Distinct in vivo functions of two macrophage subpopulations as evidenced by studies using macrophage-deficient op/op mouse. The op/op mice totally lack macrophage growth factor colony-stimulating factor (CSF)-1 and thus, by definition are completely depleted of CSF-1-dependent functions of the macrophage cell lineage. Moreover, they possess a severe and generalized macrophage deficiency. However, residual macrophages of these mice should still have normal CSF-1-independent functions. Studies designed to elucidate this issue have revealed that op/op mice are capable of normal in vivo phagocytic function and demonstrate normal humoral and cellular response postimmunization with sheep red blood cells. However, release of monokines such as tumor necrosis factor and granulocyte CSF following administration of endotoxin is severely impaired in op/op mice as compared with littermate controls. These studies suggest that the CSF-1-dependent macrophage population (absent in the op/op mouse) is primarily responsible for regulatory functions of these cells mediated by monokines, while the CSF-1-independent macrophage population (present in the op/op mouse) is primarily responsible for the classical macrophage functions in immunity such as phagocytosis, antigen processing and presentation.


Firing characteristics of vestibular nuclei neurons in the alert monkey after bilateral vestibular neurectomy. After destruction of the peripheral vestibular system which is not activated by moving large-field visual stimulation, not only labyrinthine-ocular reflexes but also optokinetic-ocular responses related to the "velocity storage" mechanism are abolished. In the normal monkey optokinetic-ocular responses are reflected in sustained activity changes of central vestibular neurons within the vestibular nuclei. To account for the loss of optokinetic responses after labyrinthectomy, inactivation of central vestibular neurons consequent on the loss of primary vestibular activity is assumed to be of major importance. To test this hypothesis we recorded the neural activity within the vestibular nuclear complex in two chronically prepared Rhesus monkeys during a period from one up to 9 and 12 months after both vestibular nerves had been cut. The discharge characteristics of 829 cells were studied in relation to eye fixation, and to a moving small and large (optokinetic) visual stimulus producing smooth pursuit (SP) eye movements and optokinetic nystagmus (OKN). Units were grouped into different subclasses. After chronic bilateral vestibular neurectomy (BVN) we have found: (1) a rich variety of spontaneously active cells within the vestibular nuclear complex, which--as far as comparison before and after BVN is possible--belong to all subclasses of neurons functionally defined in normal monkey; and (2) no sustained activity changes which are related to the activation of the "velocity storage" mechanism; this is especially true for "pure-vestibular", "vestibular-pause" and "tonic-vestibular-pause" cells in normal monkey which show a "pure", "pause" and "tonic-pause" firing pattern after BVN. Neurons which are modulated by eye position are, however, modulated with the velocity of slow eye movements with comparable sensitivity during SP and OKN. Retinal slip is extremely rarely encoded. The results of the present study do not directly answer the question why the "velocity storage" mechanism is abolished after BVN but they suggest that only a small number of central vestibular cells may be inactivated by neurectomy.


Laminin in the human embryo implantation: analogy to the invasion by malignant cells. To explore the possible similarities between the biochemical processes of embryo implantation and malignant invasion. The expression of a basement membrane (BM) glycoprotein laminin, a matrix binding cell surface receptor protein beta 1-integrin, and a BM collagen degrading metalloproteinase type IV collagenase, was studied in cultured human in vitro fertilized embryos. Eight healthy women suffering from tubal infertility were participating in the IVF program in the Department of Obstetrics and Gynecology in University Hospital of Oulu. Twenty oocytes and 110 pre-embryos that were not transferred for the fertilizations were used in this study. Fibronectin, laminin, beta 1-integrin, and type IV collagenase immunoreactive proteins were studied in embryos by immunoperoxidase staining, and type IV collagen degrading activity was measured from the culture media of the embryos. Laminin and beta 1-integrin were expressed in the early human embryos before the time of implantation. Type IV collagen degrading activity and the 72 kd-type IV collagenase immunoreactive protein were expressed at the time of implantation. Laminin supported the expression of type IV collagenase. The expression of laminin, beta 1-integrin, and type IV collagenase in vitro are temporally in good correlation with the time of the implantation in vivo. Laminin and beta 1-integrin can relate to the attachment of the embryos to the uterine BM and type IV collagenase to the degradation of the BM collagen during the implantation. Laminin can augment the process locally.


Somatotopic termination of the spino-olivary fibers in the cat, studied with the wheat germ agglutinin-horseradish peroxidase technique. Terminal sites of the spino-olivary fibers (SOFs) were examined by the anterograde transport of wheat germ agglutinin-horseradish peroxidase in the cat. The tracer was injected at various spinal cord levels from the first cervical to the caudal segments. The SOFs derived from the C1-T1 segments terminated medially in the caudal half (levels II-VIII of Brodal) of the medial accessory olive (MAO), which projects to the A zone of the cerebellar cortex, whereas the SOFs derived from the L6-S1 segments terminated laterally in the caudal half (levels I-VIII) of the MAO. No projections were found from the T2-L5 segments to the MAO. In the dorsal accessory olive (DAO), the SOFs terminated at levels III-XIV; the DAO projects to the B zone and the C1 and C3 zones of the cerebellar cortex. The SOFs derived from the C1-C4 segments terminated in the most medial part of the DAO (levels III-XIV), followed laterally by those from the C5-T1 segments. Further laterally, the SOFs derived from the T2-L5 and the L6-S1 segments terminated in the mediolateral order at levels V-XIV. The SOFs from the L6-S1 segments occupied the most lateral part of the DAO. The present study demonstrates that there is a distinct somatotopic termination of the SOFs in the mediolateral order in the caudal MAO and the DAO.


Expression of loricrin is negatively controlled by retinoic acid in human epidermis reconstructed in vitro. In epidermis, the last steps of keratinocyte differentiation are characterized by the covalent cross-linking of cornified envelope precursors such as involucrin and loricrin, a hydrophobic protein recently described in mouse and human epidermis. In situ hybridization of normal human skin sections with a human loricrin cRNA probe and immunolabeling with an antiserum directed against a synthetic peptide corresponding to the carboxyterminus of human loricrin revealed the presence of loricrin transcripts and protein in the granular layers of epidermis. In human epidermis reconstructed in vitro by growing keratinocytes on dermal equivalents, loricrin and loricrin mRNAs were also restricted to granular cells, but their amounts seemed higher than in epidermis from skin biopsies. The reactivities for both loricrin and loricrin mRNAs were abolished by a treatment of the cultures with a retinoic acid concentration (10(-6) M) provoking a complete inhibition of terminal epidermal differentiation (parakeratosis). Thus, the regulation of loricrin synthesis is different from that of another envelope precursor, involucrin, which does not seem to be significantly modulated by retinoic acid. Together with the well-documented inhibition of epidermal transglutaminase by retinoic acid, our results provide a molecular basis for the inhibition of cornified envelope formation by retinoic acid.


Effects of vitamin A on collagen metabolism by cultured rat liver cells. Conflicting results have been reported concerning the phenotypes of collagen produced by cultured Ito cells. These variations may be attributed to differences in pretreatment, i.e., with or without vitamin A to facilitate the separation of Ito cells. In the present study, the effects of vitamin A on collagen metabolism by Ito cells and hepatocytes of rats were analyzed. In cultured Ito cells, staining reactions to type I collagen increased, and those to type IV collagen and laminin decreased after pretreatment with vitamin A. The rate of collagen synthesis by Ito cells decreases significantly by treatment with vitamin A. The decrease was clearer in degraded collagen than in intact collagen. The synthesis of type I collagen increased and that of type IV collagen significantly decreased in Ito cells by treatment with vitamin A. In the hepatocytes, the staining reaction to type I collagen increased with vitamin A pretreatment. The net collagen and type III collagen synthesis in hepatocytes decreased by treatment with vitamin A. These results indicate that vitamin A modifies collagen metabolism in different cell types in different ways.


The sequential change of serum 2',5' oligoadenylate synthetase in different infectious patterns of duck hepatitis B virus in ducks in experimental transmission. Duck hepatitis B virus (DHBV) shows clear age-dependent infectious patterns like that of Hepatitis B virus, and many factors have been assumed to have a role in the persistence of the infection. In the present study, the activities of the interferon-induced enzyme 2',5' oligoadenylate synthetase (2,5AS) were observed sequentially in the serum of ducks experimentally infected with DHBV on posthatch days 1, 7 and 14. These were compared with the infectious pattern to investigate whether the endogenous interferon response after infection in ducks of different ages has a major role in its determination. The infectious pattern of DHBV in 1-day-old ducks was persistent without hepatitis and the others were transient with hepatitis. Persistently infected ducks showed significantly lower activities of 2,5AS compared with those with transient hepatitis, which resulted in a rapid elimination of DHBV. Although 1-day-old ducks showed significantly high 2,5AS compared with non-infected ducks, interferon response alone appeared to be insufficient for the elimination of DHBV. The immune response seemed necessary for the complete elimination of DHBV by way of evoking hepatitis and stimulating more interferon response during the usual infectious course. The interferon system alone did not seem to have a critical role in determining the infectious pattern. Other factors, including the immune response to the virus, seemed to have a major role in this problem.


