Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.


Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.


Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.


Mapping the three-dimensional locations of ribosomal RNA and proteins.

Seven regions of 16S rRNA have been located on the surface of the 30S ribosomal subunit by DNA hybridization electron microscopy in our laboratory. In addition, we have recently mapped the three-dimensional locations of an additional seven small ribosomal proteins by immunoelectron microscopy. The information from the direct mapping of the sites on rRNA has been incorporated into a model for the tertiary structure of 16S rRNA, accounting for approximately 40% of the total 16S rRNA. A novel structure, the platform ring, is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500-545, and occupies a region on the exterior surface of the subunit, near the EF-Tu binding site. In addition, 19 of the 21 small subunit ribosomal proteins have been mapped by immunoelectron microscopy in our laboratory. In order to evaluate the reliability of our model for the three-dimensional distribution of 16S rRNA, we have predicted which sites of rRNA are adjacent to ribosomal proteins and compared these predictions with r-protein protection studies of others. Good correlation between the model, the locations of rRNA sites, the locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins, provides independent support for this model.


Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli.

The published model for the three-dimensional arrangement of E coli 16S RNA is re-examined in the light of new experimental information, in particular cross-linking data with functional ligands and cross-links between the 16S and 23S RNA molecules. A growing body of evidence suggests that helix 18 (residues 500-545), helix 34 (residues 1046-1067/1189-1211) and helix 44 (residues 1409-1491) are incorrectly located in the model. It now appears that the functional sites in helices 18 and 34 may be close to the decoding site of the 30S subunit, rather than being on the opposite side of the 'head' of the subunit, as hitherto supposed. Helix 44 is now clearly located at the interface between the 30S and 50S subunits. Furthermore, almost all of the modified bases in both 16S and 23S RNA appear to form a tight cluster near to the upper end of this helix, surrounding the decoding site.


Comparison of dissimilarity patterns of E coli, yeast and mammalian tRNAs.

A number of experimental approaches have been developed for identification of recognition (identity) sites in tRNAs. Along with them a theoretical methodology has been proposed by McClain et al that is based on concomitant analysis of all tRNA sequences from a given species. This approach allows an evaluation of nucleotide combinations present in isoacceptor tRNAs specific for the given amino acid, and not present in equivalent positions in cloverleaf structure in other tRNAs of the same organism. These elements predicted from computer analysis of the databank could be tested experimentally for their participation in forming recognition sites. The correlation between theoretical predictions and experimental data appeared promising. The aim of the present work consisted of introducing further improvements into McClain's procedure by: i), introducing into analysis a variable region in tRNAs which had not been previously considered; to accomplish this, 'normalization' of variable nucleotides was suggested, based on primary and tertiary structures of tRNAs; ii), developing a new procedure for comparison of patterns for synonymous and non-synonymous tRNAs from different organisms; iii), analysis of 3- and 4-positional contacts between tRNAs and enzymes in addition to a formerly used 2-positional model. A systematic application of McClain's procedure to mammalian, yeast and E coli tRNAs led to the following results: i), imitancy patterns for non-synonymous tRNAs of any amino acid specificity and from any organisms analysed so far overlap by no more than 30%, providing a structural basis for discrimination with high fidelity between cognate and non-cognate tRNAs; ii), the predicted identity sites are non-randomly distributed within tRNA molecules; the dominant role is ascribed to only two regions--anticodon and amino acid stem which are located far apart from one another at extremes of all tRNA molecules; iii), the imitancy patterns for synonymous tRNAs in lower (yeast) and higher (mammalian) eukaryotes are similar but not identical; iv), distribution of predicted identity sites in the cloverleaf structure in prokaryotes and eukaryotes is essentially different: in eubacterial tRNAs the major role in recognition plays anticodon and/or amino acid acceptor stem, whereas in eukaryotic (both unicellular and multicellular) tRNAs the remaining part of the molecules is also involved in recognition; v), the imitancy patterns of synonymous tRNAs from prokaryotes and eukaryotes are dissimilar, this observation leads to the prediction that the tRNA identity sites for the same amino acid in prokaryotes and eukaryotes may differ.


Determination of tRNA(Phe) recognition nucleotides for phenylalanyl-tRNA synthetase from Thermus thermophilus.

The tRNA(Phe) recognition nucleotides for phenylalanyl-tRNA synthetase from an extreme thermophile Thermus thermophilus were investigated. Using yeast tRNA(Phe) T7 transcripts with various point mutations it was shown that four recognition points (G34, A35, A36 from anticodon and A73 from acceptor stem) are important for aminoacylation at 37 degrees C. In the case of the 73rd discriminator base A----U, but not A----C, substitution suppresses aminoacylation. Position 20, which proved to be essential for all phenylalanyl-tRNA synthetases so far studied, is not involved in the recognition of tRNA(Phe) by the T thermophilus enzyme.


Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation.

To identify the proteins of the 30S ribosomal subunit of E coli that neighbor mRNA in the ternary initiation complex (mRNA*30S subunit*tRNA(fMet), we used an affinity cross-linking approach in which photoactivated groups were attached to different positions along the mRNA chain. A series of mini-genes originating from the 5'-end region of the cro gene of lambda bacteriophage were constructed as templates for mini-mRNA synthesis. Two strategies were used to introduce photo-reactive agents into the message. According to the first, two transcripts were isolated from E coli and chemically derivatized at their 5'-ends with a photoinducible diaziril group. One of these messages allowed for localization of the 5'-end of the Shine-Dalgarno sequence while the other one allowed for labeling of the ribosome at the 5'-end side of the initiation AUG codon in the P site. According to the second approach, 5-azidouridine (5N3U) was randomly incorporated into mRNA transcripts during a T7 RNA polymerase catalyzed reaction by using a mixture of 5N3UTP and UTP. A message that had U residues at either -4, -3, -1, +2 and +14, +19, +20 positions was used (A from cro AUG is +1). Whereas cross-links with the 5N3U transcripts were essentially 'zero-length', the 5'-derivatized transcripts were covalently attached to ribosomal components about 14 A from the 5'-end. We found that proteins S1, S7, S5, S3 and S4 compose, or were close to, the ribosomal mRNA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)


Stabilization of standard platelet concentrates and minimization of the platelet storage lesion by a prostacyclin analogue.

Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss of platelet sensitivity to aggregating agents. To mimic the situation after transfusion and to assess the reversibility of platelet inhibition, platelets were washed during and after storage and resuspended in fresh-frozen autologous plasma. The Iloprost-treated and washed platelets exhibited an increased sensitivity to the aggregating agents, compared with the control platelets (p less than 0.01). Post-storage recovery of the synergistic aggregation was more than 80% of prestorage aggregation. Beta-thromboglobulin (beta TG) release and thromboxane B2 (TXB2) formation were significantly inhibited in Iloprost-treated platelets (p less than 0.01). After the second centrifugation step, beta TG release was 0.7% +/- 0.3%, compared with 2.7% +/- 0.9% for the controls. TXB2 was 99 +/- 91 pg/ml, compared with 495 +/- 356 pg/ml for the controls. Platelet morphology and ultrastructure were well preserved during 5-day storage. In addition, Iloprost exerted a cytoprotective effect, as evidenced by the significant reduction in lactate dehydrogenase leakage. Post-storage LDH was 378 +/- 159 and 415 +/- 239 U/l respectively by the two Iloprost concentrations, compared with 1180 +/- 937 U/l for the control platelets. The inhibitory and cytoprotective effects of Iloprost were sustained throughout storage, in contrast to the effect of PGE1 (Prostin) which was limited to the early phase of storage.


Bayesian restoration of single-channel patch clamp recordings.

The technique of patch clamp recording makes possible the measurement of current flowing through a single ion channel in a cell membrane. Examination of such recordings suggests that the current is quantal in nature, alternating in a seemingly random manner between "on" and "off," but the recordings are corrupted by noise from a variety of sources. In this paper we propose and illustrate methods for restoring the underlying quantal signal from such noisy measurements. The methods use a Markov chain prior distribution for the underlying quantal process and base the restoration on the resulting posterior distribution.


Hematopoietic growth factors upregulate the p65 type II interleukin-1 receptor on bone marrow progenitor cells in vitro.

Having previously shown that interleukin-1 (IL-1) induces the expression of IL-1 receptors (IL-1Rs) on bone marrow (BM) cells in vivo through an indirect mechanism, we studied whether hematopoietic growth factors (HGFs) could induce the expression of IL-1R on BM cells in vitro. In vitro treatment of light-density murine BM (LDBM) cells with either IL-3, IL-6, granulocyte--colony-stimulating factor (CSF), or granulocyte-macrophage--CSF caused a 5- to 10-fold upregulation of IL-1R expression, whereas IL-1, IL-5, IL-7, and macrophage-CSF had no effect. Scatchard analysis showed one class of IL-1Rs on LDBM cells with an average of 66 +/- 20 sites per cells. After 24 hours of treatment with IL-3, the number of IL-1Rs increased to 413 +/- 125, without effecting the affinity. This effect required protein synthesis, but was independent of cell division. Purified lineage-negative progenitor cells (Lin-) did not express detectable levels of IL-1R, but 24 hours of treatment with IL-3, GM-CSF, and G-CSF stimulated IL-1--specific binding. Autoradiographic analysis of Lin- cells showed that IL-1R induction by IL-3 occurs on undifferentiated blast cells. Affinity labeling of Lin- cells treated with HGFs showed an increase in a 65-Kd IL-1 binding protein that did not bind or compete with an anti-type I IL-1R antibody, suggesting that these cells expressed type II IL-1R. These data suggest that IL-1 stimulation of myelopoiesis occurs by a mechanism involving IL-1R upregulation on hematopoietic progenitor cells by HGFs.


