obtained in only 6 patients. Seven patients died 1 to 3 years after the onset of the disorders. Studies on low-density lipoprotein metabolism are in progress to determine the mechanism of clinical manifestations unexplainable by embolism.


Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.


Humanization of an anti-p185HER2 antibody for human cancer therapy.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.


Development of antibodies against the rat brain somatostatin receptor.

Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).


Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade.

We tested a theoretical prediction that patterns of excitatory input activity that consistently fail to activate target neurons sufficiently to induce synaptic potentiation will instead cause a specific synaptic depression. To realize this situation experimentally, the Schaffer collateral projection to area CA1 in rat hippocampal slices was stimulated electrically at frequencies ranging from 0.5 to 50 Hz. Nine hundred pulses at 1-3 Hz consistently yielded a depression of the CA1 population excitatory postsynaptic potential that persisted without signs of recovery for greater than 1 hr after cessation of the conditioning stimulation. This long-term depression was specific to the conditioned input, ruling out generalized changes in postsynaptic responsiveness or excitability. Three lines of evidence suggest that this effect is accounted for by a modification of synaptic effectiveness rather than damage to or fatigue of the stimulated inputs. First, the effect was dependent on the stimulation frequency; 900 pulses at 10 Hz caused no lasting change, and at 50 Hz a synaptic potentiation was usually observed. Second, the depressed synapses continued to support long-term potentiation in response to a high-frequency tetanus. Third, the effects of conditioning stimulation could be prevented by application of NMDA receptor antagonists. Thus, our data suggest that synaptic depression can be triggered by prolonged NMDA receptor activation that is below the threshold for inducing synaptic potentiation. We propose that this mechanism is important for the modifications of hippocampal response properties that underlie some forms of learning and memory.


Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.


Organization and evolution of the human epidermal keratinocyte transglutaminase I gene.

Transglutaminases (TGases; protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent crosslinking enzymes that modify proteins posttranslationally. Several distinct types of TGases have been identified, which appear to be encoded by a family of closely related genes. We isolated the gene encoding human keratinocyte-specific type I TGase (TGase I) and characterized its chromosomal organization. The TGase I gene consists of 15 exons separated by 14 introns and exhibits a restriction fragment length polymorphism. Exons appear to encode functional and/or structural domains: exon I and part of exon XV encode untranslated regions, whereas exons VII and XI contain the active site and a presumptive calcium-binding domain, respectively. Interestingly, exon VI of TGase I contains a consensus Arg-Gly-Asp tripeptide sequence whose presence suggests an intriguing extracellular function for the enzyme. We present a likely phylogenetic tree for seven known members of the TGase family based on amino acid sequence similarity. Arguments presented suggest that the active enzyme evolved first and the structural human erythrocyte membrane protein 4.2 (band 4.2) has undergone a rapid change in amino acid sequence. It follows that band 4.2 evolved from the type II TGases, whereas factor XIII subunit a evolved from the type I group.


Cloning of the yeast FAS3 gene and primary structure of yeast acetyl-CoA carboxylase.

We have isolated and determined the nucleotide sequence of the yeast FAS3 gene, which encodes acetyl-CoA carboxylase (EC 6.4.1.2). The sequence has an open reading frame of 6711 bases coding for a protein of 2237 amino acids with a calculated molecular weight of 250,593. The presence of the unique biotin-binding site, Met-Lys-Met, and the known CNBr peptide and COOH-terminal sequences confirmed the nucleotide-derived amino acid sequence. The yeast, chicken, and rat carboxylases have an overall sequence identity of 34%, suggesting that the eukaryotic carboxylase evolved from a single ancestral gene. The amino acid sequences of yeast fatty acid synthase subunits are least homologous with the animal synthase sequences, whereas carboxylase sequences are highly conserved. The sequences of the ATP, HCO3-, and CoA binding sites of the carboxylases are also well conserved (approximately 50% identical). The sequences surrounding the biotin binding site are poorly conserved, suggesting that this sequence may not be critical as long as the biotin is available for carboxylase reactions. On the basis of this sequence identity, we have defined the putative biotin carboxylase and transcarboxylase domains.


Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance.

Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.


Actions of dopamine and dopaminergic drugs on cloned serotonin receptors expressed in Xenopus oocytes.

Using electrophysiological techniques, we studied interactions of dopamine and selected dopaminergic drugs with serotonin (5-hydroxytryptamine; 5-HT) receptors expressed in Xenopus oocytes by RNAs transcribed from cloned cDNAs. Oocytes showing strong expression of 5-HT1c and 5-HT2 receptors became weakly responsive to the neurotransmitter dopamine, which, like 5-HT, elicited Cl- currents through activation of the phosphatidylinositol/Ca2+ messenger pathway. The two types of 5-HT receptors showed similar sensitivity to dopamine; threshold responses were activated at concentrations as low as 1 microM. However, maximum dopamine responses were only 5-20% of maximum responses activated by 5-HT. The dopamine D1 receptor antagonist SCH 23390 was a potent agonist on 5-HT1c and 5-HT2 receptors. SCH 23390 elicited currents at concentrations as low as 1 nM, but maximum responses were again only 5-20% of those activated by 5-HT. Fenoldopam, a dopamine D1 receptor agonist, also interacted with 5-HT1c and 5-HT2 receptors, eliciting threshold responses between 10 and 20 nM. Our experiments raise the possibility that low micromolar concentrations of dopamine can cause weak activation and concomitant desensitization of serotoninergic systems in vivo and demonstrate that benzazepines can interact with 5-HT receptors at nanomolar concentrations.


Molecular identification of Wolbachia, the agent of cytoplasmic incompatibility in Drosophila simulans, and variability in relation with host mitochondrial types.

Sequences of a segment of the 16S ribosomal DNA of Wolbachia, a rickettsia-like microorganism responsible for cytoplasmic incompatibility in Drosophila simulans, have been obtained after polymerase chain reaction (PCR) amplification. Their comparison with other eubacterial sequences allows us to assign these endosymbionts to the alpha subdivision of purple bacteria. Four related sequences have been obtained for microorganisms carried by eight isofemale lines representative of the three mitochondrial types of D. simulans. Their phylogeny and level of divergence do not parallel that of the mitochondrial DNA, suggesting that several independent infections occurred. There is no direct relation between bacterial phylogeny and formerly identified incompatibility types.


Exocytosis by synaptic terminals innervating the adrenal gland of the goldfish reveals multiple domains within the plasmalemma.

The adrenal chromaffin gland of the goldfish has typical synaptic terminals embedded in its surface which are homologues of the cholinergic fibres innervating the mammalian adrenal medulla. The terminals contain both lucent synaptic vesicles and larger secretory granules with dense cores, known to be storage sites for transmitters and peptides, respectively. Three domains are present within the terminal plasmalemma. Exocytosis of vesicles is thought to be associated with a 'synaptic domain' marked by synaptic thickenings around which the vesicles cluster. Exocytosis of granules, stimulated by high K+ and visualized with the aid of tannic acid, is almost exclusively associated with areas of the membrane adjacent to chromaffin cells, and in particular with unspecialized regions which constitute the 'parasynaptic domain', creating a pattern of targeted secretory discharge. Sites of release within the 'non-synaptic domain', which is sheathed in glial cell lamellae, are extremely rare, despite the expansive character of this domain and the close association of granules with the plasmalemma within it. The pattern of secretory release described may be correlated with the position of the terminals at the surface of the innervated organ.


Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH.

We have used three-microelectrode voltage clamp in conjunction with the ammonium prepulse method to investigate the effects of lowered intracellular pH (pHi) on resting potassium currents of frog skeletal muscle fibres. Potassium currents were recorded in 40 mM K+, Cl(-)-free solution in response either to voltage steps or ramps. An ammonium prepulse (2 h) reduced pHi to 6.45 from a control value of 7.19. The intracellular ATP concentration, measured with high-pressure liquid chromatography (HPLC), was unchanged by this procedure. Mean outward potassium currents were larger in low pHi than in control fibres, being about twice as large at +40 mV, whereas mean inward currents were very similar in control and low-pHi fibres. The sulphonylurea glibenclamide blocked single KATP channels in excised patches with a Kd of 3 microM. In intact fibres 50 microM glibenclamide had no effect on K+ currents in controls but reduced currents in low-pHi fibres. In the presence of glibenclamide, K+ currents in low-pHi fibres were not significantly different from those in control fibres. We suggest that reduced pHi in intact skeletal muscle fibres opens ATP-dependent potassium channels (KATP channels), as has been shown to occur in excised patches of membrane.


Early changes in the development of dopaminergic neurotransmission after maternal exposure to cannabinoids.