Circulating tumor-associated antigens detected by monoclonal antibodies against the polypeptide core of mucin--comparison of antigen MUSE11 with CA15-3. The antigen MUSE11 detected by a monoclonal antibody (MAb) is an adenocarcinoma-associated antigen, while CA15-3 is a representative breast cancer-associated antigen detected by MAbs 115D8 and DF3. MAb MUSE11 showed higher binding activity to a synthetic peptide corresponding to the tandem repeat motif of the mucin core protein than that of MAb DF3, although MAb DF3 also had a significant binding activity indicating that MAbs MUSE11 and DF3 could recognize an identical polypeptide core. The reactivity of MAb DF3 to a breast cancer cell line MRK-neu-1 was completely abolished by neuraminidase treatment whereas that of MAb MUSE11 was partly conserved. The simultaneous measurement of the antigens MUSE11 and CA15-3 in sera from 35 cancer patients demonstrated that the incidence of abnormal serum level of CA15-3 was lower than that of antigen MUSE11. These data suggest that at least a part of the structural basis for the difference between the serum levels of antigen MUSE11 and CA15-3 could be carbohydrate side chains including sialic acids.


Involvement of cyclic adenosine 3',5'-monophosphate in methylation during 1-methyladenine production by starfish ovarian follicle cells. Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). With respect to 1-MeAde production, the effect of GSS on follicle cells results in the receptor-mediated formation of cyclic AMP (cAMP). It has also been reported that methylation is involved in 1-MeAde production by GSS. This study was undertaken to determine whether cAMP is the agent responsible for mediating methylation in 1-MeAde biosynthesis by isolated follicle cells of the starfish Asterina pectinifera. Methionine and selenomethionine enhanced 1-MeAde production by GSS in follicle cells. These stimulatory effects were dependent on the GSS concentration. Production of 1-MeAde by GSS was inhibited by ethionine and selenoethionine, competitive inhibitors of methionine. Like GSS, 1-MeAde production induced by concanavalin A, trypsin, and 3-isobutyl-1-methylxanthine (IBMX), which stimulated cAMP accumulation in follicle cells, was influenced by methionine and its related compounds. In contrast, although 1-methyladenosine (1-MeAde-R) induced 1-MeAde production by follicle cells without increasing cAMP levels, methionine and its related compounds had no effect. Use of [methyl-14C]methionine showed that a radiolabel was incorporated into 1-MeAde during incubation with GSS and IBMX, but not with 1-MeAde-R. These results strongly suggest that cAMP plays an important role in the process of methylation during 1-MeAde biosynthesis induced by GSS.


Neurotransmitters and stimulation of fluid reabsorption in migratory locust rectal cells. Several biogenic amines enhance fluid reabsorption and the accumulation of cyclic adenosine-monophosphate (cAMP) in the rectum of the migratory locust but only 5-hydroxytryptamine (5-HT) acts in a dose-dependent manner at low concentrations (between 10(-8) and 5.10(-7) M). Cyclic AMP is a second messenger of 5-HT, and its actions on fluid reabsorption are calcium-dependent. Polymyxin B (a protein kinase C inhibitor) mimics the actions of 5-HT on fluid reabsorption and on calcium-dependent cAMP accumulation. This suggests the presence of other sources of calcium and a possible relationship between several transduction systems within different rectal cells. The second messenger system mediating the 5-HT antidiuretic message differs from those involved in the transduction of the known locust antidiuretic hormones.


Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas. Phenotypic and genetic studies were performed on some atypical aeromonas strains of uncertain taxonomic position. 16S rRNA gene sequence analysis revealed that these strains represent a hitherto unknown genetic line within the genus Aeromonas, for which the name Aeromonas allosaccharophila sp. nov. is proposed. The type strain is CECT 4199.


A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization, cloning and targeted mutagenesis. DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J. Bact. 159, 36-41, and 1984b, Gene 31, 109-116).


Nuclear and mitochondrial ribosomal RNA variability in the obscura group of Drosophila. Parts of 28S (nuclear) and 12S (mitochondrial) ribosomal RNA of Palearctic, Nearctic and African species of the obscura group have been sequenced by the direct method of sequencing. Rates of nucleotide substitutions in both molecules were compared. The nucleotide divergence is higher in the mitochondrial rRNA. Average distances of species taken in pairwise were compared to results obtained with the melanogaster subgroup: the divergence of nuclear rRNA appears lower, that of the mtDNA higher whereas genetic distances (allozymes) and sncDNA distances are similar. Noticeable variability of evolutionary rates can be observed even in low taxonomical levels. Phylogenetic trees for the obscura group are in general agreement with those obtained with other characters.


Transplantation of organs between species. The shortage of organs from human donors for transplantation has stimulated a renewed interest in the use of organs from animals or xenografting. Such grafts can be transplanted between closely related species--concordant, or distantly related species--discordant. In this mini review the immune processes which cause rejection for each of these categories is described. Data demonstrating methods by which this rejection can be controlled are summarised and a possible approach to clinical application is described.


Interaction of lectins with human IgE: IgE-binding property and histamine-releasing activity of twelve plant lectins. We examined the IgE-binding reaction and the histamine-releasing response of basophils to a panel of 12 lectins: concanavalin A (Con A), Lens culinaris hemagglutinin (LcH), Pisum sativum agglutinin (PSA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), Bauhinia purpurea agglutinin (BPA), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA-I), Lotus tetragonolobus agglutinin (Lotus A), Ulex europeus agglutinin I (UEA-I), phytohemagglutinin E (PHA-E) and phytohemagglutinin L (PHA-L), IgE from allergic patients bound with high affinity to Con A, LcH, PSA, RCA-I and PHA-E, and with lower affinity to WGA, BPA, Lotus A and UEA-I, but they did not bind to SBA, PNA or PHA-L. There was no apparent individual difference in the reactivity of IgE to these lectins between 10 IgE preparations from allergic patients. The binding to these lectins, except Lotus A and UEA-I, were competitively inhibited by the lectin-specific sugars or glycopeptide. Upon stimulation by Con A, LcH, PSA, WGA, RCA-1 and PHA-E, leukocytes from allergic patients showed a significant release of histamine, but cells from IgE-deficient subjects did not respond to these lectins. The histamine-releasing responses by these lectins were also inhibited by specific sugars or glycopeptides.


Mast cell heterogeneity: evidence that mast cells isolated from various connective tissue locations in the rat display markedly graded phenotypes. The present study has extended histochemical and functional investigations into rat mast cell heterogeneity using isolated mast cells from four connective tissue locations; the peritoneum, mesentery, lung and skin. On histological examination, mast cells from these locations displayed a range of phenotypes following formalin fixation and staining with Alcian blue/safranin O, suggesting the existence of both chondroitin sulphate and heparin proteoglycans in varying proportions in these cell types. Functional studies using the structurally diverse polycationic secretagogues, compound 48/80, the polyamino acids, polymyxin B, substance P, ACTH1-24, mastoparan, protamine sulphate, histone, d-tubocurarine and ranitidine confirmed the existence of such phenotypic gradation. This investigation highlights the inappropriate usage of the terms CTMC and MMC which represent two phenotypic extremes between which a gradation of phenotypes clearly exists.


Effects of adenosine on histamine release from human lung fragments. The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A23187 or with concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyloxy]-phenyl]-1, 3-dipropylxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence of XAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a site antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.


Effect of NZ-107, a newly synthesized pyridazinone derivative, on antigen-induced contraction of human bronchial strips and histamine release from human lung fragments or leukocytes. The effects of a newly synthesized pyridazinone derivative, NZ-107, 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone, and two well-known antiasthmatic drugs, amlexanox (orally active disodium cromoglycate-like drug) and disodium cromoglycate (DSCG) on antigen-, histamine- and leukotriene C4 (LTC4)-induced constriction of isolated human tracheal muscle, and histamine release from human lung tissues and leukocytes were investigated in vitro. In some experiments, salbutamol was used as a reference drug. NZ-107 inhibited antigen-, histamine- and LTC4-induced contraction of tracheal muscle. Amlexanox and DSCG did not affect the contractile response of tracheal muscle caused by each stimulant. Salbutamol inhibited antigen-induced contraction of tracheal muscle. NZ-107, amlexanox, DSCG and salbutamol clearly inhibited the antigen-induced release of histamine and LTC4 from human lung tissue. The antigen-induced histamine release from atopic human leukocytes was inhibited by NZ-107 and amlexanox, but not by DSCG. Pretreatment with IL-3 did not alter antigen-induced contraction of tracheal muscle and histamine release from lung tissue, but antigen- or calcium ionophore A 23187-induced histamine release from leukocytes was clearly enhanced. Amlexanox inhibited the IL-3-induced enhancement of histamine release from leukocytes in the case of both stimuli, but NZ-107 and DSCG had no effect. These data suggest that NZ-107 has potent anti-allergic actions based on the inhibition of antigen-induced contraction of human tracheal muscle and mediator release from human lung tissue and leukocytes.