Specific repression of granulocyte-macrophage and granulocyte colony-stimulating factor gene expression in interleukin-1-stimulated endothelial cells with antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) have been used to effect the specific inhibition of cellular gene expression. We have evaluated the application of this approach to the inhibition of interleukin-1 (IL-1)-induced granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in cultured human umbilical vein endothelial cells. Antisense ODNs or control ODNs (sense ODNs or missense ODNs containing random base substitutions) were added to cultures of endothelial cells, the cells were induced with IL-1 alpha, and the conditioned media were assayed for GM-CSF and G-CSF by quantitative bioassays and for immunoreactive GM-CSF by enzyme immunoassay. Antisense ODNs complementary to the first 15 or 18 bases of the translation start sites of GM-CSF or G-CSF mRNAs inhibited, in a concentration-dependent fashion, the IL-1-stimulated expression of the corresponding factor, but did not affect expression of the other factor. Control ODNs did not affect GM-CSF or G-CSF expression. Exposure to a GM-CSF antisense ODN, but not a control ODN, substantially reduced cytoplasmic GM-CSF mRNA levels in IL-1-stimulated endothelial cells. Neither ODN affected levels of endothelial leukocyte adhesion molecule (ELAM)1 or glyceraldehyde-3-phosphate dehydrogenase mRNAs. We conclude that antisense ODNs complementary to the translation start sites of GM-CSF or G-CSF mRNAs inhibit expression of the corresponding factor in a sequence-specific fashion and this effect is mediated, at least in part, by reduction in the cytoplasmic level of the targeted mRNA. Moreover, IL-1-induced GM-CSF or G-CSF expression does not depend on expression of the other factor.


Type beta transforming growth factors promote interleukin-3 (IL-3)-dependent differentiation of human basophils but inhibit IL-3-dependent differentiation of human eosinophils.

Basophils and eosinophils share a common differentiation pathway. Factors regulating terminal commitment toward one cell type, however, have so far not been defined. Interleukin-3 (IL-3) is a potent differentiation factor for both human eosinophils and basophils. In the present study, the effects of various recombinant human (rh) growth regulators on IL-3-dependent growth of eosinophils and basophils were studied in a bone marrow (BM) suspension culture system (normal donors, n = 13). We found that type beta transforming growth factors (TGFs) lead to a significant increase in the absolute numbers of basophils in BM cultures grown in the presence of IL-3 (day 14 of culture; IL-3: 133 +/- 20 v IL-3 + TGF-beta 1: 231 +/- 28 x 10(3)/mL [P less than .01]) and to an increase in the total histamine values (IL-3: 72.6 +/- 22.2 v IL-3 + TGF-beta 1: 142.9 +/- 37.3 ng/mL [P less than .015]) compared with rhIL-3 alone. In contrast, type beta TGFs were found to inhibit the IL-3-dependent growth of eosinophils (IL-3: 170.4 +/- 37.2 v IL-3 + TGF-beta 1: 16.7 +/- 5.2 x 10(3)/mL [P less than .01]) and formation of eosinophil cationic protein in the same culture system. The effect of TGF-beta 1 (and TGF-beta 2) on IL-3-dependent differentiation of basophils and eosinophils was dose- and time-dependent (maximum effects observed with 1 to 10 ng/mL of rhTGF-beta 1 or TGF-beta 2) and could be neutralized by an antibody specific for TGF-beta 1. In contrast to the TGFs, interferon-alpha (IFN-alpha) and IFN-gamma were found to downregulate IL-3-dependent formation of both basophils (IL-3: 167 +/- 33 v IL-3 + IFN-alpha: 67 +/- 25 v IL-3 + IFN-gamma: 65 +/- 33 x 10(3)/mL [P less than .01]) and eosinophils (IL-3: 239 +/- 5 v IL-3 + IFN-alpha: 81 +/- 4 v IL-3 + IFN-gamma: 67 +/- 17 x 10(3)/mL [P less than .05]) in our culture system. Type beta TGFs as well as the IFNs failed to directly induce differentiation of human basophils or eosinophils in the absence of other growth factors. Together, these results show that type beta TGFs and IFNs are potent regulators of cytokine-dependent growth and differentiation of human allergic effector cells.


Stem cell factor induction of in vitro murine hematopoietic colony formation by "subliminal" cytokine combinations: the role of "anchor factors".

The high levels of hematopoietic growth factors required for in vitro and in vivo activity raise questions as to their role in normal hematopoietic maintenance. We hypothesize that the use of combinations of cytokines to stimulate hematopoietic progenitors might allow individual factors to exert their influence at lower, more physiologically relevant concentrations. Growth factor combinations were assessed by their ability to stimulate both total colonies and high proliferative potential colony-forming cells (HPP-CFC), an early murine hematopoietic progenitor, in double-layer agar cultures. Very-low-level combinations of colony-stimulating factor (CSF)-1, granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin (IL)-1 alpha, and IL-3 had little or no clonogenic capacity. Plateau levels of rr stem cell factor (rrSCF), a c-kit ligand, used alone also had negligible clonogenic capacity, but when combined with the low-level combination of the other five factors produced total colony and HPP-CFC growth approaching that produced by all factors at plateau levels. Delayed addition experiments suggest that this effect may represent sequential activity of SCF and the other factors. We propose a model of the normal hematopoietic microenvironment in which SCF at locally high concentration on the stromal cell surface "anchors" the hematopoietic stem cell's response to multiple other cytokines at physiologically relevant levels.


Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo.

Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon-gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G-CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.


Induction of colony-stimulating factor receptor expression on hematopoietic progenitor cells: proposed mechanism for growth factor synergism.

In many cells systems, the cellular interaction between two or more humoral factors leads to a synergistic response in terms of cellular growth and function. In particular, the growth and differentiation of hematopoietic progenitor cells involves numerous synergistic interactions between colony-stimulating factors (CSFs) that individually stimulate hematopoiesis (granulocyte-CSF, granulocyte-macrophage-CSF, and interleukin-3 [IL-3]), as well as between these factors and other cytokines that individually have no proliferative effect on progenitor cell growth (IL-1 and IL-6). The present study investigated whether hematopoietic growth factor (HGF) synergy could be mediated by upregulation of CSF receptors. Synergistic effects on bone marrow (BM) progenitor cell colony formation, regardless of the combination of factors used, were consistently preceded by increased CSF receptor expression on highly enriched BM progenitor cells, but not on unfractionated BM cells. Induction of CSF receptors preceded detectable differentiation and did not require cell division because nocodazole, an inhibitor of mitosis, blocked CSF-mediated cell proliferation, but not receptor upregulation. Furthermore, combinations of cytokines that did not synergize also failed to affect the level of CSF receptors on BM progenitors. These results have led us to propose a model for HGF synergy whereby one mechanism of action the investigated synergistic cytokines might be the ability to induce increased expression of CSF receptors.


Differentiation and functional activity of human eosinophilic cells from an eosinophil HL-60 subline: response to recombinant hematopoietic growth factors.

We studied the effect of hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte [G]-CSF, interleukin (IL)-1, IL-3, IL-5, IL-6, and macrophage [M]-CSF) on differentiation and functional activity of human eosinophilic HL-60 cells (Eos-HL-60) and compared them with effects on parental HL-60 promyelocytic leukemia cells. Purified biosynthetic GM-CSF and IL-5 enhanced cell proliferation and induced eosinophilic differentiation in the eosinophilic subline in both liquid and agar cultures. IL-3 and IL-6 stimulated cell proliferation but had no effect on cell differentiation, whereas IL-1 and G-CSF affected neither differentiation nor proliferation of Eos-HL-60 cells under the conditions tested. GM-CSF-, IL-3-, and IL-5-treated Eos-HL-60 cells showed increased O2- production in response to phorbol esters (PMA), enhanced phagocytosis of Candida albicans, and release of the enzymes arylsulfatase, beta-glucuronidase and eosinophil peroxidase (EPO). The degranulation of eosinophils induced by GM-CSF, IL-5, and IL-3 may have relevance to the potential clinical toxicity of these hematopoietins, which also stimulate eosinophilopoiesis. G-CSF had no effect on enzyme release, oxidative metabolism, or phagocytic capacity of Eos-HL-60 cells. IL-5 did not affect proliferation, differentiation, or enzyme release in promyelocytic HL-60 cells. These results indicate the specificity of IL-5 for the eosinophil lineage, confirm the effects of GM-CSF and IL-3 on eosinophilopoiesis and mature eosinophil function in a model system, and indicate the absence of G-CSF and IL-1 stimulation of eosinophils. The Eos-HL-60 line is a useful model for studying human eosinophil responses to cytokines.


Molecular cloning and analysis of in vivo expression of murine P-selectin.

