Immunoreactive beta-core-like material in normal postmenopausal urine: human chorionic gonadotrophin or LH origin? Evidence for the existence of LH core.

Material with the immunochemical properties of the beta-core of human chorionic gonadotrophin (hCG) can be found in the urine of normal postmenopausal women. However, we have been unable to detect intact hCG (using an assay which is specific for the alpha-beta heterodimer of intact hCG) in serum of such subjects. The levels of serum LH and urinary beta-core were compared in matched samples from 28 women (serum LH: median 27 U/l, range 4-70 U/l, urinary beta-core: median 0.27 microgram/l, range less than 0.05-0.645 microgram/l). Urine (4 litres) from three postmenopausal women was concentrated, dialysed and subjected to gel exclusion chromatography on Sephadex G-100. Fractions were analysed by specific assays for LH, intact hCG, total beta-hCG (free beta-subunit and intact hCG), free alpha-subunit and beta-core. Material eluting at the expected position of the beta-core fragment of hCG was detected in all three samples by the beta-core, beta-hCG and LH assays, despite the fact that the LH antibody does not recognize the authentic beta-core of pregnancy. Electrophoresis and Western blotting of the concentrated urines revealed that material of the same molecular size as beta-core was recognized by the antibody to LH but not by a monoclonal antibody raised to free beta-hCG which also recognizes the beta-core molecule of hCG. We conclude that the predominant core-like material identified in postmenopausal urine is probably derived from the beta-subunit of LH.


Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type 1 and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA 1 as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the down-regulation of latent antigens can be reversed.


Cloning and expression of Trypanosoma cruzi ribosomal protein P0 and epitope analysis of anti-P0 autoantibodies in Chagas' disease patients.

Chagas' disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a major cause of heart failure in endemic areas. Antigenic mimicry by T. cruzi antigens sharing epitopes with host macromolecules has been implicated in the pathogenesis which is thought to have a significant autoimmune component. We report herein on the cloning and characterization of a full-length cDNA from a T. cruzi expression library encoding a protein, TcP0, that is homologous to the human 38-kD ribosomal phosphoprotein HuP0. The T. cruzi P0 protein shows a clustering of residues that are evolutionarily conserved in higher eukaryotes. This includes an alanine- and glycine-rich region adjacent to a highly charged COOH terminus. This "hallmark" domain is the basis of the crossreactivity of the highly immunogenic eukaryotic P protein family. We found that T. cruzi-infected individuals have antibodies reacting with host (self) P proteins, as well as with recombinant TcP0. Deletion of the six carboxy-terminal amino acids abolished the reactivity of the T. cruzi infection sera with TcP0. This is similar to the specificity of anti-P autoantibodies described for a subset of patients with systemic lupus erythematosus (SLE) (Elkon, K., E. Bonfa, R. Llovet, W. Danho, H. Weissbach, and N. Brot. 1988. Proc. Natl. Acad. Sci. USA. 85:5186). These results suggest that T. cruzi P proteins may contribute to the development of autoreactive antibodies in Chagas' disease, and that the underlying mechanisms of anti-P autoantibody may be similar in Chagas' and SLE patients. This study represents the first definitive report of the cloning of a full-length T. cruzi antigen that mimics a characterized host homologue in structure, function, and shared antigenicity.


CD31 expressed on distinctive T cell subsets is a preferential amplifier of beta 1 integrin-mediated adhesion.

The CD31 (platelet endothelial cell adhesion molecule-1 [PECAM-1]/endothelial cell adhesion molecule [endoCAM]) molecule expressed on leukocytes, platelets, and endothelial cells is postulated to mediate adhesion to endothelial cells and thereby function in immunity, inflammation, and wound healing. We report the following novel features of CD31 which suggests a role for it in adhesion amplification of unique T cell subsets: (a) engagement of CD31 induces the adhesive function of beta 1 and beta 2 integrins; (b) adhesion induction by CD31 immunoglobulin G (IgG) monoclonal antibodies (mAbs) is sensitive, requiring only bivalent mAb; (c) CD31 mAb induces adhesion rapidly, but it is transient; (d) unique subsets of CD4+ and CD8+ T cells express CD31, including all naive (CD45RA+) CD8 T cells; and (e) CD31 induction is selective, inducing adhesive function of beta 1 integrins, particularly very late antigen-4, more efficiently than the beta 2 integrin lymphocyte function-associated antigen-1. Conversely, CD3 is more effective in inducing beta 2-mediated adhesion. Taken together, these findings indicate that unique T cell subsets express CD31, and CD31 has the capacity to induce integrin-mediated adhesion of T cells in a sensitive and selective fashion. We propose that, in collaboration with other receptors/ligands, CD31 functions in an "adhesion cascade" by amplifying integrin-mediated adhesion of CD31+ T cells to other cells, particularly endothelial cells.


Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin.

Nitric oxide (NO) is a short-lived biologic mediator that is shown to be induced in various cell types and to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of the inducible type of the NO synthase (NOS). However, there is limited evidence for the existence of such inducible NOS in a human cell type. We show here the induction of NO biosynthesis in freshly isolated human hepatocytes (HC) after stimulation with interleukin 1, tumor necrosis factor (TNF), IFN-gamma, and endotoxin. Increased levels of nitrite (NO2-) and nitrate (NO3-) in culture supernatants were associated with NADPH-dependent NOS activity in the cell lysates. The production of NO2- and NO3- was inhibited by NG-monomethyl L-arginine and was associated with an increase in cyclic guanylate monophosphate release. The data presented here provide evidence for the existence of typical inducible NO biosynthesis in a human cell type.


In a small multideterminant peptide, each determinant is recognized by a different V beta gene segment.

Given the vast potential for diversification of the T cell receptor (TCR) repertoire and the fact that V(a) beta mice exist in the wild, it would have been predicted that in spite of the absence of 10 TCR V beta gene segments, V(a) beta mice would still have been able to produce an antigen-specific T cell response to all determinants. We have recently shown that Vb beta mice, with a wild-type TCR V beta repertoire, respond to peptide 110-121 of sperm whale myoglobin, with a majority of T cells expressing TCR V beta 8.2 and restricted to a hybrid I-A(d)/I-E(d) major histocompatibility complex molecule, and a smaller number of T cells expressing TCR V beta 8.1 and restricted to the I-A(d) molecule. However, V(a) beta mice, lacking members of the TCR V beta 8 gene family, responded only with I-A(d)-restricted T cells. Thus, it appeared that the I-A(d)-restricted response was less constrained, or more plastic. We now show that the two separate panels of I-A(d)-restricted T cell hybrids derived from V(a) beta or Vb beta mice in fact recognize distinct determinants within the same peptide 110-121. The determinant recognized by V(a) beta T cells is NH2 terminal (core: 110-118) with an absolute requirement for the residue Ala-110 for a successful interaction with TCRs. On the other hand, Vb beta T cells recognize the COOH-terminal region (core: 112-118) on the same peptide with an absolute requirement for COOH-terminal residue 118. In the dominance hierarchy displayed by the three distinct determinants of peptide 110-121, V(a) beta mice cannot recognize the two most dominant: the hybrid I-A(d)/I-E(d)-restricted determinant and the COOH-terminal, I-A(d)-restricted determinant. They instead respond with T cells specific for a third, distinctly NH2-terminal determinant. Our results show a strict association between recognition of a particular specificity and TCR V beta usage. This evidence suggests that even when a small peptide induces a heterogenous group of TCR V beta S, this need not be considered evidence for plasticity. Rather, at the level of individual determinants within the peptide, the results can point in the opposite direction, towards serious constraints in recognition at the level of V beta expression.


Identification of T cell epitopes occurring in a meningococcal class 1 outer membrane protein using overlapping peptides assembled with simultaneous multiple peptide synthesis.

The meningococcal class 1 outer membrane protein (OMP) plays an important role in the development of protective immunity against meningococcal infection, and is therefore considered to be a promising candidate antigen (Ag) for a meningococcal vaccine. The induction of an effective antibody response entirely depends upon T helper cells. To identify T cell epitopes of the OMP, we prepared 45 overlapping synthetic peptides representing the entire sequence of the class 1 protein of reference strain H44/76. Fully automated simultaneous multiple peptide synthesis (SMPS) was used to assemble the 45 twenty mer which overlapped by 12 amino acid residues on a 12 mumol scale. The peptides were tested for recognition by peripheral blood mononuclear cells (PBMC) obtained from 34 volunteers. Surprisingly, all synthetic peptides induced proliferative responses of PBMC isolated from one or more human histocompatibility leukocyte antigen (HLA)-typed immune adults. With PBMC from seven nonimmune donors, no proliferative response was observed. Immunodominant regions were found, recognized by PBMC from many volunteers, irrespective of their HLA type. Most of the immunodominant T cell epitopes are located outside the variable regions and, thus, will be conserved among different meningococcal (and gonococcal) strains. Furthermore, the overlapping peptides could be used to identify the epitopes recognized by OMP-specific T cell clones with known HLA restriction. It is interesting that the epitopes defined with the clones occur in highly conserved areas, shared by all neisserial porin proteins. In summary, this analysis of the T cell response to the meningococcal class 1 OMP constitutes a complete study of reactivity to a foreign protein, and illustrates some important features of Ag recognition by T cells. Our data demonstrate unexpected diversity in the T cell recognition of the OMP, and imply that the T cell repertoire against foreign Ag may be greater than previously assumed. This observation is supported by recent data on the interaction of peptide and major histocompatibility complex (MHC) class II, the latter being much less selective than MHC class I. Finally, a comparative analysis pointed out the limitations of algorithms predicting T cell determinants, and the importance of the empirical methodology provided by SMPS.


Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothelial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (alpha 4 beta 1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosclerosis, and tumor cell metastasis. The major form of VCAM-1 (VCAM-7D) has seven extracellular immunoglobulin (Ig)-like domains, of which the three NH2-terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. By functional analysis of VCAM-7D relative to VCAM-6D (a minor 6-domain form of VCAM-1 in which domain 4 is deleted because of alternative splicing), and chimeric constructs between VCAM-1 and its structural relative intercellular adhesion molecule-1 (ICAM-1), we show that either the first or the homologous fourth domain of VCAM-1 is required for VLA-4-dependent adhesion. Either of these binding sites can function in the absence of the other. When both are present, cell binding activity is increased relative to monovalent forms of the molecule. The homologous binding regions appear to have originated by internal duplication of a portion of a monovalent ancestral gene, before the mammalian radiation. Thus VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication. We have also produced a new class of anti-VCAM-1 monoclonal antibodies that block domain 4-dependent adhesion, and demonstrate that both binding sites participate in the adhesion function of VCAM-1 on endothelial cells in vitro. Therefore both sites must be blocked in clinical, animal, or in vitro studies depending on the use of anti-VCAM-1 antibodies to inactivate the VCAM-1/VLA-4 adhesion pathway.


Development of an antiserum to the midportion of substance P: applications for biochemical and anatomical studies of substance P-related peptide species in CNS tissues.

This report describes the generation and biochemical characterization of a high-affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4-10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high-affinity COOH-terminally directed anti-substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P-like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus anti-substance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P-like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti-substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC-fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P-glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohistochemical analyses employing affinity-purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.


Growth factor (M-CSF) and antigenic properties of macrophages in meningioma.

In meningiomas, transformed meningeal cells can share morphological aspects (in hemangiopericytic meningioma) and antigenic properties (i.e.: HLA-DR antigens expression) with elements of the monocyte/macrophage lineage. In this report, we describe a case of a highly vascular meningioma where numerous tumor cells, studied with immunohistochemical methods, present phenotypic properties of macrophages. Moreover, the cerebrospinal fluid (CF) analysis disclosed, using a biological assay, a high level of a growth factor for monocytic elements, the Macrophages Colony Stimulating Factor (M-CSF). Our findings may confirm that transitional aspects between different mesenchymal cells could be present in meningiomas.


Spatiotemporal increases in epidermal growth factor receptors following peripheral nerve injury.

Non-neuronal cells of peripheral nerve respond to axonal injury with a series of cellular changes that facilitate neuronal regeneration. To characterize the potential role of the epidermal growth factor (EGF) family of proteins in this response, we monitored the expression of EGF receptor mRNA and protein in the injured rat sciatic nerve. EGF receptor mRNA is synthesized in both primary cultured fibroblasts and Schwann cells, and Schwann cells express EGF receptor-like immunoreactivity. In situ hybridization and immunocytochemistry revealed that EGF receptor mRNA and protein are expressed in Schwann cells and fibroblasts of the sciatic nerve in vivo, and that receptor levels increase following nerve injury. Thirty-six hours postlesion, EGF receptors were expressed in gradients along the nerve both proximal and distal to the lesion, with the highest levels localized adjacent to the transection site. By 72 hr, receptor levels were maintained in a gradient in the proximal segment, but were uniformly increased throughout the portions of the distal segment that were analyzed. These changes were similar to those observed for low-affinity NGF receptor mRNA and protein, with transection causing increased expression in both Schwann cells and fibroblasts. Northern blots confirmed that primary cultured fibroblasts express low-affinity NGF receptor mRNA. To determine whether spatiotemporal gradients were a general characteristic of the nerve injury response, we monitored expression of the mRNA encoding the major myelin protein P0. Levels of P0 mRNA decreased initially in cells immediately adjacent to the transection site and, by 72 hr, were uniformly decreased throughout the distal segment. These data suggest that members of the EGF family of proteins may play a role in the peripheral nerve response to injury, and demonstrate a generalized gradient of cellular responses that commence at the transection site and progress distally in the nerve in the absence of intact axons.


Olfactory receptor neurons from antennae of developing male Manduca sexta respond to components of the species-specific sex pheromone in vitro.

Male-specific olfactory receptor neurons, dissociated from developing antennae of the moth Manduca sexta and grown in long-term primary cell culture, can respond to at least one component of the female moth's sex-pheromone blend with the opening of a nonspecific cation channel. This response does not require the coapplication of pheromone-binding protein.


NGF induces the expression of the VGF gene through a cAMP response element.

NGF is a peptide growth factor that plays a key role in the differentiation and survival of neurons in both the PNS and CNS. NGF acts through both transcription-dependent and transcription-independent mechanisms to regulate the differentiation of PC12 cells. To better understand the regulation of gene expression by NGF, we have defined a cis-acting sequence that is immediately upstream of the transcription start site of the VGF (a2/NGF33.1) gene that is required for induction by NGF. Within this sequence is a consensus cAMP response element (CRE) embedded in a 14 base pair palindrome. Mutations in this CRE eliminate induction of the VGF gene both by NGF and by agents that act via cAMP. Although this sequence confers transcriptional induction by both NGF and cAMP, it is not sufficient to allow induction by epidermal growth factor, acidic or basic fibroblast growth factor, or phorbol 12-myristate 13-acetate (PMA). Thus, this sequence defines an element that is selectively activated by NGF and cAMP. Promoter fragments from the VGF gene that include the core CRE efficiently bind the inducible transcription factor CREB, while fragments bearing mutations that eliminate NGF and cAMP inducibility fail to do so. Sequence comparisons and hybridization studies indicate that there are at least two alternatively spliced forms of VGF mRNA, and the accumulation of both of these forms is similarly regulated by NGF and cAMP.


Tumor necrosis factor modulates Ca2+ currents in cultured sympathetic neurons.

The effect of recombinant human tumor necrosis factor-alpha (rhTNF) on calcium currents of cultured neurons from neonatal rat superior cervical ganglia (SCG) was studied using whole-cell patch-clamp technique. We found that rhTNF-treated SCG neurons exhibited increased calcium current density without significant alteration in the steady-state parameters of activation and availability. The fraction of the current sensitive to dihydropyridines and omega-conotoxin also remained unchanged. Recovery from slow inactivation of the current, but not recovery from fast inactivation, was prolonged in rhTNF-treated cells when compared to that of control cells. We conclude that immune peptides such as rhTNF can alter cellular functions of sympathetic neurons via modulating ionic conductances. However, these changes observed in calcium currents of SCG neurons cannot account for the effect of rhTNF on norepinephrine secretion observed in a previous study. It is proposed that rhTNF exerts an additional effect at a later event in the exocytotic process.


The lack of effect of basic and acidic fibroblast growth factors on the naturally occurring death of neurons in the chick embryo.

In vivo treatment of developing chick embryos with acidic and basic fibroblast growth factors (aFGF and bFGF) failed to affect the differentiation and survival of several populations of developing neurons in the CNS and PNS. All of the neuronal populations examined are known to undergo naturally occurring cell death, and they include spinal and cranial motoneurons, dorsal root ganglia, sympathetic ganglia, nodose ganglia, ciliary ganglia, and sympathetic preganglionic neurons in the PNS, as well as the accessory oculomotor nucleus, the isthmo-optic nucleus, and the brainstem auditory nuclei laminaris and magnocellularis in the CNS. Despite the lack of effect of bFGF on neuronal survival and differentiation, in vivo treatment increased the serum levels of bFGF and stimulated the proliferation of non-neuronal cells in the spinal cord. Therefore, although the administration of exogenous FGF to the developing chick embryo in vivo clearly has some biological activity in the CNS, it was nonetheless ineffective in promoting neuronal survival or differentiation. These data do not support the idea that FGF is a physiologically relevant neurotrophic agent in the developing avian nervous system.


Intrinsic lattice connections of macaque monkey visual cortical area V4.

We made focal iontophoretic as well as larger pressure injections (n = 30; 19 used for most analyses) of the tracer biocytin in visual area V4 of six macaque monkeys. The resulting transported label enabled mapping of intrinsic inter- and intralaminar connections within the region. We found that pyramidal neurons of layers 2 and 3 make extensive lateral projections within area V4, with oval or circular patches of terminals in layers 1-3. Any small patch of tissue (approximately 250 microns wide) injected in the superficial layers appeared to connect reciprocally to patches scattered up to 3 mm around the injection. The patches of terminal label measure 250-450 microns across, spaced roughly 600 microns (range, 450-1300 microns) center to center, and where most densely packed they occupy 33% of the cortical area with approximately 3-4 patches/mm2. Small injections in layers 4 and 6 did not produce contributions to these patchlike lattice connections, while injections in layer 5 gave very weak rising contributions to the superficial layer patch system. Large pressure injections of biocytin gave wider spread and more densely labeled patches, but their size and spacing appeared much the same as with small injections. The V4 pattern of label was compared to the patterns seen in areas V1 and V2 after similar-sized injections of biocytin; patches in V1 and V2 were slightly smaller (roughly 250-300 microns across) and more closely spaced (425-450 microns center to center), consistent with earlier measures in these areas using HRP. The peak areal occupancy of patches was approximately the same, 27-33% in each region. We interpret these findings as indicating a functional repeat distance of 450-600 microns in area V4 (fixed tissue measures) with a patchy, discontinuous layout. Given the dimensions of the V4 intrinsic connectional system demonstrated here, it seems unlikely that it relates directly to the topography of specific afferent terminations or efferent neuron groups, which appear from other studies to have a larger scale of repeat (2-3 mm). However, patches in V4 tend to aggregate, forming clusters extended in the mediolateral direction, suggesting a possible relation to coarser connection zones.


A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus.