Responses of isolated Japanese monkey tracheal muscle to allergic mediators. The responsiveness of isolated Japanese monkey (Macaca fuscata) tracheal muscle to antigen, carbachol, histamine, leukotriene C4 (LTC4), U-46619 and substance P (SP) was compared to that of isolated human trachea. Weak but persistent contraction was observed after the addition of antigen to isolated Japanese monkey tracheal muscle passively sensitized with monkey serum containing IgE antibody against Japanese cedar (Cryptomeria japonica) antigen. Unlike monkey tracheal muscle, a fair amount of contraction was caused by the antigen in human tracheal muscle passively sensitized with human atopic serum. When chopped, passively sensitized monkey or human lung tissue was challenged with antigen, a significant level of histamine was released from these tissues. In Japanese monkey tracheal muscle, histamine and SP produced no contraction of tracheal muscle, whereas carbachol, LTC4 and U-46619 caused contraction in a dose-dependent fashion. Contrary to the Japanese monkey, histamine and carbachol caused distinct contraction in tracheal muscle obtained from the cotton-headed tamarin (Saguinus oedipus). In human tracheal muscle, all test substances (carbachol, histamine, LTC4, U-46619 and SP) induced clear contraction. In lung parenchyma obtained from Japanese monkey, histamine induced a weak contraction, and this histamine-induced contraction was also inhibited by pyrilamine (H1 receptor antagonist). These results indicate that antigen-induced contraction of isolated Japanese monkey tracheal muscle, passively sensitized with monkey atopic serum, is not a useful model for human allergic bronchoconstriction in vitro because of the unresponsiveness of tracheal muscle to histamine and SP.


Ru 41.740 triggers human mononuclear blood cells to release tumor growth inhibitory factors in vitro. Ru 41.740 (Biostim) is an immunostimulating drug of microbial origin which may stimulate human mononuclear blood cells (mainly monocytes) to release soluble factors which inhibit replication of several tumor cell lines in vitro. Since this effect may be of clinical importance in the treatment of cancer a number of tests have been conducted in order to find methods to augment this secretion. In vitro tests suggested that this non-specific antitumor activity of Biostim may not be enhanced by concomitant treatment of patients with inhibitors of cyclo-oxygenase and lipoxygenases or by interferons alpha, beta, gamma or the hemopoietic growth factors GM-CSF and G-CSF.


LY 186655, a phosphodiesterase inhibitor, inhibits histamine release from human basophils, lung and skin fragments. LY 186655 (Tibenelast, Lilly) is a new phosphodiesterase inhibitor, not derived from the xanthine, possessing bronchodilating activity in animals. The aim of this work was to study the effect of LY 186655 and theophylline on histamine release from human leukocytes, skin and lung fragments. Histamine was measured using a spectrofluorometric method. Both drugs (3 x 10(-5)-3 x 10(-3) M) exhibited a dose-dependent inhibition on anti-IgE (1/2000)-induced histamine release from human leukocytes. At 3 x 10(-3) M, theophylline was significantly more effective than LY 186655 (mean inhibition 94 and 42%, respectively). On lung fragments, theophylline and LY 186655 (3 x 10(-5)-3 x 10(-3) M) caused strong and comparable inhibitory effects on anti-IgE (1/500)-induced histamine release with a mean inhibition reaching maximally 65%. Histamine release induced by compound 48/80 (1 mg/ml) on sliced human foreskin was reduced with both drugs (3 x 10(-3) M) by about 37%. We conclude that LY 186655 inhibits in vitro immunological histamine release from human lung and cutaneous mast cells as well as basophils with a similar pattern of activity to theophylline.


Transurethral resection of 1250 bladder tumours. A total of 1250 bladder tumours were subjected to transurethral resection (891 curative, 107 palliative operations, 252 TUR biopsies). Complication rate was 9.9%, mortality rate 0.8%. In patients with primary tumours the 1-year recurrence rate after TUR was 23.8%, the 3-year rate was 36.6%, with an increase in proportion to stage. The 5-year survival rate was 66.5%. Within five years 9% of the patients died from tumour generalization, also with a rising tendency in proportion to stage. TUR as a curative method is suitable mainly for the removal of Ta and T1, under circumstances also of T2 G1-G2 tumours.


Development of urethral stricture after transurethral prostatectomy: a retrospective study. A total of 103 patients who were diagnosed to have benign prostatic hyperplasia (BPH) without preoperative urethral stricture and underwent transurethral prostatectomy (TURP) were evaluated retrospectively from patient charts. The incidence of urethral stricture development was calculated as 11.65% (12 out of 103 patients). Among the aetiologic factors analyzed, the most important ones appeared to be postoperative infection, age of the patient, duration of postoperative catheterization and histology of the disease, in the order of significance in the development of urethral stricture after TURP.


Language intervention and disruptive behavior in preschool children with autism. Disruptive behaviors are often exhibited by children with severe disabilities during difficult teaching tasks. Because learning verbal communication can be a difficult task for nonverbal children with autism, disruptive behaviors are common during such interventions. The purpose of this experiment was to assess whether the incorporation of parameters of natural language interactions and motivational techniques might reduce disruptive behavior during language teaching tasks. Within a repeated reversals design with order of conditions and number of sessions varied within and across children, treatment was conducted for two language teaching conditions. During one condition trials were presented serially in a traditional analog clinical format where the therapist presented instructions, prompts, and reinforcers for correct responses. The other condition incorporated parameters of natural language interactions and motivational techniques, such that stimulus items were functional and varied; natural reinforcers were employed; communicative attempts were reinforced; and trials were conducted within a natural interchange. Results showed that greater improvements in responding and considerably less (often negligible) disruptive behavior occurred during the natural language teaching conditions. Results are discussed with respect to their implications for improving language interventions, and with respect to reducing disruptive behavior without the need for specialized or severe interventions focused specifically on the disruptive behavior.


A comparative study of development and symptoms among disintegrative psychosis and infantile autism with and without speech loss. To investigate clinical pictures and the validity of disintegrative psychosis (DP) as defined in ICD-9, 18 cases of DP were compared with 51 and 145 cases of infantile autism (IA) with and without speech loss, respectively, on clinical variables. The DP cases showed clearer regression after more satisfactory development than the IA cases with speech loss. Around age 7, about 4 years after regression, those with DP were significantly more severely retarded than those with IA, yet both were similar in autistic symptomatology. EEG abnormalities and mothers 30 or older at delivery were significantly more common in the histories of those with DP than of those with IA. DP may be linked with IA having speech loss with regression in mental development as a common denominator.


Optimal administration schedules of the newly synthesized platinum analog NK121 and bleomycin analog NK313 in nude mice with squamous cell carcinoma. To determine the optimal administration schedules of the newly synthesized, less nephrotoxic platinum analog NK121 and less pneumotoxic bleomycin analog NK313, the antitumor effects of 2 different injection schedules, (1) single injections on days 1, 5, and 9, (2) continuous infusion for 7 days, were compared in nude mice bearing human squamous cell carcinoma. Tumor growth delay was employed as the experimental endpoint of antitumor activity. Body weight and hematologic changes in the mice were also investigated as indications of drug toxicity after the combination treatment of NK121 and NK313. The antitumor effects of the 2 analogs indicated higher responses with the single injections of NK121 and continuous infusion of NK313. In combination chemotherapy with NK121 and NK313, the highest antitumor effect was observed when mice were given NK121 by single injections followed by NK313 by continuous infusion. This sequence was also less toxic than a simultaneous treatment schedule, which was of weak clinical significance because of its low antitumor activity, severe weight loss, slight myelosuppression, and renal toxicity.


Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system. The Bacillus subtilis glucose starvation-inducible transcription units, gsiA and gsiB, were characterized by DNA sequencing, transcriptional mapping, mutational analysis, and expression in response to changes in environmental conditions. The gsiA operon was shown to consist of two genes, gsiAA and gsiAB, predicted to encode 44.9- and 4.8-kDa polypeptides, respectively. The gsiB locus contains a single cistron which encodes a protein of unusual structure; most of its amino acids are arranged in five highly conserved, tandemly repeated units of 20 amino acids. The 5' ends of gsiA and gsiB mRNAs were located by primer extension analysis; their locations suggest that both are transcribed by RNA polymerase containing sigma A. Expression of both gsiA and gsiB was induced by starvation for glucose or phosphate or by addition of decoyinine, but only gsiA was induced by exhaustion of nutrient broth or by amino acid starvation. Regulation of gsiA expression was shown to be dependent upon the two-component signal transduction system ComP-ComA, which also controls expression of genetic competence genes. Mutations in mecA bypassed the dependency of gsiA expression on ComA. Disruption of gsiA relieved glucose repression of sporulation but did not otherwise interfere with sporulation, development of competence, motility, or glucose starvation survival. We propose that gsiA and gsiB are members of an adaptive pathway of genes whose products are involved in responses to nutrient deprivation other than sporulation.


Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16. Two hundred thirty-nine base pairs upstream from acoXABC, which encodes the Alcaligenes eutrophus H16 structural genes essential for cleavage of acetoin, the 2,004-bp acoR gene was identified. acoR encodes a protein of 668 amino acids with a molecular mass of 72.9 kDa. The amino acid sequence deduced from acoR exhibited homologies to the primary structures of transcriptional activators such as NifA of Azotobacter vinelandii, NtrC of Klebsiella pneumoniae, and HoxA of A. eutrophus. Striking similarities to the central domain of these proteins and the presence of a typical nucleotide-binding site (GETGSGK) as well as of a C-terminal helix-turn-helix motif as a DNA-binding site were revealed. Between acoR and acoXABC, two different types of sequences with dual rotational symmetry [CAC-(N11 to N18)-GTG and TGT-(N10 to N14)-ACA] were found; these sequences are similar to NtrC and NifA upstream activator sequences, respectively. Determination of the N-terminal amino acid sequence of an acoR'-'lacZ gene fusion identified the translational start of acoR. S1 nuclease protection assay identified the transcriptional start site 109 bp upstream of acoR. The promoter region (TTGCGC-N18-TACATT) resembled the sigma 70 consensus sequence of Escherichia coli. Analysis of an acoR'-'lacZ fusion and primer extension studies revealed that acoR was expressed at a low level under all culture conditions, whereas acoXABC was expressed only in acetoin-grown cells. The insertions of Tn5 in six transposon-induced acetoin-negative mutants of A. eutrophus were mapped within acoR. On the basis of these studies, it is probable that AcoR represents a regulatory protein which is required for sigma 54-dependent transcription of acoXABC.


Regulation of plasmid virulence gene expression in Salmonella dublin involves an unusual operon structure. The 80-kb plasmid pSDL2 of Salmonella dublin Lane is essential for lethal systemic infection in experimental mice. A cluster of five plasmid genes, designated spvR, spvA, spvB, spvC, and spvD, is sufficient to express the plasmid-related virulent phenotype. The spvR gene product has recently been identified as a positive regulator of spvB expression in the stationary phase of bacterial growth (F. C. Fang, M. Krause, C. Roudier, J. Fierer, and D. G. Guiney, J. Bacteriol. 173:6783-6789, 1991). In this study, we evaluated the role of SpvR in the transcription of the downstream virulence genes spvABCD. Analysis of mRNA synthesis revealed that SpvR promotes transcription of the downstream spvABCD genes in the stationary growth phase. Transcript mapping of the spv region demonstrated an unusual operon structure involving messages for spvA, spvAB, spvABC, and spvABCD. Quantitative measurement of transcription and of gene expression by use of translational spv-lacZ fusions suggested that SpvA, SpvB, SpvC, and SpvD are produced in decreasing abundance. Primer extension assays identified two transcriptional start sites 70 and 98 bp upstream of the start codon of spvA, but none upstream of spvB, spvC, or spvD. Deletion of a 320-bp EcoRI-ApaI segment that contains both start sites abolished expression of the downstream spvB and spvC genes. Our results establish a central function of SpvR as a positive regulator of the downstream spvABCD genes in the stationary phase of bacterial growth and indicate that the primary mechanism of regulation is by activation of promoters upstream of spvA.


TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization. We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene. Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene. We describe three kinds of elements for making each fusion type. Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance. The third kind is transposition defective (Tnp-) and encodes kanamycin resistance. In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions. Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances. Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype. Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments. Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription. This feature is especially useful in the determination of operon structures. Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described.


Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide. Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.


The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. Subcutaneous implants of a recombinant human form of the bone-inducing protein rhBMP-2 (recombinant human bone morphogenetic protein-2) in rats have resulted in the local induction of endochondral bone formation. To test the osteoinductive activity of rhBMP-2 in an osseous location, we created five-millimeter segmental defects in the femora of forty-five adult male Sprague-Dawley rats. Two doses of lyophilized rhBMP-2 (1.4 or 11.0 micrograms) were implanted in each defect, together with guanidine-hydrochloride extracted demineralized rat-bone matrix as a carrier, and the results were compared with those in rats that had implantation of guanidine-hydrochloride extracted demineralized rat-bone matrix only. The formation and healing of bone were determined by radiographic, histological, and mechanical analysis. Both doses of rhBMP-2 induced formation of endochondral bone in the osseous defects in a dose-related manner. Implantation of 11.0 micrograms of rhBMP-2 yielded significant (p less than 0.05) bone formation, resulting in radiographic, histological, and mechanical evidence of union. Despite new-bone formation in the defects that had received 1.4 micrograms of rhBMP-2, no instances of union were observed.


Progesterone receptor determined by immunocytochemical and biochemical methods in human breast cancer. Immunocytochemical assay (ICA) of the progesterone receptor (PgR) was performed on 152 patients with stage I-II breast cancer. We employed the rat monoclonal antibody KD-68 and a peroxidase/antiperoxidase displaying system. The results obtained by ICA (Pg(RICA)) were compared with those by the biochemical dextran-coated charcoal assay (PgRDCC). Comparing the two methods we found an overall agreement (accuracy) of 77.5%, a PgR(ICA) sensitivity of 83.5% and a specificity of 73%. Both methods were significantly associated with oestrogen receptor expression, detected by DCC (P less than 0.001 for PgRDCC and P = 0.0014 for PgR(ICA)). No significant association was found between PgR(ICA) or PgRDCC and the other clinicopathological features analysed. After a median follow-up of 36 months, the overall survival probability was 91% in PgRDCC-positive versus 81.5% in PgRDCC-negative patients (log-rank test, chi 2 = 0.91) compared to 87.5% in PgR(ICA)-positive versus 82% in PgR(ICA)-negative ones (log-rank test, chi 2 = 0.93). Disease-free survival probability was 74.5% in both PgRDCC-positive and PgRDCC-negative patients (log-rank test, chi 2 = 0.02) compared to 78% in PgR(ICA)-positive versus 71.5% in PgR(ICA)-negative cases (log-rank test, chi 2 = 0.37). The present study demonstrates that ICA is a reliable method to detect PgR, correlating well with the DCC assay. Moreover, the ICA assay seems to provide clinical information complementary to the biochemical method. The definition of its prognostic value in operable breast cancer needs additional studies, particularly in node-negative patients.


Length polymorphisms in tRNA intergenic spacers detected by using the polymerase chain reaction can distinguish streptococcal strains and species. Intergenic tRNA spacers from strains of streptococcal groups A, B, and G were amplified by using the polymerase chain reaction (PCR) at low stringency with consensus tRNA gene primers. Cloning and sequencing showed that many of the homologous intergenic spacers differed in length between species. The sequences of the tRNA genes that flank these polymorphic spacers were determined and used to synthesize fully complementary primers. With these primers at high stringency, PCR products which varied in lengths from 53 to 71 bp, depending on the species or strain, were obtained from streptococcal DNAs, even in the presence of a 1,000-fold mass excess of human DNA. PCR products, the lengths of which could also be used for classification, were obtained at high stringency from a few genera closely related to Streptococcus. No products were obtained from genomic DNAs from more distantly related genera. Production of species- or strain-specific tRNA intergenic length polymorphisms with primers that generate characteristic products from a variety of species within the same genus should be applicable to many organisms, including those that would otherwise be difficult to culture or identify.


Detection of Mycoplasma hyopneumoniae by using rRNA-oligodeoxynucleotide hybridization. A system that uses rRNA-oligodeoxynucleotide hybridization was developed for the detection of Mycoplasma hyopneumoniae. Synthetic oligonucleotide MHP1 was hybridized specifically with M. hyopneumoniae. Furthermore, the detection of M. hyopneumoniae in clinical samples, such as bronchoalveolar lavage fluid and lung lesions from experimentally infected pigs, was evaluated by this assay. The evidence obtained from the assay indicated that the system can be used to efficiently diagnose mycoplasmal pneumonia of swine. Additionally, a nonradioisotopic system with chemiluminescence detection was tested. This system was 10-fold less sensitive than a test that used radioisotopes.


