Application of BrdU/Hoechst-ethidium bromide two parameter flow cytometry in studying synchronous and non-synchronous mouse cells. BrdU/Hoechst-EB bivariate flow cytometry has a wide application in the study of factors controlling cell cycle for asynchronous cells such as embryonic stem cells (ES), and for synchronous cells such as stimulated resting B cells (Bo). The technique allows one to calculate the average cell cycle duration time. ES cells are found to cycle every 8-10 h, and most B cells are 11-12 h, but there is a small subset of B cells with a cycle time of only 6-7 h. Using this technique, we also study the roles of different T lymphocytes on B cell activation when B cells are stimulated with anti-IgM antibodies (commonly used, anti-mu). Exposure to anti-mu recruits small B cells into the cell cycle, but arrests them in the G1 phase of the second cycle. Interleukin (IL) 4 is a costimulator of anti-mu. In addition, IL-4 is an S-phase progression factor. Contrary to that seen when B cells are stimulated by other mitogens, very few cells are in the G2 compartments after anti-mu plus IL-4 stimulation. This phenomenon is reminiscent of embryonic cells. Our findings provide strong evidence to propose that there are two restriction points for B cell activation: at the transition from G0 to G1 and at the transition from G1 to S phase.


Properties of soluble CR2 in human serum. A soluble form of complement receptor number 2 (sCR2) found in human serum closely resembles that produced in culture by B lymphoblastoid cells. Epitope analysis with a panel of CD21 monoclonal antibodies revealed only minor differences between antigen from the two sources. Purified sCR2 from both sources bound to C3dg prepared from human or mouse serum and to u.v.-inactivated Epstein-Barr virus. SDS-PAGE analysis of culture supernates of B-lymphoid cells labelled by growth in medium containing 35S-methionine revealed a major component of molecular weight approximately 130 kDa and another band at 30 kDa. Incubation with endoglycosidase F reduced the size of the high molecular weight component. Gel filtration of untreated serum or culture supernate revealed that, in its native state, sCR2 behaved as a molecule or complex of apparent molecular weight 320 kDa. Possible explanations are discussed.


The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species. Immunization with Anaplasma marginale outer membranes induced immunity against clinical disease which correlated with antibody titer to outer membrane proteins, including a 19-kDa protein (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). This 19-kDa protein, designated major surface protein 5 (MSP-5), was encoded by a single-copy 633-bp gene. The molecular mass of MSP-5, defined in immunoblots by binding to monoclonal antibody ANAF16C1, was conserved among all recognized species of Anaplasma: A. marginale, A. centrale, and A. ovis. Recombinant MSP-5, which absorbed the antibody reactivity of bovine immune serum to native MSP-5, was recognized by anti-A. marginale and anti-A. centrale immune sera in a competitive inhibition assay with monoclonal antibody ANAF16C1. The presence of antibody to the epitope defined by monoclonal antibody ANAF16C1 in all postinfection sera tested indicates that this epitope is a potential diagnostic antigen for use in identifying persistently infected cattle.


Modulation of endotoxin-induced monokine release in human monocytes by lipid A partial structures that inhibit binding of 125I-lipopolysaccharide. We have previously shown that the synthetic tetraacyl precursor Ia (compound 406, LA-14-PP, or lipid IVa) was not able to induce the production of tumor necrosis factor, interleukin-1, and interleukin-6 in human monocytes but strongly antagonized lipopolysaccharide (LPS)-induced formation of these monokines. This inhibition was detectable at the level of mRNA production. To achieve a better understanding of molecular basis of this inhibition, we investigated whether lipid A precursor Ia (LA-14-PP), Escherichia coli-type lipid A (LA-15-PP), Chromobacterium violaceum-type lipid A (LA-22-PP), and synthetic lipid A partial structures and analogs (LA-23-PP, LA-24-PP, and PE-4) were able to influence the binding of 125I-LPS to human monocytes and compared this inhibitory activity with the agonistic and antagonistic action in the induction of monokines in human monocytes. 125I-LPS (20 ng per well) was added to human monocytes in the presence or absence of unlabeled rough Re mutant-derived LPS (Re-LPS) or lipid A compounds, and specific LPS binding was determined after 7 h. This binding was found to be dependent on CD14 as shown by the use of an anti-CD14 monoclonal antibody. Compound LA-14-PP was found to inhibit the binding of 125I-LPS to the cells in a similar dose-response to that of unlabeled LPS. This shows that the inhibitory capacity on LPS binding does not correlate with the monokine-inducing capacity because Re-LPS is active in inducing tumor necrosis factor, interleukin-1, and interleukin-6, while LA-14-PP is not. The strong capacity of LA-14-PP to inhibit 125I-LPS binding, however, correlates with the strong inhibitory capacity of this compound on LPS-induced monokine production. Compounds LA-15-PP, LA-23-PP, and LA-24-PP were active in the inhibition of 125I-LPS binding but were 5- to 10-fold weaker than Re-LPS and LA-14-PP. Of all lipid A structures tested, compound LA-22-PP expressed the weakest inhibitory capacity on LPS binding. These compounds showed again that the activity of binding inhibition does not correlate with the monokine-inducing capacity. We assume that the inhibitory effects of lipid A partial structures on LPS-induced monokine production have their origin in a competitive inhibition between LPS and the lipid A partial structures for the same binding site on the cell membrane.


Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30-kilodalton-region proteins and the secreted antigen 85 complex and demonstration of antigen 85B on the Mycobacterium leprae cell wall surface. Proteins of the antigen 85 complex in the 30-kDa region secreted by live mycobacteria are important in the immune response against mycobacterial infections and may play an important biological role in the host-parasite interaction. In the present study, we have characterized epitopes of the 30-kDa-region proteins and the antigen 85 complex by using a panel of 13 monoclonal antibodies (MAbs) reacting with these antigens, 6 of which have not been described before. By using five previously characterized related secreted proteins of Mycobacterium tuberculosis, MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27 kDa), and MPT64 (26 kDa), we have identified at least 10 different MAb-reactive epitopes on the proteins of the antigen 85 complex. A heterogeneous distribution of epitopes was observed within the components of the antigen 85 complex. Two distinct epitopes specific for antigen 85B and two other epitopes restricted to the 85A and 85B components were recognized. Two of them were shared with a previously unidentified 27-kDa protein present in M. tuberculosis culture fluid from which all MPT proteins were derived. The rest of the MAb-reactive epitopes were found to be present mostly in antigens 85A and 85B and to a lesser extent in antigen 85C. None of these MAbs recognized component 85C alone nor did they bind to the related MPT51 and MPT64 proteins. Interestingly, most of the MAbs reacted with purified native proteins of the antigen 85 complex but not to them in their denatured forms. In contrast, reactivity of the MAbs with the cytosol fraction of M. tuberculosis in immunoblotting revealed that they bound to a closely related cytosolic 30-kDa protein(s) even when they were denatured. Heterogeneity of these MAb-reactive epitopes of the antigen 85 complex was further evident as they were found to be distributed in various patterns among 19 different mycobacterial species. By using fusion proteins of the Mycobacterium leprae 30/31-kDa antigen 85 complex, we have localized at least six different epitopes within amino acid residues 55 to 266 of the M. leprae antigen 85 complex. Finally, by immunohistochemical analysis, we have demonstrated the in situ expression of one of the novel MAb-reactive epitopes specific for antigen 85B on the cell wall surface of M. leprae within macrophages in lepromatous leprosy lesions and thus provide direct evidence for the presence of the B component of the antigen 85 complex on the surface of intact M. leprae.


Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae. The P2 porin protein is the most abundant outer membrane protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene.


Enhancement of host defense by Y-25510, (+-)-3-[4-(2-dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3,4 -b] pyridine-1-acetic acid, a novel synthetic compound. A comparison with recombinant human granulocyte colony-stimulating factor in 5-fluorouracil-treated mice. The effect of a novel synthetic compound, Y-25510, (+-)-3-[4-(2-dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3,4 -b] pyridine-1-acetic acid, on recovery from long-lasting leukopenia induced by 5-fluorouracil was compared with that of recombinant human granulocyte colony-stimulating factor (rhG-CSF). When mice were administered i.p. with 5-FU (200 mg/kg) on days 0 and 7, intravenous administration of Y-25510 (100 and 1000 micrograms/kg) prevented the decrease in the peripheral leukocyte and neutrophil number and accelerated the recovery from leukopenia. Subcutaneous administration of rhG-CSF (50 micrograms/kg) did not prevent leukopenia but accelerated the recovery from leukopenia. In particular, peripheral neutrophil number increased over a normal level. The administration of Y-25510 (10, 100 and 1000 micrograms/kg) restored the decrease in the number of bone marrow cells, spleen cells, lymphocytes, neutrophils and monocytes. The administration of rhG-CSF (50 micrograms/kg) restored the decrease in the number of bone marrow cells, spleen cells, and neutrophils but not that of lymphocytes and monocytes. In fractions of bone marrow cells on day 21, the administration of Y-25510 (1000 micrograms/kg) showed a tendency of restoring the decrease in neutrophil number. In conclusion, the administration of Y-25510 prevented leukopenia and accelerated the recovery from leukopenia in the 5-FU-treated mice. It is suggested that the mechanism of the restorative action of Y-25510 is different from that of rhG-CSF. In a number of immature bone marrow cells Y-25510 has a potent stimulatory effect on the recovery from the decrease in number of hematopoietic cells, keeping a balance in number of each blood cell.


