Characterization of the high molecular weight insulin-like growth factor complex in term pregnancy serum. Insulin-like growth factors (IGFs) circulate in human adult serum predominantly as a high mol wt complex of 150 kilodaltons (kDa), which is made up of three subunits: alpha, the acid-labile subunit, a glycoprotein of around 85-100 kDa; beta, the acid-stable IGF-binding protein subunit, which is the 40-kDa GH-dependent glycoprotein IGF-binding protein-3 (IGFBP-3); and gamma, the 7.5 IGF-I or -II peptide subunit. During human pregnancy, all three subunits are elevated when measured by RIA. However, recent ligand blotting studies have shown that IGFBP-3 is markedly reduced during pregnancy. When pregnancy serum is acidified and neutralized to inactivate endogenous alpha-subunit, ternary complex formation is normal with exogenous radiolabeled alpha-subunit, indicating functional IGFBP-3. We have attempted to clarify the status of IGFBP-3 and the high mol wt (alpha beta gamma) complex in human term pregnancy serum by further analyses. Term pregnancy (TP) or nonpregnancy (NP) pooled sera were fractionated on a S-300 neutral column. The high mol wt (125-150 kDa) and the low mol wt (30-40 kDa) IGF-IGFBP complexes were identified by both RIA for IGFBP-3 and ligand blotting; each was pooled separately. Half of each pool was lyophilized and rechromatographed in acid to separate the IGF-I peptides from their IGFBPs. The IGFBP fractions were recovered for further studies. When the IGFBPs from the high mol wt complex were cross-linked to [125I]IGF-I or -II, bands of 48, 34, 26, and 21 kDa, which were more intense with [125I]IGF-II, were observed, and all were immunoprecipitable by anti-IGFBP-3 antibody. The smaller forms were elevated in TP serum. Affinity cross-linking analysis with [125I]alpha-subunit showed that the IGFBPs from the high mol wt, but not the low mol wt, complex can reform the ternary 150-kDa complex when cold IGF-I or IGF-II is added exogenously. A smaller 130-kDa complex was also present, and it was the predominant form in TP serum. Both bands were immunoprecipitable by anti-IGFBP-3 antibody. When purified alpha-subunit or fractions from neutral Sephacryl S-300 chromatography were cross-linked to covalent [125I]IGF-II:IGFBP-3, a specific band at 150 kDa was observed with pure alpha-subunit as well as with S-300 150- to 100-kDa serum fractions. These results suggest that 1) functional alpha-subunit is present in TP serum; 2) intact as well as smaller fragments of IGFBP-3 are present in the big complex of TP serum and are able to bind IGF and complex with alpha-subunit; and 3) IGFBP-3 in TP serum has reduced binding to [125I]IGF-I, but not to [125I]IGF-II.


Human luteal cells express dipeptidyl peptidase IV on the cell surface. We previously reported that human theca interna cells and small luteal cells express membrane-bound aminopeptidase N, and suggested that membrane-bound peptidases are involved in folliculogenesis and luteal function by regulating extracellular peptide concentrations. In this study, we examined the expression of dipeptidyl peptidase IV (DPP IV), which is a membrane-bound peptidase and has its catalytic domain at extracellular sites, in human granulosa cells, thecal cells of growing, preovulatory, and atretic follicles, as well as corpora lutea. Indirect immunofluorescence staining of ovarian tissues with specific monoclonal antibodies revealed that DPP IV was present in large and small luteal cells in corpora lutea. DPP IV peptidase activity was also detected histochemically in corpora lutea. In growing, preovulatory, and atretic follicles, there was weak immunoreactivity and DPP IV peptidase activity on luteinized theca interna cells, but not on granulosa cells. The expression of DPP IV on the cell surface of large and small luteal cells was confirmed by indirect immunofluorescence staining of freshly isolated luteal cells. These results indicate that DPP IV is a useful surface differentiation marker of human luteal cells and suggest that peptidases are involved in luteal function.