Comparison of DNA fingerprints and somatic serotypes of serogroup B and E Pasteurella multocida isolates. The DNA fingerprint profiles and somatic serotypes of 71 Pasteurella multocida capsule serogroup B isolates, 13 capsule serogroup E isolates, and 16 somatic reference serotype strains were compared. Each of the 16 reference somatic serotypes had a unique DNA fingerprint profile with the HhaI restriction endonuclease. Fifty-four serogroup B isolates (isolated from classical cases of hemorrhagic septicemia) reacted with somatic serotype 2 or 5 antiserum and had DNA fingerprint profiles which resembled that of the serotype 2 reference strain. Seven DNA fingerprint profiles were found among 16 serogroup B strains representing other somatic serotypes. The DNA fingerprints of these isolates were different from the fingerprints of the 16 somatic reference serotype strains. All 13 serogroup E isolates had identical somatic serotypes and identical DNA fingerprint profiles when the HhaI endonuclease was used. The HhaI fingerprint profile of the serogroup E isolates did not match any fingerprint profile of the reference somatic serotype strains. Following DNA profiling with the HhaI endonuclease, the 13 serogroup E isolates were differentiated sequentially with HpaII restriction endonuclease. A descriptive identification epithet for P. multocida isolates was constructed. The descriptive epithet consists of serologic identification and sequential DNA profiles with restriction endonucleases HhaI and HpaII, respectively. DNA fingerprinting of P. multocida is a precise characterization method. In conjunction with serologic typing, it can further classify P. multocida isolates for epidemiologic studies.


Mapping the major antigenic domains of the native flagellar antigen of Borrelia burgdorferi. Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a monoclonal antibody raised by immunization of mice with purified flagella and pooled sera from patients with multiple erythema migrans, late Lyme borreliosis, or secondary syphilis were analyzed by Western blots (immunoblots) of peptides transferred to Immobilon polyvinylidene difluoride filters. The major epitope binding one murine monoclonal antibody (158) was localized to a carboxy-terminal domain that includes residues 300 to 336. The dominant epitopes binding human polyclonal antibodies are in the central portion of the molecule (residues 182 to 218) that is not conserved compared with other bacterial flagellins. Additional reactive epitopes were identified in the amino-terminal domain of the protein. Sera from patients with syphilis bound strongly to the amino-terminal conserved domain, providing a structural basis for cross-reactivity seen in standard enzyme-linked immunosorbent assays, but not to the central part of the molecule. Specific and cross-reactive antigenic determinants need to be considered in the design of improved immunodiagnostics for spirochetal diseases.


Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays. Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.


Ribotyping of Helicobacter pylori from clinical specimens. Ribotyping is a method used to type strains of bacteria by analyzing the restriction enzyme digestion patterns of the rRNA genes. This method was applied to 126 strains of Helicobacter pylori from 100 unrelated symptomatic patients who had endoscopies done and to 15 strains from 15 infected subjects from seven families. Analysis of the rRNA gene patterns revealed 77 distinct ribotypes from the 100 patients. From 15 of these subjects, isolates were recovered from antral mucosal biopsies at follow-up endoscopy. All follow-up isolates from the same patient, with one exception, yielded identical digest patterns. This patient had strains with two distinct digest patterns obtained from a set of three isolates cultured from biopsy specimens taken at different times. Five patients who had isolates recovered from different sites in the stomach (antrum, gastric body, duodenum, and pyloric channel) showed ribotyping patterns which were identical for each patient yet distinct between patients. In seven family groups studied, identical digest patterns were detected in members of two families, with variability in strains detected among members of the remaining families. This study demonstrates that ribotyping provides a useful, reliable, reproducible, and highly discriminatory typing scheme for the study of H. pylori infection.


Keratinization of palatal mucosa beneath metal-based removable partial dentures. Exfoliative cytology provides a non-invasive method of examining changes in epithelial differentiation beneath dentures. Cell sampling by mechanical scraping may introduce errors due to collection of deeper epithelial cells, therefore an alternative sampling method was evaluated. Cells collected from the hard palates of 24 dentate subjects using either a wooden tongue depressor or transparent, adhesive-coated tape were fixed and stained by Papanicolaou's technique. The percentage of the total epithelial cells counted that were anucleate (the orthokeratinization index) was 99.95 per cent using the tape technique but 15 per cent lower using the smear technique. It appears that the tape technique gives a more accurate representation of the orthokeratinization of superficial cell layers. The tape technique was used to examine epithelial changes beneath tooth-supported removable partial dentures (RPDs). In 26 subjects, who had worn cobalt chromium based RPDs for at least 6 months, samples taken from beneath the metal denture base had a 50 per cent lower orthokeratinization index than those from uncovered areas of the palate.


Wild mice express an Ig V lambda gene that differs from any V lambda in BALB/c but resembles a human V lambda subgroup. The lambda L chain locus in the inbred mouse strains commonly used in the laboratory contains a limited number of germ-line genes; only three V lambda and three functional J lambda-C lambda genes have been identified in BALB/c mice. Previous studies indicated that wild mice may have a considerably expanded number of C lambda genes, as judged by the number of DNA restriction fragments that hybridize to C lambda probes derived from BALB/c. In order to evaluate the expression of these putative lambda genes, we have determined sequences of cDNA encoding lambda-chains in hybridomas from wild mice of the subspecies Mus musculus musculus from two different geographic regions, Denmark and Czechoslovakia. Two of these hybridomas produce L chains with J and C regions that are very similar to those of BALB/c lambda 1 chains, but the V regions of these L chains are only approximately 40% identical in amino acid sequence to the known murine V lambda. Indeed, these wild mouse V lambda are closer in sequence to human V lambda than they are to BALB/c V lambda, especially to human V lambda of subgroup VI, with which they share an unusual two-residue insertion in framework 3; L chains bearing V regions of this rare human type have a marked tendency to enter into amyloid deposits. These findings suggest that similar V lambda may be widespread in mammalian populations, although analysis by Southern blotting indicates that they are not found in BALB/c mice. A third hybridoma produces a L chain whose V lambda resembles BALB/c V lambda 1. The J lambda and C lambda segments of the cDNA encoding all three hybridoma L chains are identical; evidently, of the several putative genes that hybridize to C lambda 1 probes, one is expressed preferentially.


Diversity in MHC class II ovalbumin T cell epitopes generated by distinct proteases. It is generally accepted that a limited number of T cell epitopes are generated by APC from an immunogenic protein. To ascertain the number of determinants on OVA recognized in the context of the H-2s haplotype, we generated 19 T-T hybridomas against OVA and H-2s and we synthesized 46 overlapping peptides spanning the entire protein. Eighteen T-T hybrids were stimulated by eight different peptides. The peptide recognized by one T cell hybrid was not identified. The effect of four protease inhibitors on the processing and presentation of OVA by the LS.102.9 B cell hybridoma seemed to implicate several groups of proteases in the processing of this Ag. Alkylation of cysteine residues with iodoacetic acid showed in a few cases a dramatic decrease in the capacity of OVA to stimulate T-T hybrids recognizing cysteine-free peptides. In contrast, two T-T hybrids recognizing cysteine containing peptides were not affected by the alkylation, suggesting that alkylation inhibited the processing of OVA without affecting peptide interaction with class II MHC molecules. These data demonstrate that the repertoire of peptides generated by APC from OVA is not limited to one or few immunodominant peptides, and results from the activity of several endopeptidases and/or exopeptidases. In addition, the structure of the Ag (native or denatured) was shown to affect the efficiency with which different epitopes are generated.


Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition. The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.


Enhancing the immunogenicity of a permissive binding T cell epitope derived from the simian immunodeficiency virus-encoded negative regulatory factor. Using the murine system we have analyzed an immunogenic T cell peptide epitope corresponding to amino acids 96-112 of the simian immunodeficiency virus-negative regulatory protein sequence. This epitope was unusual in that it was strongly immunogenic in mice of five of the six H-2 haplotypes tested. We generated a T cell hybridoma (SVNF) specific for this peptide in order to determine how manipulating the peptide might alter its immunogenicity. Substitution analysis showed that His 103, Pro 104, Val 106, and Pro 107 were important amino acids for stimulating SVNF because substitutions at these positions diminished the reactivity of SVNF. However, we also found that substituting an Ala for a Val at position 100 or a Val for an Ala at position 110 enhanced reactivity of SVNF. We were able to further enhance the immunogenicity of this epitope by extending the carboxyl terminus two amino acids and making the resulting carboxyl-terminal Lys an amide and by adding a Glu to the amino terminus. These modifications shifted the in vitro activity of SVNF at least two orders of magnitude. We also compared the ability of this modified peptide and the wild-type SIV nef 96-112 to prime a T cell response in vivo. We primed mice with various doses of either the wild-type or the modified peptide and looked at the ability of the draining lymph node cells to proliferate to wild-type peptide. We found that the modified peptide was 10- to 100-fold better at priming a T cell response than the wild-type peptide. Therefore, we were able to create a peptide that was more immunogenic than the wild-type peptide in vivo as well as in vitro. Manipulations such as these that enhance the immunogenicity of T cell epitopes must be considered in developing peptide vaccines against HIV or other infectious agents.