P-selectin (CD62) is a rapidly inducible cell surface adhesion molecule that is expressed on platelets and endothelial cells and mediates their interaction with leukocytes. In vitro studies have suggested that this receptor may play an important role in hemostasis and in inflammatory response to tissue injury. We report the molecular cloning and sequencing of murine cDNA for P-selectin. The lectin, epidermal growth factor (EGF)-like, transmembrane, and cytoplasmic domains are highly conserved between mouse and human, with an overall amino acid identity of 79%. To further investigate the biology of this adhesion molecule in vivo, we analyzed mRNA levels for P-selectin in mice after injection with endotoxin. Northern blot data indicate that the cellular response in vivo includes a rapid increase in the level of mRNA, presumably for new synthesis of P-selectin. The increase in mRNA is maximal at 4 hours, and turnover is relatively rapid, with levels of RNA having decreased substantially by 6 hours following stimulation with endotoxin. After administration of endotoxin, the highest levels of mRNA expression were detected in liver, lung, kidney, and heart.


Fetal hemoglobin levels in sickle cell disease and normal individuals are partially controlled by an X-linked gene located at Xp22.2.

Fetal hemoglobin (Hb F) production in sickle cell (SS) disease and in normal individuals varies over a 20-fold range and is under genetic control. Previous studies suggested that variant Hb F levels might be controlled by genetic loci separate from the beta-globin complex on chromosome 11. Using microscopic radial immunodiffusion and flow cytometric immunofluorescent assays to determine the percentage of F reticulocytes and F cells in SS and nonanemic individuals, we observed that F-cell levels were significantly higher in nonanemic females than males (mean +/- SD, 3.8% +/- 3.2% v 2.7% +/- 2.3%). F-cell production as determined by F reticulocyte levels in SS females was also higher than in SS males (17% +/- 10% v 13% +/- 8%). We tested the hypothesis that F-cell production in both normal and anemic SS individuals was controlled by an X-linked locus with two alleles, high (H) and low (L). Using an algorithm to determine the 99.8% confidence interval of a normal distribution in nonanemic individuals, we estimated that males and females with at least one H allele had greater than 3.3% F cells. Comparisons of male-male or female-female SS sib pairs with discordant F reticulocyte levels distinguished two phenotypes in SS males (L, less than 12%; H, greater than 12%) and three phenotypes in SS females (LL, less than 12%; HL, 12% to 24%, HH greater than 24%). Linkage analysis using polymorphic restriction sites along the X chromosome in eight SS and one AA family localized the F-cell production (FCP) locus to Xp22.2, with a maximum lod score (logarithm of odds of linkage v independent assortment) of 4.6 at a recombination fraction of 0.04.


Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3-3'-diaminobenzidine and immunocytochemistry.

We describe the application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver. For automatic detection by light and electron microscopy peroxisomes must be stained with the alkaline DAB procedure for catalase. There is a good agreement between the results obtained by conventional morphometric techniques and by automatic image analysis of DAB-stained electron microscopic preparations. Moreover, the image analyzer may be used in conjunction with a light microscope for evaluation of semithin sections (1-0.25 microns), provided the section thickness factor is taken into consideration. This latter approach has proven highly efficient in estimation of peroxisome proliferation. The limitations of this method and the relevance of volume density as a reliable morphometric parameter for evaluation of peroxisome proliferation are discussed. In the second part of this study we present the application of image analysis for quantitation of alterations of individual peroxisomal enzyme proteins after treatment with bezafibrate in immunogold stained ultrathin sections. There is good agreement between the results of quantitative immunocytochemistry and Western (immuno) blot analysis of highly purified peroxisomal fractions. In our experience quantitative immunoelectron microscopy provides a versatile, highly sensitive, and efficient method for detection of modulations of various proteins in peroxisomes. Finally the limitations and prospects of quantitative immunocytochemistry for investigation of peroxisomal proteins are discussed.


Comparative mapping of mouse chromosome 4 and human chromosome 9: Lv, Orm, and Hxb are closely linked on mouse chromosome 4.

The genes for orosomucoid (ORM-1 and ORM-2), delta-aminolevulinate dehydratase (ALAD), and hexabrachion or tenascin (HXB) all map to the q31-qter region of human Chromosome (Chr) 9. The mouse homolog of each of these genes has been mapped to Chr4, but hexabrachion has not previously been mapped by linkage analysis. We have now ordered Orm-1, Lv (the mouse homolog of ALAD), and Hxb in an interspecific backcross panel, by use of tyrosinase related protein-1, Tyrp-1, whose human homolog maps to 9p13-pter (Abbott et al., Genomics 1991) as a reference locus. No recombinants were identified in 124 animals between Lv and Orm-1. Hxb was found to be 1.6 cM distal to Lv and Orm-1, and 4.8 cM proximal to Tyrp-1, or b. These data therefore contribute to our knowledge of the conserved synteny between HSA 9q and MMU 4.


Effects of chronic amitriptyline administration on saliva from the parotid and submandibular glands of the rat.

The effect of prolonged treatment with amitriptyline on the secretory activity of rat salivary glands evoked by parasympathetic nerve stimulation and isoprenaline administration has been studied. Low doses of amitriptyline (10 mg/kg per day for 2 or 4 weeks), did not significantly affect salivary flow evoked by either parasympathetic nerve or isoprenaline stimulation. Higher doses of amitriptyline (50 mg/kg/day for 2 or 4 weeks) however, markedly decreased parasympathetic-evoked salivary secretion (flow and volume) from both parotid and submandibular glands, while isoprenaline-evoked secretions were unaffected. Sodium, potassium, and calcium concentrations of nerve-elicited or isoprenaline-evoked saliva were not significantly altered by amitriptyline treatment. Protein concentration and amylase activity of nerve-elicited parotid saliva were, however, greatly increased by chronic amitriptyline administration. Possible mechanisms for drug-induced increase in nerve-elicited salivary protein concentration include changes in cholinergic receptor binding, release of neuropeptides and variations in phosphatidylinositol turnover, which need further study.


Reprocessing data to form QALYs.

To determine whether reprocessing data from published sources into quality adjusted life years (QALYs), as recommended in The QALY Toolkit, is a useful method of helping purchasing authorities to determine the most cost effective pattern of care to buy for their populations. United Kingdom. The method was tested for six elective surgical conditions; data from published studies were reprocessed into the Rosser index, to obtain values for change in quality of life. These were then used to form QALYs. A small validation exercise was carried out. QALYs formed from the Rosser index. Published data could not be found for three interventions (cataract surgery, inguinal hernia repair, varicose vein surgery). For the remainder (prostatectomy, hip replacement, and knee replacement) data were found which could be reprocessed to form QALYs, though it was often hard to compare data from different studies and many assumptions had to be made. The value of QALY results obtained by this method is questionable, given the large number of assumptions which had to be made. For many interventions published data are unlikely to be available.


Causes and consequences of intrauterine growth retardation in Latin America.

The purpose of this article is to present a critical review of the literature on the causes and consequences of intrauterine growth retardation (IUGR) and of body proportions at birth among IUGR infants. IUGR is generally defined as a birth weight below the tenth percentile of a reference weight distribution according to gestational age. Chronic maternal malnutrition and other poverty-related factors are likely to be important determinants of IUGR in developing countries. IUGR has been associated with increased risks of neonatal morbidity and mortality and alterations in physical and mental development during early childhood. IUGR newborns can be classified as "symmetric" or "asymmetric" in terms of body proportionality. The available literature indicates that the risk of perinatal morbidity is higher among asymmetric newborns than among their symmetric counterparts, while symmetric newborns confront a higher risk of impaired physical and mental development. Obviously, the clinical and statistical usefulness of these IUGR and body proportionality assessments depends on the accuracy of the birth measurements (length, weight, and estimated gestational age) from which they are derived. Latin America now has the hospital infrastructure needed to obtain reliable birth data of this kind. It is important to use these resources effectively in order to improve the health assessment of newborns and gain a better understanding of the causes and consequences of IUGR.


Open or transurethral surgery for the large prostate gland.

A comparative study of transurethral (TUR) and open prostatectomy for the large prostate gland has been carried out. Over a 5-year period 94 transurethral resections and 73 open procedures were performed for prostate glands weighing more than 50 g. There was a single death in each group, giving relative mortality rates of 1.1% TUR and 1.4% open. The post-operative hospital stay was significantly shorter in the TUR group. The open group suffered significantly more complications in the first week but the TUR patients, although having fewer immediate postoperative complications, had significantly more in the first year, so that overall complication rates were similar (35% vs 36%). Patient satisfaction was equal in both groups.


Laser ablation of the prostate in patients with benign prostatic hypertrophy.

Seventeen patients with bladder neck obstruction due to benign prostatic hypertrophy have been treated with the Neodymium: YAG laser. We review our experience since the first patient was treated in September 1990. Using a prototype deflecting gold alloy tip on a quartz laser fibre (Lateralase TM), we ablated obstructing prostatic adenoma and constricting bladder neck tissue. Experience with this technique has enabled a patient population to be defined in whom laser therapy for prostatic obstruction may be effective. The treatment is relatively simple, speedy and attended by virtually no blood loss. Laser ablation therapy may offer some advantages over conventional transurethral resection of the prostate (TURP) in a selected subgroup of patients. The advent of new delivery systems may make laser ablation therapy a practical alternative to TURP.


The development of new techniques in the assessment and monitoring of recovery from severe head injury: a preliminary report and case history.

The case of a 19-year-old male who sustained a severe brain injury is described. This patient was initially given a very gloomy prognosis, leading to poor expectations for recovery. Additionally, like many such patients in the United Kingdom, he was cared for by a ward team who were without specialized training in head injury rehabilitation. Despite these factors the patient went on to make a good recovery. The issues of specialized care and the need for more appropriate patient assessment procedures are raised. The MRC Head Injury Assessment Project is described.


P53 and Ki-67 immunoreactivity in human prostate cancer and benign hyperplasia.