Induction of persistently demyelinated lesions in the rat following the repeated adoptive transfer of encephalitogenic T cells and demyelinating antibody. A chronic relapsing model of demyelinating experimental allergic encephalomyelitis (EAE) was induced in Lewis rats by the repeated co-transfer of encephalitogenic, myelin basic protein (MBP)-specific T cells in combination with a demyelinating monoclonal antibody (mAb) specific for the myelin oligodendrocyte glycoprotein (MOG). In controls, repeated injections of 5 x 10(5) MBP-specific T cells at intervals of 18-21 days resulted in an increasing resistance to the induction of further episodes of EAE. However, intravenous injection of the mAb 4 days after each T cell transfer overcame this 'vaccination' effect and induced severe clinical relapses associated with an increasing and persistent neurological deficit. Histological examination revealed that four cycles of treatment with T cells and mAb were sufficient to result in the formation of large plaques of demyelination in the spinal cord that failed to undergo significant remyelination within 60 days of the final injection of mAb. These lesions consisted of a matrix of astrocytic scar tissue traversed by numerous naked axons. These observations demonstrate that the formation of large, persistently, demyelinated lesions in a T cell-mediated model of EAE in the Lewis rat is dependent on the presence of an appropriate anti-myelin autoantibody response.


Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures. Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.


Repair of a myelin lesion by Schwann cells transplanted in the adult mouse spinal cord. In multiple sclerosis and experimental demyelination, oligodendrocytes and Schwann cells are able to repair myelin lesions of the central nervous system. However, spontaneous myelin repair is often insufficient. Several approaches to enhance remyelination have been considered and transplantation of myelin-forming cells has been proposed as one of them. In this paper, we present results which confirm the ability of transplanted Schwann cells to remyelinate an induced demyelinated lesion of the spinal cord. Schwann cells were either purified Schwann cells isolated from 1-2-day-old rat sciatic nerves, or immortalized Schwann cells (MSC80) arising from a purified culture of 7-day-old mouse sciatic nerves. They were transplanted into or at a distance from a lysolecithin-induced lesion of the Shiverer spinal cord. Labelling of the Schwann cells with the fluorochrome Hoechst 33342 enabled us to trace them after transplantation in their host and evaluate their ability to reach and to repair the demyelinated lesion. Using the Hoechst-Shiverer model, we show that when transplanted in the lesion, cultured Schwann cells, even immortalized, are able to remyelinate such a lesion efficiently. In addition, when transplanted at a distance from the lesion, they are able to reach and repair the lesion in time frames which allow them to compete actively with host oligodendrocytes.


Interleukin 4 inhibits in vitro proliferation of leukemic and normal human B cell precursors. In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells.


Epitope regions on U1 small nuclear RNA recognized by anti-U1RNA-specific autoantibodies. Autoantibodies specifically directed to U1RNA were found in patients suffering from systemic lupus erythematosus (SLE) overlap syndromes. To obtain more insight in the mechanism responsible for this U1RNA-specific antibody formation and to use the antibodies eventually as a tool to study U1RNA-protein (U1RNP) interactions, the B cell epitopes on U1RNA were mapped. Using in vitro synthesized domains of U1RNA, the main epitope regions were found in stemloops II and IV. Furthermore, 3'-end or 5'-end truncation of both stemloop II and stemloop IV showed that the conformation of the stemloops is critical for antibody recognition. Mutant studies on both stemloops indicated that in the case of stemloop II the stem is the main antigenic region, whereas in stemloop IV, the loop (E-loop) is a main target. The results of this study support the idea that the anti-U1RNA autoantibody could be the result of a process driven by the human U1RNP complex itself (antigen-driven process).


Ion channels in human erythroblasts. Modulation by erythropoietin. To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].


Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3. IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to it receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs of these mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor.


Macrophages, T cell receptor usage, and endothelial cell activation in the pancreas at the onset of insulin-dependent diabetes mellitus. Current knowledge of the phenotype of mononuclear cells accumulating in pancreatic islets in insulin-dependent diabetes (IDDM) and factors determining their homing into the pancreas is limited. Therefore, a pancreas obtained at the onset of IDDM was studied in detail. Cryostat sections were stained for mononuclear cell types, T cell receptor subtypes, and adhesion molecules of vascular endothelium and studied by immunofluorescence microscopy, and peripheral blood mononuclear cells were phenotyped using flow cytometry. Monocytes/macrophages (lysozyme- or CD 14-reactive cells) were identified among other mononuclear cell types in islet infiltrates. V beta 8-positive T cells were overrepresented, but T cells with other V beta s studied (V beta 5, V beta 5.1, V beta 6, V beta 12) were also found. The vascular endothelium of the islets and many small vessels nearby islets strongly expressed intercellular adhesion molecule-1, whereas vascular cell adhesion molecule-1 and E-selectin were totally absent. We conclude: (a) that increased expression of intercellular adhesion molecule-1 on vascular endothelium may increase endothelial adhesion of mononuclear cells and enhance their accumulation in the pancreas during diabetic insulitis; (b) that T cells with certain T cell receptors can be enriched in infiltrated pancreatic islets; and (c) that macrophages and antigen-specific CD 8-positive T cells are involved in pancreatic beta cell destruction at the onset of IDDM.