Protective effect of ren-shen-yang-rong-tang (Ninjin-youei-to) in mice with drug-induced leukopenia against Pseudomonas aeruginosa infection. When 200 mg/kg of cyclophosphamide (CY) or 5-fluorouracil (5-FU) were given subcutaneously to mice, severe reduction of leukocyte numbers in the peripheral blood was observed on day 4 and from day 4 to day 8 after the treatment, respectively. Daily administration of ren-shen-yang-rong-tang (Japanese name: Ninjin-youei-to, NYT), (1 g/kg/day) and recombinant human granulocyte-colony stimulating factor (rhuG-CSF, 2 micrograms/mouse/day) either from day 0 to day 4 after treatment with CY or from day 0 to day 8 after treatment with 5-FU accelerated the recovery of peripheral leukocytes. Administration of NYT and rhuG-CSF inhibited decreases in the number of colony forming units in the spleen (CFU-S) in drug-treated mice. In mice infected intraperitoneally with a virulent strain of Pseudomonas aeruginosa 4 days and 8 days after treatment with CY and 5-FU, respectively, lethal doses were much lower (approximately 1/1000) than that in normal mice. Administration of NYT and rhuG-CSF to drug-treated mice inhibited the enhanced bacterial growth in the peritoneal cavity. Administration of NYT or rhuG-CSF to drug-treated mice enhanced the recovery of accumulation of leukocytes into the infected peritoneal cavity from their depressed state. Administration of NYT was more effective for improving the host resistance of 5-FU-treated mice than for improving that of CY-treated mice in contrast to rhuG-CSF which exhibited stronger effect in CY-treated mice than in 5-FU treated mice.


A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies. A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60 degrees C to 22 degrees C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.


Human carcinoma cells synthesize and secrete tenascin in vitro. Tenascin is an extracellular matrix glycoprotein produced in response to epithelial-mesenchymal interactions that initiate fetal organogenesis, and it is also found in the stroma of benign and malignant neoplasms. Thirty-five human cell lines representing a variety of cancers were examined by immunoprecipitation and polyacrylamide gel electrophoresis of radiolabeled tenascin proteins from conditioned media. Two forms of tenascin with relative molecular masses of 190,000 and 250,000 were identified. Eight cell lines produced both forms. With the exception of myeloid and lymphoid leukemias and Burkitt's lymphoma, all of the mesodermal and neuroectodermal tumor lines were found to synthesize tenascin. Unexpectedly, tenascin was secreted by several mammary and colonic adenocarcinomas as well as by a line derived from normal mammary epithelial cells, and in some cases increased production was induced by transforming growth factor beta in serum-free medium. Cells producing fibronectin but not tenascin attached and spread on plastic culture dishes, while those producing tenascin alone remained suspended in the medium or were rarely attached. Tenascin also inhibited fibronectin-mediated adhesion of MCF7 breast carcinoma cells in vitro. The results suggest that tenascins synthesized and secreted by some cancer cells, especially those of epithelial origin, may have specific roles in determining tumor cell adhesion and ultimately the ability to form invasive outgrowths.


Influence of glucocorticoid, aspirin, and gamma globulin on interleukin-6 production in the hepatoma cell line. The current study was designed to observe the effects of drugs used for the treatment of inflammatory diseases (glucocorticoid, aspirin, and gamma globulin) on interleukin 6 (IL-6) production in the liver which may be involved in acute phase protein stimulation. The HUH-7 hepatoma cell line was used in which the IL-6 gene is expressed and produces IL-6 in vitro. The cells were cultured in RPMI 1640 supplemented with 1% fetal calf serum for 12 h prior to addition of these drugs. We collected the cells and supernatants at 24 h, 48 h, and 72 h after addition of glucocorticoid, aspirin, and gamma globulin. IL-6 mRNA in the cells was measured by Northern blot hybridization, while the IL-6 activity in culture supernatants was measured by enzyme-linked immunosorbent assay. At 48 h after addition of glucocorticoid and aspirin, there was a suppression of IL-6 activity in the culture supernatants, which was associated with a decrease of mRNA level in the cells. However, suppression by gamma globulin was not observed. These data demonstrate that glucocorticoid and aspirin are potent regulators of IL-6 synthesis in the hepatoma cell. These components may decrease acute phase protein production via the suppression of IL-6 production in the liver during inflammation.


Influence of staurosporine, a more selective derivative CGP 41,251 and an inactive analogue CGP 42,700 on histamine release from isolated rat mast cells. The influence of staurosporine, a potent but nonselective inhibitor of protein kinase C, on rat mast cell histamine release, was compared with that of two derivatives, CGP 41,251 with a high degree of selectivity for protein kinase C and the related CGP 42,700 which is without activity. Staurosporine was a more potent inhibitor of mast cell responses than CGP 41,251, in accordance with their reported potencies. CGP 42,700 was investigated in the same concentration range as CGP 41,251 and served as a control for unspecific effects. Antigen induced histamine release was more effectively inhibited by staurosporine than by CGP 41,251, and responses to compound 48/80 were only modestly affected by both drugs. Responses to the ionophore A23187 were unaffected by staurosporine whereas CGP 41,251 was an effective inhibitor at suboptimal ionophore concentrations. In contrast, responses to combinations of the phorbol ester TPA and subthreshold concentrations of the ionophore could be potently inhibited by staurosporine but were under certain conditions moderately enhanced by lower concentrations of the drug, whereas CGP 41,251 was only inhibitory. Except for a slight inhibition of ionophore responses CGP 42,700 was without effect. The results demonstrate that the actions of staurosporine cannot be ascribed solely to inhibition of protein kinase C, whereas the influence of CGP 41,251 appears to be consistent with an inhibition of this kinase.


Optimal electric conditions in electrical impulse chemotherapy. The relationship was examined between the tumoricidal effect and the electrical variables of in vivo electrical impulse chemotherapy. Donryu rats subcutaneously inoculated with AH-109AY hepatocellular carcinomas were given a single high-voltage electrical impulse of varying voltage and duration, 30 min after an intramuscular injection of 4 mg/kg bleomycin. As the voltage (V) was increased from 0 to 5 kV, the tumoricidal effect (E) increased in proportion to the square of the voltage. As the pulse duration (D) was increased from 2.5 to 5.8 ms, the tumoricidal effect increased in direct proportion to it. Combining these results yielded the formula, E = gamma V2D, which indicates that tumoricidal efficacy was proportional to the applied electrical energy. When the electrical energy was kept constant while varying the voltage and duration of pulse, the anticancer effect was the same, confirming this relationship for lower energy levels.


Cytochemical localisation and characterisation of proteoglycans (glycosaminoglycans) in the epithelial-stromal interface of the seminal vesicle of the guinea pig. The proteoglycans (PGs) in the guinea pig seminal vesicle were demonstrated ultrastructurally by both cuprolinic blue (CB) and ruthenium red (RR) staining. The PGs appeared as electron-dense granules with RR, but were filamentous following CB staining using the critical electrolyte concentration method. Three major types of PGs (T1, T2, T3) have been described according to their different locations and sizes. T1 filaments were short and were found mostly on both sides of the lamina densa of the basal lamina of the glandular epithelium (40-60 nm long) and also on the basal laminae of smooth muscle cells and capillary endothelial cells (20-30 nm long). In the epithelial basal lamina they were regularly spaced at an interval of 40-60 nm. T1 filaments in the lamina densa were smaller and more randomly distributed. Cytochemical characterisation of these PGs by various GAG degrading enzymes showed that T1 PGs are rich in heparan sulphate. T2 filaments were 30-40 nm long and closely associated with the collagen fibrils. They were arranged perpendicular to the long axis of collagen fibrils, also at intervals of about 60 nm. T2 filaments were removed by chondroitinase (Ch)-ABC, Ch-ABC plus Streptomyces (S)-hyaluronidase and pronase, but resistant to nitrous acid, heparitinase, heparinase, neuraminidase and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulphate. T3 filaments (60-100 nm) were widely distributed in the stroma at sites such as the interstitial spaces of the lamina propria, the reticular layer below the basal lamina, around individual collagen fibrils or bundles of such fibres, and on the cell surfaces of fibroblasts. The T3 filaments were removed by Ch-ABC, Ch-AC and pronase but were resistant to heparitinase, heparinase, S-hyaluronidase, neuraminidase and nitrous acid. They are therefore rich in chondroitin sulphate.


Oncogene.

Oncogene. Recent studies have shown that activation of oncogenes, genes concerned with cell growth, and inactivation of anti-oncogenes may be responsible for uncontrolled cell proliferation leading to malignancy. These oncogenes code for products or proteins which are closely similar to growth factors or receptors of growth factors. Alterations in lipid metabolism in the form of excess formation of inositol triphosphate and relocation of protein kinase C, the second messengers of the mitotic process, can initiate cell division. Oncogenes can be activated by chromosomal aberrations induced by chemicals, viruses and drugs. The identification of oncogenes and their products may have relevance to the development of new therapeutic strategies in cancer.