Perinatal exposure to cannabinoid derivatives has been shown to affect brain development. In this work, we studied the changes induced by maternal exposure to cannabinoids during gestation and lactation on the dopaminergic activity in the prosencephalic area of offspring of several days of development. This brain area contains an increasing population of dopaminergic terminals from the different dopaminergic pathways that become differentiated in the adult rat. We measured the endogenous content of dopamine and its intraneuronal metabolite, L-3,4-dihydroxyphenylacetic acid, and the activity of tyrosine hydroxylase as indices of dopaminergic activity. Results showed that perinatal exposure to cannabinoids caused several changes in the evolution of the dopaminergic indices studied. These changes were mainly observed in males. The only alteration in females occurred on the tenth day of development: An increase in dopamine content was observed with no changes in either the content of L-3,4-dihydroxyphenylacetic acid or tyrosine hydroxylase activity. In males, the content of both dopamine and L-3,4-dihydroxyphenylacetic acid were decreased on the day previous to birth in the animals exposed to cannabinoids. Although the reduction in its metabolite disappeared on the fifth day, the decrease in dopamine was maintained and it was correlated with a decrease in tyrosine hydroxylase activity. However, this decrease in the activity of tyrosine hydroxylase was followed by an increase on the tenth day. These results allow us to conclude that perinatal exposure to cannabinoids produces changes in the normal development of several indices of the activity of dopaminergic neurons in the brain area containing the most important population of dopaminergic endings. These changes were mainly observed in males. They could be responsible for a long-term alteration in the neurological processes in which these neurons are involved in the adult.


Dopaminergic influences on male sexual behavior of rhesus monkeys: effects of dopamine agonists.

Previous research has demonstrated that the mixed D1/D2 dopamine receptor agonist, apomorphine, and the specific D2 dopamine receptor agonist, quinelorane, facilitated penile erections and masturbatory behavior of male rhesus monkeys when they were tested in the presence of a female monkey that they could see, hear, and smell but not physically contact. The present study was designed to further examine dopaminergic influences on male sexual behavior of rhesus monkeys by evaluating male copulatory behavior following administration of these dopaminergic agents, as well as a D1 agonist, CY 208-243. Apomorphine and quinelorane treatment produced dose-dependent effects on male sexual responding. Compared to vehicle-based performance, postejaculatory intervals were shortened following treatment with either 100-200 micrograms/kg apomorphine or 2.5-10 micrograms/kg quinelorane. Higher doses of apomorphine or quinelorane did not reliably influence the postejaculatory interval. Ejaculation latency, intromission frequency, and number of thrusts/intromission increased following administration of 200-400 micrograms/kg apomorphine and 25 micrograms/kg quinelorane, indicating that dopaminergic stimulation in this dose range raised the monkeys' ejaculatory threshold. No behavioral effects of the D1 agonist, CY 208-243, were observed in this testing situation. These experiments provide further evidence that dopaminergic mechanisms may play a role in the regulation of male sexual behavior of rhesus monkeys and, in particular, demonstrate that the direction of the effect depends on the dopamine receptor subtype and dosage of the dopamine agonist being administered.


Effect of a new anxiolytic, DN-2327, on learning and memory in rats.

The effects of a new anxiolytic, (2-(7-chloro-1,8-naphthyridin-2-yl)-3- [(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-carbonylmethyl] isoindolin-1-one (DN-2327), on the execution of step-through passive avoidance and delayed spontaneous alternation tasks were assessed and compared with those of diazepam (DZP) and buspirone. DN-2327 and buspirone (both 10 and 20 mg/kg, PO) impaired performance in the 48-h passive avoidance recall test when given prior to the test session, but not when given before the training trial. DZP impaired the performance at doses of more than 5 and more than 10 mg/kg PO when given prior to the test session and when given before the training trial, respectively. The action of DZP (10 mg/kg PO) when given before the training trial was antagonized by flumazenil (20 mg/kg, IP) and tended to be antagonized by DN-2327 (10 and 30 mg/kg, PO), but was not affected by buspirone. No evidence for possible amnesic effects of DN-2327 or buspirone on working memory was found in the delayed spontaneous alternation task, but DZP (3 and 10 mg/kg, PO) caused significant impairment of working memory. Electroshock sensitivities detected by flinch, jump, and vocalization thresholds were not influenced significantly by DN-2327 (30 and 100 mg/kg, PO), DZP (10 and 30 mg/kg, PO) or buspirone (30 and 100 mg/kg, PO).(ABSTRACT TRUNCATED AT 250 WORDS)


Atipamezole, an alpha 2 antagonist, stabilizes age-related high-voltage spindle and passive avoidance defects.

The present study investigates the effects of an alpha 2 antagonist, atipamezole (Ati), on the high-voltage spindle (HVS; Ati at 0.1, 1.0, and 3.0 mg/kg) activity, passive avoidance retention (PA; Ati at 3 mg/kg; injected before retention trial), and water maze (WM; Ati at 3 mg/kg; injected after daily training trials) acquisition of young and aged rats. PA retention trial performance defect of aged rats was partially alleviated by Ati at a 3-mg/kg dose. Ati at 3 mg/kg had no effect on the PA performance of young rats. Retention trial performance of nonshocked young or aged rats was not altered by a 3-mg/kg Ati dose. WM acquisition was not affected by posttraining Ati injections. Age-related increase of HVS was stabilized by Ati at 1 or 3 mg/kg. Ati at 1 and 3 mg/kg completely suppressed HVS of young rats. Ati at 0.1 mg/kg had no effect on HVS of young or aged rats. The results suggest that alpha 2-antagonist-administration-induced increase in noradrenergic activity may stabilize age-related HVS activity increase and PA performance defect.


Transglutaminase activity in human brain tumors.

Transglutaminase (TG) activity was measured in tissue samples of 45 human brain tumors (pituitary adenomas, meningiomas and gliomas) obtained during neurosurgery. Biochemical analysis and histopathological classification were carried out in the same samples. Mean enzyme activity was highest in non-glial tumors, but due to a high variability of values no significant changes were found between the various histological groups. Thus, TG activity, although considered to play a role in neoplastic growth, does not represent a biochemical marker of malignancy in human brain tumors.


Is it still adequate to study the nervous system using methods of catalytic enzyme histochemistry?

The frequent use of methods different from enzyme histochemistry evokes the question, whether it is still useful to apply methods of catalytic enzyme histochemistry to study the nervous system. In this brief overview it is shown, that catalytic enzyme histochemistry can still contribute to a better understanding of nervous system function. This was enabled by methodological progress, i.e., the modification of already existing procedures or the development of new techniques for the visualization and measurement of enzymes in tissue sections using their catalytic properties. The methods for acetylcholinesterase, monoamine oxidase as well as certain exoglycosidases and phosphatases will be given as examples. The application of these procedures as well as of methods for proteases, oxyradical-generating oxidases and enzymes involved in the metabolism of amino acid and other transmitters will illustrate, that qualitative (localization) and quantitative (measurement) catalytic enzyme histochemistry can still contribute to a better understanding of nervous system function.


Pertussis toxin non-sensitive G protein mediates cholinergic stimulation for secretion of pancreastatin and somatostatin from QGP-1N cells.

To clarify the possible role of a guanine nucleotide-binding protein (G-protein) in the signal transducing system activated by carbachol, actions of carbachol on human pancreastatin producing cell line (QGP-1N) were compared with those of fluoride, a well-known activator of stimulatory (Gs) or inhibitory (Gi) G protein. 10(-5) M of carbachol as well as 20 mM of NaF stimulated secretion of pancreastatin and somatostatin and intracellular Ca2+ mobilization. These secretion and Ca2+ mobilization were not modified by pertussis toxin, an inhibitor of Gi protein. These results suggest that pancreastatin and somatostatin secretions from QGP-1N are regulated by acetylcholine through a muscarinic receptor coupled to the activation of polyphosphoinositide breakdown by a G protein, which appears to be fluoride sensitive but is other than a Gi-like protein.


The source of calcium for CCK-induced contraction of the guinea-pig gall bladder.

The sources of calcium for cholecystokinin octapeptide (CCK-OP)-induced gallbladder smooth muscle contraction are considered both extracellular and intracellular, but the relative need for intracellular calcium especially at low, physiological concentrations is not clear. To better define the calcium sources responsible for guinea-pig gallbladder contractions in vitro, we inhibited calcium influx using the calcium channel blocker, methoxyverapamil, and a calcium-free Krebs' solution. Availability and release of intracellular calcium stores were depleted by strontium substitution and ryanodine. CCK-OP was compared to bethanechol and potassium chloride (KCl). Preventing calcium influx with 10(-5) M methoxyverapamil depressed the responses to CCK-OP, bethanechol and KCl. Methoxyverapamil, however, had little effect on the time-dependent generation of tension to CCK-OP, but significantly reduced the response to bethanechol and KCl, each at ED50. The duration of the contractile response in the calcium-free Krebs' solution to CCK-OP was longer than that for bethanechol. Strontium (2.5 mM) significantly attenuated the response to CCK-OP and bethanechol, but not to KCl. Ryanodine significantly reduced contractions induced by CCK-OP but not for bethanechol, both at low dose ED25. These results indicate that contraction of the guinea-pig gallbladder induced by CCK-OP, bethanechol and KCl requires extracellular calcium influx. Further, the initiation and maintenance of contraction by CCK-OP and bethanechol necessitates calcium mobilisation from intracellular stores. CCK-OP may have a greater penchant for these calcium stores, particularly at physiological doses.