The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells. The resistance of mice to lethal infection by murine CMV (MCMV) is under complex host genetic control with contributions from both H-2 and non-H-2 genes. We have previously shown that an autosomal, non-MHC encoded gene, Cmv-1, controls MCMV replication in the spleen. We have investigated the mechanism by which the Cmv-1 resistance gene confers protection against MCMV infection. Using H-2 compatible irradiation bone marrow chimeras, the enhanced resistance to MCMV infection that is associated with the Cmv-1l allele in the C57BL background was shown to be mediated by an irradiation-sensitive bone marrow-derived cell population, or a factor produced by these cells. The lack of correlation between serum IFN titers and the strain distribution pattern of Cmv-1 in CXB recombinant inbred mouse strains suggests that IFN does not mediate resistance conferred by this gene. Similarly, the lack of effect of in vivo depletion of mature CD4+ and CD8+ T cells on virus replication in C57BL/6J mice indicates that T cells are unlikely to be involved. In contrast, in vivo depletion of NK cells by injection of the anti-NK1.1 mAb PK136 abrogated restricted splenic virus replication in C57BL/6J----BALB.B chimeric mice and in the Cmv-1l CXB strains. These data indicate that the effect of the Cmv-1 gene is mediated by NK cells. The significant augmentation in NK cell activity after MCMV infection of the susceptible Cmv-1h strains (BALB/cBy), CXBG/By, CXBH/By, CXBI/By, and CXBK/By) indicates the existence in these mice of NK cells that are functionally and phenotypically distinct from those in Cmv-1l strains. NK cells present in the Cmv-1h strains are unable to restrict efficiently splenic MCMV replication in vivo, possibly due to a lack of specificity for virus-infected target cells. Finally, flow cytometric analysis of NK1-1 expression in CXB and BXD RI mice together with MCMV replication studies in the BXD RI strains indicate that Cmv-1 is closely linked to NK1.1 and other loci that reside on a distal segment of murine chromosome 6 in a region that has recently been defined as the natural killer complex.


Involvement of beta 2-integrins in the migration of human natural killer cells. Human large granular lymphocytes with the NK cell phenotype (CD16+ or CD56+CD3-) were greatly enriched among the cells which migrated spontaneously through untreated or albumin-coated, 3-microns pore size polycarbonate filters for 1 to 8 h. Three days of rIL-2 treatment (300 IU/ml) and 3 to 5 wk of rIL-2 treatment (100 IU/ml) generated a 2.7 +/- 0.9-fold and 5.6 +/- 0.8-fold increase in cell migration, respectively. The adhesion and subsequent migration of freshly isolated NK cells was mainly mediated by CD11b/CD18, because migration could be inhibited by 80 +/- 8% anti-CD11b (Mac-1) antibodies but not with antibodies against CD11a (LFA-1) or CD11c (p150,95), the other alpha-chains of the beta 2-integrins. After rIL-2 activation, however, CD11a/CD18 was the major receptor utilized in migration, inasmuch as anti-CD11a antibody caused a 69 +/- 8% reduction in the number of migrated cells. Anti-CD11b antibody decreased migration by 43 +/- 12%, and together these antibodies inhibited migration by 82 +/- 7%. Anti-CD11a alone did not have any effect on adhesion, but CD11a/CD18 cooperated in the adhesion because anti-CD11b decreased adhesion by 40 +/- 11% and together these antibodies inhibited adhesion by 74 +/- 6%. The ability of large granular lymphocytes to rapidly utilize beta 2-integrins and unidentified ubiquitous ligands for binding and migration may be significant for their capacity to function in the first line of immune defense under highly variable conditions.


The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils. The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)


Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion. Rat basophilic leukemia (RBL-2H3) cells are a useful in vitro model for studies of mast cells and basophils. We examined the adherence of RBL-2H3 cells to different extracellular matrix proteins and the effect of such attachment on secretion. The cells bound to fibronectin-coated surfaces with maximum binding by 1 h at 37 degrees C. There was less attachment to laminin, collagen type I, and collagen type IV. There was no adherence to uncoated surfaces or in the absence of Ca2+. Binding to fibronectin was blocked by a synthetic peptide containing the sequence Arg-Gly-Asp. Therefore, the binding of RBL-2H3 cells to fibronectin may be mediated by surface molecules that belong to the integrin family. Adherence to fibronectin-coated surfaces resulted in cell spreading, a reorganization of the cytoskeletal elements, and a redistribution of the secretory granules. Attachment to fibronectin also dramatically enhanced high affinity IgE receptor-mediated histamine release. This enhancement was maximum by 1 h of adherence and lasted for at least 6 h. There was also enhanced secretion by the Ca2+ ionophore A23187. Thus, adherence to fibronectin can enhance both receptor and non-receptor-mediated release. Addition of soluble fibronectin to RBL-2H3 cells in suspension had no effect on secretion. Therefore, enhanced histamine release required cell attachment to immobilized fibronectin. These results suggest that secretion from mast cells/basophils may be modulated by their interaction with the extracellular matrix.


RANTES, a monocyte and T lymphocyte chemotactic cytokine releases histamine from human basophils. Chemotaxis of different populations of cells and release of proinflammatory mediators in response to antigenic stimulation are important processes in allergic diseases. These lead to the late phase response, a hallmark of chronic allergic diseases. Recombinant RANTES, a member of the "intercrine/chemokine" family of cytokines, has been previously shown to be chemotactic for monocytes and T cells of memory/helper phenotype. In this manuscript, we show that it is capable of inducing histamine release from human basophils at concentrations as low as 10(-10) M and compare its activity with that of monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), another intercrine/chemokine. RANTES (10(-7) M) caused histamine release from the leukocytes of 26 of 33 donors tested (mean 21.8 +/- 3.1%). In the same group of donors, MCAF/MCP-1, goat anti-human IgE (anti-IgE; 1 microgram/ml), and FMLP (10(-5) M) released 41.1 +/- 2.9%, 40.5 +/- 4.6%, and 44 +/- 3.1% histamine, respectively. The percent histamine release by RANTES in atopic vs nonatopics was 30.3 +/- 6.7 and 16.5 +/- 2.4, respectively (p less than 0.05), and histamine release by RANTES correlated significantly with histamine release by MCAF (r = 0.69; p less than 0.001) but not with histamine release by anti-IgE (r = 0.29; p greater than 0.05). Histamine release by RANTES and MCAF/MCP-1 was extremely rapid, reaching a maximum within 1 min. RANTES was also shown to activate highly purified basophils (80% pure), and its activity was inhibited by a polyclonal anti-RANTES antibody. At a suboptimal concentration (6 x 10(-9) M), RANTES did not prime basophils to enhance histamine release by secretagogues such as anti-IgE, C5a, or FMLP. On the other hand, preincubation of basophils with RANTES or MCAF/MCP-1 desensitized basophils to either factor but not to anti-IgE, C5a, or FMLP. Preincubation of basophils with pertussis toxin markedly diminished the basophil response to either RANTES or MCAF/MCP-1. These results suggest that RANTES and MCAF/MCP-1: 1) are potent activators of basophils; 2) may function via the same, or a closely related, receptor system in basophils; and 3) may represent a link between activation of monocytes, lymphocytes, and basophils in inflammatory disorders such as the late phase allergic reaction.


Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site. The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.


Soluble forms of CD40 inhibit biologic responses of human B cells. We have expressed the CD40 surface Ag as both a soluble 28-kDa molecule and a 57-kDa Fc fusion protein containing the human IgG1 Fc region. Soluble CD40 and the Fc fusion protein inhibited the proliferative response of anti-IgM-activated human B cells to the CD40 mAb G28-5. Similarly, G28-5- and IL-4-induced IgE secretion from PBMC depleted of T cells was effectively blocked by both forms of soluble CD40. Although the soluble constructs of CD40 had only a minimal inhibitory effect on IL-4-mediated proliferation of anti-IgM-activated B cells, IL-4-induced soluble CD23 shedding from both PBMC and T cells depleted of PBMC, and IgE secretion from PBMC, were significantly reduced in a concentration-dependent manner when soluble CD40 was present in the culture. The data presented demonstrate that both soluble forms of the CD40 molecule are biologically active, and suggest that the ligand for CD40 is inducible in IL-4-stimulated cultures and that it mediates both shedding of sCD23 and IgE secretion.