Detection of HTLV-I in clinical specimens. The American Red Cross, which collects 50% of blood for transfusion in the United States, now tests all prospective blood donors for HTLV-I and HTLV-II antibodies. It will therefore be important to recognize the significance and clinical spectrum of diseases associated with these viruses, and to become familiar with the current methods used to diagnose infection. This review summarizes the techniques currently in use to screen for HTLV-I/II antibodies, as well as methods to detect viral genome and/or gene products in blood and tissue specimens.


A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates. We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.


Genetic heterogeneity within Mycobacterium fortuitum complex species: genotypic criteria for identification. A 1.5-kb segment of the DNA that encodes 16S rRNA was amplified by polymerase chain reaction, and 880 nucleotide positions were determined from each of the described biovariants of Mycobacterium fortuitum. Signature sequences which allow rapid identification of M. fortuitum strains at the biovariant level are described. Our data demonstrate a close phylogenetic relationship between Mycobacterium senegalense and M. fortuitum and indicate that the described biovariants of M. fortuitum represent genetically distinct taxa.


Infection with a plasmid-free variant Chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection. Clinical samples in transport media from 40 patients exhibiting pathologies potentially caused by Chlamydia trachomatis infection were analyzed for chlamydial nucleic acid, and the results were compared with those of culture. Chlamydial culture was performed by a shell vial centrifugation method with HeLa 229 host cells. Polymerase chain reaction (PCR) assays were used to detect either regions on a 7.5-kb plasmid characteristic of C. trachomatis (plasmid-PCR) or a segment of the 16S rRNA genes (rRNA-PCR). All PCR results were confirmed by hybridization with probes for the specific amplified products in either a Southern or a dot blot format. An RNase protection (RNP) assay was used to detect genus-specific chlamydial 16S rRNA directly from the clinical samples. The PCR assays detected C. trachomatis but not other bacteria, including Chlamydia spp. C. trachomatis was isolated from six samples which were positive by the rDNA-PCR and plasmid-PCR assays. Five of the culture-positive specimens were positive by the RNP assay. Twenty-two samples were negative by all criteria. Surprisingly, nine samples were positive by rRNA-PCR and RNP assays only. Nucleic acid sequencing of the rRNA-PCR-amplified products indicated a close relationship between the variants and C. trachomatis. The data may indicate an unrecognized process in C. trachomatis infection or that these patients were infected by a variant strain of C. trachomatis which lacks the C. trachomatis-specific plasmid.


Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. We have determined and compared partial 16S rRNA sequences from 23 Lyme disease spirochete isolates and aligned these with 8 sequences previously presented. The 16S rRNA signature nucleotide compositions were defined for each isolate and compared with the genomic species signature nucleotide sets previously established. To identify positions truly indicative of species classification which could serve as targets for polymerase chain reaction species-specific identification primers, 16S rRNA-based phylogenetic analyses were conducted. On the basis of the identified signature nucleotides, we designed polymerase chain reaction primer sets which (i) amplify all spirochete species associated with Lyme disease and (ii) differentiate between these species. The primer sets were tested on 38 Borrelia isolates associated with Lyme disease and were found to be sensitive and specific. All Lyme disease isolates tested were amplification positive. These primers allow for the rapid species identification of Lyme disease isolates.


Identification of a genetically distinct subspecies of Mycobacterium kansasii. To assess the usefulness of a specific DNA probe for Mycobacterium kansasii, 105 isolates from Australia, Belgium, Japan, South Africa, and Switzerland were collected and analyzed. Twenty of these isolates were probe negative, of which 18 were from Belgium and Switzerland. Analysis of all isolates by Southern blot hybridization indicated a lack of variability among probe-positive isolates, while probe-negative isolates were clearly distinct and showed greater diversity. Sequence analysis of the 250 nucleotides at the 5' end of the 16S rRNA gene revealed that 19 of the 20 probe-negative isolates had a sequence different from that of M. kansasii. A total of five nucleotide differences were present in a cluster consisting of two nucleotide deletions and three nucleotide substitutions. These results suggest the existence of a genetic subspecies of M. kansasii.


Isolation of a fastidious Mycobacterium species from two AIDS patients. Two strains of fastidious mycobacteria were isolated from two patients with AIDS and clinical disease suggestive of Mycobacterium avium complex infection. Acid-fast bacilli were isolated from blood and bone marrow of both patients in BACTEC 12B and/or 13A media. The acid-fast bacilli failed to grow on subculture to routine Löwenstein-Jensen medium containing pyruvate and egg yolk agar. After several attempts, the strain from one patient was finally cultured on Middlebrook 7H9 medium with agar, charcoal, and yeast extract 13 months after the initial specimens were received in the laboratory. The second patient's strain was cultured on the same medium 6 weeks postinoculation with fresh BACTEC fluid and 5 months after specimen collection. Routine biochemical and growth tests were performed on these isolates but failed to give definitive identifications. 16S rRNA gene sequencing suggested that the organisms share at least 98.9% homology with M. simiae. Even greater homology (99.86%) was found with the recently described species "M. genavense." Recognition of the fastidious nature of some mycobacteria that infect AIDS patients is important in the treatment of infections in these patients and in understanding the epidemiology of atypical mycobacterial infections. It is suggested that a liquid culture medium such as BACTEC be employed for primary isolation of mycobacteria from AIDS patients and that subculture to the charcoal medium described here be carried out for those organisms that fail to grow on subculture to routine media.


Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9. The study described here was carried out to further characterize reference strains of Serpulina (Treponema) hyodysenteriae representing serotypes 8 and 9. Results obtained from restriction fragment length polymorphism analysis, enteropathogenicity testing, and endotoxin profiles confirmed their identifications. Electron microscopy indicated that both strains were covered with a thin layer of capsule-like material. Immunoblot analysis indicated that an antigen in the 19-kDa region of proteinase K-digested whole cells reacted only with homologous antiserum. The serotype-specific antigens were sensitive to periodate oxidation but resistant to proteinase K digestion and migrated in the same region as purified lipopolysaccharides. Immunoblotting with proteinase K-digested whole cells appeared as useful as immunodiffusion with extracted lipopolysaccharide for the serological classification of S. hyodysenteriae. Immunogold labeling of whole cells and purified periplasmic flagella showed strong cross-reactions between S. hyodysenteriae and Serpulina innocens. Outer membrane preparations of strains representing serotypes 8 and 9 contained four major proteins which reacted with antisera against both species, and one major protein with a molecular mass of 46 kDa which reacted only with antisera against S. hyodysenteriae, irrespective of the serotype. Our findings suggest that periplasmic flagella and some outer membrane proteins are antigens common to both S. hyodysenteriae and S. innocens, whereas a 46-kDa outer membrane protein may be a species-specific antigen of S. hyodysenteriae. Finally, we propose immunoblotting as an alternative method to immunodiffusion for the serotyping of S. hyodysenteriae.


Differentiation of Shigella flexneri strains by rRNA gene restriction patterns. We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolates with each of the five restriction endonucleases BamHI, EcoRI, HindIII, PstI, and SalI generated reproducible restriction patterns. However, HindIII produced the optimum digestion pattern of the rRNA genes and was more useful than the other enzymes used in differentiating strains. Analysis of the 72 isolates showed 11 different HindIII cleavage patterns of their rRNA genes. Four of these HindIII-generated ribotypes could be further differentiated into two to four subribotypes by using PstI. The results indicate that ribotyping has an application for differentiation of S. flexneri strains and can complement serotyping. Definition of strains in terms of both serotype and ribotype may be of greater use in epidemiological studies.


Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi. Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equi merozoite antigen 1 (EMA-1), was evaluated in the CI ELISA. Recombinant EMA-1 bound antibody from the sera of B. equi-infected horses from 18 countries. The antibody response to EMA-1 was then measured in horses experimentally infected with B. equi via transmission by the tick vector Boophilus microplus or by intravenous inoculation. Anti-EMA-1 antibody was detected 7 weeks post-tick exposure and remained, without reexposure to B. equi, for the 33 weeks of the evaluation period. The data indicate that recombinant EMA-1 can be used in the CI ELISA to detect horses infected with B. equi.


Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes. A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.


Comparison of whole-cell antibodies and an antigenic flagellar epitope of Borrelia burgdorferi in serologic tests for diagnosis of Lyme borreliosis. A recombinant protein (p41-G) of an antigenic region of flagellin was used in a standard and amplified enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Comparable sensitivities (88 to 94%) were noted when sera from 17 persons who had erythema migrans and antibodies to whole-cell B. burgdorferi were tested against the p41-G antigen. In tests of a second study group of 36 persons who had erythema migrans but no detectable antibodies to whole-cell B. burgdorferi, 3 (8%) were positive when the p41-G antigen was used. Assay specificity likewise increased when the p41-G fragment was included in an ELISA with human sera containing treponemal antibodies. Recombinant flagellar proteins of B. burgdorferi, such as the p41-G antigen, can be used in an ELISA and may help confirm Lyme borreliosis during early stages of infection and improve specificity.