Analysis of pokeweed mitogen-induced in vitro proliferative and antibody responses of bovine lymphocytes.

The functional role of subpopulations of bovine cells in the regulation of pokeweed mitogen (PWM)-induced proliferative and antibody responses of peripheral blood mononuclear cells (PBM) was analysed. Subpopulations of bovine PBM identified by specific monoclonal antibodies (mAbs) were isolated by sorting them through the fluorescence activated cells sorter (FACS). The depletion from PBM of T cells bearing the CD4 marker, recognised by mAb IL-A12, resulted in a reduction of PWM-induced responses, which could be partly reversed by the addition of CD4 positive T cells. The depletion of cells belonging to the macrophage/monocyte lineage also resulted in a reduction of PWM-induced proliferative responses. PBM depleted of CD8 positive T cells, recognised by mAb IL-A51, showed increased PWM-induced responses, which in turn were reduced by the addition of mAb IL-A51 positive cells. Proliferative and antibody responses were also obtained by PWM stimulation of FACS-purified B cells, in the presence of bovine T cell growth factor.


Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.

Germ-line mutations of the APC gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominantly inherited disease in humans. Patients with FAP develop multiple benign colorectal tumors. Recently, a mouse lineage that exhibits an autosomal dominantly inherited predisposition to multiple intestinal neoplasia (Min) was described. Linkage analysis showed that the murine homolog of the APC gene (mApc) was tightly linked to the Min locus. Sequence comparison of mApc between normal and Min-affected mice identified a nonsense mutation, which cosegregated with the Min phenotype. This mutation is analogous to those found in FAP kindreds and in sporadic colorectal cancers.


Microdomains of high calcium concentration in a presynaptic terminal.

Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion, supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation of a low-affinity calcium-binding site at the active zone.


Modulation of single-unit activity in the rat medial amygdala by neurotransmitters, estrogen priming, and synaptic inputs from the hypothalamus and midbrain.

The medial amygdala (m-AMG) appears to act as an integrative center for sensory, synaptic, and endocrine signals important in the regulation of reproductive function. Extracellular single-unit recordings from anesthetized, ovariectomized female rats were used to investigate neuropharmacological, hormonal, and synaptic modulation of neurons in the m-AMG. Electrical stimulation of the ventromedial hypothalamus (VMH) elicited excitatory or inhibitory orthodromic responses in 72% and antidromic responses in 7% of m-AMG neurons, whereas stimulation of the midbrain central gray (MCG) induced orthodromic responses in 43% of m-AMG neurons. Interestingly, most cells that were influenced by MCG stimulation were also orthodromically driven by the VMH, as 40% of all m-AMG cells responded orthodromically to both the VMH and MCG. Furthermore, the majority of these cells tended to be modulated by both areas in the same direction. Iontophoretic application of glutamate, GABA, ACh, and LHRH could modulate the spontaneous firing rate of m-AMG neurons. In particular, ACh had a predominantly excitatory action, which was more effective on m-AMG neurons that were orthodromically driven by the VMH and that were from estrogen-primed animals. In addition to increasing chemical responsiveness to ACh, estrogen priming of ovariectomized animals also increased the spontaneous firing rate of m-AMG neurons and decreased the number of silent cells. These modulatory actions on m-AMG neurons may be important in the medial amygdala's regulation of the behavioral and endocrine aspects of reproductive function in the female rat.


Contribution of the amygdala and nucleus accumbens to ventral pallidal responses to dopamine agonists.

Neurons recorded from ventral pallidum/substantia innominata (VP) of the basal forebrain respond to dopaminergic agonists that activate either the D1 or D2 the receptor subtype. Major afferent systems to the VP originate within amygdaloid nuclei (AMN) and the nucleus accumbens (NA). Since both the AMN and the NA are dopaminoceptive, the present study sought to analyze the contribution of these afferent systems to VP responses to dopaminergic agonists. Single VP neurons were electrophysiologically recorded in vivo from chloral hydrate-anesthetized rats, and the following determinations were made. 1) Effects of pharmacologic inactivation of an afferent system were assessed by monitoring VP neurons during intracerebral microinjections of the local anesthetic procaine, administered directly into either the AMN or the NA. 2) With procaine-induced VP rate changes used to indicate an afferent influence on the recorded neuron, VP responses to apomorphine (an agonist that acts at D1 and D2 receptor subtypes), SKF38393 (a D1 agonist), or quinpirole (a D2 agonist) were determined and compared with responses in rats not receiving the procaine pretreatment. Following pharmacologic inactivation of either the AMN or the NA, approximately 80% of the VP neurons monitored demonstrated rate changes, illustrating that spontaneous neuronal firing in the Vp is dependent on tonically input systems. Following afferent cessation, responses to apomorphine and quinpirole remained intact, suggesting that the AMN or NA is not necessary for VP responding to the systemic administration of dopaminergic agonists that act at D2 receptors. In contrast, the number of neurons that responded to SKF38393 was diminished follow intra-AMN (but not intra-NA) procaine. This suggests that D1-induced VP responses are mediated, at least in part, via the AMN.


Effect of various neurotransmitters and neuropeptides on the release of corticotropin-releasing hormone from the rat cortex in vitro.

Corticotropin-releasing hormone (CRH), in addition to its neuroendocrine role, may act as a central neurotransmitter. Cerebral cortical CRH may have an important role in behavioral and neurodegenerative disorders. To gain an understanding of factors that may influence cortical CRH, we investigated the effect of several neurotransmitters and neuropeptides on the release of immunoreactive CRH (iCRH) from various cerebral cortical regions [frontal (FC), parietal (PC), temporal (TC), and occipital (OC)] in vitro. The hypothalamic release of iCRH was also evaluated under the same experimental conditions. Basal release of iCRH was approximately 2-fold, and KCl-stimulated iCRH release was approximately 4-fold higher in the hypothalamus than in any of the cortical regions. Cortical iCRH release was stimulated by 10 nM somatostatin (SRIF) in PC and 1 nM neuropeptide Y (NPY) in TC. Cortical iCRH release was inhibited by 1 and 10 nM acetylcholine (ACh), 0.1 microM glutamate, and 10 nM NPY. These effects were confined to the FC and/or PC. Hypothalamic iCRH release was stimulated by 1 and 10 nM ACh, 10 microM GABA, and 1 and 10 nM serotonin but was inhibited by 10 nM SRIF and 1 microM GABA. Growth hormone-releasing hormone did not affect cortical or hypothalamic iCRH release. These results demonstrate that CRH release from the cerebral cortex and the hypothalamus are under different regulatory mechanism(s). Furthermore, they indicate that the release of CRH in various cortical regions may be regulated differentially by the same neurotransmitter.


Criteria for judging the relative toxicity of chemicals from developmental toxicity data: a workshop summary.

In summary, participants of this workshop confirmed that many criteria need to be considered when interpreting the results of developmental toxicity studies. All aspects of developmental toxicity are of interest, but their importance to the consideration varies along the continuum from hazard detection to risk estimation. As with many other manifestations of toxicity, potential developmental risk to humans is a function of exposure and developmental toxicity, the latter reflecting the inherent potential of a substance to cause an adverse effect under some defined condition. All of the criteria discussed in this workshop were considered to be of some importance in characterizing the developmental toxicity of a substance, their relative importance being a function of the question under consideration. Proper interpretation of developmental toxicity data, which must include prenatal as well as postnatal observations to be considered complete, should take into account the differential toxicity to the mother and the conceptus, the nature of the developmental toxicity observed, and the consistency of response between species. The proximity of human exposure levels to dose levels that are developmentally toxic in animals is a major determinant of the potential for hazard to humans. Mechanistic and pharmacokinetic knowledge can modulate interpretation of descriptive data and refine the prediction of human hazard. Recognizing that in vitro studies will never completely recapitulate the results of in vivo studies, the committee to update the "Smith list" will move forward taking the discussions of this workshop into account.


Muscle relaxation with Norcuron in patients undergoing obstetrical-gynaecological operation.

The observations with Norcuron (Richter) injection used for muscle relaxation in the course of 204 obstetrical-gynaecological operations have been discussed. In the majority of cases Caesarean section was performed, the other interventions were major operations. Norcuron proved to be a neuro-muscular blocking agent which has no cardiovascular side-effects, has a medium duration of action, and may be repeatedly administered according to requirement without risk of cumulation. It may be combined with other anaesthetics, its action may be successfully antagonized, and the drug has no postoperative after-effects. The Apgar scores of the newborns remained within the normal ranges.


Transfection with gamma-glutamyl transpeptidase enhances recovery from glutathione depletion using extracellular glutathione.