CD34+ bone marrow cells are infected with HIV in a subset of seropositive individuals. Individuals infected with HIV frequently develop cytopenias and suppressed hematopoiesis. The role of direct HIV infection of hematopoietic progenitor cells in this process has not been defined. In this study, purified CD34+ bone marrow progenitor cells from 74 Zairian and American patients were studied by both coculture viral isolation and polymerase chain reaction for evidence of HIV infection. A total of 36.5% of Zairian and 14% of American patients had HIV infection of the CD34+ cell subset, with as many as 1 in 500 CD34+ cells infected. Most of the Zairian patients in this study had advanced HIV infection and markedly decreased CD4/CD8 T lymphocyte ratios (mean 0.160 +/- 0.08), and no laboratory value predicted the presence of infection in the CD34+ subset of a given Zairian individual. In contrast, American patients with CD34+ cell infection had total CD4 cells less than 20/mm3 and a greater decrease of the CD4/CD8 T lymphocyte ratio compared to seropositive Americans without CD34+ cell infection (p = 0.003). Hematopoiesis, studied by methylcellulose colony assays, was depressed in all seropositive patients studied with no significant further suppression when CD34+ cells were infected. Thus, CD34+ bone marrow progenitor cells are infected in vivo in a subset of seropositive individuals and may serve as an additional reservoir of virus in HIV-infected individuals.


Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.


Drug-specific T cells derived from patients with drug-induced allergic hepatitis. Drug-induced allergic hepatitis is a tissue-specific inflammatory disease caused by hypersensitivity to a particular drug. Although the frequency of drug-induced allergic hepatitis appears to increase in proportion to the medicine, the mechanism by which tissue specificity is determined is still to be elucidated. In this study, we established CD4+ T cell clones specific for particular drugs from patients with drug-induced allergic hepatitis accompanied with mild blood eosinophilia and analyzed the possible role of liver protein as a directing factor of liver-specific inflammatory reactions. All CD4+ T cell clones obtained from two patients with this disease proliferated in response to a combination of the particular drug plus liver specific protein (LSP), which consists of over 30 proteins. Some T cell clones were responsive to an antigenic conformation consisting of the 200-kDa glycoprotein (partly purified LSP), a component of LSP, plus the causal drug. In contrast, all CD4+ T cell clones from a patient with simple drug-induced eosinophilia responded to the causal drug in the absence of LSP and partly purified LSP. These data suggested that LSP or partly purified LSP of the appropriate Ag is the target that leads to liver-specific inflammation in drug-induced allergic hepatitis. Furthermore, T cell lines derived from patients with drug-induced allergic hepatitis and simple drug-induced eosinophilia produced large amounts of IL-5 after the appropriate antigenic stimulation, whereas CD4+ T cell clones from donors with a normal amount of peripheral blood eosinophils secreted a much less IL-5. Taken together, these results indicate that overproduction of IL-5 by the allergen-sensitized T cells may result in blood eosinophilia.


Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. Synthetic constructs were assembled as multiple Ag peptide systems containing repetitive sequences of Plasmodium falciparum and Plasmodium berghei, the causative agents of human and murine malaria respectively, and two universal human tetanus toxin T cell epitopes 830-843 and 947-967. These constructs were tested for antibody production in mice and for their capacity to stimulate human PBL and tetanus toxin-specific T cell clones. A high antibody titer can be obtained in mice when multiple Ag peptide systems are injected in various adjuvants or in PBS alone. Furthermore, all constructs can activate PBL from every donor tested. However, a variable response was obtained when different clones specific for the two tetanus toxin universal epitopes were used. These constructs may represent possible candidates for a malaria vaccine.


A novel cell-surface molecule expressed by human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the Ig superfamily. cDNA isolated from a human lymphocyte library were analyzed and shown to encode a novel cell-surface glycoprotein, termed HB15, expressed by dendritic cell subsets and activated lymphocytes. The predicted mature 186 amino acid protein was composed of a single extracellular V-type Ig-like domain, a transmembrane region, and a 39-amino acid cytoplasmic domain. In contrast to most Ig-like domains, analysis of a partial genomic DNA clone revealed that the extracellular Ig-like domain of HB15 was encoded by at least two exons. Northern blot analysis revealed that HB15 derived from three mRNA transcripts of approximately 1.7, 2.0, and 2.5 kb expressed by lymphoblastoid cell lines. Two mAb reactive with HB15 were produced and used to show that HB15 is expressed as a single chain cell-surface glycoprotein of M(r) 45,000. HB15 expression was specific for lymphoblastoid cell lines and mitogen-activated lymphocytes, and HB15 was not expressed at detectable levels by circulating leukocytes. Immunohistologic analysis revealed that HB15 had a unique pattern of expression, being found predominantly in hemopoietic tissues with strong expression by scattered interfollicular interdigitating reticulum cells and weak expression by germinal center cells. HB15 was also expressed by Langerhans cells within the skin. HB15 therefore serves as a unique marker for the subset of dendritic cells represented by Langerhans cells and interdigitating reticulum cells. Thus, the HB15 glycoprotein represents a newly identified member of the Ig superfamily that may play a significant role in Ag presentation or the cellular interactions that follow lymphocyte activation.


Therapeutic approaches to varicella-zoster virus infections. Varicella-zoster virus (VZV) infections, the cause of chickenpox and shingles, are usually benign but are associated with morbidity and mortality, especially in immunocompromised hosts. Significant advances have been achieved in the treatment of VZV infections. In immunocompromised patients, both vidarabine and acyclovir have proved useful for the therapy of chickenpox and herpes zoster. Acyclovir, administered intravenously, is the treatment of choice for these infections. Both chickenpox and herpes zoster in the normal host are amenable to therapy with orally administered acyclovir. For older individuals with herpes zoster, acceleration of cutaneous healing can be accomplished at dosages of 800 mg five times a day for 10 days. Acyclovir therapy of chickenpox is recommended for adolescents and young adults with infection. In the future, improved therapies for VZV infections may include such newer antiviral drugs as bromovinyl arabinosyl uracil and acyclovir prodrugs.


Have we looked beyond the physical and psychosocial? Palliative care can easily focus on physical and psychosocial problems but, for various reasons, ignore the spiritual needs of patients. There is confusion between the words 'religious' and 'spiritual,' the latter being the search for existential meaning, something done by all men and women--particularly as they approach death. This paper looks at excuses for not looking at spiritual issues but suggests the attending professional cannot avoid this aspect of care.


Transfer of molecules from glia to axon in the squid may be mediated by glial vesicles. Although the transfer of glial proteins into the squid giant axon is well documented, the mechanism of the transfer remains unknown. We examined the possibility that the transfer involved membrane-bound vesicles, by taking advantage of the fact that the fluorescent compound, 3,6-acridinediamine, N,N,N,',N'-tetramethylmonohydride [acridine orange (AO)], rapidly and selectively stains vesicular structures in glial cells surrounding the giant axon. We labeled cleaned axons (1-3 cm long) by incubation for 1 min in filtered seawater (FSW) containing AO. Because the AO was concentrated in glial vesicular organelles, these fluoresced bright orange when the axon was examined by epifluorescence microscopy. To look for vesicle transfer, axoplasm was extruded from such AO-treated axons at various times after labeling. During the initial 15 min, an increasing number of fluorescent vesicles were observed. No further increases were observed between 15 and 60 min post AO. The transfer of the fluorescent vesicles into the axoplasm seemed to be energy dependent, as it was inhibited in axons treated with 2 mM KCN. These results suggest that a special mode of exchange exists between the adaxonal glia and the axon, perhaps involving phagocytosis by the axon of small portions of the glial cells.


Differential effects of serotonin depletion on sensitization and dishabituation in the leech, Hirudo medicinalis. The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.


Estradiol pulses induce progestin receptors selectively in substance P-immunoreactive neurons in the ventrolateral hypothalamus of female guinea pigs. Low doses of estradiol, administered as pulses, are as effective as higher doses for priming ovariectomized (OVX) guinea pigs to display progesterone-facilitated lordosis. High doses of estradiol, administered by constant-release implants, induce progestin receptors in many substance P-immunoreactive (SP-IR) neurons in the ventrolateral hypothalamus (VLH), a site at which estradiol primes OVX guinea pigs to respond behaviorally to progesterone. To test the hypothesis that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P-containing neurons in the VLH, OVX females received estradiol implants 1 week prior to perfusion, or two pulses of estradiol-17 beta, injected 39 and 11 h before perfusion. Colchicine was administered intracerebroventricularly prior to perfusion. No significant differences were observed in the total number of progestin receptor-immunoreactive (PR-IR) or substance P-immunoreactive cells in the VLH and VLH/ventromedial hypothalamus (VMH), respectively, of females receiving the two estradiol treatments. However, the percentage of PR-IR cells in the VLH also immunoreactive for SP was significantly higher in the estradiol pulse-treated (53%), than in the estradiol capsule-implanted animals (36%). These data suggest that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P-containing neurons in the VLH and are consistent with the hypothesis that substance P is involved in progesterone-facilitated lordosis in guinea pigs.