The presynaptic terminal and axon of neurons can undergo structural changes in response to environmental signals. Since these changes require protein synthesis in the cell body, the needs of the periphery must somehow be communicated to the cell soma. To look for such a mechanism, we used artificial protein constructs with properties expected of a signal that is transported from the axon to the nucleus. One construct consisted of the nuclear import signal peptide (sp) of the SV40 large T antigen, coupled to human serum albumin (HSA) and rhodamine (r). When injected into the axoplasm of Aplysia californica neurons in vitro, the rHSA-sp was transported in the retrograde direction through the axon to the cell body and then into the nucleus. Little, if any, moved in the anterograde direction toward growth cones. The retrograde movement of injected rHSA-sp was rapid (greater than 25 mm/d) and depended upon intact microtubules. The sp portion of rHSA-sp provided access to both the retrograde transport system and the nuclear import apparatus. Thus, rHSA was not transported at all, but accumulated in organelles near the injection site. Also, rHSA-sp containing an sp with a Lys to Thr substitution, which is known to reduce nuclear import markedly, was transported only poorly. To look for endogenous molecules that use this system, we affinity-purified a rabbit polyclonal antibody to the signal sequence. The antibody recognized an 83 kDa polypeptide on Western blots of Aplysia nervous tissue. These data indicate that Aplysia neurons contain the machinery to convey macromolecules from the axon periphery to the nucleus.


Developmental changes in calcium conductances contribute to the physiological maturation of cerebellar Purkinje neurons in culture.

The relationship between calcium conductances and developmental changes in the active and passive membrane properties of cerebellar Purkinje neurons from rats was studied in a culture model system by using current-clamp and voltage-clamp techniques. These cultures, at 6-21 d of age, represented the main period of morphological and physiological development of the Purkinje neuron. In the current-clamp studies, input resistance decreased and the current-voltage curve became more S-shaped as the neurons matured in culture. Spike-generating properties also changed. Immature Purkinje neurons without dendritic structure produced repetitive, fast TTX-sensitive simple spikes when stimulated electrically. The simple spike frequency increased with maturation. In older neurons (greater than or equal to 12 d in vitro) with well-developed dendritic structure, a burst event, the complex spike, preceded the repetitive simple spike firing. Magnesium (10 mM) and cadmium (50-100 microM), calcium channel blockers, antagonized the repetitive simple spike firing in both young and old neurons. The complex spike of the older neurons was also antagonized by magnesium (10 mM) but was resistant to cadmium (50-100 microM), suggesting that a pharmacologically distinct calcium conductance mediated this spike event. Whole-cell voltage-clamp recordings showed that the older Purkinje neurons expressed two calcium currents, a low-threshold rapidly inactivating calcium current resistant to cadmium (50-100 microM) and a high-threshold slowly inactivating calcium current antagonized by cadmium (50-100 microM). In young Purkinje neurons without dendritic structure (6-9 d in vitro), only the high-threshold calcium current was evident. The amplitude of this current increased approximately 50% during development. These results indicate that the developmental expression of calcium conductances plays a prominent role in the physiological maturation of the cultured Purkinje neurons, which closely simulate the physiologic cells they model. The high-threshold calcium conductance is expressed early in development and contributes to repetitive simple spike firing of both the young and old neurons. The low-threshold calcium conductance appears later in development, coincident with dendritic expression, and plays a major role in the generation of the complex spike.


Immunological analysis of proteoglycan structural changes in the early stage of experimental osteoarthritic canine cartilage lesions.

Specific modifications of the proteoglycan (PG) structure of osteoarthritic (OA) dog cartilage in relation to endogenous metalloprotease activity were examined using murine anti-proteoglycan monoclonal antibodies (MoAbs). OA lesions were induced over a period of 8 weeks in crossbred dogs (Pond-Nuki model). The articular cartilage was removed and homogenized in a Tris buffer, pH 7.5, and then divided into four groups: direct PG extraction, no addition, presence of 1 mM p-aminophenyl mercuric acetate (APMA), and presence of 1 mM APMA and 10 mM o-phenanthroline, incubated for 42 h at 37 degrees C followed by PG extraction. MoAbs reactive with PG protein and carbohydrate epitopes included 1C6, 3B3, 5D4, D1B2, and m4D6. The results showed marked alterations induced by APMA activation of the endogenous metalloproteases. PG changes were apparent at at least three sites: one was either in the hyaluronic acid-binding region or between the hyaluronic-binding region and the G2 globular domain, another was between the keratan-sulfate-rich domain and the chondroitin sulfate-attachment domain, and a third was in the chondroitin sulfate-attachment domain. Constitutive metalloprotease activity resulted in less marked PG alterations with preservation of functional PG aggregation to hyaluronan.


Immunohistochemical localization of beta 1-integrins in anterior cruciate and medial collateral ligaments of human and rabbit.

The integrins are a family of adhesion-mediating cell surface receptors that play critical roles in cell-extracellular matrix interactions and have been shown to be important in the healing response in several tissues. We have studied integrin expression in normal human and rabbit anterior cruciate (ACL) and medial collateral (MCL) ligaments of the knee as a preamble to studies of beta 1-integrin expression in healing ligaments. Histologic sections of human and rabbit ACL and MCL were probed for integrin expression utilizing integrin-specific monoclonal antibodies (mAbs) followed by immunoperoxidase detection. Staining of human specimens with mAbs revealed the presence of beta 1-, alpha 1-, and alpha 5-integrin chains on the tissue fibroblasts of both ACL and MCL, while staining of rabbit specimens with rabbit integrin-reactive monoclonals revealed the presence of beta 1- and alpha 5-integrin on these ligaments. Equivalent amounts of the integrins studied were present on normal ACL and MCL. We conclude that the rabbit is an appropriate model for analyzing the expression and functional role of integrins in ligament wound healing.


Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.


Oesophageal replacement in paediatric patients.

Over a 5-year period from January 1986 to December 1990, 24 children aged between 16 months and 12 years with undilatable oesophageal stricture had oesophageal replacement with isoperistaltic colonic conduit. All the strictures followed accidental corrosive burns. The procedure was well tolerated; all the patients were able to swallow within 3 weeks of surgery. Major postoperative complications were threatening pneumothorax (two cases), gastric outlet obstruction due to Ascaris lumbricoides (two cases) and cervical fistula (eight cases) which closed spontaneously in each case. There were no operative or postoperative deaths. Twenty-two patients have been followed up for 2-59 months. Children tolerate oesophageal replacement well. The short-term and medium-term results are good, but anxiety over the fate of the retained native oesophagus is noted.


Variation among trainee surgeons in interpreting diagnostic peritoneal lavage fluid in blunt abdominal trauma.

Diagnostic peritoneal lavage is useful in selected patients who have sustained blunt abdominal trauma. The way in which 50 trainee surgeons (37 registrars and 13 senior registrars) perform and interpret diagnostic peritoneal lavage was investigated in this study by a simple questionnaire followed by assessment of simulated lavage fluid. This exposed misconceptions about interpretation of diagnostic peritoneal lavage fluid and a reliance upon visual assessment. Assessment of the simulated lavage fluid revealed a wide range of thresholds for a positive result (2460-48,700 red blood cells/mm3), although 49 surgeons (98%) had lower thresholds than that generally recommended. Senior registrars had significantly higher thresholds than registrars, implying a learning curve involving unnecessary laparotomies. In order to avoid this situation and to detect injuries that might otherwise be missed, trainee surgeons are recommended to perform cell count analysis routinely on diagnostic peritoneal lavage fluid.


Diverticular disease of the colon in black Africa.

Diverticular disease of the colon, previously believed to be rare among Africans, is now an emerging disease entity in many areas of the African tropics. Fifteen patients, the majority of whom were overweight (73%), under 50 years of age (73%) and who have remained on the bulky diet traditional to much of Africa, are reported. The findings suggest that factors previously uncommon in the area may now be operating to cause the disease in the population, and the highly processed food products of the supermarkets may be an important contributor to the development of this new disease entity.


Antibiotic prophylaxis in acute non-perforated appendicitis in children: single dose of metronidazole and gentamicin.

In a 3-year study, 103 children with acute non-perforated appendicitis who underwent appendicectomy were randomized for either a single preoperative dose of gentamicin and metronidazole or three doses of gentamicin and metronidazole given before and after the operation. The overall wound infection rate was 1.9%. There was no significant difference between wound infection rates of the single-dose group (2.1%) and the three-dose group (1.8%). The mean(s.d.) hospital stays of the single-dose and three-dose groups were similar: 6.6(2.2) days and 6.4(2.7) days. This study shows that a single preoperative dose of gentamicin and metronidazole is as effective as three doses of gentamicin and metronidazole in the control of post-appendicectomy wound sepsis.


Computed tomography for oesophageal carcinoma: its value to the surgeon.

Between January 1988 and June 1991, 75 patients with carcinoma of the oesophagus or gastro-oesophageal junction were evaluated by computed tomography (CT). Fifty of these patients underwent operation, allowing 48 cases to have a detailed surgical and pathological verification of CT features. For thoracic oesophageal tumours the accuracy of CT was 59% for fat plane status, 86% for aortic contact, 81% for tracheobronchial tree compression and 66% for direct local invasion. CT was 69% accurate for identifying lymph nodes, of which only 38% contained metastatic deposits. For gastro-oesophageal junction tumours, CT was 74% accurate for fat plane status and 90% accurate for direct local invasion. Accuracy for detecting lymph node involvement was 63%, metastatic tumour being present in 91% of these nodes. By pathological staging, only 15% of all resections could be considered potentially curative. The value of CT was found to be in predicting a palliative or curative resection, and in warning the surgeon about possible infiltration of specific mediastinal or abdominal structures that would be encountered during operative dissection.


Surgery in collective protection against chemical warfare.

In the 3 months from October 1990 to January 1991, 110 operations were performed at 33 Field Hospital, Royal Army Medical Corps at Al Jubayl, Saudi Arabia in collective protection against chemical warfare. The evolution of improvements in this environment is presented in the context of the first operative series undertaken.


Estimating trauma centre workload.

To estimate the volume of major trauma seen in the UK in the light of the recent report from the Royal College of Surgeons of England, all publications relating to UK trauma practice published since the report were analysed. Major trauma represents one in every 1000 accident and emergency department attendances on average. Although this is less than in the USA, the severity of injury is at least equivalent to North American practice.


New technique for assessing muscle damage after trauma.