Glutathione (L-gamma-glutamyl-L-cysteinylglycine) is an important constituent of the antioxidant and detoxifying mechanisms of cells. The plasma membrane bound enzyme, gamma-glutamyl transpeptidase (GGT), catalyzes the first step in the degradation of extracellular glutathione, the components of which are then used for de novo glutathione synthesis. We tested the hypothesis that an increase in GGT activity would enhance the utilization of extracellular glutathione by cells challenged with a glutathione-depleting agent. A eukaryotic system stably overexpressing GGT (nearly 200-fold) was developed by transfection of NIH-3T3 fibroblasts with a human placental GGT cDNA. These cells and controls were incubated for 30 min with 1 mM diethyl maleate, which caused approximately 80% intracellular glutathione depletion. Glutathione was added to the medium and cells were allowed to resynthesize intracellular glutathione. The transfected cells used extracellular glutathione much more efficiently than controls in terms of both the concentration dependence and the rate of glutathione resynthesis. Serine-borate, a competitive inhibitor of GGT, blocked the restoration of intracellular glutathione. The results support the hypothesis that the increase in GGT activity that occurs in some toxicologic or pathologic conditions could provide protection against glutathione depletion.


Two polyhydroxylated steroids from the Chinese soft coral Sinularia microclavata.

Two polyhydroxylated steroids have been isolated from the South China Sea soft coral Sinularia microclavata, and their structures were established as 24-methylenecholestane-1 alpha,3 beta,5 alpha,6 beta-tetrol and 1 alpha,3 beta,5 alpha-trihydroxy-24-methylenecholestan-6-one from spectral evidence and from comparison with two reference compounds, numersterol A and 24-methylenecholestane-1 alpha,3 beta,5 alpha,6 beta, 25-pentol, which were isolated from the soft corals, Simularia numerosa and Sarcophyton glaucum, respectively.


Advantages and guidelines for using alpha-2 agonists as anesthetic adjuvants.

Xylazine and medetomidine produce reliable sedation, muscle relaxation, and analgesia in dogs and cats. In addition, alpha-2 agonists have proved very effective as sedative-analgesic adjuncts when coadministered with benzodiazepine or opioid agonists. Alpha-2 agonists should not be classified as monoanesthetics. They are excellent anesthetic adjuncts when combined with dissociatives and opioids. Because of the acute alterations in cardiopulmonary function commonly induced by alpha-2 agonists, it is suggested that their use be restricted to the young healthy patient undergoing routine surgical or diagnostic procedure. The development of more specific and selective alpha-2 agonists will continue to enhance the safety and reliability of this novel class of compounds. The unique spectrum of anesthetic properties induced by alpha-2 agonists has assured them of an increasingly prominent role in the development of new and sophisticated ways of achieving anesthesia.


Precautions when using alpha-2 agonists as anesthetics or anesthetic adjuvants.

Alpha-2 adrenergic agonists have some unique properties that could theoretically make them useful in the perianesthetic period: they reduce the MAC of inhaled anesthetics, and they are reversible. They also have properties that may limit their usefulness, such as marked decreases in cardiac output. Their clinical utility awaits further studies.


Anatomy of the spinal cord and how the spinal cord is affected by local anesthetics and other drugs.

The spinal cord can be affected by several categories of drugs: local anesthetics, opiate agonist, alpha 2 adrenergic agonists, and ketamine. The mechanism of action, side effects, and usefulness of these injections vary with the type of drug used.


Absence of selection of HIV-1 variants in vivo based on transcription/transactivation during progression to AIDS.

The activity of the human immunodeficiency virus type 1 (HIV-1) transactivation protagonists tat and TAR has been analyzed from sequential primary material. The sequences were amplified from uncultured peripheral blood mononuclear cells. Despite fluctuations within the tat and TAR quasispecies there was no obvious selection for a variant encoding more powerful transactivation components either in vivo or ex vivo, indicating that this system is not exploited during disease progression. The basal levels of the natural promoters were, depending on the cell line, two- to fourfold higher than that of the reference promoter, itself derived from ex vivo adapted HIV-1 Lai.


Protective role of cytotoxic lymphocytes against murine leukemia virus-induced neurologic disease and immunodeficiency is enhanced by the presence of helper T cells.

We examined the role of T cells and their separated subsets in providing immunity against ts1 (a mutant of the Moloney murine leukemia virus) induced paralysis and immunodeficiency. Adoptive transfer of syngeneic total T cells from immunized mice protected newborn mice, at least partially, from ts1-induced disease syndrome. In infected mice who received total immune T cells, virus replication was reduced and the mice survived longer. When only separated immune CD8+ T cells were transferred to infected mice, similar protection, albeit to a lesser extent, was observed. Transfer of separated immune CD4+ T cells alone gave no protection. However, when recombined CD4+ and CD8+ cells were transferred together, an immune response similar to that when total T cells were transferred was observed. Cytotoxic assays from ts1-immunized mice revealed the presence of virus-specific CD8+ cytotoxic T lymphocytes that could lyse virus-expressing cells at a high effector/target ratio. We conclude that CD8+ T cells alone can provide immunity against ts1-induced paralysis and immunodeficiency and that the simultaneous presence of CD4+ T cells can also significantly enhance the immune response.


[C-erbB-2 oncogene amplification in breast cancer in correlation to steroid and epidermal growth factor receptor].

The amplification grade of oncogene c-erbB-2 was examined by the polymerase-chain-reaction-method in DNA's of 56 primary mammary carcinomas. 26 (46.4%) of these showed the amplified oncogene c-erbB-2. In the strongly amplified cases, the expression of the c-erbB-2 oncoprotein was verifiable immunohistochemically. Between the progesterone receptor status (PR) and the amplified c-erbB-2 oncogene there was a statistically proven dependency. No correlation was observed between the amplified c-erbB-2 oncogene and the epidermal growth-factor receptor (EGFR).


Candida albicans grown in glucose-free media contains serum-independent chemotactic activity.

Infection of skin with Candida albicans is usually followed by infiltration of neutrophil granulocytes (PMN). So far, chemotaxins for PMN have been isolated from C. albicans cultures grown in the presence of glucose. However, since glucose is not present in skin in vivo, a contribution of such factors to Candida-triggered cutaneous inflammation would appear unlikely. In order to clarify this question, chemotactic activity was measured in extracts from three different strains of C. albicans which were grown in five different peptone media free of carbohydrates and serum. In addition, four culture systems were supplemented with lipids normally present in human stratum corneum, including a triglyceride, cholesterol, and sphingomyelin. In all sugar-free grown cultures, serum-independent chemotactic activity was detected by use of the Boyden chamber technique. Since, as shown here in vitro, production of neutrophil chemotaxins by C. albicans is independent of glucose-feeding, a possible role of Candida-chemotaxins in the pathophysiology of cutaneous candidosis can no longer be excluded.


Werner's syndrome: no difference in in vitro life span of dermal fibroblasts from proximal and distal parts of the body.

Recently it has been reported that fibroblasts from distal parts of the body of a patient with Werner's syndrome grew poorly in vitro as compared with those from the proximal part of the same patient. To confirm this observation, cultures of fibroblasts from different parts of the body were set up in 2 cases of Werner's syndrome, but no significant difference in life span was observed. Fetal calf serum (FCS) and fibroblast growth factor (FGF) stimulated growth of fibroblasts from different body parts equally well. These data indicate that there is no difference in growth activity of fibroblasts from proximal and distal body parts in patients with Werner's syndrome. Moreover, the growth rate of epidermal outgrowths did not differ significantly between proximal and distal parts of these patients.


A comparison of morphoea and lichen sclerosus et atrophicus in vitro: the effects of para-aminobenzoate on skin fibroblasts.

To study the effects of para-aminobenzoate on the dermis, fibroblast cell lines derived from lesions of lichen sclerosus et atrophicus, from morphoea and from normal skin were incubated with Potaba in vitro. Monolayer cultures containing Potaba showed a dose-dependent inhibition of proliferation beginning at 1,000 micrograms/ml with total inhibition at 10,000 micrograms/ml. Mean ID50 values for the three groups were not significantly different. There was a similar dose dependent inhibition of glycosaminoglycan secretion in all 3 groups, except at 10,000 micrograms/ml where secretion by lichen sclerosus et atrophicus and morphoea fibroblasts was significantly more inhibited than normal lines. Inhibition of the glycosaminoglycan secretion at 10-1,000 micrograms/ml was a direct effect of the drug rather than an indirect effect of changes in cell density, and lichen sclerosus et atrophicus fibroblasts produced about 40% more GAG than the morphoea or normal lines growing at similar densities. Collagen synthesis was increased in both lichen sclerosus et atrophicus and morphoea cell lines, with increased non-collagenous protein in morphoea lines. These results confirm that there are differences between lichen sclerosus et atrophicus and morphoea, and suggest glycosaminoglycan secretion as a possible target for the therapeutic action of Potaba.


Hypertrichosis lanuginosa acquisita.

Hypertrichosis lanuginosa acquisita was observed in a female patient with stage IV malignant melanoma and also diffuse melanosis of the skin. The patient died within 2 months after the appearance of HL.


Extraocular sebaceous carcinoma.