Antigenicity of Helicobacter pylori lipopolysaccharides. An investigation of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of lipopolysaccharides (LPSs) extracted from seven strains of Helicobacter pylori revealed that these molecules were silver stainable and exhibited a high degree of variability in their patterns. Two strains synthesized a variety of sizes of LPS molecules such that fractionation by SDS-PAGE resulted in a stepwise gradation of bands which extended from the top to the bottom of the silver-stained gel. The LPSs from the remaining five strains were made up of molecules which were more homogeneous in size and clustered around two separate areas of the gel. Antigenic analyses of phenol-water-extracted LPSs by immunoblotting and the passive hemagglutination assay suggested that, in addition to strain-specific antigens, all of the LPSs carried a common antigen. Antibodies to this common antigen could be removed from antisera by absorption, and the resulting antisera were used to differentiate strains on the basis of their O antigens by the passive hemagglutination assay technique. The finding that LPSs from 3 of 10 clinical isolates reacted specifically in one or two of the typing antisera suggested that the development of a scheme for differentiating H. pylori on the basis of O antigens is feasible.


Laboratory aspects of "Mycobacterium genavense," a proposed species isolated from AIDS patients. "Mycobacterium genavense" is a proposed new species recently reported to cause disseminated infections in 18 patients with AIDS in Europe. We have recovered "M. genavense" as slowly growing fastidious mycobacteria in blood cultures of seven patients with AIDS. In the original studies of "M. genavense," the fastidious organism grew only in BACTEC 13A vials. The Seattle, Washington, isolates of "M. genavense" also failed to grow when subcultured from 13A vials to routine solid media, but dysgonic colonies were produced on Middlebrook 7H11 agar supplemented with mycobactin J. The mycolic acid pattern of patients' isolates closely resembled that of the type strain of Mycobacterium simiae when analyzed by one- and two-dimensional thin-layer chromatography and by high-performance liquid chromatography. Whole-cell fatty acid analyses by gas-liquid chromatography distinguished the isolates from M. simiae but misidentified them as Mycobacterium fortuitum. Sequence determinations of the hypervariable regions of the 16S rRNA gene indicate that these organisms belong to the recently proposed new species "M. genavense." Growth from Middlebrook 7H11 agar supplemented with mycobactin J consistently yielded positive tests for catalase (semiquantitative and at 68 degrees C), pyrazinamidase, and urease which enable mycobacteriology laboratories to presumptively identify "M. genavense" without nucleic acid analyses. The failure of "M. genavense" to grow on conventional mycobacterial solid media suggests that mycobacterial blood cultures should include a broth medium incubated for at least 8 weeks.


Purification of Pneumocystis carinii trophozoites and identification of their circulating antigens. We have developed a new method to isolate Pneumocystis carinii trophozoites from experimental rat bronchoalveolar lavage specimens by using a Percoll discontinuous gradient and have identified the circulating antigens in experimental rat P. carinii pneumonia. The antigenic components of the trophozoites were compared with those of cysts by immunoblotting. A major immunoreactive band of 90,000 Da and some others of lower molecular mass were found in trophozoites. On the other hand, bands of 110,000, 50,000, and 45,000 Da were observed in cysts. The band of 50,000 Da was not identified when antitrophozoite rabbit serum preabsorbed with P. carinii-infected rat serum was used for immunoblotting. These results suggest that the molecule of 50,000 Da is the major circulating antigen in P. carinii-infected rats.


CD43 expression in B cell lymphoma.

CD43 expression in B cell lymphoma. To determine the expression of CD43 in frozen sections in a range of B cell lymphomas. The monoclonal antibody WR14, clustered provisionally in the Fourth Leucocyte Typing Workshop as a CD43 reagent, was investigated by epitope blocking studies on formalin fixed reactive lymph node tissue, using the established CD43 antibody MT1, to validate its use as a CD43 reagent. CD43 expression was studied in 131 immunophenotypically defined B cell lymphomas, including lymphocytic lymphoma (Lc, n = 13), centrocytic lymphoma (Cc, n = 14), and a range of follicle centre cell lymphomas (FCC) including centroblastic/centrocytic follicular (CbCcF, n = 48), centroblastic diffuse (CbD, n = 39), centroblastic/centrocytic diffuse (CbCcD, n = 4), centroblastic follicular and diffuse (Cb FD, n = 3) and centroblastic/centrocytic follicular and diffuse (CbCc FD, n = 1). Nine lymphomas of mucosa associated lymphoid tissue (MALT) were also examined. Epitope blocking studies showed that WR14 is a CD43 reagent that binds to an epitope identical with or close to that recognised by MT1. Eleven of 13 (84%) cases of Lc and 11 of 14 (78%) cases of Cc expressed CD43; 87 of 95 (91%) cases of FCC did not. All eight low grade lymphomas of MALT were negative. One high grade lymphoma, transformed from a low grade MALT lymphoma, was positive for CD43. The expression of CD43 by tumours of B cell lineage was associated with the expression of CD5 (p < 0.001) although either antigen could occasionally be found in the absence of the other. CD43 reagents can be used in conjunction with CD5 antibodies for the immunophenotypic discrimination of follicle centre cell lymphomas from non-follicle centre cell lymphomas.


Neurogenic inflammation and the oral mucosa. Since the rôle of neurogenic inflammation in various pathological conditions is well-established, we presume that it also has great importance in the development of several inflammatory processes in the oral mucosa. In the course of experimentally-induced neurogenic inflammation of the oral mucosa in rats, vasodilation and plasma extravasation occur in the area supplied by unmyelinated capsaicin-sensitive fibres. Upon activating these fibres in the oral mucosa, mediators such as histamine, substance P (SP) and calcitonin gene related peptide (CGRP) are released from the peripheral terminals of afferent nerves causing characteristic symptoms of neurogenic inflammation. By histochemical techniques, SP- and CGRP-immunoreactive fibres have occurred in the area of the rat oral mucosa stimulated mostly in the free and attached gingiva around the molar teeth in the lower jaw. Capsaicin pretreatment performed neonatally or at adult age causes a loss of SP-immunoreactive fibres and prevents the vasodilatory responses, as well as the increase in vascular permeability elicited by the antidromic stimulation of the inferior alveolar nerve. As the transection of inferior alveolar nerve decreased the extravasation of Evans blue on the ipsilateral side and did not affect the capsaicin-induced enhancement in blood flow, we suggest that the two symptoms of the inflammation, i.e., the increased vascular permeability and decreased vascular resistance, should be produced by different mechanisms. The results of all the morphological and functional studies seem to confirm the possibility that there is an important neurogenic component of the inflammatory alterations caused by different mechanical and chemical stimuli in the oral mucosa.


Characteristics of ruminant mammary epithelial cells grown in primary culture in serum-free medium. Cells were obtained from the mammary glands of sheep and cows by collagenase-hyaluronidase digestion. Characterization of cells as epithelial was by reaction with a monoclonal antibody to cytokeratin. A subpopulation of spindle-shaped or stellate cells reacted with a monoclonal antibody to desmin and may be related to myoepithelial cells. The development is described of a simple serum-free culture system for these cells on gels of rat tail (type 1) collagen. A commercial medium (M199) was used, buffered with Hepes and with bovine serum albumin as the sole protein supplement, plus fibronectin for the first 18 h only as an attachment factor. The cell cultures showed stimulated DNA synthesis in response to mitogens on attached gels and also responded as floating cultures to lactogenic hormones with production of alpha-lactalbumin.


'Usage' testing of root canal sealing materials--a personal view. A number of systems for the biological testing of dental materials have been suggested. This paper reviews the protocols suggested in the ISO standards for the in-use or 'usage' tests for root canal sealing materials.


A comparison of four in vitro marginal leakage tests applied to root surface restorations. Marginal leakage associated with a variety of restorative materials, placed in root surface cavities of extracted teeth, was assessed by immersing the teeth in either acidified gelatin, eosin dye, silver nitrate or a solution of radiocalcium. The allocation of a score, dependent on the depth of tracer penetration at the interface, allowed comparisons to be made between the leakage tests employed. The rank order of the four leakage tests was generally consistent. Eosin resulted in the most severe leakage, followed by silver nitrate and radiocalcium, whilst acidified gelatin was the least sensitive method for demonstrating marginal leakage. Differences in leakage scores were not, however, always statistically significant. None of the four tests investigated was ideal, although eosin dye was considered to be the most appropriate method of demonstrating marginal leakage associated with root restorations.


Canine left ventricular hypertrophy predisposes to ventricular tachycardia induction by phase 2 early afterdepolarizations after administration of BAY K 8644. The purpose of this study was to test the hypothesis that the longer duration of ventricular action potentials in hypertrophied hearts predisposes to the development of early after-depolarizations and triggered ventricular tachyarrhythmias. For unknown reasons, the incidence of sudden death is greater in patients with myocardial hypertrophy. We measured left ventricular monophasic action potentials in normal dogs and dogs with left ventricular hypertrophy before and after administration of the calcium agonist BAY K 8644 and the potassium channel blocker cesium. We demonstrated longer action potential durations in dogs with than in those without left ventricular hypertrophy. Also, BAY K 8644 produced phase 2 early afterdepolarizations and ventricular tachyarrhythmias more frequently in the dogs with than in those without left ventricular hypertrophy. Phenylephrine, an alpha agonist, further increased the action potential duration in hypertrophied hearts and the propensity to develop early afterdepolarizations and ventricular tachyarrhythmia after administration of BAY K 8644. Control and hypertrophied hearts developed early afterdepolarizations and ventricular tachyarrhythmia equally when exposed to cesium. Although in vitro studies have shown that fibers of hypertrophied ventricular myocardium can develop triggered activity as a result of both early and late afterdepolarizations, the present study is the first to show in vivo that the hypertrophied ventricular myocardium compared with the normal ventricle is predisposed to develop phase 2 early afterdepolarizations that appear to trigger ventricular tachyarrhythmia. It is possible that such a mechanism contributes to the development of ventricular tachyarrhythmia and sudden cardiac death in patients with left ventricular hypertrophy. If this is shown to be true, specific pharmacologic interventions can be suggested.