We report a new technique which is a useful objective method for assessing the extent and severity of traumatic muscle damage using scintigraphic imaging. Radionuclide indium-111 antimyosin was used in 13 patients with trauma. The increased uptake was confined to the injured muscles and the intensity was related to the severity of damage.


Relationships between surgical ability ratings and spatial abilities and personality.

Twenty-two trainee surgeons were tested on high level tests of intelligence, a detailed battery of paper-and-pencil and computerized spatial ability tests, and personality tests. They were also rated for operating ability, clinical decision-making ability, and for overall performance by consultant surgeons who knew them well. Ability ratings were high, indicating very little dissatisfaction with the performance of trainee surgeons. There were no significant correlations between surgical ability ratings and intelligence test scores. The only significant correlation between spatial ability and ability ratings was with one subtest, and was in the opposite direction to that expected. Trends in the data suggested that those trainees rated as superior tended to be more introverted and conscientious, and tended to have better stereoscopic depth perception. The discussion questions the necessity for aptitude testing in surgery and points out some statistical pitfalls in the area. It is concluded that little advance may be made with the application of aptitude testing in the selection of surgeons until there are more objective criteria of surgical ability in different surgical specialties.


Aetiological role of otolaryngological disease in paediatric intracranial abscess.

Recent reports suggest that congenital heart disease has supplanted otolaryngological disease as the major aetiological factor in the development of paediatric intracranial abscess. A survey of intracranial abscess identified from the records of the Information and Statistics Division of the Scottish Health Service Common Services Agency and the departmental records of the regional neurosurgical units in Scotland for the period 1981-1985 was undertaken to test this hypothesis. A total of 22 cases in individuals under the age of 16 years confirmed at surgery or autopsy were classified aetiologically on the basis of localization, clinical and investigative findings. Three were cardiogenic, four otogenic and three rhinosinugenic in origin; the others were due to miscellaneous causes. The mean(s.d.) age was 9(4.5) years. The overall mortality rate was 18%. Otolaryngological disease is the major aetiological factor in paediatric intracranial abscess in the UK.


Cryotherapy for retinopathy of prematurity in a regional neonatal intensive care unit.

Ninety-six low birth-weight premature infants at risk of retinopathy of prematurity (ROP) were screened over a 25-month period, yielding 30 eyes of 15 infants with 'treatable' disease. The response and complications of cryotherapy were prospectively evaluated in these infants over a 3-24-month period. Six eyes of three infants progressed to cicatricial disease despite treatment. One infant developed angle closure glaucoma but neither eye showed cicatricial disease. In 24 eyes of 12 infants regression of active ROP was observed after treatment. The local and systemic complications associated with cryotherapy are discussed.


Parotid gland tumours: a 15-year experience.

Between 1973 and 1988, 302 patients underwent surgical operations for parotid gland swelling. Primary operations were performed in 293 cases, while secondary operations for recurrent tumour were performed in nine cases. A total of 244 patients (80.8%) were found histologically to have either a benign parotid tumour or a tumour-like lesion, while 58 patients (19.2%) were diagnosed as having malignant tumours. Two hundred and eighty-nine patients underwent superficial and total conservative parotidectomy and in 13 cases a radical parotidectomy was performed. The median follow-up was 5 years (range 1-15 years). Permanent facial nerve palsy, tumour recurrence, Frey's syndrome and parotid fistula were recorded as 0.7, 0.7, 2.1 and 0.4%, respectively. Thus, with full understanding of the surgical anatomy of the parotid gland and correct tumour identification, preservation of the facial nerve and serious postoperative complications can be minimized following superficial and/or total conservative parotidectomy.


PDS II (polydioxanone) is the monofilament suture of choice for subcuticular wound closure following breast biopsy.

In a randomized study of 120 patients having a local anesthetic breast biopsy, wounds closed with subcuticular PDS II were cosmetically superior to those closed by Prolene.


Paraoesophageal hiatal hernias: when to operate.

Between 1979 and 1988, 29 cases of paraoesophageal hernia presented to one surgeon (F.D.S.). There were 23 women and six men and the mean(s.e.m.) age was 66.3(4.1) years. All were symptomatic and 13 hernias (45%) were complicated by gastric volvulus, haemorrhage or perforation. Ten (34%) had evidence of gastro-oesophageal reflux, suggesting a sliding component in these cases. Operation, mostly transthoracic, consisted of hernial reduction, crural repair and, if indicated, an antireflux procedure. There were three deaths. Two occurred as a result of spontaneous, intrathoracic perforation of the hernia. The third followed dilatation of a benign stricture 2 months after surgery. The only major complication was a pulmonary embolus with full recovery. The mean(s.e.m.) follow-up time was 47.6(7.8) months and there were no recurrences. This series confirms that symptomatic paraoesophageal hernias warrant early repair because of the frequency and severity of associated complications. Although debate continues as to whether this policy should be extended to asymptomatic paraoesophageal hernias, we suggest that this should be so.


Case-control study of patient satisfaction with day-case and inpatient inguinal hernia repair.

A case-control study has been carried out to assess patient satisfaction with day-case or inpatient inguinal hernia repair using matched pairs of patients undergoing the same technique of inguinal hernia repair under general anaesthesia. Patients completed a single-page questionnaire 3-6 months after surgery. Although there was no objective difference in recovery, nearly half of the patients felt that they were discharged too early and the majority would prefer inpatient treatment. Day-case patients required significantly more medical attention after discharge and most of the reported wound complications were not known to the hospital. It is concluded that the introduction of day-case surgery in a district general hospital is not without problems and requires changes in working practices and resource availability with careful monitoring of outcome during its implementation.


Abnormalities of T lymphocyte subsets in Behçet's disease demonstrated with anti-CD45RA and anti-CD29 monoclonal antibodies.

We assessed T cell subpopulations using 2-color flow cytometry with phycoerythrin conjugated anti-CD45RA and anti-CD29 and fluorescein conjugated anti-CD4 and anti-CD8 monoclonal antibodies, on peripheral blood lymphocytes from 19 patients with Behçet's disease (BD) and 18 healthy control subjects. The percentage of CD4+ cells was significantly lower in patients with BD (34 +/- 2%) than in control subjects (46 +/- 3%) (p less than 0.001). Among CD4+ cells, the percentage of suppressor-inducer (CD4+CD45RA+) cells was significantly lower in patients with BD (31 +/- 4%) than in control subjects (45 +/- 2%) (p less than 0.01), while the percentages of helper-inducer (CD4+CD29+) cells were similar in patients and controls. The percentage of CD8+ cells was significantly higher in patients with BD (36 +/- 2%) than in control subjects (26 +/- 2%) (p less than 0.001) involving both CD45RA+ and CD29+ subpopulations. Within CD4+ cells, the percentage of suppressor-inducer (CD4+CD45RA+) cells was significantly decreased in patients with active BD (26 +/- 3%) compared with control subjects (45 +/- 2%) (p less than 0.001), whereas in patients with inactive BD the difference was statistically insignificant. Our results suggest that the defective suppressive function in patients with active BD may be related to the decreased suppressor-inducer subpopulation (CD4+CD45RA+).


HLA associations with rheumatoid arthritis: a piece of the puzzle.

HLA molecules play a central role in the immune response by presenting processed antigenic peptides to T cells. In the case of rheumatoid arthritis (RA), a specific sequence present within the peptide binding cleft of HLA class II molecules has been implicated in genetic susceptibility to this disease. Several mechanisms could account for this finding. One possibility, designated determinant selection, rests on the possibility that HLA molecules associated with RA may bind a distinct set of foreign or self antigens that can trigger a T cell response in the joint. Alternatively, HLA molecules may predispose to RA by shaping the T cell repertoire during thymic differentiation in neonatal and early adult life. These 2 mechanisms are not mutually exclusive, and it must be remembered that several other susceptibility genes as well as environmental factors remain to be identified before a comprehensive understanding of RA is achieved.


Cystic fibrosis mutations delta F508 and G542X in Jewish patients.

We have screened our CF patients for mutations in exons 10 and 11 of the CFTR gene. Two mutations, delta F508 and G542X, have been found in 66 Jewish CF patients. The average frequency of the delta F508 mutation in the Jewish population is 33.8%. The G542X mutation accounts for 13% of the Ashkenazi CF mutations and has been found in three out of seven chromosomes of Jewish patients from Turkey (probably descended from Ashkenazi immigrants). The G542X mutation was not found in any of the other non-Ashkenazi patients. All the G542X bearing chromosomes have the same haplotype. Based on these observations it is concluded that the G542X mutation was introduced into the Jewish people after the split into Ashkenazi and non-Ashkenazi.


The 14th C. L. Oakley Lecture. Candida albicans HSP 90: link between protective and auto immunity.

Heat shock proteins (HSP) are thought to play a role in the aetiology of autoimmune diseases, but are also common targets for the immune response to many infections. Patients recovering from systemic candidosis produce antibodies to Candida albicans HSP 90, both to species-specific epitopes and, more commonly, to epitopes shared with human HSP 90. One such autoreactive antibody was protective in a mouse model of systemic candidosis.


Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence.

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.


Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene.

We have analyzed P-element-transformed lines carrying hsp26/lacZ transgenes with various deletions and substitutions within the Drosophila melanogaster hsp26 promoter region in order to identify the sequences required for the formation of the DNase I hypersensitive sites (DH sites). DH sites are generally found associated with promoters and enhancer elements of active and inducible eukaryotic genes, and are thought to be nucleosome-free regions of DNA that interact with regulatory proteins and the transcriptional machinery. There are two major DH sites located within the promoter region of the hsp26 gene, centered at -50 and at -350 (relative to the hsp26 transcription start site). The sequences from -135 to -85, which contain (CT)n.(GA)n repeats, contribute significantly to the formation of the DH sites in the hsp26 promoter region. Deletion or substitution of this (CT)n region drastically reduces the accessibility of the DNA at these sites to DNase I. This reduction in accessibility was quantified by measuring the susceptibility of the DNA within nuclei to cleavage at a restriction site within the DH site. In addition to the (CT)n region and the promoter at -85 to +11 (region P), one of two other regions must be present for effective creation of the DH sites: sequences between -351 and -135 (region A), or sequences between +11 and +632 (region D). Disruption of the wild-type chromatin structure, as assayed by the loss of accessibility to the DH sites, is correlated with a decrease in inducible transcriptional activity, even when the TATA box and heat shock regulatory elements are present in their normal positions.