Expression and localization of proteins of the complement system in human skin. The complement system participates in the immune recognition of foreign antigens, many of which may penetrate the skin by physical injury or transcutaneous adsorption. In this study, we examined the presence of complement components and complement regulatory proteins in the human skin and cultured human keratinocytes. Immunofluorescence studies showed C3, Factor B, decay accelerating factor, the C3b receptor (CR1), and C3d receptor (CR2), distributed among cells of the epidermis as well as on cultured keratinocytes. Immunoblot analysis of keratinocytes supernatants showed the presence of C3 with a molecular weight of approximately 180 kD. The decay accelerating factor was localized as previously reported on elastic fibers; additionally it was observed in the basement membrane zone. In situ hybridization studies suggest the expression of CR1 and CR2 mRNA in human epidermis. These results show the presence in the human epidermis of complement components that are capable of generating the initial C3 convertase of the alternative pathway. The presence of complement regulatory proteins could endow keratinocytes with immune functions such as the regulation of complement activation and endocytosis of C3 opsonized particles.


Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.


Immunochemical characterisation of a murine monoclonal anti-idiotypic antibody. Y7, a murine monoclonal IgG1 kappa antibody against a human monoclonal IgM lambda DJ molecule, was affinity purified on an IgM lambda immunoaffinity column. As detected by enzyme-linked immunosorbent assay (ELISA) the isolated Y7 monoclonal antibody was shown to be not cross-reactive with human IgG, human secretory IgA, mu chain, lambda + kappa chains and another human monoclonal IgM lambda BR. Binding to the polyclonal human IgM standard in the same assay was about 30 percent. The epitope specificity of affinity purified and biotinylated Y7 MoAb was localized only in the nonreduced pepsin Fab fragments of IgM lambda DJ immunogen. As the immunogen was determined to be a specific antibody to phosphorylcholine, the specificity of Y7 MoAb was further ascertained in its capacity to induce 95% inhibition of immunogen binding for phosphorylcholine.


Amyloid in prostatic corpora amylacea.

Amyloid in prostatic corpora amylacea. To determine the presence and nature of amyloid in prostatic corpora amylacea using immunohistological studies. Prostatic tissue from 18 transurethral and two open resection specimens was studied. Paraffin wax embedded tissue sections were stained with haematoxylin and eosin and the alkaline Congo red method with and without previous treatment with potassium permanganate. Sections were also stained with antibodies to amyloid A, beta 2 microglobulin, lambda and kappa light chains, prealbumin IgA, G, M, S100 protein, prostatic specific antigen, amyloid P component and CAM 5.2 (control and blocking studies were performed). The prostatic corpora amylacea universally showed the presence of amyloid. In all instances this contained beta 2 microglobulin. Prostatic corpora amylacea represents a localised amyloidosis of beta 2 microglobulin origin that is unrelated to chronic renal failure and haemodialysis.


Subclinical ischaemic episodes during the steady state of sickle cell anaemia. To determine the clinical, haematological, biochemical and rheological changes that occur in the asymptomatic steady state of sickle cell anaemia. Patient self-assessment visual analogue scores (for wellbeing and tiredness), the blood concentration of acute phase proteins (C-reactive protein, orosomucoid, and fibrinogen), and blood rheology (percentage of dense cells and the number of sickled cells that occluded pores 5 microns in diameter) were studied longitudinally on 10 occasions in each of 20 outpatients with sickle cell anaemia. Patients in the steady state showed fluctuation in visual analogue scores, in concentration of acute phase proteins, and in rheological parameters consistent with minor episodes of tissue injury. Significantly more variation in acute phase proteins occurred in the steady state of 14 of the 20 patients who developed one or more vaso-occlusive crises during the 16 month study period. Rheological fluctuation in the steady state simulated rheological change during crisis, namely a transient rise and then fall in the number of dense and poorly filterable cells. The term "steady state" is a misnomer, being characterised by biochemical and rheological fluctuation consistent with minor episodes of microvascular occlusion that are insufficient to cause the overt tissue infarction of painful crisis.