Mutation of the p53 gene is one of the commonest genetic abnormalities found in solid human tumours. This gene is probably concerned with the control of cellular proliferation and in view of this we carried out a study of human prostate cancer and benign prostatic hyperplasia, comparing the expression of mutated p53 with measurement of growth fractions as assessed by staining with Ki-67. A series of 29 patients with prostate cancer (CaP) were compared with 34 men with benign hyperplasia (BPH); 22 of 29 prostate cancers (76%) contained Ki-67 immunoreactivity compared with 10 of 34 (29%) BPH. With respect to p53 staining, significantly more prostate cancers (17%) were stained than BPH (0%). The mean Ki-67 score in cancers positive for p53 (4.3%) was greater than that found in cancers negative for p53 (1.2%), but no statistically significant relationship was found between tumour grade and Ki-67 staining. The use of Ki-67 and p53 staining may allow identification of tumours with a higher rate of cell growth and may permit development of prognostic factors.


Neural changes in acute arthritis in monkeys. III. Changes in substance P, calcitonin gene-related peptide and glutamate in the dorsal horn of the spinal cord.

The effects of an experimentally induced arthritis on immunoreactivity of putative primary afferents neurotransmitter/neuromodulators were examined. Immunoreactive staining for substance P (SP), calcitonin gene-related peptide (CGRP) and glutamate (Glu) in the monkey dorsal horn was examined following inflammation of one knee joint induced by injection of 5% kaolin and 5% carrageenan. Spinal cords were examined at different time periods after induction of arthritis (2.5, 4, 6 and 8 h). Side to side differences in immunoreactivity were determined by a computer assisted quantitation system. A significant overall decrease in immunoreactivity of the lumbar versus the cervical dorsal horn was found for SP. The decrease for SP showed maximal changes of 68.3% at 4 h and 54.7% at 6 h. Immunoreactivity for CGRP was decreased 31.5% at 8 h and variable at other time points. Immunoreactivity for Glu, showed an ipsilateral increase of 31.4% at 4 h, 33.7% at 6 h, 39.9% at 8 h and a significant effect for lumbar versus cervical. Repetitive peripheral stimulation of the joint was shown to be important for changes in SP and Glu immunoreactivity. Without frequent peripheral stimulation in the early stages of the development of arthritis, SP showed no quantitative side to side differences. Increases in Glu immunoreactivity were present but not as prominent with minimal joint manipulation. These studies suggest that Glu may be involved in the aching pain of inflammation at rest whereas SP, CGRP and Glu may mediate pain induced by joint movement.


Survey of urological centres and review of current practice in the pre-operative assessment of prostatism.

A survey of 24 urological centres has shown a wide variation in the routine pre-operative assessment of patients being considered for prostatectomy. Imaging of the urinary tract by intravenous urography (IVU) or ultrasound (US) is performed in 21/24 centres (79%) and plain films in 16/24 (67%). Post-micturition residual volume (PMRV) is estimated quantitatively in 10/24 centers (42%). Although there is little agreement on what constitutes a significant PMRV, a large PMRV leads to increased likelihood of operation, and earlier operation. Peak urine flow rate (Q max) is measured in 19/24 centres (79%). The significance of these findings is discussed.


Is pre-operative imaging of the urinary tract worthwhile in the assessment of prostatism?

The reports of routine pre-operative imaging investigations performed on patients presenting with symptoms of uncomplicated benign prostatic hypertrophy were compared with management decisions and clinical outcome. In 175 patients with prostatism no urological abnormality which altered management was discovered on plain films of the abdomen and pelvis and ultrasound of the urinary tract which were performed routinely. Post-micturition residual volume (PMRV), estimated by ultrasound, was compared with the maximum urine flow rate (Q max) in 57 patients. PMRV showed negative correlation with Q max. Both high PMRV and low Q max were associated with the urologist's decision to operate, but multiple logistic regression revealed that ultrasound residual volume was not a significant predictor of operation when adjusted for Q max. Urologists in this hospital therefore use flow rates as the primary indication of the need to operate. We suggest that no routine pre-operative imaging need be performed in patients presenting with prostatism.


Urinary flow rates in patients with benign prostatic hypertrophy following treatment with alfuzosin. DUALF Group.

In order to document further the onset of action of alfuzosin, a selective alpha-1 blocker, 93 symptomatic patients with benign prostatic hypertrophy were randomly allocated to a single oral dose of either alfuzosin 1.25 mg or 2.5 mg, or placebo, after a 1-week placebo lead-in period. The effects on flow rates were assessed 1 h 30 min after administration. Peak and mean flow rates were significantly increased in the alfuzosin groups, as compared with placebo, in a dose-dependent manner. After a single intake of placebo, the mean values of these 2 parameters showed little change. The effect on the cardiovascular system (heart rate and blood pressure) was mild. This study indicates that the action of alfuzosin is already present 1 h 30 min after administration.


Finasteride in the treatment of benign prostatic hyperplasia. A urodynamic evaluation.

A group of 69 men with bladder outflow obstruction due to benign prostatic hyperplasia (BPH) were treated in a double-blind, placebo-controlled study with finasteride (Proscar, MK-906), a 5-alpha reductase inhibitor, 5 mg or 10 mg/day or placebo for 3 months; subsequently, 20 patients received finasteride 5 mg/day for a further 9 months in an open extension study. In treated patients dihydrotestosterone declined by over 60%, remaining unchanged with placebo. Symptom scores fell significantly in all 3 groups. Mean maximum flow rates fell slightly in placebo-treated patients but improved by 1.5 ml/s in the 10 mg group and by 3.3 ml/s in the 5 mg group. After 1 year's treatment, the reduction in symptom score and increase in flow rate were well maintained; the mean prostate volume was reduced by 14% and prostatic specific antigen declined by 28%. It was concluded that finasteride shows some efficacy in the treatment of BPH, with minimal toxicity, but 12 months of therapy or longer may be necessary to achieve maximal effect.


Calcitonin gene-related peptide enhances substance P-induced behaviors via metabolic inhibition: in vivo evidence for a new mechanism of neuromodulation.

The present study examined the effects of intrathecal (i.t.) injection of calcitonin gene-related peptide (CGRP) on caudally directed biting and scratching induced by i.t. substance P (SP), bombesin (BBS), strychnine (STR), and kainic acid (KA). CGRP alone (5.25, 10.5 and 21 nmol) had no effect on these behaviors, but CGRP pretreatment produced a dose-related enhancement of behaviors induced by SP or BBS, but not by KA or STR. 2-Amino-5-phosphonovaleric acid (APV, 25 nmol), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, did not block the CGRP potentiation of SP and BBS induced behaviors. CGRP, however, failed to enhance scratching and biting induced by a SP analogue [pGlu5-Mephe8-MeGly9]SP(5-11) (Dime-C7) that is resistant to enzymatic degradation by SP endopeptidase. These findings demonstrate that CGRP potentiates SP induced behavioral responses via inhibition of neuropeptide degradation and that this mechanism may serve as a physiological mechanism of SP modulation.


Galanin-evoked acetylcholine release in the rat striatum is blocked by the putative galanin antagonist M15.

The effect of the putative galanin (GAL) antagonist M15 on the Gal-evoked release of acetylcholine (ACh) was investigated in the rat striatum using microdialysis and HPLC techniques. GAL (3 nmol/10 microliters), applied in the lateral ventricle (i.c.v.) or perfused through the microdialysis membrane into the striatum, was found to enhance basal ACh release. The GAL-evoked release of striatal ACh was completely blocked by M15 (3 nmol/10 microliters), administered either i.c.v. or into the straitum. This finding suggests that GAL stimulates the basal ACh release in the rat striatum by a direct action of the peptide on striatal GAL receptors.


Modulation of synaptic efficacy in field CA1 of the rat hippocampus by forskolin.

Activation of cAMP-dependent protein kinase (kinase A) has recently been shown to enhance responses evoked by stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in cultured hippocampal pyramidal neurons. Here we report results of experiments designed to determine if activation of the cAMP cascade potentiates synaptic strength in field CA1 of rat hippocampal slices. We find that bath application of the direct adenylate cyclase activator forskolin (50 microM) enhances the field excitatory postsynaptic potential (EPSP) slope and population spike amplitude evoked by stimulation of Schaffer/commissural afferents. This effect is potentiated by the phosphodiesterase inhibitor and adenosine receptor antagonist 3-isobutyl-1-methylxanthine (IBMX). The enhancement produced by forskolin is suppressed in the presence of adenylate cyclase inhibitors and is not mimicked by the inactive forskolin analogue 1,9-dideoxyforskolin, indicating that, indeed, activation of adenylate cyclase mediates the effects of forskolin in field CA1. Our observations support the idea that changes in intracellular cAMP levels can modulate synaptic efficacy of excitatory glutamatergic synapses in the mammalian hippocampus.


In vivo studies on the enhancement of serotonin reuptake by tianeptine.

The present study investigates the in vivo effects of the serotonin uptake enhancer tianeptine. The serotonin metabolite, 5-hydroxy-indolacetic acid (5-HIAA) was measured by in vivo voltammetry and carbon fiber electrodes chronically implanted in different brain areas of freely moving rats. Tianeptine (10 mg/kg i.p.) increased extracellular 5-HIAA in the hippocampus and hypothalamus. The interaction between tianeptine and drugs known to interfere with the uptake or release of serotonin (sertraline, buspirone, D-norfenfluramine) was then studied and, to ascertain the in vivo pharmacological relevance of tianeptine's effects, its ability to reduce the serotoninergic syndrome was evaluated. Both the biochemical and behavioral data indicate that in vivo tianeptine's effects on the serotoninergic system are likely to be due to serotonin uptake enhancement.