Structure, expression, and function of Ng-CAM, a member of the immunoglobulin superfamily involved in neuron-neuron and neuron-glia adhesion.

The neuron-glia cell adhesion molecule (Ng-CAM) mediates neuron-neuron adhesion by a homophilic mechanism and neuron-astrocyte adhesion by a heterophilic mechanism. The protein is expressed on neurons and Schwann cells but not on astrocytes. It is most prevalent during development on cell bodies of migrating neurons and on axons during formation of nerves. Ng-CAM expression is greatly increased following nerve injury. Anti-Ng-CAM antibodies inhibited migration of granule cells along Bergmann glia in cerebellar explants and fasciculation of neurites in outgrowths from explants of dorsal root ganglia. The combined results indicate that Ng-CAM on neurons binds to Ng-CAM on adjacent neurons and to as yet unidentified ligands on astrocytes. Ng-CAM is synthesized in chicken neurons from a 6 kb mRNA as Mr approximately 200,000 forms which are cleaved to yield two components of Mr 135,000 and 80,000. It is glycosylated and can be phosphorylated. Amino acid sequence analysis indicates that it contains six immunoglobulin domains, five fibronectin type III repeats, a transmembrane domain and a cytoplasmic region. Structural analyses indicate that Ng-CAM is most closely related to the mammalian glycoprotein L1 but significant differences between them strongly suggest that they are not equivalent molecules. The recent identification of another structurally related molecule in the chicken called Nr-CAM underscores the notion that these molecules are members of a subfamily of neural cell adhesion molecules within the immunoglobulin superfamily that have related or complementary functions in the nervous system.


Substance P immunoreactivity of rat brain stem neurons in primary culture.

Substance P (SP)-ergic neurons from 16/17 day-embryonic rat brain stem in primary culture were identified by immunocytochemistry using biotinylated avidin and phosphatase alkaline methods with affinity-purified anti-SP antibodies. An average of 84% of neurons contained SP from day 9 to day 21 after plating. These in vitro data show that SP-containing neurons develop in our culture conditions. SP may act as a maturation factor as well as a neurotransmitter.


Preparation of a protein-free total brain white matter lipid fraction: characterization of liposomes.

A method of preparing a total lipid extract (TLE), free of protein, by extracting brain white matter with tetrahydrofuran is presented. The optimal conditions of extraction were found to be 50 ml of THF per gram of lyophilized tissue, though fresh tissue can also be used if larger volumes of solvent are employed. The method allowed, in a short time and in a single step, a yield of TLE of 50% on a dry weight basis. Its analytical characterization revealed a qualitative and quantitative composition very similar to the lipid composition of CNS myelin, including all the phospholipid and galactolipid species, cholesterol and gangliosides, but it contained only traces (0.1%) of protein. TLE has been used to prepare liposomes, either multilamellar (MLVs) or unilamellar (LUVs, SUVs), characterized by freeze-fracture electron microscopy. A multilayered, heterogeneous population of liposomes is observed in the MLVs preparation. When these samples were submitted to a freezing and thawing procedure the resulting liposomes were single-walled, and their intravesicular volume was increased. They were quite impermeable to the monovalent cation 86Rb+ and, by contrast, rather permeable to 45Ca+ +. Their complex lipid composition, together with their permeability properties and their response to ionophores, make them very useful to study protein-lipid interactions occurring within the myelin membrane as well as the functional properties of myelin proteins in reconstitution experiments.


Expression of the neu oncogene under the transcriptional control of the myelin basic protein gene in transgenic mice: generation of transformed glial cells.

We have taken a transgenic approach in an effort to specifically transform oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). Transgenic mice were generated with a DNA construct that contained the activated neu oncogene under the transcriptional control of the myelin basic protein (MBP) gene. The MBP/c-neu transgenic animals have experienced a low incidence of brain tumors that express molecular markers specific to oligodendrocytes, providing a mouse model to study the formation and progression of oligodendrocyte tumors. A tumor from a transgenic animal has been dispersed in culture, and transformed cells that express properties of oligodendrocytes and astrocytes have been maintained. The degree to which these cells express phenotypic characteristic of oligodendrocytes or astrocytes is influenced by culture conditions. These transformed cells should serve as a valuable resource with which to study various molecular and biochemical aspects of the myelination process, as well as the lineage interrelationship of CNS glial cells.


Effect of dexamethasone on the nerve growth factor-induced increase in the NILE glycoprotein in PC12 cells.

The long-known and well-documented increase in the NILE glycoprotein produced by nerve growth factor in PC12 cells is prevented by simultaneous treatment with dexamethasone. The absence of this surface marker on the fully differentiated cells has been demonstrated by both glucosamine incorporation and immunohistofluorescence.


Cellular immune crossreactivity between myelin basic protein and synapsin in rats with experimental allergic encephalomyelitis.

We previously demonstrated that antibodies against myelin basic protein (MBP) obtained from animals with experimental allergic encephalomyelitis (EAE), induced with MBP and purified by affinity chromatography, have the property to recognize a neuronal protein, synapsin Ia and Ib. To investigate whether this crossreactivity also occurs at the cellular level, we purified spleen and lymph node mononuclear cells from rats sensitized with MBP or synapsin using polystyrene plates coated with the respective antigen. We observed that animals injected with MBP have T lymphocytes that bind both antigens. Using the same system, each purified cell population was confronted again to the studied antigens. The anti-MBP cells recognized once more epitopes of MBP and about 40% of them also recognized synapsin. On the other hand, cells that first were attached to synapsin, in the second exposure to antigens bound to MBP and synapsin in similar amounts. Double immunofluorescent labeling of the mononuclear cells isolated from animals injected with bovine myelin or MBP showed that the same lymphocyte was able to recognize MBP as well as synapsin. In both experimental systems the quantitative results were similar indicating that in bovine myelin- or MBP-sensitized animals practically all the cells that recognize synapsin are anti-MBP cells, and of the total cells raised against MBP, around 40% of them show this crossreactivity. On the contrary, animals injected with synapsin have cells that bind to this protein but not to MBP indicating that the described crossreactivity, as observed at humoral level, is only in one way.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of hypertonic saline dextran resuscitation on oxygen delivery, oxygen consumption, and lipid peroxidation after burn injury.

We compared the effects of lactated Ringer's (LR) and hypertonic saline dextran (HSD) on postburn cardiovascular function, O2 consumption, lipid peroxidation, and bacterial translocation. Miniature pigs with 40% total body surface area (TBSA), third-degree burns received, 30 minutes postburn, either Parkland resuscitation (LR group, n = 8) or HSD, 10 mL/kg/30 minutes, followed by LR, 4 mL/kg/%burn over the next 23 hours (HSD group, n = 8). The HSD prevented the early decrease in cardiac index (CI); the early increase in the resistance of the systemic, mesenteric, celiac, and renal vascular beds; and the decrease in mesenteric O2 consumption seen after burns when LR alone is used for resuscitation. The HSD also moderated the systemic and mesenteric lipid peroxidation. Bacterial translocation was less in the HSD group (3 of 8 animals) compared with the LR group (5 of 8 animals), but was not statistically different. Hypertonic saline dextran may be beneficial in improving the postburn microcirculation and attenuating postburn oxidant-induced lipid peroxidation in the systemic tissues and the gut.


Improved indication and followup in transurethral resection of the prostate using the computer program CLIM: a prospective study.

Analysis of preoperative and postoperative detrusor pressure-flow measurements with the aid of the IBM compatible software package CLIM in 29 patients who underwent transurethral prostatic resection indicated that CLIM supplies reliable obstruction parameters that are recommended for preoperative assessment and postoperative followup, and that approximately a third of the patients who present with prostatism are nonobstructed, suggesting that the indication for transurethral resection of the prostate is over assessed.


Prevention of acrolein-induced bladder injury by pentosanpolysulfate.

The active metabolite of cyclophosphamide, acrolein, which is capable of damaging the transitional epithelium of the bladder, was evaluated in both in vivo and in vitro models to determine if its damaging effect could be reduced by the presence of a sulfated polysaccharide pentosampolysulfate. It was discovered that in all models pentosanpolysulfate was capable of reducing transitional cell injury due to acrolein.


False positive prostate specific antigen values in the sera of women with renal cell carcinoma.

The assessment of serum prostate specific antigen (PSA) with a polyclonal radioimmunoassay in 22 women with renal cell carcinoma resulted in measurable values in 6 patients, with the values ranging from 0.5 to 27.0 ng./ml. Tumor stage was T3 in all of these patients and the morphological features showed an eosinophilic subtype according to the German nomenclature. Additional measurements with a monoclonal radioimmunoassay in 3 of these 6 patients were negative as were all subsequent measurements postoperatively, although performed with the polyclonal radioimmunoassay. Immunohistochemical staining was negative for PSA. However, positivity for alpha-1-antichymotrypsin was detected, which was reported to occur in complexes with PSA and other proteases. Cross reaction between the polyclonal antibodies of the radioimmunoassay and 1 of these complexes is concluded. As far as clinical considerations are concerned, such cross reaction must be considered just as likely in male patients. Thus, it is recommended that therapeutic considerations in patients who underwent radical prostatectomy, for example, should not be exclusively based on PSA measurement performed with a polyclonal assay.


The effect of digital rectal examination on the serum prostate specific antigen concentration: results of a randomized study.