Specificity of colon specific antigen and colon ovarian tumour antigen. Sections (5 microns thick) from 101 primary adenocarcinomas (including ovarian, colorectal, gastric, breast, oesophageal, prostatic, pancreatic, endometrial and gall bladder) were incubated wtih anticolon specific antigen (CSA) and anticolon ovarian tumour antigen (COTA) antibodies using the peroxidase antiperoxidase technique with positive and negative controls. Anti-CSA positivity was seen in 19 of 20 colonic adenocarcinomas, but it was also seen in a large number of the other tumours. While anti-COTA staining was positive in 16 of 20 colonic adenocarcinomas and 20 of 30 ovarian adenocarcinomas, it was also positive in a large number of the tumours. Anti-CSA and anti-COTA are not adequately specific in the identification of a colonic or ovarian origin of an adenocarcinoma and cannot reliably be applied to the identification of a metastatic adenocarcinoma of unknown primary site.


Effect of Tisseel on healing after periodontal flap surgery. The purpose of this investigation was to evaluate the effect on healing of fast and slow absorbable Tisseel in combination with periodontal flap surgery. Mucoperiosteal flaps were raised on the buccal aspect of maxillary premolars and mandibular premolars and first molars in 4 beagle dogs. The underlying buccal, interproximal and inter-radicular bone was then removed to a level of approximately 5 mm apically to the original bone crest and half way into the interdental spaces and bifurcations. The exposed root surfaces were curetted in order to remove the periodontal ligament tissue, and a notch was made in the root surface at the base of the defects. On the control teeth, the flaps were sutured immediately after creation of the defects, while on the test teeth, a layer of fast (group I) or slow (group II) absorbable Tisseel was applied between the curetted roots and the subsurface of the flaps prior to suturing. Postoperatively, the teeth were brushed 2 x weekly. The dogs were sacrificed after 4 months. Histological analysis revealed that the amounts of new attachment and bone regrowth were similar in the test and control groups, although the results tended to be most favorable for the group of teeth treated with fast absorbable Tisseel (Group I).


Substance P-containing pyramidal neurons in the cat somatic sensory cortex. Light and electron microscopic immunocytochemical methods were used to verify the possibility that neocortical pyramidal neurons in the first somatic sensory cortex of cats contain substance P. At the light microscopic level, substance P-positive neurons accounted for about 3% of all cortical neurons, and the vast majority were nonpyramidal cells. However, 10% of substance P-positive neurons had a large conical cell body, a prominent apical dendrite directed toward the pia, and basal dendrites, thus suggesting they are pyramidal neurons. These neurons were in layers III and V. At the electron microscopic level, the majority of immunoreactive axon terminals formed symmetric synapses, but some substance P-positive axon terminals made asymmetric synapses. Labelled dendritic spines were also present. Combined retrograde transport-immunocytochemical experiments were also carried out to study whether substance P-positive neurons are projection neurons. Colloidal gold-labelled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase was injected either in the first somatic sensory cortex or in the dorsal column nuclei. In the somatic sensory cortex contralateral to the injection sites, a few substance P-positive neurons in layers III and V also contained black granules, indicative of retrograde transport. This indicates that some substance P-positive neurons project to cortical and subcortical targets. We have therefore identified a subpopulation of substance P-positive neurons that have most of the features of pyramidal neurons, are the probable source of immunoreactive axon terminals forming asymmetric synapses on dendritic spines, and project to the contralateral somatic sensory cortex and dorsal column nuclei. These characteristics fulfill the criteria required for classifying a cortical neuron as pyramidal.


Raphespinal and reticulospinal axon collaterals to the hypoglossal nucleus in the rat. Neurons in the medial tegmental field project directly to spinal somatic motoneurons and to cranial motoneuron pools such as the hypoglossal nucleus. The axons of these neurons may be highly collateralized, projecting to multiple levels of the spinal cord and to many diverse regions at different levels of the neuraxis. We employed a double fluorescent retrograde tracer technique to examine whether medial tegmental neurons that project to the spinal cord also project to the hypoglossal nucleus. Injections of Diamidino Yellow into the hypoglossal nucleus and Fast Blue into the spinal cord produced large numbers of double labeled neurons in the medial tegmental field, particularly in the caudal raphe nuclei and adjacent ventromedial reticular formation. In these structures the number of neurons projecting to both the hypoglossal nucleus and the spinal cord was equivalent to the number of neurons projecting to multiple levels of the spinal cord observed in control animals. Fewer neurons projecting to both the hypoglossal nucleus and the spinal cord were observed in several other nuclei and subregions of the medial tegmental field, while almost no such neurons were observed in the lateral tegmental field or other pontomedullary structures. These results demonstrate that neurons of the caudal raphe nuclei and adjacent ventromedial reticular formation project to both the spinal cord and the hypoglossal nucleus, and support the concept that the diffuse projections to motoneuron pools from the medial tegmental field globally modulate both spinal and cranial somatic motoneuron excitability.