The effect of chronic atypical antipsychotic drugs and haloperidol on amphetamine-induced dopamine release in vivo.

The effect of chronic administration of antipsychotic drugs (21 days in drinking water followed by 3 days drug washout) on the D-amphetamine (1.0 mg/kg, s.c.)-induced increase in dopamine (DA) release in the striatum and the nucleus accumbens of awake, freely-moving rats was investigated with microdialysis. Chronic administration of haloperidol, a typical antipsychotic, (0.5 mg/kg/day), decreased basal extracellular DA release in the striatum and the nucleus accumbens but did not affect D-amphetamine-induced DA release in either region. In marked contrast, chronic administration of three atypical antipsychotic drugs: amperozide (2 mg/kg/day), clozapine (10 mg/kg/day) and melperone (2 mg/kg/day) increased basal extracellular DA and enhanced D-amphetamine-induced DA release in the striatum. In the nucleus accumbens, basal extracellular DA was decreased by chronic amperozide, unchanged by chronic clozapine and increased by chronic melperone. Most significantly, D-amphetamine-induced DA release was inhibited by chronic amperozide or clozapine, but unaffected by chronic melperone in this region. These results suggest that atypical antipsychotic drugs can alter DA release in a region specific manner. In particular, attenuation of amphetamine-like stimulation of DA release with reduced basal DA release in the nucleus accumbens could contribute to the antipsychotic action of amperozide which has a very weak affinity for D2 DA receptors.


Monoamine innervation of bed nucleus of stria terminalis: an electron microscopic investigation.

Immunocytochemical studies showed distinctive monoamine input to the bed nucleus of the stria terminalis (BST). A comparison of axons immunoreactive (IR) for a catecholamine synthetic enzyme [tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH) or phenylethanolamine-N-methyl transferase (PNMT)] or serotonin (5-HT) was performed. TH-IR axons had a greater density in the lateral BST, but DBH-IR and 5-HT-IR axons had a greater density in the medial BST. PNMT-IR axons were dense in the intermediate BST. TH-IR axons had a greater density than DBH- and PNMT-IR axons in the dorsolateral BST, but DBH-IR axons had the greatest density in the ventrolateral BST. Ultrastructural studies revealed that TH-IR terminals formed synapses with soma, dendrites, spines, and axons in the dorsolateral BST. DBH-IR terminals formed synapses with dendritic shafts and spines, and 5-HT-IR terminals formed synapses with dendrites in the ventrolateral BST. Only some 5-HT-IR axons were myelinated. The medial vs. lateral organization of the noradrenergic and dopaminergic afferents in the BST of the rat brain is now evident and is similar to the human brain. The medial-lateral functional subdivision of the BST is supported by the pattern of dopaminergic, noradrenergic, and serotonergic afferents. This demonstration of epinephrine-producing afferents in the BST is the first detailed description of adrenergic input to the BST and aided the determination that catecholaminergic innervation of the ventrolateral BST is predominantly noradrenergic as has been proposed for many years. However, the additional demonstration of rich dopaminergic innervation of the dorsolateral subnucleus suggests further division of the BST into dorsal and ventral functional subgroups.


Hematopoietic colony-stimulating factors. Uses in combination with standard chemotherapeutic regimens and in support of dose intensification.

Three hematopoietic colony-stimulating factors (CSF) are currently approved for clinical use and several more are being used in clinical trials. These factors have impressive quantitative and/or functional effects on their respective target cell lines, but their best clinical uses have not been defined. The literature regarding the use of CSF in standard chemotherapy is reviewed data and from early Phase I-II studies the use of granulocyte-macrophage CSF (GM-CSF) in support of dose intensification without progenitor cell replacement are presented. Dose intensive cyclophosphamide (5000 mg/m2), etoposide (1500 mg/m2), and cisplatin (150 mg/m2) (DICEP) were administered during 3 days, followed by CSF until hematologic recovery was achieved. Patients had advanced types of cancer unlikely to respond to standard chemotherapy. CSF can shorten the duration of leukocyte nadirs when given with standard chemotherapy, but a benefit to patients in terms of improved survival or quality of life has not been shown. Dose intensification has increased partial and complete response rates in several tumor types. The higher complete remission rate may translate into improved survival; no convincing data have been published to date supporting that possibility. The duration of severe leukopenia (absolute neutrophil count, less than 100/microliters) after DICEP can be shortened from 8.5 days to 5-6 days with either granulocyte CSF (G-CSF) or GM-CSF. GM-CSF has been shown to decrease the duration of hospital stay from 18.7 to 9.6 days and to decrease the need for readmittance to the hospital for cytopenic fever. A high complete remission rate (35%) was seen in patients with breast cancer and durable complete remissions were achieved in patients with refractory non-Hodgkin lymphoma. CSF shortens the duration of postchemotherapy leukocyte nadirs and allows maintenance of dose when this is essential for a beneficial outcome. Substantial dose escalations are also possible with CSF support and can produce a high complete response rate that offers hope for improved survival in selected patients.


The interferons.

In the 10 years since the interferons (IFN) entered large-scale clinical trials, much has been learned much about their uses as single agents. alpha-IFN, the most widely studied, has shown antitumor and antiviral efficacy against various tumors and tumor-related viruses; it has been approved by the Food and Drug Administration for the treatment of patients with hairy cell leukemia, acquired immune deficiency syndrome-related Kaposi sarcoma, and condylomata acuminata. Although IFN are effective as single agents in certain clinical situations, increasing experience with these cytokines suggests that their greatest therapeutic potential may be in combination with other biologic response-modifying, cytotoxic, or antiviral drugs. Trials combining alpha-IFN with 5-fluorouracil to treat colorectal carcinoma or with zidovudine to treat acquired immune deficiency syndrome have shown the significant impact that IFN administered in conjunction with other carefully selected agents can have. To design the most effective combination regimens, better preclinical models that clarify the mechanisms of action of IFN and define their biochemical interactions with other agents are needed.


The role of interferons in the treatment of solid tumors.

Originally described as antiviral agents, interferons (IFN) were investigated as potential anticancer agents because of their antiproliferative and cytotoxic effects, their ability to activate specific components of the immune system, and their relatively modest toxicities. Interest intensified when durable complete remissions were observed in patients with hairy cell leukemia after IFN treatment; modest, but reproducible activity also was found against tumors such as melanoma and renal cell carcinoma which are unresponsive to conventional chemotherapy. Observations of synergy between IFN and cytotoxic drugs in vitro and in vivo suggested that IFN may have additional utility as modulating agents. Reports of major clinical trials using IFN either alone or in combination with other agents were reviewed. Activity was identified by disease site. Correlations were made with important preclinical studies. IFN has reproducible, but modest, single-agent activity against melanoma, renal cell carcinoma, and acquired immune deficiency syndrome-related Kaposi sarcoma. IFN may be useful in the treatment of a number of benign, in situ, or low-grade tumors. IFN in combinations with cytotoxic agents have demonstrated activity in solid tumors. Clinical trials using combinations of IFN and 5-fluorouracil or dacarbazine suggested a potential benefit for the combination compared with single-agent chemotherapy. These are preliminary findings that require confirmation, but they suggest that combination therapy should be investigated further. In early preclinical and clinical studies, combinations of IFN and other biologic agents, hormonal agents, and radiation therapy appear to be interesting. The role of IFN in the treatment of solid tumors may be evolving from that of single-agent therapy to combination therapy with other active agents. Additional studies are required to determine the optimal doses, schedules, and sequencing of these combination therapies.


Development of glutathione S-transferase placental form (GST-P) stained foci during hamster buccal pouch mucosa carcinogenesis.

The expression of the placental form of glutathione S-transferase (GST-P) using anti-rat liver GST-P antibody was investigated in hamster buccal pouch mucosa (HBPM) treated with 0.5% dimethylbenz[a]anthracene (DMBA) biweekly for 12 weeks. This preliminary study showed that the anti-rat liver GST-P antibody is applicable to the HBPM model and that DMBA treatment induced GST-P positive foci. These foci are randomly distributed and frequently involved the hyperplastic and dysplastic segments of the epithelium, as well as squamous cell carcinoma. Further study is needed to explore the kinetics of these GST-P positive foci.


Biochemical modulation of tumor cell energy: regression of advanced spontaneous murine breast tumors with a 5-fluorouracil-containing drug combination.

This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide plus 5-fluorouracil, in CD8F1 mice bearing spontaneous, autochthonous, breast tumors or first-passage advanced transplants of these spontaneous tumors. The combination and sequence of administration of these drugs were selected on the basis of known potentiating biochemical interactions. High performance liquid chromatography and nuclear magnetic resonance spectroscopy measurements of biochemical changes resulting from treatment with N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide indicated a severe depletion of cellular energy levels in the treated tumors. 6-Aminonicotinamide produced a severe block of the pentose shunt, and 5-fluorouracil severely inhibited both thymidylate synthase and thymidine kinase in the treated tumors. This quadruple drug combination, administered on a 10-11-day schedule, produced an impressive partial tumor regression rate of 67% of large, spontaneous, autochthonous, murine breast tumors and a tumor regression rate of 74% of first-passage transplants of the spontaneous breast tumors.