To determine the effect of digital rectal examination on the serum prostate specific antigen (PSA) concentration a prospective, randomized, controlled trial involving 143 patients was conducted. Of the patients 86 (60%) had benign prostatic hyperplasia (BPH), 47 (33%) had prostate cancer and 10 (7%) had chronic prostatitis. The study group consisted of 71 men, all of whom had a serum PSA determination followed by a digital rectal examination and then a second serum PSA determination. The control cohort consisted of 72 men, all of whom had 2 serum PSA determinations without an intervening digital rectal examination. The median change in the serum PSA level for the study group was 0.4 ng./ml. compared to -0.1 ng./ml. for the control cohort (p less than 0.0001). For 76% of the study patients the second serum PSA level was greater than the initial value; only 32% of the control patients exhibited a higher second serum PSA level than the initial level (p less than 0.0001). However, only 4 patients with an initial PSA value in the reference range (0.0 to 4.0 ng./ml.) had a post-digital rectal examination value greater than 4.0 ng./ml. and only 1 patient whose presenting serum value was less than 10.0 ng./ml. had a serum PSA level greater than this cutoff point after digital rectal examination. This minimal change in serum PSA after digital rectal examination was independent of the diagnosis (BPH, cancer or chronic prostatitis), initial serum PSA concentration and examiner. Thus, although digital rectal examination had a statistically significant effect on the serum PSA concentration, the clinical significance of a 0.4 ng./ml. median increase appears inconsequential. Based on these findings, physicians should be confident that the serum PSA concentration in the immediate post-digital rectal examination period is accurate and does not compromise clinical use of the tumor marker.


[Anti-platelet antibody and platelet function].

The effect of anti-platelet antibodies, including murine monoclonal antibodies, autoantibodies and alloantibodies, on platelet function was analyzed. The target antigen of these antiplatelet antibodies, investigated in the present study, was a glycoprotein IIb/IIIa, which is a receptor of fibrinogen and plays an important role in platelet aggregation. Some of these antibodies inhibited agonist-induced platelet aggregation. The target antigen of one murine monoclonal antibodies, designated OP-G2, was a glycoprotein IIb/IIIa and interestingly, this antibody induced platelet aggregation, which required divalent cation and fibrinogen. We compared the epitope of these antibodies by inhibition assay and found the epitope of these antibodies to be very close. The binding of OP-G2 to the platelets required Ca2+. These data suggest that OP-G2 recognizes an epitope at or in very close proximity to the fibrinogen binding site of GPIIb/IIIa, as compared with other antibodies.


[Regulation of nitric oxide synthase].

Nitric oxide (NO) synthase is known to be widely distributed in various cells. We characterized this enzyme using partially purified enzyme fractions from rat neutrophils, macrophages, and cerebellum. The cerebellar fraction required Ca(2+)-calmodulin, while that from macrophages required neither calmodulin nor Ca2+. The neutrophil fraction required Ca2+. The enzyme was inhibited by analogues of the substrate, L-arginine. N omega-nitro-L-arginine (NNA) was 20 times more potent than L-NG-monomethyl-arginine (NMA) in blocking the cerebellar enzyme. In contrast, NNA and NMA were about equipotent against neutrophil and macrophage enzymes. These data suggest that the enzyme is regulated by 3 different mechanisms in these cells, with differences between the cerebellar and neutrophil or macrophage enzyme in the catalytic binding site.


[Detection of circulating activated platelets by anti-GMP-140 monoclonal antibodies].

The clinical significance and detection methods for circulating activated platelets are reviewed. It has been recently demonstrated that three kinds of platelet granules, dense granule, alpha-granule and lysosome, have specific membrane proteins; granulophysin, GMP-140 (PADGEM) and CD63 antigen, respectively, and that these specific granule-membrane proteins become exposed on the surface of the activated platelets. It is believed that detection of circulating activated platelets, using monoclonal antibodies specific to these granule-membrane, may be reliable and diagnostic value in thrombotic and prethrombotic diseases. Preliminary clinical studies using 2T60, a newly developed anti-GMP-140 monoclonal antibody, was presented.


Protective effects of calcium antagonists in human renal transplantation.

To test the hypothesis that calcium antagonists decrease the incidence and severity of delayed graft function, we conducted three separate, prospective, randomized trials. In these trials, we investigated the effects of diltiazem and those of the prostacyclin analogue iloprost. In the first study, 22 control patients and 20 diltiazem patients received grafts perfused with either vehicle or diltiazem 20 mg/liter in the Euro-Collins solution. Subsequently, the diltiazem subjects were given the drug as a bolus of 0.28 mg/kg, followed by a continuous infusion of 0.002 mg/min/kg for the following two days. Thereafter, diltiazem 60 mg was given to the treated subjects orally for up to four years. In the second study, 11 control subjects and 10 diltiazem subjects received the same postoperative regimen, but all grafts were harvested without addition of diltiazem to the perfusion solution. In the third protocol, four groups were studied as follows: 19 control subjects who received no specific treatment, 16 subjects who received diltiazem, 16 subjects who were given iloprost, and 14 subjects who received both iloprost and diltiazem. The donor kidney of treated patients was perfused with either diltiazem, iloprost, or both drugs. Primary graft function occurred more commonly in the groups receiving diltiazem. Further, in the first study the number of hemodialyses per patient was reduced in those patients with delayed graft function. Fewer rejection episodes occurred in patients receiving diltiazem. Plasma levels of soluble interleukin-2 receptors decreased significantly during diltiazem treatment. Moreover, renal biopsies showed less severe signs of Cyclosporin A (CsA) nephrotoxicity in diltiazem-treated patients compared to controls, even though these patients also exhibited higher CsA trough levels.(ABSTRACT TRUNCATED AT 250 WORDS)


[Loss of ganglion cells in the retina secondary to vincristine therapy].

We report the case of a 25-year-old black female from Zaire with AIDS diagnosed 2 years earlier. Nine months before her death, she was treated for a disseminated Kaposi sarcoma with vincristin, adryamycin and bleomycin. At that time, visual acuity was normal and ophthalmologic examination was unremarkable except for the presence of bilateral Drusen and a cotton wool spot OS. Three months after the onset of chemotherapy, the patient complained of progressive visual field constriction, which progressed to blindness within a 4 month period. Five months after the onset of the tri-therapy a bilateral CMV retinitis developed, which was successfully treated by intravitreous injections of ganciclovir. This therapy was stopped as soon as blindness was established, with subsequent massive bilateral recurrence of the CMV retinitis. Histologic examination showed complete atrophy of the retinal ganglion cells and areas of CMV retinitis. The optic nerve was demyelinated and exhibited astrocytic gliosis. Immunohistochemistry confirmed the presence of CMV in infected retina and revealed the absence in the optic nerve of the class III beta-tubulin isotype and of the 200 kd neurofilament subunit. In contrast, oculomotor nerves appeared intact. The presence of HIV in the eye and in the optic nerve was excluded using PCR technique. The retinal ganglion cell loss and optic nerve atrophy appeared to be purely degenerative in nature, since there was no evidence of vascular occlusion, inflammation or retrobulbar compressive process. We therefore conclude that blindness was caused by vincristine therapy. The patient actually received 22 mg of vincristin intravenously in 11 courses over 7 months, although discontinuation was recommended by us after 5 months.(ABSTRACT TRUNCATED AT 250 WORDS)


Atherosclerosis, cell motility, calcium, and calcium-channel blockers.

Three key players in the humoral-cellular interactions that occur during the early development of atherosclerosis are presented as they activate platelets and vascular smooth muscle cells but eventually can be corrected by calcium-channel blockers. Platelet-activating factors via phospholipase C and phosphoinositides increase cytosolic calcium and phosphorylate contractile proteins, thereby inducing a change--aggregation and the secretory response of platelets. Low-density lipoprotein (LDL) has a similar hormone-like action and activates the signal transfer cascade that eventually leads to platelet aggregation as well as vascular smooth muscle cell proliferation. These effects can be greatly reduced by high-density lipoproteins. Platelet-derived growth factor stimulates the transcription of the LDL-receptor gene as well as the HMG-CoA reductase gene. The latter is inhibited by calcium-channel antagonists while the former is further enhanced. Thus, calcium-channel antagonists interfere with the stimulus-response coupling not only via slow calcium-channel influx inhibition but also by an additional membrane action and interference with gene activation.


Calcium, calcium antagonism, atherosclerosis, and ischemia.

Calcium is a ubiquitous cation involved in a wide variety of physiological processes. Normally, cytosolic calcium is maintained within narrow limits but under certain conditions the levels rise--either because of excess calcium entry, internal release, or failure of the extrusion mechanisms. Such conditions include hypertension and myocardial ischemia. Calcium ions are also involved in the formation of atherosclerotic lesions. Hypertension, ischemic heart disease, and atherosclerosis are all amenable to calcium antagonist therapy. The efficacy of this class of drugs in the management of such a wide spectrum of disorders is in accord with the central role played by calcium in the etiology of these disorders. To some extent, however, the disorders are interrelated, with hypertension being a major risk factors for ischemic heart disease and atherosclerosis.


The development and regression of vascular hypertrophy.

Genetic hypertension [essential hypertension (EH) in humans and spontaneous hypertension in rats (SHRs)] is associated with an altered vascular structure, expressed in resistance vessels (small arteries) as an increased media:lumen ratio. The vascular smooth muscle cells appear normal and there is no cellular hypertrophy. The amount of smooth muscle may be increased in SHR resistance vessels (i.e., there is evidence for hyperplasia). Present evidence in EH indicates that there is no increase in vascular smooth muscle mass, suggesting that the altered structure is due to "remodeling," a rearrangement of a normal amount of normal material. With antihypertensive therapy, reduction in mean blood pressure does not necessarily normalize vascular structure, and there are few grounds for supposing that one drug is better than another in this respect. Preliminary evidence suggests that normalization of the pulse pressure may be more important than normalizing mean blood pressure, if vascular structure is to be corrected. Although vascular structure may not per se be an important determinant of blood pressure, reduction in blood pressure without correcting vascular structure is undesirable. Thus, improvement of the means for correcting vascular structure may be an important way of improving prognosis with antihypertensive treatment.