A 79-year-old man with no history of previous irradiation presented with a large ulcerated tumour of the cheek. The histological features favoured a true sebaceous carcinoma. Neither squamous nor basal differentiation were seen. Sudan stainings were positive. Tumour cells expressed suprabasal keratins and were negative for carcinoembryonic antigen, vimentin and S 100 protein. Extraocular sebaceous carcinomas occurring without previous irradiation are rare tumours which behave aggressively. Treatment regimens are discussed.


Schnitzler's syndrome (urticaria and macroglobulinemia) associated with pseudoxanthoma elasticum.

Schnitzler's syndrome, first described in 1974, is defined by chronic non-pruritic urticaria, osteocondentation, and a monoclonal IgM dysproteinemia, but without criteria of lymphoproliferative disease. We report a patient with chronic urticaria and macroglobulinemia. In addition, he had double monoconal dysproteinemia IgM kappa (31.3 g/l) and IgA lambda, osteocondensation, and some cutaneous lesions of pseudoxanthoma elasticum. Only 20 cases of Schnitzler's syndrome have been reported hitherto. This is the first case associated with pseudoxanthoma elasticum, which was localized and discovered at the same time as Schnitzler's syndrome. We discuss the possible role of monoclonal immunoglobulin in the occurrence of localized elastorrexhis.


Coeliac-type dental enamel defects in patients with dermatitis herpetiformis.

The teeth of 30 adult patients with dermatitis herpetiformis and 66 sex- and age-matched healthy controls were examined for dental enamel defects. Sixteen of the patients (53%) with dermatitis herpetiformis, opposed to only one (2%) of the healthy controls (p less than 0.001), were found to have coeliactype permanent-tooth enamel defects. The grades of these defects were milder than those described for severe coeliac disease. There was no correlation between the degree of enamel defects and jejunal villous atrophy. The present finding of frequent coeliactype dental enamel defects in adults with dermatitis herpetiformis suggests that these patients were already suffering from subclinical gluteninduced enteropathy in early childhood, at the time when the crowns of permanent teeth develop.


Clinical and non-invasive evaluation of 12% ammonium lactate emulsion for the treatment of dry skin in atopic and non-atopic subjects.

Clinical dryness of the leg skin is a common problem among dermatological patients. The efficacy and safety of 12% ammonium lactate emulsion (Keratisdin) for the treatment of dry skin on the legs of atopic and non-atopic subjects has been assessed by clinical criteria and by five different non-invasive methods. These methods measure biophysical parameters such as electrical capacitance of stratum corneum, skin surface lipids, transepidermal water loss (TEWL), skin surface topography (scanning electron microscopy and image analysis) as well as the biomechanical properties of the skin. Treatment with the test emulsion significantly reduced the severity scores for dryness, desquamation and pruritus when measured 15 days later. All patients tested showed a significant increase in electrical capacitance, skin surface lipids, extensibility and firmness of the skin, and an improvement in the skin barrier function and skin surface topography. This study showed that non-invasive techniques are excellent complementary tools in clinical studies.


The in vivo effect of UVB radiation on skin bacteria in patients with atopic dermatitis.

Fourteen patients suffering from atopic dermatitis under treatment with UVB radiation were subjected to aerobic bacterial cultures in order to investigate whether this ultraviolet waveband has any in vivo germicidal effects, and, if so, whether there is a correlation with clinical improvement. Treatments were given 3 times a week for 8 weeks. Bacterial samples were collected before, midway and after the termination of therapy. On the latter two occasions, cultures were performed 30 min and 24 h post-UVB irradiation. The main bacteria found were Staphylococcus epidermidis and S. aureus. S. aureus carriage was found in 12 patients in lesional, dermatitic skin, and in 11 patients in clinically non-lesional skin. UVB radiation was found to have an antimicrobial effect primarily concerning S. aureus. Bacterial counts of this organism in lesional skin were decreased from a mean of 1.3 x 10(3) to 1.2 x 10(1) bacteria per cm2 skin at the 8-week 30-min count (p less than 0.01) and 7.5 x 10(1) at the 8-week 24-h count (p less than 0.05). The treatment yielded a statistically significant clinical improvement.


Atypical presentation of co-existent Haemophilus ducreyi and Treponema pallidum infection in an HIV-positive male.

A 25-year-old homosexual black male presented with asymmetrical perianal ulceration of uncertain clinical origin. Indepth microbiological examination revealed the combined presence of Haemophilus ducreyi and Treponema pallidum. The atypical clinical appearance may have been due to the changed immunological status of the host's being infected with Human Immunodeficiency Virus.


Non-specific immunity in patients with primary anogenital warts treated with interferon alpha plus cryotherapy or cryotherapy alone.

Combination treatment of primary anogenital warts with subcutaneous interferon alpha 2a plus cryotherapy was no more efficacious than cryotherapy alone. Patients with primary AG warts showed no in vitro or in vivo suppression of non-specific immunity. In patients treated with interferon plus cryotherapy non-specific cellular immunity was stimulated, both in vitro and in vivo compared with patients treated with cryotherapy alone.


Transepidermal water loss related to volar forearm sites in humans.

The aim of this study was to demonstrate differences in human skin transepidermal water loss (TEWL) in vivo related to site on volar forearm. Fourteen healthy volunteers entered the study and seven sites were tested. After randomization of both forearm and measure order, TEWL measurements were performed using the Servo Med Evaporimeter. TEWL values next to the wrist were found statistically greater than on the other sites. Hence wrist region should preferably be excluded from TEWL measurements on forearm.


Lichen planus: an unusual cause of phimosis.

We report on a 47-year-old man with oral and genital lichen planus. After some months of the disease, increasing phimosis developed which had not been present before. Retraction of the foreskin was now impossible and sexual intercourse was painful. Treatment with triamcinoloneacetonide and etretinate ameliorated the phimosis but the patient was still not comfortable and circumcision was performed. Histology from the foreskin revealed the typical picture of lichen planus. No features of lichen sclerosus et atrophicus were present. This is the first published observation of phimosis as a result of lichen planus.


Moisturizers prevent irritant dermatitis.

The purpose of this study was to investigate the ability of eight different moisturizers to prevent irritant dermatitis. Twelve healthy female students washed the outer aspect of their upper arms with a liquid detergent for one minute twice a day for one week. Seven skin creams and one skin oil were applied to 3 x 7 cm areas of the left upper arm just after each washing, while the right upper arm was left untreated. Transepidermal water loss (TEWL) (mean) increased from 7.1 to 9.3 g/m2/h (p less than 0.001) and laser-Doppler flowmetry (LDF) value (mean) decreased from 11.8 to 10.8 arbitrary units (N.S.) in the left upper arm, but there was no statistical difference between the eight moisturizers. During the second week of the study, the test subjects did not continue washing their arms. Eight areas (3 x 7 cm) of the right upper arm were treated with the moisturizers twice a day. The mean TEWL value decreased from 20.3 to 8.6 (p less than 0.0011) over 7 days, but there were no significant differences between the individual moisturizers. The laser-Doppler values showed the same trend as the TEWL values. In conclusion, regular use of emollients prevented irritant dermatitis from a detergent.


Nickel contact sensitivity in the guinea pig. An efficient open application test method.

Nickel contact sensitivity was successfully induced in guinea pigs using an open epicutaneous application method. Immediately after pretreatment with 1% aqueous sodium lauryl sulfate, upper back skin was treated daily for 4 weeks with 0.3%-3% nickel sulfate in either a 1% lanolin cream (Vaseline, pH 5 SAD crème) or hydroxypropyl cellulose. Weekly intradermal injections with aluminium potassium sulfate were used as adjuvant. The animals were challenged twice with a one week interval, with nickel sulfate 2% in water and 1% in petrolatum, respectively. The response rates in the test groups treated with nickel sulfate 1% or 3% in the lanolin cream or 1% in hydroxypropyl cellulose were significantly different from the response rate in the control group. Considering both readings at both challenges, the frequency of sensitization was 57-93% (8 of 14 to 13 of 14 animals) in the group treated with 1% in the lanolin cream, 60-100% (9/15 to 15/15 animals) in the group treated with 3% in the lanolin cream, and 67-75% (8/12 to 9/12 animals) in the group treated with 1% in hydroxypropyl cellulose. Rechallenge of initially sensitized animals 10 weeks later confirmed that a lasting contact allergy had been obtained.


Drug-triggered pemphigus in a predisposed woman.

A 31-year-old woman with three pemphigus-prone antigens in her HLA haplotype (B7, DR4, DQw7) developed the disease soon after taking a pyrazolone derivative, viz. feprazone. The pemphigus lesions persisted despite withdrawal of the drug and worsened appreciably when she used ceftriaxone (a new cephalosporin with three sulphur atoms) for a bout of acute pharyngitis. Thiol groups formed from the metabolic breakdown of ceftriaxone are thought to have promoted acantholysis via a biochemical route. Genetic predisposition alone ('the soil') may be essential, though not per se sufficient for outbreak of pemphigus; the intervention of exogenous, heterogeneous factors ('the seed') often seems decisive in triggering full-blown disease.


Bullous impetigo caused by group A streptococci. A case report.