Chick embryo exposure to carbamates alters neurochemical parameters and behavior. Recent evidence has shown that exposure to pesticides can lead to long-term neurophysiological and functional deficits. We have demonstrated previously that locomotion in chicks exposed to some organophosphates and carbamates could be altered persistently without concomitant central or peripheral esterase inhibition. Furthermore, histopathology of the ataxic chickens showed no lesions in either the central or peripheral systems. In this study, we examined whether locomotion alterations seen in chicks exposed in ovo to carbaryl and aldicarb are accompanied by perturbations in particular central neurotransmitter systems. Carbaryl and aldicarb were injected in ovo on day 15 of incubation at 6, 16 and 65 mg kg-1 egg weight and at 0.2, 0.4 and 35 mg kg-1 egg weight, respectively. Neurotransmitter levels (assayed by high performance liquid chromatography with electrochemical detection) and locomotion were measured at various times (1-43 days) after dosing. At the lower doses of both carbaryl and aldicarb, a trend towards prolonged decreases in cerebral dopamine and homovanillic acid was seen. The high dose of carbaryl significantly reduced dopamine and the high dose of both compounds significantly decreased homovanillic acid and 5-hydroxyindoleacetic acid. Persistent locomotion alterations were observed only at the higher doses of both carbaryl and aldicarb when the specific neurotransmitters measured returned to normal levels.


[New approaches to the diagnosis of carbohydrate metabolic disorders]. A bioenergetic conception of insulin-independent diabetes mellitus (IIDM) is put forward. Molecular mechanisms of carbohydrate and fat metabolism are discussed which result in a vicious regulatory cycle to provoke a transition to stable pathology. Previously unknown role of human saliva in oral cavity in this regulatory cycle is revealed. New diagnostic tests are developed on the basis of the conception on IIDM pathogenesis. The results of clinical trials are presented which have been obtained during population screening.


Is your audiovisual teaching effective?

Is your audiovisual teaching effective? Medical illustrators produce vast numbers of images used in audiovisual programmes for medical teaching. We know that students can learn by using audiovisual teaching materials but do not always know how well they learn, particularly in comparison with other teaching methods. To find out how effective audiovisual teaching is, it must be evaluated or measured. This paper looks at the evaluation of a range of medical audiovisual teaching programmes and their effectiveness by observation, questionnaire, tests and examinations.


Immunodiagnosis of human dirofilariasis by enzyme-linked immunosorbent assay using recombinant DNA-derived fusion protein. An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-Dirofilaria immitis recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human dirofilariasis. D. immitis-infected human sera reacted strongly with FP that was immobilized with anti-beta-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other filariasis. In detection of anti-D. immitis IgG antibody. ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.


Use of an aceto-carmine procedure to examine the excysted metacercariae of Echinostoma caproni and E. trivolvis. A simple aceto-carmine procedure was developed to relax, fix, stain, and clear excysted metacercariae of Echinostoma caproni and E. trivolvis. This procedure allowed for morphologic details on whole metacercariae comparable to those seen with more elaborate staining procedures. Differences in dimensions and staining intensities between the two species of metacercariae are described.


Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation. Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.


Loss of laminin epitopes during glomerular basement membrane assembly in developing mouse kidneys. Kidney glomerular basement membranes (GMBs) originate in development from fusion of a dual basement membrane between endothelial cells and primitive epithelial podocytes. After fusion, segments of newly synthesized matrix, derived primarily from podocytes, appear as subepithelial outpockets and are spliced into GBMs during glomerular capillary loop expansion. To investigate GBM assembly further, we examined newborn mouse kidneys with monoclonal rat anti-mouse laminin IgGs (MAb) conjugated to horseradish peroxidase (HRP). In adults, these MAb strongly label glomerular mesangial matrices but bind only weakly or not at all to mature GBMs. In contrast, anti-laminin MAb intensely bound newborn mouse GBMs undergoing initial assembly. After intraperitoneal injection of MAb-HRP into neonates, dense binding occurred across both subendothelial and subepithelial pre-fusion GMBs as well as forming mesangial matrices. Considerably less MAb binding was seen, however, in post-fusion GBMs from more mature glomeruli in the same section, although mesangial matrices remained positive. In addition, new subepithelial segments in areas of splicing were negative. These results conflict with those obtained previously with injections of polyclonal anti-laminin IgGs into newborns or adults, which result in complete labeling of all GBMs. Although epitope masking cannot be completely excluded, we believe that decreased MAb binding to developing GBM reflects actual epitope loss. This loss could occur by laminin isoform substitution, conformational change, and/or proteolytic processing during GBM assembly.


Simultaneous visualization of immunodetected antigens and tissue components revealed by non-enzymatic histochemical stains. We explored the possibility of simultaneous application of histochemical and immunohistochemical staining techniques on the same paraffin-embedded human tissue section. Conventional histological stains (PAS, Alcian, Alcian-PAS, Van Gieson, Gomori silver impregnation, and Giemsa) were used in association with a battery of markers (keratins, leucocyte common antigen, S-100 protein, Factor VIII-related antigen) that are widely employed in diagnostic and experimental studies. We found that the best procedure was to perform immunostaining before the histochemical reaction, as this enables all the other possible combinations to be carried out. In addition, several detection systems, such as peroxidase-anti-peroxidase (PAP), alkaline phosphatase-anti-alkaline phosphatase (APAAP), and avidin-biotin complex (ABC), were tested and all gave consistent results. Some minor modifications of the histological staining methods were necessary, but the current immunohistochemical techniques could be used as established. Preliminary findings indicate that immunohistochemistry can be combined with histochemistry techniques by means of a relatively simple procedure whose only disadvantage is the time required to carry out the double staining.


Amylase-producing IgD-type multiple myeloma. We describe the case of a 63-year-old woman with an IgD-type multiple myeloma and hyperamylasaemia. The evolution of the amylase concentration, the immunohistochemical data and the intracellular amylase contents of the plasma cell were consistent with secretion of amylase by the malignant clone. Moreover, cytogenetic analysis of the bone marrow revealed two structural rearrangements involving chromosome 1 near the amylase locus. Multiple myeloma should be added to the amylase-secreting tumours. This rare entity is not confined to Japan, where it was first recognized.


A promising interferon plus four-drug chemotherapy regimen for metastatic melanoma. The goal of this study was to determine the therapeutic efficacy and toxicity of a four-drug chemotherapy regimen combined with natural leukocyte interferon alfa (IFN-alpha) for metastatic melanoma. Between December 1988 and December 1991, 48 consecutive unselected patients with metastatic melanoma were entered onto this phase II study. Forty-five of these patients were assessable for response and 46 for toxicity. The four-drug chemotherapy regimen was as follows: dacarbazine (DTIC) 200 mg/m2 days 1 to 5, vincristine 1 mg/m2 days 1 and 4, bleomycin 15 mg days 2 and 5 intravenously (IV), and lomustine (CCNU) 80 mg day 1 orally. IFN-alpha, initiated day 8, was administered 3 x 10(6) IU/d, subcutaneously (SC) for 6 weeks, followed by 6 x 10(6) IU three times per week. A small protocol modification was adopted from the 21st patient onwards whenever there was more than 2 months' stabilization or progression with the original protocol: IFN therapy was split into 2-week treatment periods interrupted by a 2-week rest period. Among the 45 assessable patients, the objective response rate was 62% (95% confidence limit, 48 to 77); six patients (13%) achieved a complete response (CR) and 22 (49%) a partial response (PR). Metastases in such sites as liver also responded favorably (one CR, six PR, one stable disease [SD], two progressive disease [PD]). After splitting IFN therapy for nonresponders, in two patients PD and in another two patients SD changed into regression. Three of the six patients with a CR have suffered a relapse, but the other three have been off treatment for 7, 18, and 31 months without recurrence. Most of the symptomatic patients also experienced rapid relief of symptoms. Overall toxicity of this mainly outpatient regimen seemed to be acceptable. One patient died of a septic fever with grade 4 leukopenia and thrombocytopenia. The most frequent side effects were transient fever, nausea/vomiting, fatigue, and grade I/II hematologic toxicity. Results demonstrate a remarkably high response rate in combining IFN-alpha and four chemotherapeutic agents. The apparent schedule-dependency of responses must be further explored in a controlled phase III study.


Granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor infusion makes high-dose etoposide a safe outpatient regimen that is effective in lymphoma and myeloma patients. This study assessed the efficacy of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human granulocyte colony-stimulating factor (rhG-CSF) in ameliorating the extent and duration of hematologic toxicity after high-dose etoposide cancer therapy. Thirty-two non-Hodgkin's lymphoma and myeloma patients were treated with 2 to 2.4 g/m2 etoposide infused intravenously (IV) during a 10- to 12-hour period, followed 72 hours later by subcutaneous administration of rhGM-CSF or rhG-CSF. Hematologic toxicity was compared with that observed in 29 patients who were treated with high-dose etoposide without growth factors. The median duration of grade 4 neutropenia in growth factor-treated patients was 3 days, and granulocyte counts never decreased to less than 100/microL in approximately half of the patients. The corresponding figures in the control patients were 8 and 3 days, respectively (P < .0001). No effect was observed in platelet and RBC recovery. Growth factor-treated patients became eligible to receive additional myelotoxic chemotherapy a median of 5 days earlier than controls. Nonhematologic toxicity was minimal. Grade 1 mucositis was observed in two of 61 patients (3%). Antitumor activity assessed within 1 month after etoposide administration was documented in 58% of 38 assessable patients. Finally, high-dose etoposide expanded and mobilized the pool of peripheral-blood hematopoietic progenitors. The use of rhGM-CSF or rhG-CSF makes high-dose etoposide a safe outpatient regimen and should encourage the inclusion of this highly effective and well-tolerated drug in novel treatment strategies that use high-dose therapy early in the clinical course of chemosensitive tumors.


Leucine excess under conditions of low or compensated aminoacidemia does not change skeletal muscle and whole-body protein synthesis in suckling lambs during postprandial period. We determined the effect of a 4-h leucine infusion, leading to 15-fold elevated plasma leucine concentrations, on skeletal muscle and whole-body protein synthesis in suckling lambs during the postprandial period. The [3H]phenylalanine large dose method was validated and used to quantify the fractional rates of protein synthesis (Ks in %/d) at the end of the leucine infusion. In the first experiment leucine infusion lowered plasma amino acid concentrations but did not change the Ks, the capacity for protein synthesis (Cs, mg RNA/g protein) or the efficiency of translation [g protein synthesized/(d-g RNA)] in any muscles studied or the whole body. In the second experiment the leucine-induced decreases in plasma amino acid concentrations were compensated by the simultaneous infusion of substantial amounts of amino acids. Again leucine excess did not significantly change Ks, Cs and efficiency of protein synthesis. These results indicated that leucine excess in suckling lambs during the postprandial period lowered aminoacidemia without any change of the protein synthesis rates in skeletal muscles or the whole body.


Angiogenic activity of fibrin degradation products is located in fibrin fragment E. The source of angiogenic activity of fibrin degradation products has been sought in a series of experiments, applying degradation products from different types of fibrin and fibrinogen to the chick chorioallantoic membrane. The presence of platelets or fibronectin during clotting was not essential for activity, and neither was crosslinking. Fibrinogen degradation products were non-stimulatory, as was serum. Molecular sieve column chromatography indicated a range of active fragments. Admixture of active fibrin degradation products with antifibrin fragment E, but not D, antiserum neutralized activity. Preparations containing only fibrin fragment E retained activity. A commercial preparation of fibrinogen fragment E was inactive until treated with thrombin. These experiments point to fibrin fragment E being the source of angiogenic activity, with thrombin cleavage being the essential step in generating activity.


The "plaque-free zone" in health and disease: a scanning electron microscope study. A "plaque-free" zone has been described on the enamel surface of healthy extracted teeth. This study examined this zone on chronic adult periodontitis-affected teeth (CAPT). Ten healthy controls and 16 CAPT were collected immediately after extraction, fixed, dehydrated, critical point dried, coated, and viewed by scanning electron microscopy (SEM). The "plaque-free" zone was observed in both groups as an area with few bacteria between the apical plaque border and the coronal limit of an epithelial layer on the root surface, extending to the residual periodontal ligament. On the healthy specimens, the apical plaque border consisted mainly of cocci and short rods, while on the CAPT specimens spirochetes predominated. Isolated or small groups of microorganisms were always present in the "plaque-free" zone and at its apical limit, close to or in contact with junctional epithelial cells. This zone is therefore not completely free of plaque, as suggested. It was concluded that a tissue complex, analogous to that in health on enamel, persists on the root surfaces of CAPT throughout the disease process. It comprises a discrete plaque border, a dental cuticle with sparse organisms, and an epithelium analogous to junctional epithelium. Its main function would appear to be to prevent bulk access of plaque to the surrounding tissues, including direct contact of bacteria with underlying ligament.


Retardation of epithelial migration in monkeys using a carbon dioxide laser: an animal study. Obtaining a connective tissue attachment to the root surface following a mucoperiosteal flap surgery has been a goal of periodontal therapy for a long time. The objective of this study was to examine whether controlled de-epithelialization with the CO2 laser would retard the apical migration of the epithelium and thereby increase the amount of connective tissue attachment. Elastics were placed on the maxillary premolars and incisors of 7 cynomolgus monkeys to create periodontal defects. Open flap debridement was performed on each side. On the experimental side, the oral epithelium was removed by CO2 laser irradiation. This report describes the 3 specimens at 7, 14, and 28 days respectively. On the control side sulcular epithelium was seen at 14 days. Sulcular epithelium was first seen on the test side at 28 days. For all specimens over 7 days, there was a trend to less epithelium and more connective tissue attachment on the experimental side than on the control side. However, no statistical analysis was possible on this histologic study. The CO2 laser may be a useful tool to retard epithelium and thereby enhance new connective tissue attachment.


Effect of terfenadine on the plasma concentrations of substance P and vasoactive intestinal polypeptide in volunteers. The effect of terfenadine on the plasma concentrations of substance P and vasoactive intestinal polypeptide (VIP) was studied in 7 healthy subjects and 8 subjects with the common cold. Before terfenadine administration, the mean plasma substance P concentration of the subjects with the common cold was significantly higher than that of the healthy subjects. The increased mean plasma substance P concentration of the subjects with the common cold was decreased after terfenadine administration. In the healthy subjects, the mean plasma substance P concentration was unchanged by terfenadine administration. The mean plasma VIP concentration of the subjects with common cold was slightly higher than that of the healthy subjects before and after terfenadine administration, with no significant difference.


Effects of long-term laxative treatment on neuropeptides in rat mesenteric vessels and caecum. In order to investigate the toxic effects of long-term treatment with anthraquinone laxatives, rats were fed either chocolate alone, or chocolate adulterated with senna or danthron (1,8-dihydroxyanthraquinone) for 5 months. Mesenteric blood vessels and the outer muscle layers of the caecum, together with the myenteric plexus, were examined using ultrastructural, histochemical, immunohistochemical and immunoassay techniques. There was no ultrastructural evidence of degeneration in either the mesenteric vessels or the caecum. In the mesenteric vessels, levels of neuropeptide Y were significantly reduced in the danthron-fed rats, but levels of substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were unaffected by all treatments. In the caecum, VIP-, SP- and CGRP-immunoreactivity and catecholamine-fluorescence were unchanged by the laxative treatments.


Comparative immunogenicity of gp120-derived proteins and their induction of anti-V3 loop region antibodies. This report compares the immunogenicity of three different preparations of gp120 [the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) virus]: (a) native, fully glycosylated gp120; (b) a nonglycosylated, denatured form of gp120; and (c) a nonglycosylated peptide representing the "immunodominant" third hypervariable region of the gp120 molecule. Results indicate that the native glycosylated form of gp120 induces a maximal anti-HIV response in which a majority of B cells bind to conformation-dependent epitopes but less than one-fifth recognize the V3 loop region.


Replication and apical budding of HIV-1 in mucous-secreting colonic epithelial cells. The human colonic adenocarcinoma cell line HT29 can be infected with various isolates of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). In some cases, the virus was able to perform its complete cycle of replication as demonstrated by the persistent production of mature viral particles in the cell-free culture supernatant. We have cultured HT29 cells chronically infected with the replicative strain HIV1-NDK in a chemically defined serum-free medium. Under these conditions, the cells were able to maintain a high level of viral replication, as demonstrated by reverse transcriptase activities and in situ hybridization studies. By indirect immunofluorescence labeling and electron microscopy, we observed that serum starvation was associated with the differentiation of HIV-1-infected HT29 cells into mucous-secreting cells resembling epithelial goblet cells of the colonic mucosa. These mucous-secreting cells, which accounted for 50% of the overall population, produced mature particles of HIV through their apical membrane in the vicinity of mucous granules. These data suggest that HIV-infected goblet cells in the colonic mucosa may produce the virus in the colorectal lumen; this could explain the route of transmission of HIV in the case of anal intercourse.


Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm. It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.