Identification and characterization of new antigenic fragments related to carcinoembryonic antigen in adult feces.

Antigens in human adult feces related to carcinoembryonic antigen (CEA) were analyzed with respect to their molecular masses, CEA domain compositions, and N-terminal amino acid sequences. By avoiding perchloric acid treatment, new fecal antigens related to CEA were identified. The fecal antigens revealed by Western blot were M(r) 78,000, 70,000, 60,000, 50,000, 44,000, 36,000, 33,000, and 25,000 and a species M(r) less than or equal to 14,000. Unlike native CEA, all of the fecal antigens were very poorly soluble in perchloric acid and did not bind to concanavalin A, suggesting that they had undergone significant deglycosylation in the digestive tract. The major fecal antigens were purified by immunoaffinity chromatography and their N-terminal amino acid sequences determined. FA78, FA60, FA33, and the M(r) less than or equal to 14,000 antigen had the N-terminal amino acid sequence of the CEA N-domain, and FA44 and FA25, the sequence of the CEA A2 domain. The CEA domain compositions of the fecal antigens were investigated by probing them with anti-CEA monoclonal antibodies of known domain specificities. The N-terminal amino acid sequences, immunoreactivities with anti-CEA monoclonal antibodies, and apparent molecular masses of the fecal antigens allowed the following domain assignments (based on CEA as N-A1B1-A2B2-A3B3): FA78, N-A1B1-A2B2-A3B3; FA60, N-A1B1-A2B2; FA44, A2B2-A3B3; FA33, N-A1B1; and FA25, A2B2. The M(r) less than or equal to 14,000 antigen was shown to be the N-domain of CEA or nonspecific cross-reacting antigen. FA36 was assigned the N-AB domain structure of nonspecific cross-reacting antigen. The results suggested that FA78, FA60, FA44, FA33, and FA25 were degradation products (including deglycosylation and proteolysis) of CEA and that FA36 was a degradation product of nonspecific cross-reacting antigen.


Correlation between in vivo toxicity and preclinical in vitro parameters for the immunotoxin anti-B4-blocked ricin.

Anti-B4-blocked ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody and the protein toxin, "blocked ricin." In blocked ricin, the galactose-binding sites of the ricin B-chain which mediate nonspecific binding to cells are blocked by covalently linked affinity ligands prepared from N-linked oligosaccharides of fetuin. Blocked ricin consists of two species, one with two covalently attached ligands and one with three covalently attached ligands. In a Phase I dose escalation clinical trial, Anti-B4-bR was administered to patients with relapsed and refractory B-cell neoplasms by 7-day continuous infusion. Although several different lots of Anti-B4-bR had similar IC37 values as determined by in vitro cytotoxicity testing on cultured human cell lines, these lots differed in their in vivo toxicity when administered to patients. Thus, IC37 values alone were not sufficient to predict in vivo toxicity. We report that the degree of cell kill at concentrations of drug that saturate the B4 antigen and murine 50% lethal dose values provide additional parameters that may be predictive of in vivo cytotoxicity. Furthermore, we performed detailed cytotoxicity studies of the ricin species containing two and three covalently attached ligands, respectively. In vitro cytotoxicity testing using these samples revealed that Anti-B4-bR made with blocked ricin containing two covalently attached ligands is capable of depleting five logs of target cells in an in vitro cytotoxicity assay, while Anti-B4-bR comprised of blocked ricin with three ligands can deplete only one log of cells. Log cell kill at antigen saturating concentration, murine 50% lethal dose and biochemical analysis of the composition of blocked ricin are therefore important considerations for establishing the potential efficacy and safety of Anti-B4-bR.


Chondrocytic differentiation of peripheral neuroectodermal tumor cell line in nude mouse xenograft.

We have established a cell line (KU-SN) from a peripheral neuroectodermal tumor originating in the left scapula of a 4-year-old girl. The original tumor was immunoreactive with antibodies for neurofilament proteins, neuron-specific enolase, vimentin, S100 protein, and beta 2-microglobulin. Dense core granules, 50-150 nm in diameter, were identified by electron microscopy. The cell line was established from tumor cells in metastatic lung fluid. KU-SN cells were immunoreactive with the antibodies for neurofilament proteins, vimentin, neuron-specific enolase, S100 protein, glial fibrillary acidic protein, cytokeratin, and carcinoembryonic antigen. Besides these neuronal features, KU-SN cells express type 2 collagen and insulin-like growth factor 1 receptor. The addition of insulin-like growth factor 1 (100 ng/ml) increased the growth rate of KU-SN cells 2.1-fold over control. Some cells were positive for Alcian blue and alkaline phosphatase staining. Cytogenetic analysis of KU-SN cells disclosed a reciprocal chromosomal translocation [t(11,22)]. Northern blot analysis of KU-SN cells demonstrated amplified expression of the c-myc gene but not the N-myc gene. When tumor cells were transplanted into nude mice, cartilage was formed. The cartilage was immunoreactive with the antibody for HLA-ABC, indicating that it was derived from the tumor cells, not from mouse tissue. Chondrocytic differentiation was not observed in xenografts of Ewing's sarcoma cell lines SK-ES or RD-ES or the peripheral neuroectodermal tumor cell line SK-N-MC. These results indicate that KU-SN cells represent primitive neural crest cells having the potential for chondrocytic differentiation.


Squamous metaplasia of normal and carcinoma in situ of HPV 16-immortalized human endocervical cells.

The importance of cervical squamous metaplasia and human papillomavirus 16 (HPV 16) infection for cervical carcinoma has been well established. Nearly 87% of the intraepithelial neoplasia of the cervix occur in the transformation zone, which is composed of squamous metaplastic cells with unclear origin. HPV DNA, mostly HPV 16, has been found in 90% of cervical carcinomas, but only limited experimental data are available to discern the role of HPV 16 in this tissue specific oncogenesis. We have initiated in vivo studies of cultured endocervical cells as an experimental model system for development of cervical neoplasia. Using a modified in vivo implantation system, cultured normal endocervical epithelial cells formed epithelium resembling squamous metaplasia, whereas those immortalized by HPV 16 developed into lesions resembling carcinoma in situ. In contrast, their ectocervical counterparts formed well differentiated stratified squamous epithelium and a lesion with mild dysplastic change, respectively. The HPV 16-immortalized cells showed in vivo cytokeratin expression patterns similar to their respective normal counterparts, confirming their different origins. Thus, this study provides direct experimental evidence for the transformation of simple epithelial cells of endocervical origin into stratified squamous metaplasia and indicates the differential susceptibility of endo- and ectocervical epithelial cells for conversion to cancer by HPV 16.


Reversal of divergent differentiation by ras oncogene-mediated transformation.

In embryogenesis, ovarian surface epithelial cells and ovarian granulosa cells arise through divergent differentiation from a common mesenchymal precursor, the urogenital ridge. In the adult rat, ovarian surface epithelial cells are nonsteroidogenic and keratin positive, while ovarian granulosa cells are steroidogenic and keratin negative. In culture, Kirsten murine sarcoma virus-transformed, tumorigenic ovarian surface epithelial cells continued to express keratin but also became steroidogenic. Transformed ovarian granulosa cells remained steroidogenic but also acquired keratins. Mesodermally derived cells from other sources did not show these differentiation-related changes in response to transformation. The results suggest that v-ras oncogenes may cause the reversion of adult, developmentally related cells to the phenotype of a common, multipotential precursor. They also demonstrate the capacity of v-ras to either induce or reduce the same differentiated characteristic, depending on the developmental history of the target cells.


Haemodynamic sequelae of pulmonary fibrosis following intratracheal bleomycin in rats.

Intratracheal instillation of bleomycin in rats has been extensively used as an animal model of pulmonary fibrosis in humans, although it produces a patchy, airway based response. We proposed that the haemodynamic sequelae of the bleomycin model might be less severe than those associated with more diffuse lung injury. Pulmonary and systemic haemodynamic indices were examined in adult Sprague-Dawley rats (approximately 400 g, n = 10) both at rest and during submaximal exercise on a rodent treadmill, approximately 60 days after intratracheal bleomycin (6 units.kg-1), and the results compared to a control group (n = 6). Compared to controls, mean pulmonary artery pressure (PPA) was increased by intratracheal bleomycin (p = 0.02) both at rest [24.5 (SEM 2.6) v 18.2(0.9) mm Hg] and during exercise [34.7(3.0) v 26.7(0.7) mm Hg]. PPA-pulse product was also increased, with a similar trend in right ventricular work index, but cardiac index was not altered. Right ventricular hypertrophy was noted on necropsy examination. Consistent with pulmonary fibrosis, lung dry weight, total protein, and hydroxyproline were also raised, and these values correlated strongly with (mean) PPA at rest (r2 = 0.86, 0.81, 0.69, respectively) and during exercise (r2 = 0.81, 0.79, 0.65, respectively). Packed cell volume was increased by intratracheal bleomycin, at 49(1) v 45(1)%, p = 0.02. CaO2 tended to decrease with exercise in the bleomycin group, although this was not statistically significant, while systemic oxygen delivery and consumption were not altered. Pulmonary hypertension and right ventricular hypertrophy occur in this model of lung fibrosis, and correlate with the severity of fibrosis. However, these sequelae were less severe than those previously demonstrated in association with crotalaria ingestion. We suggest that the haemodynamic sequelae of the intratracheal bleomycin model are consistent with patchy, airway based fibrosis, but reflect less well the haemodynamic sequelae of more diffuse fibrotic injury associated with systemic processes.