Bullous impetigo is considered to be a staphylococcal disease. Staphylococcus aureus, phage type 71, produces an epidermolytic toxin, assumed to be the cause of bullous formation in the skin. We present a case of bullous impetigo. Microbiological tests suggested beta-hemolytic streptococci, group A, M-type 3, as the etiological agent. Group A streptococci were isolated from the throat of the patient's mother and brother. The strains were shown to be identical, by means of DNA-'fingerprinting' and M-typing.


Morphometry in clinical dermatology.

Rapid and simple methods are presented which allow the estimation of areas, area fraction and contour lengths in clinical dermatology. They are based on point counting and intercept measurements using simple grids made on transparent film or on overhead foils. Because of their ease of use, these grids allow the determination of the size as well as the irregularity of skin lesions even during daily clinical work. The applications presented here include the area determination of naevi, leg ulcers, wheal and flare reactions and migration areas in lymphocyte and macrophage migration assays. Additionally, the determination of area fraction in psoriasis and sebutape evaluation is described. Other possible applications such as estimation or contour length, and determination of irregularity and estimation of the volume of tumours are discussed.


Birthmarks in 4346 Finnish newborns.

We examined all babies born live (4346) at two Finnish hospitals in the course of one year to determine the frequency of birthmarks, specially pigmented lesions, among Finnish newborns. All birthmarks excluding common salmon patches on the forehead and neck were recorded and photographed at birth. The babies were re-examined at the age of three months. Various birthmarks were recorded for 241 of 4346 babies, i.e. for 5.5% of all newborns. Ninety-one (2.1%) infants had congenital pigmented skin lesions, 167 (3.8%) had various vascular lesions and 21 (0.5%) had other birthmarks. The frequency of congenital melanocytic naevi was 1.5%. Most of the naevi were less than 20 mm in diameter. Only one child had a giant naevus. The frequency of congenital naevi in our study was the same or somewhat higher than previously described (1-8) but fewer other pigmented skin lesions were found than in previous studies perhaps due to racial differences.


Relapsing bullous staphyloderma.

Relapsing eruptions of bullae rapidly turning into pustules were seen in a 69-year-old woman of good general health. At different times during several months of observation, strains of S. aureus were grown from various lesions, including one (phage group III) producing enterotoxin C. Systemic involvement except for high BSR was absent and repeated blood cultures were negative. Histopathological findings resembled impetigo. Antibiotic treatment was effective. As this disease does not fit into any of the well-known pustular infectious dermatoses, we suggest calling it relapsing bullous staphyloderma.


Evaluation of port wine stain perfusion by laser Doppler imaging and thermography before and after argon laser treatment.

Thirteen patients with port wine stains (PWS) were treated with argon laser therapy. Before and at different points in time following treatment, skin blood perfusion and temperature were mapped with laser Doppler imaging and thermography. In nine patients no elevation in blood perfusion was observed in the PWS in comparison with the surrounding normal skin before treatment. In the remaining four patients a significantly (p less than 0.01) higher blood flow was recorded within the PWS. Immediately after treatment nine patients showed elevated perfusion within the PWS. During the first two days following treatment, all patients showed a gradually decreasing hyperperfusion in the borderline between the PWS lesion and surrounding skin. Immediately after treatment 10 patients had a significantly (p less than 0.01) higher temperature in the PWS than in normal skin. During the first 24 h following treatment, an elevated perfusion was in general accompanied by a tissue temperature increase. Three and a half months after argon laser treatment, three patients showed excellent clinical results with no remaining PWS spots or scarring. Two of these patients had had both elevated perfusion and temperature in the PWS prior to treatment.


Allogeneic cultured keratinocytes in the treatment of leg ulcers. A pilot study.

Forty-two patients (10 males and 32 females) with 52 chronic leg ulcers were treated with sheets of cultured allogeneic keratinocytes. Sixty-five % of the ulcers healed completely and the healing rate differed between various diagnostic groups. The best results were obtained in patients with venous ulcers and wounds with mixed etiology, whereas less improvement was observed with ischaemic ulcers. Rheumatic ulcers also responded well in combination with oral corticosteroids. The overall impression was that the grafting procedure markedly enhanced wound healing.


A new micronized 5-methoxypsoralen preparation. Higher bioavailability and lower UVA dose requirement.

A new tablet of micronized 5-methoxypsoralen (5-MOP) and a commonly used tablet in therapy (Psoraderm 5) were compared in 12 healthy subjects. Each subject ingested 1.2 mg/kg body weight of each formulation on different days. Bioavailability and phototoxicity of 5-MOP were compared. The results showed that serum and suction blister concentrations were significantly higher and occurred earlier after the oral intake of the micronized preparation. A series of graduated UVA doses were administered, one dose each time the concentration serum peaked, in order to determine the minimum phototoxic dose for each formulation. The micronized preparation induced greater photosensitivity than the unmicronized one. The micronized 5-MOP tablet may thus allow lower doses of UVA to achieve therapeutic results in photochemotherapy and a shortened waiting period following ingestion of drug.


Peptide T in the treatment of severe psoriasis.

We investigated the effect of treatment with peptide T on severe psoriasis in 5 patients. Within 2 months, peptide T led to complete remission of all lesions in 1 patient and to good improvement in 3 others. In 1 patient, no effect was observed.


Successful treatment of chronic skin diseases with clobetasol propionate and a hydrocolloid occlusive dressing.

The lesions of 141 patients with chronic skin diseases unresponsive to therapy were treated once a week with clobetasol propionate lotion left under the completely occlusive patch Duoderm. In 131 patients the lesions resolved completely, while partial remission was observed in the remaining 10. The mean interval to complete remission was: for chronic plaque psoriasis, 12 days; psoriasis on palms and soles, 2.5 weeks; palmoplantar pustulosis, 2.2 weeks; skin lesions of Reiter's syndrome, 3 weeks; chronic lichenified eczema, 2.0 weeks; neurodermatitis, 3.1 weeks; breast eczema, 9 days; discoid lupus erythematosus, 3.7 weeks; lichen planus, 2.8 weeks; sarcoidosis, 4 weeks; and lichen sclerosus et atrophicus, 2 weeks. Other conditions benefitting from the treatment were pompholyx, necrobiosis lipoidica, granuloma annulare and pretibial myxedema. The amount of topical corticosteroids needed was reduced to at most 1/20 and to as little as 1/100, compared with common topical steroid preparations.


Anthralin is a potent inhibitor of pityrosporum orbiculare/ovale in vitro.

Two strains of Pityrosporum orbiculare/ovale were grown in a liquid medium and exposed to different concentrations of the imidazoles ketoconazole and clotrimazole as well as anthralin, liquor carbonis detergens and salicylic acid. With regard to growth inhibition of yeast cells, the efficacies of anthralin and the imidazoles were similar, a half-maximal inhibition being achieved with an anthralin concentration of 7 mg/l. Liquor carbonis detergens and salicylic acid also inhibited growth of Pityrosporum orbiculare/ovale, but only at much higher concentrations. The response to salicylic acid was mainly due to its acid pH.


Treatment of pityriasis versicolor with a single dose of fluconazole.

Twenty-four patients with extensive or recurrent pityriasis versicolor were treated with a single oral dose of 400 mg of fluconazole. Twenty-three patients returned for follow up. Seventeen or 74%, were free of lesions 3 weeks after treatment and no recurrences were seen 6 weeks after treatment. The majority of the patients found the treatment effective, safe and convenient.


Monocytes appearing repeatedly after chemotherapies had an identical rearrangement pattern of immunoglobulin with leukemic blasts in a patient with CD13+ acute lymphoblastic leukemia.

We recently encountered a patient with acute lymphoblastic leukemia (ALL) who showed temporal monocytosis of an unusually high cell count (5,000-30,000 monocytoid cells/microliter) five times after treatment with different chemotherapies. The leukemic cells expressed B-cell-associated antigens, CD19 and CD10, E-rosette receptor, CD2 and monocyte/myeloid antigen, CD13 simultaneously. They were peroxidase-negative. One week after the initiation of conventional chemotherapy for ALL, the leukemic blasts had disappeared. Alternatively, monocytoid cells appeared along with the recovery from nadir status. They showed several features of monocytes; they were weakly dot-positive for nonspecific esterase, reactive with CD14 and CD13 and Fc gamma-receptor-positive. Furthermore, they migrated into a fungally infected joint space. Features incompatible with normal monocytes were the absence of peroxidase reactivity, the expression of B-cell-associated antigens, CD19 and CD10 and E-rosette receptor, CD2. Southern blot hybridization analysis revealed an unexpected result that HindIII digested DNA from both leukemic blasts and monocytoid cells had the same rearranged band of IgH. Thus, an identical clonality of monocytoid cells, temporally appearing after chemotherapies and leukemic lymphoblasts, was determined in this patient with CD13+ ALL.


Growth inhibition of RPMI 8226 human myeloma cells by peripheral blood lymphocytes.