Light and electron microscopic immunolocalization of sperm proteins identified by monoclonal antibodies from the World Health Organization Task Force on Sperm Antigens. Sperm antigens recognized by monoclonal antibodies (mAbs), S19, S69, S71, S72 and S77 submitted to the World Health Organization (WHO)-Sponsored Sperm Monoclonal Antibody Workshops were immunolocalized by light (LM) and transmission electron microscopy (TEM). S19 was surface reactive while mAbs S69, S71, S72 and S77 recognized internal antigens. Indirect immunofluorescence staining of permeabilized sperm with the mAbs revealed that S69 recognized an internal tail antigen, while S71, S72 and S77 recognized acrosomal proteins. Preservation of immunoreactivity after fixation in various combinations of glutaraldehyde, paraformaldehyde and tannic acid was evaluated for mAbs S69 to S77 using immunofluorescence microscopy. The epitopes recognized by these mAbs were adversely affected by these fixatives; therefore, pre-embedding immunogold staining was employed, prior to fixation, osmication, dehydration and embedding. Using this approach, the antigen recognized by mAb S19 was found associated with the plasmalemma of the head and tail of intact sperm. Monoclonal antibody S69 localized to the fibrous sheath. The mAbs S71, S72 and S77, which required sperm permeabilization to expose their acrosomal locus by LM, did not immunoreact with the plasmalemma at the TEM level. Ultrastructural examination of acrosome-reacted sperm revealed the localization of S71 and S77 on the inner and outer acrosomal membranes and with acrosomal matrix. The S72 antigen was associated with the inner and outer acrosomal membranes.


Sustained food vacuole formation by axenic Paramecium tetraurelia and the inhibition of membrane recycling by Alcian Blue. It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.


Unexplained positive/elevated maternal serum alpha-fetoprotein associated with placenta increta. A case report. Maternal serum alpha-fetoprotein (MSAFP) is a regularly utilized antenatal screening test for the identification of pregnancies at increased risk for a variety of genetic and nongenetic abnormalities. Complete mid-trimester evaluation of the patient with a positive screening test may fail to reveal an etiology for a positive MSAFP value. This case report concerns an unexplained positive/elevated MSAFP screening test for a patient found at delivery to have abnormal placentation.


Nitric oxide synthase is critical in mediating basal forebrain regulation of cortical cerebral circulation. This study sought to determine whether the activity of nitric oxide synthase (NOS) is an important physiological link required to mediate increases in cortical cerebral blood flow (CBF) elicited by electrical microstimulation of the basal forebrain (BF). Changes in cortical CBF were assessed in urethane anesthetized rats using laser-Doppler flowmetry. Microstimulation of the BF elicited stimulus-locked increases in CBF that were dependent on frequency and current intensity (up to 280% of control at 50 Hz). Infusion of the potent NOS inhibitor NG-nitro-L-arginine (L-NNA) resulted in significant dose-related reductions in the BF-elicited response at 50 Hz (3.75-60 mg/kg, i.v.), significant elevation in resting mean arterial pressure (MAP) from 106 to 160 mmHg, and modest 21% reductions in resting CBF. The stereoisomer NG-nitro-D-arginine (D-NNA) was without any effect on CBF, although at higher concentrations MAP was elevated to levels comparable to those obtained with L-NNA. Infusion of arginase was also without effect on resting or BF-elicited CBF responses. In contrast, L-arginine (100-400 mg/kg, i.v.) significantly potentiated the BF-elicited response up to an additional 38%, without affecting resting CBF or MAP. This study suggests that NO, or a related nitroso precursor formed by NOS, has a critical role in mediating regulation of cortical CBF by BF neurons.


DM20 mRNA splice product of the myelin proteolipid protein gene is expressed in the murine heart. Northern blot analysis of poly A(+) RNA isolated from mouse heart revealed the expression of 3.3 and 2.4 kb mRNAs that hybridized with a cDNA for the mouse proteolipid protein (PLP). In order to examine the relationship of these RNAs to the myelin PLP/DM20 mRNAs, a mouse heart cDNA library was prepared and screened with a mouse PLP cDNA. A cDNA was isolated, sequenced, and found to encode the DM20 variant of PLP. Polymerase chain reaction (PCR) analysis of heart cDNA with three sets of primers confirmed the presence of DM20 mRNA in mouse heart and indicated that it is the major splice product of the PLP gene expressed in that tissue. In situ hybridization localized the expression of the DM20 mRNA to the myocardial cells. Northern blot analysis indicated that expression of the DM20 mRNA is developmentally regulated in the murine heart, increasing significantly in concentration after 12 days postpartum. Northern analysis also revealed the expression of the DM20 mRNA in the hearts of the jimpy and quaking mutants. These results indicate that the PLP gene is expressed in tissues other than brain and support the concept that products of the PLP gene may have some biological role other than as structural components of myelin.


Endogenous aFGF expression and cellular changes after a demyelinating lesion in the spinal cord of adult normal mice: immunohistochemical study. Fibroblast growth factors (FGFs) are known to act on glial cells in vitro. At the present time, their involvement in the remyelinating process of the adult central nervous system (CNS) is still unknown. In the present study, using immunohistochemistry (IHC), we investigated the evolution in time and space of acidic FGF (aFGF) expression and CNS cell changes occurring after a chemically induced demyelinating lesion. In a first early period, aFGF immunostaining was shown to decrease around the demyelinated area. A dramatic increase was then observed and was accompanied by an increase of cell density around and inside the lesion. This was correlated with the beginning of remyelination. Late after demyelination, while remyelination was still in progress, aFGF immunostaining of the lesion and unlesioned spinal cord were comparable. A role of aFGF in remyelination is proposed.


Mitotic potential of adult rat oligodendrocytes in culture. The proliferation of adult oligodendrocytes (OLGs) was examined in response to membrane bound and soluble mitogens. OLGs were isolated according to Vick, et al., J Neurosci Res 25:524-534, 1990, and co-cultured with dorsal root ganglia (DRGs). Less than 5% of the total cells incorporated a 48 hr pulse of 3H thymidine during the first 4 days of co-culture. From day 4 to day 6 there was a dramatic increase in proliferation which reached a plateau at 40% and gradually decreased to 25% from days 10 to 20 of co-culture. Axolemma-enriched fractions (AEF) were weak mitogens for OLGs (less than 5% proliferation after 7 days of stimulation), however, heparin extracts of AEF were five-fold more mitogenic than the AEF from which they were derived. Basic and acidic fibroblast growth factor were effective mitogens for the adult OLGs (labelling indices of 28% and 12%, respectively) provided that the cells were treated for 7 days with the growth factor and that the cells had been in culture for at least 14 days. Other soluble growth factors (IL-2 and PDGF) gave no mitotic response. We conclude that adult OLGs are mitotically responsive to mitogens provided that (1) the adult OLG has been cultured for sufficient time (14 days, acidic or basic fibroblast growth factor); (2) the axonal mitogen is allowed to interact with the OLG for a sufficient time (neuritic mitogen); and (3) the axonal mitogen is presented to the OLG in an activated form (AEF-heparin extract).


Developmental expression of major myelin protein genes in the CNS of X-linked hypomyelinating mutant rumpshaker. Rumpshaker (rsh) is an X-linked mutation causing hypomyelination of the CNS of mice and has recently been identified as an allele of jimpy (jp). The mutation (known as jprsh) differs in several respects from other X-linked myelin mutants, including jp, in that mice have normal longevity, oligodendrocyte numbers are not decreased, and cell death is not a feature. Myelin sheaths are deficient in immunostainable PLP protein. The present study examines the developmental expression of the major myelin protein genes and translatability of PLP and MBP mRNA. Differences between the spinal cord and brain of mutants are evident in that mRNA levels are more markedly decreased in the brain. Protein levels are severely reduced in both locations and to a proportionately greater extent than the mRNA, particularly in the spinal cord where PLP RNA and protein are approximately 80% and 10-20%, respectively, of age-matched wild type mice. DM-20 protein, the other major product of the PLP gene, is disproportionately expressed in rumpshaker as is a 10 kDa proteolipid. In vitro translation studies indicate a marked decrease in PLP translation products from mutant RNA. There is no deficiency in the number of PLP mRNA-expressing oligodendrocytes although the abundance per cell is reduced. The data suggest that the phenotypic effects of the mutation may be associated with reduced translation of major myelin proteins, in particular PLP and its incorporation into compact myelin. However, the mutation is compatible with survival of oligodendrocytes and their differentiation to the stage of expressing PLP/DM-20 mRNA.


Isolation of activated adult Schwann cells and a spontaneously immortal Schwann cell clone. Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.


Developmental changes in nerve growth factor level in rat serum. In serum, nerve growth factor (NGF) forms a complex with alpha 2-macroglobulin (alpha 2M), which formation inhibits the immunoreactivity between NGF and its antibodies. For measuring the serum level of NGF, it is thus necessary to liberate NGF from the NGF-alpha 2M complex and prevent reformation of such complex. The pretreatment of rat serum with 1 M guanidine hydrochloride for a few hours and operation of the enzyme immunoassay (EIA) in the presence of guanidine hydrochloride provided a reliable means for determination of the NGF level in serum. By this procedure we followed the serum NGF level in rats developmentally. It increased from prenatal day 2 to postnatal day 5 and decreased slightly at postnatal week 3, thereafter remaining constant throughout adulthood. In pregnant rats, the NGF level in serum increased threefold to fivefold before birth and then decreased rapidly. These data suggest that serum NGF level may reflect the demand for this molecule during establishment of the peripheral nervous system.