Effects of nifedipine-GITS on left ventricular mass and left ventricular filling.

Sixteen patients with initial diastolic blood pressure greater than or equal to 120 mm Hg were treated for 1 year with extended-release nifedipine [nifedipine-GITS (gastrointestinal therapeutic system)]. Serial changes in left ventricular mass index and associated alterations in left ventricular systolic function, left ventricular filling, plasma renin activity, atrial natriuretic peptide, and catecholamines were evaluated. Blood pressure was significantly reduced from 200 +/- 8/122 +/- 3 mm Hg (mean +/- SEM) to 144 +/- 5/89 +/- 2 mm Hg (p less than 0.0001) at 1 year. Eleven patients (69%) required only nifedipine-GITS for blood pressure control and 5 (31%) required the addition of chlorthalidone. After 6 months, the left ventricular mass index was significantly reduced by 19% from 121 +/- 8 to 96 +/- 7 g/m2 and this reduction was sustained at 1 year. Septal and posterior wall thicknesses were reduced from 13.4 +/- 0.1 to 11.2 +/- 0.04 mm and from 12.8 +/- 0.1 to 10.0 +/- 0.03 mm (p less than 0.001), respectively. Prevalence of left ventricular hypertrophy decreased from 63 to 25%. Left ventricular fractional shortening increased from 34 to 42% (p less than 0.05) and the relationship between fractional shortening and end-systolic stress did not change. Over the year of sustained blood pressure reduction, the peak velocity of early filling increased from 58 to 63 cm/s (p = 0.07), the peak velocity of late filling did not change, and the ratio of late to early peak velocity of left ventricular filling significantly decreased (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Nitrendipine in the therapeutic management of elderly hypertensive patients: results of a multicenter trial. Andalousian Hypertension Group.

Two hundred forty-seven [142 women (57.49%)] elderly patients with essential hypertension (diastolic blood pressure between 95 and 114 mm Hg) and an average age of 67.4 +/- 6 years were included in an open multicenter ambulatory trial. One hundred thirty-seven had some kind of associated disease. After a 15-day washout period, the patients began nitrendipine therapy (10 mg o.d.). After 1 month, the dose was increased to 20 mg o.d. in patients with diastolic blood pressure (DPB) greater than or equal to 95 mm Hg, and thereafter 5 and 10 mg o.d. of bisoprolol was added to the maximal dose of nitrendipine (20 mg o.d.) in the case of patients with DBP greater than or equal to 95 mm Hg at the end of the second and third months, respectively. At the end of the 6-month follow-up period, the systolic and diastolic pressures had dropped -35 and -21 mm Hg, respectively, without any change in heart rate or Quetelet index. In 210 patients (84.9%), blood pressure control was achieved: 26 (10.5%) with 10 mg of nitrendipine, 149 (60.3%) with 20 mg of nitrendipine, and 35 (14.1%) by adding bisoprolol. The lipid profile, glucose, potassium, uric acid, or creatinine did not change negatively. Sixty-six (26.72%) patients reported clinical side effects, although these were mild; only 15 (6.07%) patients were excluded because of side effects. Nitrendipine has been shown to have a high therapeutic efficacy and biochemical tolerance for first-line treatment of elderly patients with mild-to-moderate essential hypertension with or without associated diseases.


Antihypertensive and metabolic effects of nitrendipine in non-insulin-dependent diabetes mellitus with hypertension.

Fifteen non-insulin-dependent diabetes mellitus hypertensive patients received nitrendipine (20-40 mg) for 24 weeks. Mean systolic and diastolic blood pressure decreased significantly from 177/102 mm Hg before treatment to 153/86 mm Hg (p less than 0.001) after treatment. Meanwhile, the heart rate, body weight, indices of glycemic control (glucose, glycosylated hemoglobin, fructosamine, and serum C peptide levels), and serum lipid fractions did not change. It is concluded that nitrendipine does not impair glucose and lipid metabolism in diabetic patients while exerting its antihypertensive effect.


Oxidized low-density lipoproteins in atherogenesis: possible mechanisms of action.

The increased atherogenic potential of oxidized low-density lipoprotein (ox-LDL) is well documented. In the present study, we investigated possible mechanisms of action of the difference to native LDL. In vitro oxidation of LDL was determined by measurement of thiobarbituric acid-reacting substances and absorption at 234 nm. Copper (5 mumol/L) induced significant (p less than 0.01) oxidation in vitro. Furthermore, LDL isolated from atherosclerotic patients was slightly but significantly (p less than 0.05) more oxidized than LDL from normal controls (2.81 +/- 0.08 vs. 3.21 +/- 0.16 nmol of TBARS/mg of LDL protein). Ox-LDL caused significantly (p less than 0.01) more pronounced contractions of rat aortic rings in vitro compared to nonoxidized LDL expressed as a percentage of maximal contractions induced by 40 mmol/L of KCl (29.0 +/- 5.4% vs. 61.1 +/- 7.2%). Lysolecithin, which is a principal component of ox-LDL formed during oxidation, induced a dose-dependent increase in intracellular free calcium in vascular smooth muscle cells cultured from rat aorta. Doses from 2-25 micrograms/ml were tested and caused a maximum increase of more than 500% (25 micrograms/ml). In conclusion, this study provides further evidence for a higher biological activity of ox-LDL. Lysolecithin might be one of the active components formed during oxidation of LDL.


Use of a microprocessor-equipped tablet box in monitoring compliance with antihypertensive treatment.

Compliance with antihypertensive therapy is usually monitored by questionnaire, tablet counts, or estimation of drug levels in blood or in urine. The aim of this study was to examine patient compliance by means of an "electronic monitor." After 2 weeks of run-in on placebo, 34 moderately hypertensive patients were included in an open, randomized, crossover trial examining the efficacy and tolerance of nitrendipine, 20 mg as a single daily dose (morning or evening) for 1 month. We analyzed the results in 26 patients. Patients were supplied with tablet boxes equipped with a microprocessor (MENS) that registered the timing and duration of opening of the box over both the placebo and nitrendipine periods. Compliance (%) was calculated as the ratio of the number of days that the pill box was opened to the number of days between visits. The compliance was analyzed for each treatment group, namely placebo and nitrendipine morning and evening, over 1 month. Compliance (mean + SD) was 96.5 +/- 7.4% on placebo and 94.4 +/- 10.7% in the morning and 90.6 +/- 15.4% in the evening. Nitrendipine was taken in the morning at 0700 h +/- 2 h and in the evening at 1859 h +/- 2 h 12 min. The frequency of 24 h +/- 1 h intervals between medication was 83.5% on placebo. This frequency was 72.6% for morning dosage and 71.8% for evening dosage on nitrendipine. There were no differences in compliance between the morning and evening groups when analyzed according to age and sex. There was a negative correlation with time (r = -0.57, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Acute and chronic nitrendipine administration in essential hypertension: a chronobiologic study.

To evaluate the chronobiologic pattern of the hypotensive effect of nitrendipine, 10 patients with mild-to-moderate arterial hypertension were studied. They received a randomized single dose (20 mg) of nitrendipine and placebo, and 20 mg of nitrendipine daily for 2 months. Systolic and diastolic blood pressure (SBP and DBP, respectively) and heart rate (HR) were measured for 24 h using an automatic noninvasive device. The data were analyzed by single and mean cosinor methods and by ANOVA and Student's paired t test. Chronic administration of nitrendipine resulted in a more effective lowering of the SBP and DBP mesor, compared with placebo and acute administration, preserving the circadian rhythms. The preservation of the HR circadian rhythm agrees with the lack of interference of the drug with neurohormonal mechanisms.


Effects of slow-release verapamil and nitrendipine on office and 24-hour ambulatory blood pressure in hypertensive patients.

The aim of the present study was to compare the effects of slow-release verapamil (V), 240 mg and nitrendipine (N), 20 mg, administered once daily, on office (OBP) and 24-h ambulatory blood pressure (ABP) in patients with mild-to-moderate hypertension. Twenty patients were entered into this open, randomized, two-group (V, N) parallel study. The study groups had similar age and sex distribution. The OBP (V, 155/103 +/- 19/8 mm Hg; N, 141/98 +/- 13/4 mm Hg), heart rate (HR) (V, 74 +/- 7 beats/min; N, 77 +/- 10 beats/min), daytime systolic ABP (V, 149 +/- 14 mm Hg; N, 147 +/- 13 mm Hg), and nighttime ABP of the two groups were not statistically different after a 2-week washout period. The daytime diastolic ABP (V, 99 +/- 6 mm Hg; N, 93 +/- 6 mm Hg) was slightly lower (p less than 0.05) in group N. Both the OBP (V, 136/90 +/- 19/9 mm Hg; N, 135/85 +/- 10/4 mm Hg) and daytime ABP (V, 132/85 +/- 14/4 mm Hg; N, 136/87 +/- 13/8 mm Hg) dropped in the two groups after 8 weeks of treatment. Nonparametric analysis did not show statistical differences between the groups in OBP and ABP percentage drop. There was no significant change in nighttime ABP, HR (V, 73 +/- 10 beats/min; N, 74 +/- 12 beats/min), ECG, and laboratory exams. We conclude that both verapamil SR and nitrendipine are effective in reducing blood pressure in hypertensive patients without altering the HR.


Metabolic and antihypertensive effects of nifedipine in hypertensive patients.