To clarify the components of cellular immunity responsible for defense against the clonal development of myeloma cells, we tested the capacity of human peripheral blood lymphocytes (PBLs) to inhibit the growth of 3 human myeloma cell lines (RPMI 8226, OPM-1, and OPM-2). RPMI 8226 was found to be sensitive to PBLs, showing almost complete growth arrest when cultured with PBLs for 72 h. Inhibition of the growth of RPMI 8226 cells required direct cell-to-cell contact but not presensitization of the PBLs to the target cells, and did not depend on the generation of soluble factors. CD3+, CD4-, CD8- and CD16- cells were found to be the major subset contributing to inhibition of the growth of RPMI 8226 cells, and this growth inhibition was cytostatic rather than cytotoxic. These characteristics distinguished it from growth inhibition mediated by the natural killer system. Impaired PBL-mediated growth inhibition of RPMI 8226 cells was found in patients with various hematologic diseases, including myeloma. It therefore appears that the CD3+, CD4-, CD8- and CD16- cell subset might be involved in tumor immunity in myeloma.


Successful treatment of chronic adult T-cell leukemia with ubenimex.

A 38-year-old Japanese man had suffered from trichophyton infection for several years. The white blood cell count was 20,300/mm3, including 54% abnormal lymphocytes with irregularly convoluted nuclei. Adult T-cell leukemia (ATL) was diagnosed based on proliferation of CD4-positive lymphocytes, positive anti-HTLV-I antibody and monoclonal integration of proviral DNA. By 30 mg/day of ubenimex (Bestatin), the abnormal lymphocytes positive for CD4 in the peripheral blood were gradually reduced. Complete remission was maintained for 9 months without any antineoplastic agents. Ubenimex may have suppressed the growth of ATL cells in this patient. Accordingly, ubenimex may prove useful for treating some patients with chronic-type ATL.


Secretion mode of the harderian gland of rats after stimulation by cholinergic secretagogues.

We studied the morphological changes in rat Harderian glands 30 min after injection of cholinergic secretagogues. In controls, the glands exhibited a tubuloalveolar structure with relatively wide lumina, in which some osmiophilic dense droplets exocytosed from glandular cells were observed. Also two types of glandular cells (type A cells and type B cells sometimes showing exocytotic figures of lipid-secretory vacuoles) and myoepithelial cells were recognized. After injection of carbamylcholine chloride (subcutaneously, 0.1 mg/kg body weight), which has both nicotinic and muscarinic actions, many of the alveolar lumina dilated and contained a small number of osmiophilic droplets. Exocytotic figures in both types of cells and a pronounced decrease in the number of vacuoles in the glandular cells were observed. However, there was no evidence of apocrine or holocrine secretion. The injection of the higher dose of carbamylcholine (1.0 mg/kg) caused fusion of secretory vacuoles in the apical cytoplasm and contraction of myoepithelial cells. Most alveoli showed no clear lumina; their centers were jammed with cytoplasmic fragments and accumulated secretory products. Massive discharge of cytoplasmic fragments containing some secretory vacuoles was often observed. This may be classified as apocrine secretion. Bethanechol chloride (subcutaneous injection, 1.0 mg/kg), a muscarinic agonist, stimulated the Harderian-gland secretion, and enhanced exocytosis was observed. The discharge from the glandular cells, following injection of various doses of carbamylcholine, were almost inhibited by atropine sulfate, a muscarinic antagonist. The present results suggested that the cholinergic systems regulate the secretion of rat Harderian-gland cells which have muscarinic receptors.


Identification of spotted fever group rickettsiae using polymerase chain reaction and restriction-endonuclease length polymorphism analysis.

In order to facilitate the isolation and identification of spotted fever group (SFG) rickettsiae from their tick vectors, we used the centrifugation shell vial technique or traditional isolation procedures and genotypic identification using the restriction fragment length polymorphism analysis of polymerase chain amplified fragments. The presence of Rickettsia conorii both in Rhipicephalus sanguineus ticks collected in southern France, and in Rhipicephalus simus and Haemaphysalis leachi from Zimbabwe was demonstrated. This procedure seems to be of particular interest for studying the epidemiology and ecology of SFG rickettsiae.


Alpha 2-receptor-mediated inhibition of intraluminal release of gastric somatostatin in anaesthetized rats.

The aim of the present study was to investigate how the sympathetic nervous system affects the vagally induced intragastric release of somatostatin and gastrin. Experiments were performed on anaesthetized rats in which the stomach was perfused with a dextrane solution (pH approximately 5.9) or dextrane buffer (pH 7.4). pH as well as gastrin and somatostatin levels were measured in the gastric perfusate when it had passed the stomach. Vagal stimulation caused a decrease in perfusate pH and an increase of the intraluminal output of gastrin and somatostatin when the stomach was perfused with the dextrane solution pH 5.9. Pretreatment with phentolamine (1 mg kg-1) significantly increased and pretreatment with clonidine (60 micrograms kg-1 h-1) significantly decreased somatostatin release caused by vagal stimulation, whereas gastrin levels remained largely unchanged. The effect of clonidine persisted in rats pretreated with indomethacin (5 mg kg-1), which per se potentiates the vagally induced luminal somatostatin release. When the stomach was perfused with the dextrane buffer pH 7.4, basal gastrin levels were significantly higher than during perfusion with the solution pH 5.9, whereas somatostatin levels remained unchanged. Neither somatostatin nor gastrin levels increased following vagal stimulation during gastric perfusion with the dextrane buffer pH 7.4. However, following pretreatment with phentolamine somatostatin levels increased during these conditions.


Persistence of Rickettsia prowazekii in cotton rat macrophage cultures.

Rickettsia prowazekii is able to multiply and persist for a long time in cotton rat macrophage culture (29-days observation period). Electron microscopic studies showed that the structure of Rickettsiae remained intact at different intervals post-inoculation (p.i.). In the course of persistence Rickettsiae revealed a reduced capacity to infect chick embryos and guinea pigs, however, the infectious agent could be isolated at all stages of persistence of cultured cells such as fibroblasts of the guinea pig embryo, macrophages of intact cotton rats.


The effect of monocyte-derived macrophages on the growth of Rickettsia conorii in permissive cells.

We examined whether monocyte-derived macrophages (MdM) incubated with rickettsia-infected HEp-2 or BGM cells a) affect R. conorii (Boutonneuse fever) growth, and b) secrete TNF and IL-1 alpha. BGM and HEp-2 cells were infected with R. conorii at multiplicities of infection (MOI) of 1-0.01. After 2 hr of adsorption, the cells were washed and MdM were added at an effector to target ratio of between 3 and 5. At 2, 24, 48, and 96 hr post-infection (p.i.) cells were scraped off; cell-free medium was collected and TNF and IL-1 levels were determined by ELISA and RIA, respectively. MdM caused a 50-70% reduction in the yield of R. conorii in HEp-2 cells as compared to the control (infected HEp-2 cells incubated without MdM). This reduction was more pronounced at MOI 0.1 and 0.01, than at MOI 1. In contrast, no reduction in the rickettsial yield was observed in the BGM cells incubated with MdM. TNF and IL-1 alpha levels in the cell-free medium from infected HEp-2 cells incubated with MdM were higher (2-5 fold) than those from infected BGM cells incubated with MdM. These data suggest the possibility that, and the mechanisms whereby, MdM may modulate rickettsia replication in vivo.


Serum antibodies in rickettsia patients as determined by immunoblotting technique.

The Western-blot technique (WB) was used to determine which polypeptides of Israeli spotted fever (ISF) isolates and other spotted fever group rickettssia (SFGR) reference isolates (G212, S484, A828) and two reference strains. R. Rickettsii (Sheila Smith strain) and R. conorii (Boutonneuse fever), were used as antigen sources for the WB. Immunoperoxidase assay (IPA) seropositive (titer greater than 80) and seronegative (titer less than) sera were examined with the separated polypeptides of the above strains. WB analysis of the rickettsial polypeptide-serum reactions showed that R. conorii and the three isolates of ISF reacted identically with the sera, except that in the three ISF strains a 175 kD protein was present. It was also observed that all of the IPA seropositive sera examined reacted with the following polypeptides: 18kD, 20kD, 22kD (28kD to 37 kD LPS group), while each seropositive and seronegative serum reacted differently with polypeptides 23kD, 42kD, 45kD, 46kD, 52kD, 55kD, 70kD, 82kD, 105kD, 125kD, 155kD and 175kD. Using this technique, no heat labile polypeptides (preelectrophoretic treatment: 100 degrees C for 2 min vs 37 degrees C for 20 min) were observed in SFGR strains used in this study. Our results indicate that the immunoblot technique shows no difference between R. conorii and ISF antigens except the existence of 175kD protein antigen in the latter.


Dynamics of non-specific antibacterial activity of the peritoneal cells of mice induced with Coxiella burnetii antigen.