Toxicity studies of a synthetic antioxidant, 2,2'-methylenebis (4-ethyl-6-tert-butylphenol) in rats. 1. Acute and subchronic toxicity. The acute and subchronic toxicity studies on 2,2'-methylenebis (4-ethyl-6-tert-butylphenol) (MBEBP) were conducted using male and female Wistar rats. In acute toxicity test, the LD50 values were estimated to be greater than 10 g/kg BW by oral and intraperitoneal administration in each sex. In subchronic toxicity test, groups of 10 rats of each sex were fed a diet containing 0.2, 1.0 or 5.0% of MBEBP and examined at 4 and 12 weeks. Body weight gain was significantly depressed at doses of 1.0 and 5.0% in both sexes, but the depression in the 1.0% group was severer than that in the 5.0% group in males. Hematological analysis showed slight but significant decrease of hemoglobin in the 1.0 and 5.0% groups of both sexes. Urine analysis showed no remarkable changes in all treated rats of both sexes. In biochemical analysis of serum, decrease of triglyceride level and cholinesterase activity, and increase of amylase activity were observed in treated rats. Histopathologically, testicular atrophy and decrease of spermatogenesis were observed in male rats fed 1.0 or 5.0% MBEBP for 4 and 12 weeks and vacuolization of parathyroid gland cells was observed in female rats fed 1.0 and 5.0% MBEBP for 12 weeks. In subchronic test, the lowest observable adverse effect levels for MBEBP toxicity were estimated to be 171 mg/kg BW/day in male rats and 180 mg/kg BW/day in female rats.


[Basic and clinical evaluation of the new kit for detection of serum prostate specific antigen (PSA)]. BALL-ELSA PSA is a monoclonal radioimmunometric assay kit for detection of serum prostate specific antigen (PSA) developed and generally used in Europe. Basic and clinical study of the kit was performed for evaluation of it's utility in Japanese patients. The sera from 56 patients with benign prostatic hyperplasia (BPH), 35 patients with prostate cancer (PCA) and 18 normal males were examined. Other kits such as EIKEN PSA and EIKEN PAP (prostatic acid phosphatase) were also evaluated in same sera. The results of the range in the measurement, within-assay error, between-assay error, dilution test, recovery test and others were well satisfied. In our clinical study, mean + 2SD of serum PSA values in normal males was 2.57 ng/ml. Serum PSA values determined by the two RIA kits, BALL-ELSA and EIKEN, showed a good correlation (r = 0.9909), but the BALL-ELSA PSA kit yielded values about 2.4 times higher than the EIKEN PSA kit on the same sample. We think that the difference might be largely due to differences in assigned PSA calibrators and diluents between the assay kits. Further investigations and discussions were expected on this point. In our study, the most suitable cut-off level for distinguishing PCA from BPH in BALL-ELSA PSA kit was 10.0 ng/ml.


Expression of nitric oxide synthase in kidney macula densa cells. The distribution of nitric oxide synthase (NOS), the enzyme by which NO is generated from L-arginine, was investigated in rat kidney. The indirect immunofluorescence technique using a polyclonal antibody against type I NOS was applied, followed by the histochemical NADPH diaphorase staining technique on the same sections in order to demonstrate the enzymatic activity of NOS. Macula densa cells were strongly stained by both techniques, demonstrating abundant NOS in the cytoplasm of these cells. In addition, these findings were confirmed by nonradioactive in situ hybridization, thus demonstrating the corresponding messenger RNA in macula densa cells as well. Our findings provide the morphological basis for a possible role of NO as a mediator substance in signal transfer from distal tubular fluid to glomerular arterioles.


Vascular cell adhesion molecule-1 (VCAM-1) expression in murine lupus nephritis. Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface protein which mediates adherence of inflammatory cells to target cells by binding with the beta 1-integrin ligand Very Late Antigen-4 (VLA-4) on leukocytes. The expression of VCAM-1 was investigated in a murine model of lupus nephritis, the autoimmune MRL/lpr mouse. Compared with normal mice, MRL/lpr kidneys show increased VCAM-1 expression not only in the endothelium, but also in cortical tubules and glomeruli. Increased steady-state VCAM-1 mRNA levels (approximately 3 kB) are detected in the kidney of nephritic MRL/lpr mice. Two additional transcripts (approximately 2 and approximately 1.8 kB) are present in autoimmune MRL/lpr but not in normal mice. Like ICAM-1, VCAM-1 is also upregulated in vitro in renal cortical tubular and mesangial cells by TNF-alpha, IL-1 and IFN-gamma. T cell and macrophage cell lines adhere to TNF-alpha stimulated tubular epithelial cells by using the VCAM-1/VLA-4 and the ICAM-1/LFA-1 binding pairs. Kidney tissue sections from nephritic MRL/lpr also display increased adhesiveness for T cell and macrophage cell lines, and for MRL/lpr lymph node cells when compared with normal kidney sections. This enhanced adhesiveness of MRL/lpr kidney tissue is inhibited with monoclonal antibodies targeting the VCAM-1 and ICAM-1 molecules. Thus, VCAM-1 and ICAM-1 function as renal parenchymal adhesion molecules and mediate the adherence of pathogenic inflammatory cells in murine lupus nephritis.


Demonstration of plasma proteinase inhibitors in beta 2-microglobulin amyloid deposits. beta 2-microglobulin-related amyloidosis (A beta 2M) represents a frequent complication in long-term dialysis patients. Although the pathogenetic mechanism has yet to be fully understood, it is known that amyloid fibrils usually consist of intact molecules of beta 2-microglobulin (beta 2m). Plasma proteinase inhibitors (PPI) are a broad family of glycoproteins with the function of eliminating unwanted proteolysis of serine proteases. Their role in amyloidogenesis has become a subject of intense discussion, especially since the recent identification of alpha 1-antichymotrypsin in the beta-protein amyloid deposits of Alzheimer's disease. We evaluated immunohistochemically and biochemically the presence and distribution of several PPIs (alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, antithrombin III, alpha 2-macroglobulin and tissue inhibitor metalloproteinase) and amyloid P component in A beta 2M deposits in osteo-articular and visceral tissues from dialysis patients with amyloidosis, as well as two carpal tunnel synovia from non-dialysis patients and one Alzheimer's brain as controls. The immunohistochemical study demonstrated that all but one (anti-alpha 1-antichymotrypsin) of the PPI antibodies tested showed varying degrees of positive reaction against A beta 2M deposits. All the antibodies (including anti-alpha 1-antichymotrypsin) also reacted to some extent with other non-amyloid visceral and connective tissue elements diffusely and/or selectively. Among them, only the reaction of anti-amyloid P component had significantly distinctive localization to A beta 2M deposits, which were identified in adjacent serial sections by Congo red staining and immunohistochemical reaction against anti-beta 2m.(ABSTRACT TRUNCATED AT 250 WORDS)


Adhesion of rat glomerular epithelial cells to extracellular matrices: role of beta 1 integrins. Glomerular epithelial cells (GEC) maintain glomerular permselectivity and are a target of immunological glomerular injury, which may lead to proliferation or detachment from extracellular matrix (ECM). We studied adhesion mechanisms in rat GEC in culture, focusing on adhesion molecules of the beta 1 integrin family. At early time points (1 hr after plating of cells into culture wells that had been pre-incubated with purified ECM proteins), adhesion of GEC to collagen I, collagen IV, laminin, and fibronectin was inhibited with anti-beta 1 integrin antibody. The peptide RGDS inhibited adhesion to fibronectin and laminin. Immunoprecipitation studies demonstrated the presence of alpha 2, alpha 3, and beta 1 integrins; the alpha 1, alpha 4, alpha 5, alpha 6, alpha v, and beta 3 subunits were undetectable. Adhesion to all ECM proteins was dependent on divalent cations, but the effects of individual cations varied among substrata. In rat GEC, alpha 2 beta 1 and/or alpha 3 beta 1 integrins appear to mediate adhesion to collagen I, collagen IV, and laminin. The alpha 3 beta 1 integrin is also the likely receptor for fibronectin, interacting through an RGD binding site. Furthermore, single integrins or combinations of integrins appear to have distinct ligand-binding functions that are differentially regulated by divalent cations. Characterization of GEC adhesion molecules may facilitate the understanding of mechanisms of glomerular development, and cell detachment or proliferation in immune glomerular injury.


Arginase is a major pathway of L-arginine metabolism in nephritic glomeruli. L-arginine can be metabolized to nitric oxide (NO) by nitric oxide synthase (NOS) and to urea and L-ornithine by arginase. Competition between these pathways for L-arginine in inflammatory sites has been suggested. In experimental glomerulonephritis glomeruli produce nitrite; a major source is macrophages. We hypothesized that arginase is present in glomeruli and may compete for substrate with NOS in glomerulonephritis. Therefore we examined both pathways in isolated nephritic glomeruli and peritoneal macrophages. Arginase activity was present in glomeruli, increased by > 500% in nephritic glomeruli compared to controls, and was predominant over NOS. Activity increased with L-NMMA (a NOS inhibitor), but this trend did not reach statistical significance. In macrophages both pathways were present; NOS predominated basally but this was reversed by L-NMMA. In contrast with glomeruli macrophage arginase activity increased after LPS stimulation. Levels of macrophage arginase activity could not account for activity in nephritic glomeruli, suggesting another source of arginase. This is the first demonstration of high arginase activity of nephritic glomeruli. Competition between arginase and NOS pathways suggests a regulatory mechanism of L-arginine metabolism within the glomerulus, with implications for the pathogenesis of injury and scarring in glomerulonephritis.