Progestin receptors in substance P-immunoreactive neurons in the hypothalamus of male guinea pigs after behaviorally effective estradiol pulse treatment. Pulsatile administration of estradiol effectively primes orchidectomized (ORCH) male guinea pigs to display progesterone-facilitated lordosis. In contrast, a single injection of estradiol benzoate (EB) is not behaviorally effective. In ovariectomized female guinea pigs, estradiol pulses induce progestin receptors selectively in substance P neurons in the ventrolateral hypothalamus (VLH), a site at which estradiol primes females to respond behaviorally to progesterone. To test the hypothesis that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P neurons in the VLH in males, ORCH animals received a single injection of EB 40 h before, or two pulses of estradiol-17 beta, 39 and 11 h before perfusion. Colchicine was administered intracerebroventricularly prior to perfusion. The only difference found between the two estradiol treatment groups was a higher number of progestin receptor-immunoreactive (PR-IR) cells in the rostral VLH of estradiol pulse-treated males. There were no significant differences in the number of PR-IR cells in the mid- or caudal VLH, nor in the number of substance P-immunoreactive (SP-IR) neurons in the VLH/ventromedial hypothalamus (VMH) of animals receiving the two estradiol treatments. Furthermore, the percentage of PR-IR cells in the VLH also immunoreactive for SP did not differ between the estradiol pulse- (22%-25%) and the EB-injected animals (22%-32%). These data do not support the hypothesis that administration of behaviorally effective estradiol pulses, as compared to behaviorally ineffective EB injections, induce progestin receptors selectively in substance P neurons in the VLH of male guinea pigs.


Inhibitory effects of flavonoids on Moloney murine leukemia virus reverse transcriptase activity. Several flavonoids were tested for their effects on Moloney murine leukemia virus reverse transcriptase activity. Four groups of flavonoids, namely flavones, flavanones, flavonols, and flavanonols, were studied, and it was found that flavonols and flavanonols were very active in this regard while flavones and flavanones displayed very low activity. Among the flavonoids tested, fisetin, quercetin, myricetin, kaempferol, morin, (+/-)-taxifolin, (+)-catechin, and (-)-epicatechin were shown to be highly effective in inhibiting the reverse transcriptase activity. Structure-activity relationship analysis of these flavonoids revealed that the simultaneous presence of free hydroxyl groups at positions 3 and 4' enhanced the reverse transcriptase inhibitory activity. Replacement of the 3-hydroxyl group with a monosaccharide or of the 4'-hydroxyl group with a methyl group reduced inhibitory activity. The double bond at position 2 and 3 of the flavonoid's pyrone ring is not essential for inhibiting reverse transcriptase activity. The flavonoids studied demonstrated ability to inhibit the reverse transcriptase activity using either (rA)n(dT)12-18 or (rC)n(dG)12-18 as template-primers.


Reactivity of two anti-galactosyl ceramide antibodies towards myelin basic protein. Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reacted with C-8 was 2-fold less reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C.(ABSTRACT TRUNCATED AT 250 WORDS)


Tumour necrosis factor causes an increase in axonal transport of protein and demyelination in the mouse optic nerve. An increase in fast axonal transport of protein by the optic nerve was found in mice following a single combined injection of human recombinant tumour necrosis factor alpha (rTNF) and [3H]proline into the vitreous chamber. Demyelination was observed in optic nerve fibres arising from the eyes of mice which received a single rTNF injection. No such changes were detected when heat-inactivated rTNF was injected with the label. The effects of intravitreal injection of rTNF on the pathophysiology of mouse optic nerve resembled those found in mice infected with Semliki Forest virus (SFV), an animal model of multiple sclerosis. We suggest that TNF could mediate at least some of the pathophysiological changes found in SFV-infected mice and may provide a clue concerning the disease mechanism in multiple sclerosis.


Highly sensitive enzyme-linked immunosorbent assay for marograstim (KW-2228), a mutant of human granulocyte colony-stimulating factor. An enzyme-linked immunosorbent assay (ELISA) for marograstim (KW-2228) was established. This ELISA proved to be highly sensitive with the detection limit of 0.01 ng/ml (about 0.5 fmol/ml) of KW-2228 and the assay range between 0.01 and 2 ng/ml. When 0.02 to 2 ng/ml of KW-2228 added to human plasma was determined, the variation coefficiencies of intra-day and inter-day assays were 0.4 to 7.6% and 5.2 to 15.8%, respectively, with good recoveries. These results indicate that this ELISA will be applicable to pharmacokinetic studies on KW-2228. With respect to the specificity, recombinant human granulocyte colony-stimulating factor (rhG-CSF) produced in Escherichia coli as well as KW-2228 which does not have sugar chains in its structure showed slightly less immunoreactivity toward the antibody raised against KW-2228. The rhG-CSF produced in Chinese hamster ovary cells (CHO) having sugar chains showed the lower immunoreactivity. The antigenic domains of KW-2228 were evaluated using a number of variants of hG-CSF. The variants having different amino acids from KW-2228 in the 1st to 5th residue of the N-terminus showed almost equal immunoreactivities to KW-2228. The immunoreactivities of the variants lacking 7 to 18 amino acids of N-terminus were less than 50% of that of KW-2228. No immunoreactivity was observed for the variants deleted in the area of 70th to 130th amino acids from the N-terminus. In addition, the immunoreactivity of a variant lacking the 10 amino acids from the C-terminus was 20% of that of KW-2228.(ABSTRACT TRUNCATED AT 250 WORDS)


Purification and characterization of a mouse hepatocyte growth-stimulating factor from the liver of carbon tetrachloride-treated mice. Mouse hepatocyte growth-stimulating factor (mHGSF), which increased markedly in the liver of carbon tetrachloride-treated mice, was purified 275,000-fold with 21% yield from extracts of the injured liver. The purification involves ammonium sulfate precipitation and chromatography on Affi-Gel Blue, heparin-Sepharose, and S-Sepharose. The purified factor migrated as a major band of 76,000 daltons under nonreducing conditions and two bands of 62,000 and 31,000 daltons under reduced conditions. A dose-response of this growth factor for stimulation of deoxyribonucleic acid synthesis in cultured rat hepatocytes and its maximal effects were similar to those of human hepatocyte growth factor (hHGF), which we previously purified from the plasma of patients with fulminant hepatic failure (E. Gohda et al., J. Clin. Invest., 81, 414-419 (1988)). The effect of mHGSF was additive to the maximal effect of epidermal growth factor and was synergistic with that of insulin or acidic fibroblast growth factor, but was neither additive nor synergistic with the maximal effect of hHGF. mHGSF, like hHGF, was sensitive to heat and trypsin treatments and to reduction by dithiothreitol. This factor did not react with an anti-hHGF antiserum. These results indicate that mHGSF is a hHGF-like factor, but it is immunologically different from hHGF.


Homologous desensitization of human platelet thromboxane A2/prostaglandin H2 receptors. Desensitization of platelet thromboxane (TX)A2/prostaglandin (PG)H2 receptors was induced by incubating platelet-rich plasma with the stable PGH2 analog 11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM). Iloprost, a stable prostacyclin analog, was included in the incubation to prevent platelet activation. The TXA2 mimetic, [1S-1 alpha,2 beta(5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), was used to induce platelet aggregation, shape change and increases in intracellular free calcium. The EC50 values for I-BOP-induced rise in intracellular free calcium (control = 10.2 +/- 1.5 nM; desensitized = 79.4 +/- 22.4 nM, n = 6, P less than .05), aggregation (control = 15.8 +/- 2.4 nM; desensitized = 51.7 +/- 11.9 nM; P less than .05, n = 5) and shape change (control = 172 +/- 37 pM; desensitized = 350 +/- 60 pM; P less than .05, n = 7) were increased by the preincubation with U46619. Aggregation responses to thrombin and the calcium ionophore, ionomycin, were unaltered by the preincubation with U46619. Equilibrium binding studies at pH 7.4 revealed a decrease in the number of binding sites for the receptor antagonist 9,11-dimethylmethano-11,12- methano-16(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega- tetranor-TXA2 [125I]PTA-OH) (control = 3246 +/- 509 sites/platelet, desensitized = 2198 +/- 324 sites/platelet, n = 6, P less than .05) without a change in affinity.(ABSTRACT TRUNCATED AT 250 WORDS)