Reduction of chromium(VI) to chromium(V) by rat liver cytosolic and microsomal fractions: is DT-diaphorase involved?

Incubation of rat liver cytosolic or microsomal fractions with chromium(VI) led to a dramatic decrease in chromium(VI) mutagenicity, as determined by the Ames Salmonella assay using the TA100 tester strain. The cytosol-dependent decrease in chromium(VI) mutagenicity was found to be counteracted in the presence of dicumarol, an inhibitor of the cytosolic enzyme NAD(P)H:quinone oxidoreductase (DT-diaphorase). In order to determine whether DT-diaphorase is a significant factor in enzymatic reduction of chromium(VI) in rat liver tissue, cytosolic and microsomal fractions were analyzed for NAD(P)H-dependent chromium (VI) reductase activity leading to chromium(V) formation by using electron paramagnetic resonance (EPR) spectroscopy. Reaction of chromium(VI) with NADH or NADPH in the presence of either cytosolic or microsomal fractions led to the formation of stable chromium(V)--NAD(P)H complexes. When glucose 6-phosphate (G6P) was present in the reaction as part of a NADPH-generating system, stable chromium(V)--G6P complexes were formed in addition to the chromium(V)--NAD(P)H complexes. The chromium(V) complexes had g values of 1.980-1.982 and superhyperfine splitting constants of 0.8-0.9 characteristic of bis(diol)oxochromium(V) complexes. Inhibition of 90% of the cytosolic DT-diaphorase activity by dicumarol led to only partial (20-22%) inhibition of chromium(V) formation. Visible and EPR spectroscopic studies showed that purified DT-diaphorase had no detectable chromium(VI) reductase activity and did not catalyze formation of chromium(V). Inhibition of 69% of microsomal aryl hydrocarbon hydroxylase activity by ketoconazole led to partial (10%) inhibition of chromium(V) formation. These results indicate that intracellular NAD(P)H-dependent enzymatic reduction of chromium(VI) in rat liver cannot be attributed to the activity of any one enzyme in the cytosolic or microsomal fractions. DT-diaphorase appears to play an indirect role in decreasing chromium(VI)-induced mutagenicity in Salmonella, possibly through interaction with other redox active cellular components. The involvement of diols such as sugars and pyridine nucleotides in stabilizing intracellularly generated chromium(V) is discussed.


In vitro differentiation of trout oligodendrocytes: evidence for an A2B5-positive origin.

The molecular differentiation of oligodendrocytes derived from larval trout brain was studied in dissociated cell cultures using a range of cell type and stage specific antibodies. By double-labeling immunostaining using A2B5 antibodies in conjunction with antibodies against the myelin glycoproteins IP1 and IP2 evidence was obtained that oligodendrocytes of trout in vitro originate from A2B5+ precursor cells, which in terms of morphology closely resemble 0-2A progenitors of the mammalian CNS. Most surprisingly these cells did not differentiate in vitro beyond the level of IP2 expression, which signifies the initial step of oligodendroglial development in vivo. Hence it appears that in trout oligodendrocytes the initiation of the developmental program is intrinsically regulated, whereas further maturation of the cells requires appropriate environmental stimulation.


The neonatal rabbit brain glucose transporter.

The Glut 1 (Hep G2/rat brain) isoform of glucose transporter is expressed in significant amounts in adult mammalian brain. The purpose of our present study was to determine the brain cellular localization of Glut 1 during the late newborn stage of development, when brain cellular proliferation and differentiation is highly active. Employing immunohistochemistry and in-situ hybridization in 10-day-old neonatal rabbit brain sections, we undertook cellular localization of Glut 1 expression. Glut 1 protein and mRNA were mainly noted in considerable amounts within the 10-day-old brain microvasculature. Lower concentrations of Glut 1 immunoreactivity were present in certain glial cells found within the deeper cortical layers of brain. Northern blot analysis of total RNA from isolated microvasculature-enriched preparation, isolated and cultured neuronal and glial cells, whole brain and whole brain with the exclusion of microvasculature obtained from the 10-day-old, revealed the universal presence of a approximately 2.8 kb Glut 1 mRNA with the exception of the neuron-enriched cultures. We conclude that during the neonatal period, when parenchymal cellular proliferation is at a peak, Glut 1 is localized not only to the microvasculature but also to certain cells which express glial morphological characteristics. The neuronal cells either do not express Glut 1 or express minute amounts.


IL-4 down-regulates the surface expression of CD5 on B cells and inhibits spontaneous immunoglobulin and IgM-rheumatoid factor production in patients with rheumatoid arthritis.

There is evidence to suggest that CD5+ B cells may be associated with autoimmunity, e.g. they are increased in patients with rheumatoid arthritis (RA). In this study, we found that the expression of CD5 on RA B cells increased spontaneously, following culture for up to 4 days in vitro in the absence of T cells, supporting the idea that the CD5+ B cell possesses distinctive features. The spontaneous increase of CD5 expression was down-regulated by recombinant IL-4 (rIL-4). Other cytokines studied (rIL-1 alpha, rIL-2, rIL-5, rIL-6) did not alter CD5 expression. Studies of antibody production showed that rIL-4 could reduce spontaneous production of total IgG and IgM in non-stimulated RA T plus B cell cultures. Spontaneous production of IgM rheumatoid factor (IgM-RF), measured by a newly developed avidin-biotin complex ELISA, was also reduced by rIL-4. Furthermore, rIL-4 reduced the increase in IgM-RF production observed on stimulation with Staphylococcus aureus Cowan I (SAC) or pokeweed mitogen (PWM). Thus, IL-4 might act as a regulator of the development of abnormal B cell differentiation in patients with RA.


Immunoglobulin heavy chain variable region gene utilization by B cell hybridomas derived from rheumatoid synovial tissue.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects synovial joints. Activated B lymphocytes and plasma cells are present in the synovial tissue and are thought to contribute to the immunopathology of the rheumatoid joint. To investigate rheumatoid synovial B lymphocytes, we have generated B cell hybridomas from synovial tissue of an RA patient. Here we describe the immunoglobulin VH gene repertoire of eight IgM- and 10 IgG-secreting synovial-derived hybridomas. The VH4 gene family is highly represented (38.5%) in this panel of hybridomas compared with the frequency of VH4 gene expression in circulating B lymphocytes reported previously (19-22%) and with the VH4 gene frequency we observed in a panel of hybridomas derived in the same manner from the spleen and tonsil of normal individuals (19%). The increased frequency of VH4 gene expression was not due to the expansion of a single B cell clone in vivo as none of these hybridomas was clonally related. Two synovial-derived hybridomas secreted autoantibodies; one (VH3+) secreted an IgM-rheumatoid factor (RF) and the other (VH4+) secreted IgM with polyreactive binding to cytoskeletal proteins and cardiolipin. The antibodies secreted by the remaining synovial-derived hybridomas were not reactive with the autoantigens tested. The VH gene usage in a proportion (5/17) of synovial-derived hybridomas that expressed CD5 antigen provided preliminary evidence that CD5+ B cells in RA synovium have a similar increase of VH4 gene expression reported for CD5+ B cells from normal individuals and patients with chronic lymphocytic leukaemia.


Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO.


Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.

Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.


Distending the uterus: what medium is best?

Because most diagnostic hysteroscopes take only a few minutes, the experienced hysteroscopist generally prefers to use CO2 distention. For extensive operative procedures, especially submucous myomectomy, glycine or sorbitol usually are chosen.


Effects of cold treatment and ketosis induced by starvation on interferon production in leukocytes of lactating cows.

The production of interferons in blood and milk leukocytes of three groups of cows was measured to determine the effect of 6-days cold treatment (-2 degrees to -8 degrees C) and/or starving. The first group (cold) was treated with low ambient temperature (-2 degrees C to -8 degrees C) 11 hours every day for 6 days, the second (cold and starved) was treated with low temperature and starved for 6 days. The third group (controls) was fed normally and kept in a barn at room temperature (18 degrees to 20 degrees C). The leukocytes of the control and the cold treated cows responded normally to interferon induction with Newcastle Disease Virus (NDV) and mitogens: phytohemagglutinin (PHA) and concanavalin A (ConA). The cows treated with low temperature and starved for 6 days developed biochemical blood changes of ketosis. Leukocytes of these cows with ketosis produced less interferon (p less than 0.05) than before starvation and less than leukocytes of the control cows and the cold treated cows. It can be assumed that ketosis caused by starving decreases the ability of a cow's leukocytes to produce interferons.


Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo.

Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, alpha 2(IX) collagen mRNA, and transcripts of the short form alpha 1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24-36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form alpha 1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type II and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form alpha 1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form alpha 1(IX) collagen transcripts which lack the 5' region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative polyadenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.


Angiotensin converting enzyme inhibitors versus digoxin for the treatment of congestive heart failure.

Angiotensin converting enzyme (ACE) inhibition and digoxin may be used in the management of heart failure. Digoxin increases myocardial contractility in vitro, and has a modest but durable beneficial effect in congestive heart failure due to impaired left ventricular systolic function. ACE inhibitors have clear beneficial effects in all grades of heart failure and, in addition, modify the natural history and reduce mortality. Comparative studies in mild to moderate heart failure reveal a tendency towards greater benefits and tolerability of ACE inhibitors over digoxin. ACE inhibition is indicated, in conjunction with diuretic therapy, for all grades of heart failure. Digoxin is best reserved for patients with atrial fibrillation and a rapid ventricular response, and for those whose heart failure is not controlled with an ACE inhibitor plus a diuretic. In patients with heart failure following myocardial infarction, digoxin is of modest benefit. Digoxin should be administered slowly and carefully to avoid acute vasoconstriction and toxicity. Provisional data suggest ACE inhibitors are also beneficial in these patients. However, the results of clinical trials presently in progress are required to clarify their role following myocardial infarction.