It was found that primary immunization with Coxiella burnetii antigen increased mouse resistance to Salmonella typhimurium infection as evidenced by acceleration of bacterial elimination from the peritoneal cavity and a decrease in lethality of experimental animals. The existence of two rises of bactericidal activity of mouse peritoneal cells was ascertained: the "early" on days 1 and 2, and the "late" on day 14 after C. burnetii administration. The first rise was accompanied by some increase in the number of peritoneal cells as well as by some change of their qualitative representation. The second increase of antibacterial activity was detected during the pronounced cellular and humoral immune responses to C. burnetii.


Ultrastructure of viable and inactivated Coxiella burnetii after inoculation of mice.

C3HA mice were inoculated intraperitoneally (i.p.) with viable Coxiella burnetii (C.b.) (strain Apodemus flavicollis-Luga, phase I) or C.b. antigen (formalin-inactivated C.b.); pieces of spleen, liver, omentum, kidney, and suspension of peritoneal macrophages were examined by electron microscopy at 1, 7, 14, 21, 28, and 42 days post-infection (p.i.). Two main types of viable C.b. cells--small dense (DC) and bacteria-like (BC)--were found in omentum, spleen, and peritoneal exudate cells beginning from the 1st day p.i. but maximal amount they reached in spleen at day 14. Some parasitophorous vacuoles contained damaged BC or lysing C.b. cells. C.b. corpuscular antigen was found in phagosomes in ultrathin sections of spleen macrophages until 28 days p.i. By 7 days p.i. in omentum 3 types of parasitophorous phagosomes were described which were supposed to be the sites of the antigen utilization. It is assumed that most BC are destroyed in omentum macrophages during the 1st week after i.p. injection of the antigen, and DC can persist in spleen at least for one month.


Immunoblot analysis of antibody response in mice infected with Coxiella burnetii phase I.

Mouse sera collected from day 4 to day 133 postinfection (p.i.) with phase I Coxiella burnetii strain Nine Mile were analysed by immunoblotting with phase I C. burnetii cell lysate. Antibodies of IgG class protein antigens were revealed already on day 10 p.i. (60, 49 and 27 kD proteins), followed by those to 77 kD protein from day 18 p.i. and to further 7 (from 42 to 70 kD) proteins from day 28 p.i. IgG antibody reaction was observed also with 5 antigens in 14-20 kD region corresponding to lipopolysaccharides from day 22 p.i. Surprisingly, antibodies of IgM type appeared later (from day 22 p.i.) and were directed only to protein antigens, most markedly to 60 and 77 kD proteins. Differences in immunoblot patterns observed with the serum collected on day 72 p.i. before and after absorption to phase I and phase II C. burnetii cells, and to phase I cells treated by trichloroacetic acid (TCA) or KIO4, indicate the surface localization of protein phase I antigenic epitopes, which can be destroyed partly by TCA and almost completely by KIO4 treatment.


Preliminary characterization of inflammatory infiltrates in response to Rickettsia prowazekii reinfection in man: immunohistology.

The local perivascular mononuclear cell inflammatory infiltrate (PVI) response to intradermal (ID) challenge with viable R. prowazekii was studied in a typhus-immune subject. An immunohistochemical method for specific mononuclear cell markers was used on skin punch biopsies taken 6, 24 and 48 hr after challenge. By 24 to 48 hr, the histologic findings were consistent with a delayed-type hypersensitivity reaction (DTHR). Both CD4+ and CD8+ T lymphocytes dominated the PVI at 48 h. CD8+ cells entered the PVI more rapidly than CD4+ cells. Local control of R. prowazekii challenge in an immune human subject was associated with recruitment into the PVI of T lymphocytes which are rich sources of gamma-interferon, and CD8+ T cells which are potentially cytotoxic for R. typhi-infected cells.


Protein antigens of genetically related Rickettsia prowazekii strains with different virulence.

The protein antigens of two distinct lines of genetically related strains, namely the nonpathogenic strain E and its virulent revertant EVir and of the standard virulent strain Breinl were compared in SDS-PAGE and immunoblot assay using typhus patient sera and immune rabbit sera. No differences in the polypeptide pattern as detected in SDS-PAGE were found between strain E and EVir; the Breinl strain differed in a 30 kD protein. The high immunogenicity of the protein antigens of E, EVir and Breinl strains was demonstrated by immunoblot assay with human sera, which did not show any differences between the strains studied. Immunoblot analysis with immune rabbit sera to the strain E, EVir, and Breinl showed differences in immunological response to the 70 kD and 60 kD polypeptides of low virulent strain E and those of virulent strains EVir and Breinl.


Comparison of structural polypeptides, detected by immunoblotting technique, in the sera of spotted fever group rickettsia positive cases--symptomatic versus asymptomatic.

In an attempt to characterize the nature of symptomatic versus asymptomatic spotted fever group rickettsia (SFGR) infection, the immune response to R. conorii (boutonneuse fever) structural polypeptides was studied by Western-blot immunoassay. Sera from immunoperoxidase assay (IPA), SFGR seropositive (titre greater than or equal to 80) individuals, symptomatic and asymptomatic and from SFGR seronegative (IPA titre less than 80) individuals living in a kibbutz community in the desert region of Southern Israel were examined by immunoblot. This community suffered from a very high morbidity rate due to SFGR (21-fold higher than the national reported average). The entire community (n-326) has been followed-up since 1985, with serial serum samples being examined for specific IgG antibodies by IPA. The intensity of the immunoblot reaction correlated with specific IgG antibody titres as determined by IPA. This correlation was also observed between the decrease in the IgG titre and the strength of the antibody-antigen reaction by immunoblot over time for a given individual. IPA seropositive sera from asymptomatic as well as symptomatic spotted fever cases reacted to 8 individual polypeptides. In both cases antibodies to 22 kD, 24 kD, 26 kD, 28 kD, 30 kD, 32 kD, 34 kD, and 37 kD were found. In the IPA seronegative sera, antibodies to polypeptides in the range of 24 kD to 32 kD were not detected. The lack of detectable differences by immunoblotting between SFGR symptomatic vs, asymptomatic cases might be explained by other aspects of the immune response of each infected individual, and/or it is possible that virulent and non-virulent antigenically closely related SFGR strains infected symptomatic vs. asymptomatic individuals.


The reactivity between rickettsiae and Weil-Felix test antigens against sera of rickettsial disease patients.

Of the sera which were positive to Rickettsia tsutsugamushi by indirect immunoperoxidase test, approximately 80% sera were positive to a Proteus OXK antigen by Weil-Felix test at 10 or more days after the onset of fever, while only 10% sera were positive within 9 days from the onset of fever. In ELISA using the OXK antigen, almost all of the paired sera of tsutsugamushi disease (TD) patients increased on the IgM antibody titres with the rise of their titres by Weil-Felix test, whereas the IgG antibody titres of these sera were unrelated with the titres of Weil-Felix test. We suspect that the reactivity of TD patients sera to the OXK antigen in Weil-Felix test was derived from the reactivity of the IgM antibody against the OXK antigen common with R. tsutsugamushi. The patient sera infected with a Japanese isolate of spotted fever group rickettsia (SFGR) cross-reacted with the Thai Tick Typhus (TTT) strain of SFGR by indirect immunoperoxidase test. In Weil-Felix test, the reactivity of these sera to OX2 antigen were higher than that to OX19 antigen, like the sera infected with other SFGR, except of R. rickettsii. These sera also reacted with TTT and OX2 antigens by ELISA. The titres of IgM antibody against OX2 antigen in the sera in ELISA were in parallel with the titres of the sera against OX2 antigen in Weil-Felix test, but not the titres of IgG antibody. We suggest that the reactivity of the patient sera infected with SFGR to OX2 antigen of Weil-Felix test is dependent on the IgM antibody.


Phenotypic and genotypic heterogeneity of 8 new human Coxiella burnetti isolates.

Eight new strains of Coxiella burnetii were isolated from chronic Q fever patients using centrifugationashell vial technique. Seven patients had endocarditis (including one patient with an immunodeficiency syndrome), and one had a vascular prosthesis infection. Three prototype strains, Nine Mile phase II, Q212 and Priscilla and eight new isolates were cultured in L 929 cells. Heterogeneity of their cytopathic effect was observed. DNAs of the eleven strains have been isolated and purified by standard procedures. Plasmid DNA was separated from chromosomal DNA by a low melting point gel. Electrophoresis in agarose gel showed that seven of the eight new strains had plasmids which were about 40 kb (plasmid V517 was used as size marker). Endonuclease-restriction analysis of the 8 human isolates is currently under investigation.


Immunoblot analysis of IgG subclass antibody response against Coxiella burnetii in Balb/c mice.

Balb/c mice were inoculated with live or inactivated organisms of Coxiella burnetii, strain Nine Mile, phase I. Sera collected after different time intervals were subjected to immunoblot analysis and results compared with ELISA values. Immunoblots were performed with different horseradish peroxidase labelled conjugates (goat anti-mouse IgG1, IgG2a, IgG2b, IgG3) and results monitored and analysed by laser densitometry. ELISA analysis was performed using the same peroxidase labelled goat anti-mouse subclass antibody conjugates. In addition, goat anti-mouse IgG(H+L), IgG(H), IgM, and IgA conjugates were used for ELISA tests.