We studied the effects of nifedipine on blood pressure and on clinical and analytical parameters in hypertensive patients. Seven male and eight female subjects (mean age of 46.27 +/- 5.38 years, range of 41-56 years) with essential arterial hypertension were given nifedipine (20 mg b.i.d.) for 3 months. Before and after treatment, history, blood pressure, and biochemical values were recorded [blood: Na, K, Ca, creatinine, uric acid, triglycerides, cholesterol, HDL cholesterol, antidiuretic hormone (ADH), and aldosterone; urine: Na, K, Ca, creatinine, ADH, aldosterone, and percentage fraction of Na, K, and Ca excreted]. After 3 months of treatment, we found (a) significant decreases in systolic (147 +/- 18 vs. 166 +/- 16 mm Hg, p less than 0.001) and diastolic blood pressure (90 +/- 8 vs. 107 +/- 8 mm Hg, p less than 0.0007), triglycerides (107 +/- 47 vs. 120 +/- 49 mg/dl, p less than 0.0007), and cholesterol (236 +/- 4 vs. 257 +/- 44 mg/dl, p less than 0.00075) in blood, and in K excretion (50 +/- 19 vs. 46 +/- 19 mEq/g of creatinine, p less than 0.0007) and excreted fraction of K (49 +/- 6% vs. 8 +/- 5%, p less than 0.0012) in urine; (b) significant increases in HDL cholesterol (65 +/- 13 vs. 58 +/- 13 mg/dl, p less than 0.001) in blood, and in Na (115 +/- 73 vs. 109 +/- 69 mEq/g of creatinine, p less than 0.0007) in urine; and (c) no significant change in the remaining biochemical parameters, or in heart rate. Secondary effects included flushing (34%), headache (20%), ankle swelling (17%), dizziness (13%), palpitations (4%), and pruritus (4%).(ABSTRACT TRUNCATED AT 250 WORDS)


Action of metoprolol, enalapril, diltiazem, verapamil, and nifedipine on cell growth of vascular smooth muscle cells.

The influence of nifedipine, verapamil, diltiazem, metoprolol, and enalapril on the basal and angiotensin II (Ang II)-induced elevation of [3H]thymidine incorporation into vascular smooth muscle cell (VSMC) DNA was examined. Our results from four independent experiments, each performed in triplicate, are summarized by calculating the half-maximal inhibitory concentration (IC50) of the drugs. Nifedipine, verapamil, and diltiazem had IC50 values of 2.3 +/- 0.7 x 10(-6), 3.5 +/- 0.3 x 10(-6), and 6.6 +/- 2.8 x 10(-6) M, respectively. Metoprolol had an IC50 value of 49 +/- 16 x 10(-6) M, whereas enalapril was completely ineffective. All drugs used had no influence on the basal cell [3H]thymidine incorporation. This in vitro study allows one to conclude that the calcium-entry blockers can inhibit the Ang II-induced cell growth and thus may have beneficial effects on the development and regression of vascular growth, which is associated with the pathogenesis of cardiovascular diseases.


Calcium antagonists and vascular smooth muscle cells in atherogenesis.

Vascular smooth muscle cells (SMCs) play a key role in the development of atherosclerotic lesions. Vascular smooth muscle, however, does not represent a homogeneous tissue. Using myosin as a marker of the differentiation processes in development and in vascular disease, we have been able to demonstrate the existence of distinct SMC populations in rabbit aorta. In our studies, a specific SMC population of the aortic media showing an "immature" type of myosin isoform expression accounted for the majority of SMCs present in the atherosclerotic plaque. Nifedipine, a dihydropyridine-derived calcium antagonist, was able to decrease the size of this particular SMC population and to prevent the development of atherosclerotic lesions in hypercholesterolemic rabbits. Here we report about a similar effect obtained by treating hypercholesterolemic rabbits with nitrendipine, another dihydropyridine-derived calcium antagonist. This article also summarizes the main experimental and clinical studies conducted on the antiatherogenic effect of calcium antagonists and focuses on the mechanisms underlying this effect, particularly at the vascular SMC level.


Prospective randomized clinical trial comparing brachytherapy and laser photoablation for palliation of esophageal cancer.

Twenty-three consecutive patients with advanced esophageal cancer were randomized to receive either endoluminal irradiation or laser photoablation treatment. Initial improvement in dysphagia scores was observed in 83% (brachytherapy) and 91% (laser). This improvement in dysphagia was maintained at 2 months in 75% (brachytherapy) and 81% (laser). Performance scores improved in 33% (brachytherapy) and 36% (laser). Both treatments were well tolerated, required a minimum of inpatient treatment time, and allowed patients to die without terminal admission to district referral centers. Retreatments were three times as common with laser therapy, but the frequency of treatment failures was equal. Minor complications, especially transient early dysphagia, was more common in the brachytherapy group, although the only major complication (perforation) occurred in the laser group. No procedure-related deaths occurred in either group.


The effect of basic fibroblast growth factor and omentopexy on revascularization and epithelial regeneration of heterotopic rat tracheal isografts.

Donor airway ischemia is a significant problem after clinical lung transplantation despite the use of omentopexy for accelerated local bronchial revascularization. Several growth factors have been shown to induce angiogenesis in vitro and in vivo. In the present study the quantitative effects on tracheal revascularization and epithelial regeneration of omentopexy and continuous local administration of basic fibroblast growth factor were investigated in a heterotopic rat tracheal isograft model. Tracheas were harvested from donor rats and heterotopically implanted into the omentum of syngeneic recipient rats. Animals were randomly assigned to study groups differing only in treatment of the tracheal segments: omental wrap for 2, 7, or 14 days; omental wrap plus continuous local administration of basic fibroblast growth factor for 7 or 14 days; or omental wrap plus local application of saline for 7 or 14 days. Two, 7, or 14 days after the animals were put to death, the vascularity of the tracheal segments and attached omentum and the tracheal epithelial morphology were assessed in a blinded fashion with use of light microscopy and morphometric image analysis. Vascularity in tracheal segments treated with basic fibroblast growth factor was significantly (p less than 0.05) greater than in control tracheas after 7 and 14 days. Epithelial regeneration was also improved in the basic fibroblast growth factor-treated groups at days 7 and 14 (p less than 0.05). We conclude that continuous local administration of basic fibroblast growth factor enhances early revascularization of tracheal segments induced by omentopexy and accelerates epithelial regeneration in a heterotopic rat tracheal isograft model.


Current approach to hypoplastic left heart syndrome. Palliation, transplantation, or both?

Over the past 3 years, 35 newborn infants have been referred for surgical management of hypoplastic left heart syndrome. Surgical palliation (first-stage Norwood) or cardiac transplantation was offered. Twenty-four families (68%) chose palliation and 11 families (32%) chose cardiac transplantation. Of the 11 infants listed for cardiac transplantation, five underwent transplantation. Because of a lack of donors after an average wait of 25 days (19 to 31), the remaining six infants underwent palliation, with no perioperative deaths. Of the 30 infants undergoing palliation, including crossovers, 20 (67%) survived the first operative stage. Among the last 19 infants undergoing palliation in 1990, the early survival was 84%. Risk factors determined for poor outcome were year of operation (p less than 0.001) and circulatory arrest time greater than 50 minutes (p less than 0.001). Among the 13 infants undergoing palliation with a circulatory arrest time of less than 50 minutes, there were 12 survivors (92%); among 12 having a circulatory arrest time of more than 50 minutes, there were four survivors (33%). At intermediate follow-up, six infants have undergone second-stage procedures (Glenn), with five survivors. There were eight late deaths, four caused by respiratory infections and four caused by cardiac problems, including a thrombosed shunt in one infant. Three of five infants are alive and doing well after cardiac transplantation. Size of aorta, tricuspid regurgitation, and ventricular wall thickness did not prove to be risk factors. Given the existing data, we believe these infants should be managed selectively on the basis of donor availability and family wishes.


Equivalent eighteen-hour lung preservation with low-potassium dextran or Euro-Collins solution after prostaglandin E1 infusion.

Improved techniques of pulmonary preservation would help alleviate the critical shortage of donor organs in lung transplantation and would improve early graft function. A previous study demonstrated that cold pulmonary artery flush with low-potassium dextran solution was superior to Euro-Collins solution in preservation of canine lung allografts stored for 12 hours when no pulmonary vasodilator was used before donor lung flush. The present study was designed to determine whether donor pretreatment with prostaglandin E1 would affect the superiority of low-potassium dextran as a preservation solution. Prostaglandin E1 was infused (50 micrograms/min) in 12 donor dogs until potent vasodilation was demonstrated. Low-pressure pulmonary artery flush (50 ml/kg) with either Euro-Collins or low-potassium dextran solution (n = 6 for each group) was performed at 4 degrees C in a randomized, blinded fashion. Heart-lung blocks were extracted and stored at 4 degrees C for 18 hours before left lung allografting. Inflatable cuffs were placed around each pulmonary artery, allowing independent study of the native and transplanted lungs. All 12 recipient dogs survived the 3-day assessment period. Lungs flushed and stored in Euro-Collins or low-potassium dextran solution provided equivalent gas exchange function on day 0 (arterial oxygen tension: Euro-Collins 289 +/- 105 mm Hg versus low-potassium dextran 265 +/- 111 mm Hg; mean +/- standard error of the mean) and on day 3 (Euro-Collins 516 +/- 45 mm Hg versus low-potassium dextran 354 +/- 77 mm Hg; p = 0.10). Mean pulmonary artery pressures in the transplanted lung were not significantly different in the Euro-Collins and low-potassium dextran groups on day 0 (21.4 +/- 2 mm Hg versus 33.7 +/- 5 mm Hg, respectively; p = 0.09) or on day 3 (20.2 +/- 2.7 mm Hg versus 24.2 +/- 5.1 mm Hg, respectively; p = 0.50). We conclude that there was no advantage of low-potassium dextran over Euro-Collins as a flush solution in this 18-hour canine single lung allograft model in which prostaglandin E1 was administered before pulmonary artery flush.


Allergic rhinitis.

Allergic rhinitis is a common disease with characteristic symptoms affecting the eyes, ears, and face as well as the nose. A detailed history is the foundation of a correct diagnosis. Laboratory tests may be needed to supplement this in atypical presentations. A combination of pharmacotherapy, immunotherapy, and environmental control may be required to control and prevent symptoms.