Drug treatment of tuberculosis--1992.

The impact of the acquired immunodeficiency syndrome (AIDS) pandemic has made tuberculosis an increasing worldwide problem, and the effectiveness of modern chemotherapy has been blunted by the high incidence of primary drug resistance, especially in developing countries. The prospect of finding new and highly effective drugs similar to isoniazid or rifampicin is dim, yet the maximum benefits from the existing drugs which are highly effective have not been received. A 6-month regimen of isoniazid plus rifampicin, supplemented by pyrazinamide during the first 2 months, for treatment of uncomplicated tuberculosis is highly effective and the regimen of choice. Ethambutol should be added if the risk of isoniazid resistance is increased. A regimen of isoniazid, rifampicin, pyrazinamide and streptomycin for 4 months provides effective defence against smear-negative pulmonary tuberculosis. Re-treatment of multiple drug-resistant tuberculosis remains a difficult therapeutic problem. At least 3 drugs that the patient has never previously received, and that are effective according to laboratory susceptibility testing, must be used. Preventive therapy against tuberculosis is accomplished with isoniazid for 6 to 12 months, although rifampicin plus isoniazid for 3 months has been used in the United Kingdom with success. In a mouse model, rifampicin plus pyrazinamide for 2 months is more effective than isoniazid for 6 months as preventive treatment. Patient noncompliance with medication remains the biggest problem in tuberculosis control, and is a complex issue. It can only be resolved by multiple approaches. Intermittent directly observed short course chemotherapy is a major, but not the only, possible solution.


Superficial fungal infections of the skin. Diagnosis and current treatment recommendations.

Superficial fungal infections are common. Most diagnoses of fungal infections of the skin can be made by physical examination, assisted by the use of a Wood's lamp, skin scrapings for microscopic examination, and fungal cultures. Dermatophyte infections are common at all ages, in both sexes, and they have a worldwide distribution. These infections include tinea capitis, tinea cruris, tinea pedis, tinea corporis, tinea manuum and tinea barbae. Tinea versicolor, caused by Malassezia furfur, and candidal infections are also common. Treatment modalities include oral and topical agents. Good personal hygiene is an important adjunct to antifungal therapy. Decisions regarding the appropriateness of therapy in a given patient must take into account the extent and location of the infection, the benefits and risks of each of the treatments, and cost. Oral therapies include griseofulvin, ketoconazole, and itraconazole. There are a large variety of topical treatments, including nystatin, selenium sulfide, tolnaftate, haloprogin, miconazole, clotrimazole, and sodium thiosulfate. Important to successful treatment is compliance with what is sometimes a long course of treatment, and good personal hygiene.


Current recommendations for the treatment of Lyme disease.

Lyme disease is a multisystem inflammatory disease caused by infection with Borrelia burgdorferi. Soon after the tick bite which transmits the infection, the pathognomonic skin rash erythema chronicum migrans occurs in 50 to 70% of patients, often with associated symptoms resembling a 'summer cold' or viral infection. Therapy for this stage of disease consists of 3 to 4 weeks of oral therapy. The agents currently used are: amoxicillin (500 mg 3 or 4 times daily) with or without probenecid 500 mg 3 times daily, doxycycline (100 mg twice daily), or tetracycline (500 mg 4 times daily). Longer duration therapy has never been evaluated and therefore is not currently indicated. Even patients with severe early manifestations of Lyme disease should be treated orally. Later features of Lyme disease include carditis and neurological disease, which can occur days to approximately 9 months after the onset of illness, and arthritis and neurological disease which can occur weeks to years after the onset of the illness. Treatment at this stage is with 2 to 3 weeks of intravenous antibiotics, currently cefotaxime (3 g every 12 hours), ceftriaxone (1 g every 12 hours or 2 g every day) and benzylpenicillin (14 g in divided doses). There is no evidence that longer duration therapy is indicated or more efficacious. The exception to this suggestion is the patient with isolated facial seventh cranial nerve palsy; if such a patient has no other signs or symptoms to suggest Lyme disease and has normal spinal fluid, oral therapy is usually sufficient, although some physicians will give concomitant corticosteroids to hasten the resolution of the palsy. Of major consequence to the practitioner and patient is the possibility that persistent symptoms (e.g. fibromyalgia) may be caused by a process which is no longer antibiotic-sensitive. Special care in the management of so-called 'chronic Lyme disease' is crucial lest the clinician prescribes prolonged or unending courses of antibiotics for such noninfectious problems.


Treatment options for the pharmacological therapy of neonatal meningitis.

Neonatal bacterial meningitis has a relatively low incidence in developed countries, but continues to cause morbidity and mortality despite advances in antimicrobial therapy. Bacterial pathogens commonly associated with neonatal meningitis include Group B streptococci, Escherichia coli K1 and other coliforms, Listeria monocytogenes and staphylococci. As it can be difficult to differentiate meningitis from septicaemia in neonates, empirical antibiotic therapy should be effective for both. Selection of an empirical antibiotic regimen should be based on: (a) bacterial prevalence and susceptibility; (b) drug characteristics; (c) postnatal age at the onset of disease; and (d) patient-specific factors. A penicillin in combination with an aminoglycoside or cefotaxime is commonly used in empirical therapies. The increased risk of staphylococcal infection in older neonates requires consideration of an antistaphylococcal antibiotic in the empirical therapy regimen. Once a causative organism has been identified, antimicrobial therapy should be directed towards that pathogen. Duration of therapy remains empirical, but should be at least 7 days for documented bacterial meningitis. Viral meningitis continues to have a high mortality despite the availability of antiviral agents. Adjunctive therapies may further reduce the morbidity and mortality of meningitis. While most of these therapeutic options have not been investigated in neonates, they may prove to be of benefit in the future. Anti-inflammatory agents, such as glucocorticoids, nonsteroidal anti-inflammatory agents and immunoglobulin, may modulate the inflammatory response of a meningeal infection. Other possible therapies in neonatal meningitis include cerebral blood flow modulators and disease prevention with maternal vaccines and perinatal antibiotics. Practical aspects of drug therapy such as route of administration and serum drug concentration monitoring can improve both drug therapy and patient outcome. While antibiotics have greatly improved the treatment outcome of neonatal meningitis, it is clear that additional intervention will be required to increase cure rates and reduce sequelae.


Dexfenfluramine. A review of its pharmacological properties and therapeutic potential in obesity.

Dexfenfluramine stimulates serotoninergic activity by inhibiting serotonin reuptake into presynaptic neurons and by enhancing its release into brain synapses. Based on the serotonin hypothesis of appetite control these effects would be expected to reduce food intake and thus body-weight. Studies in animal models and severely overweight patients have confirmed the effectiveness of dexfenfluramine as a weight-reducing agent which appears to be well tolerated. Permanent weight loss is the goal of weight-reducing strategies and, based on current clinical evidence, dexfenfluramine appears to exert a weight reducing effect over periods of up to 12 months without development of tolerance, a problem that has limited the long term use of other pharmacological agents used in the treatment of this disorder. Dexfenfluramine facilitated weight loss in patients who had not responded satisfactorily to other weight-reducing strategies, prevented relapse in those patients who had achieved weight reduction by other methods, and corrected disturbed eating patterns (and therefore reduced weight gain) in small studies involving patients with premenstrual syndrome, seasonal affective disorder and nicotine withdrawal syndrome. Follow-up of the longest study reported with dexfenfluramine suggests that continued therapy is required in severely overweight patients if weight loss is to be maintained. Dexfenfluramine has not been directly compared with nonpharmacological measures of weight control such as behaviour modification or exercise programmes. The decision that pharmacological means are indicated in overweight patients must be highly individualised, and must consider the many complex factors that often contribute to overweight states, as well as the anticipated magnitude of drug effect. Despite such a cautionary note, and the expected need (at this stage of its development) for an expanded clinical study programme in certain areas, dexfenfluramine is a clear advance in the pharmacological approach to improved management of overweight individuals.


Cibenzoline. A review of its pharmacological properties and therapeutic potential in arrhythmias.

Cibenzoline is a class I antiarrhythmic drug with limited class III and IV activity which can be administered orally or intravenously. An elimination half-life of about 8 to 12 hours permits twice daily administration, although age and renal function must be considered when determining dosage. Cibenzoline has some activity in ventricular and supraventricular arrhythmias, including drug-refractory ventricular tachycardia or ventricular arrhythmias following recent acute myocardial infarction, although results in patients with sustained ventricular tachycardia are less promising. In comparative trials, cibenzoline has demonstrated efficacy similar to or better than that of a variety of other class I antiarrhythmic drugs and was at least as well tolerated, with a more convenient dosage schedule. However, further studies to clarify the proarrhythmic effects of cibenzoline and its use in patients with impaired left ventricular function are required, and the use of cibenzoline (and other class I antiarrhythmic agents) in patients with other than potentially lethal ventricular arrhythmias should be avoided following the results of the CAST studies. Thus, cibenzoline is an effective antiarrhythmic agent with a favourable pharmacokinetic profile that may be considered with other class I drugs in patients requiring therapy for high risk arrhythmias.