Morphology of identified preganglionic neurons in the dorsal motor nucleus of the vagus. To determine the degree of variation of neuronal morphology both within and between the subnuclei of the dorsal motor nucleus of the vagus (dmnX), structural features of the preganglionic neurons of each of the five primary subnuclei in the rat dmnX were characterized quantitatively. Each of the columnar subnuclei was separately labeled by application of the retrograde tracer fast blue to its corresponding subdiaphragmatic vagal branch. Fixed brain slices of 100 microns thickness were then prepared in coronal, sagittal, and horizontal orientations. Next, randomly selected fast blue labeled neurons (n = 1,256) were injected with Lucifer yellow, drawn with camera lucida, and digitized. For each cell, three features of the perikaryon and twelve of the dendritic tree were measured. Dorsal motor nucleus neurons with up to eight primary dendrites, 30 dendritic segments, and seventh order dendritic branches were observed. Throughout the dmnX, the dendrites of preganglionic neurons were preferentially oriented in the horizontal plane. Consistent with an organizing role for the columnar subnuclei, most dendrites remained within their column of origin. However, between 5 and 30% of the neurons in each of the columns projected dendrites into adjacent dmnX subnuclei or other brainstem nuclei, including the nucleus of the solitary tract (NTS). The cyto- and dendroarchitectural analyses revealed systematic gradations in morphology, although they did not support the idea that the dmnX was composed of multiple distinct preganglionic types. The most parsimonious interpretation of the data is that dmnX motorneurons are variants of a single prototype, with dendrites varying widely in length and degree of ramification. The extent of an individual preganglionic neuron's dendritic field was predicted by three factors: the cell's rostrocaudal position within the dmnX, its location within a transverse plane (i.e., its coronal position within or ectopic to the dmnX), and its subnucleus of origin. Neurons at rostral and midlongitudinal levels of each column had more extensive dendritic arbors than those at caudal levels. Ectopic neurons had more extensive dendritic fields than similar cells in the corresponding columns; in fact, of all vagal preganglionic neurons, ectopics had the most extensive dendritic fields. Somata and dendrites of celiac column neurons were more extensive than those of hepatic and gastric column cells. These differential regional distributions of vagal preganglionics suggest that their structure and function are correlated.


Lamina I spinocervical tract terminations in the medial part of the lateral cervical nucleus in the cat. The terminations of spinocervical tract fibers in the lateral cervical nucleus (LCN) of the cat were examined with anterogradely transported Phaseolus vulgaris leucoagglutinin (PHA-L) in order to analyze their organization relative to the most medial part and the main body (the lateral two-thirds) of the LCN, which have differential projections and physiological characteristics. Iontophoretic injections of PHA-L in laminae I-V of the spinal dorsal horn yielded dense labeling in somatotopically appropriate regions of the main body of the LCN, and, as seen previously with horseradish peroxidase, additional terminations were present in the medial LCN after injections at either cervical or lumbar spinal levels. The morphological characteristics of the PHA-L labeling in these two parts of the LCN were different. Terminations in the lateral LCN consisted of dense clusters of thick fibers bearing large numbers of boutons. The terminal axons in the medial part of the LCN displayed a reticulated network of longitudinally oriented, fine fibers with well-spaced varicosities. Some of the fine fibers in the medial LCN appeared to be collaterals of thicker fibers that terminated in the lateral LCN. Injections of PHA-L that were restricted to lamina I resulted in terminal labeling only in the medial LCN. The labeling was more sparse than that observed in the medial LCN after larger dorsal horn injections but displayed the same morphological characteristics. Lamina I terminations were seen in the medial LCN after cervical or lumbar injections on both the ipsilateral and contralateral sides. The PHA-L observations were corroborated by the presence of many retrogradely labeled lamina I cells at both cervical and lumbar spinal levels, following injections of cholera toxin subunit b or rhodamine-labeled microspheres in the medial LCN. In addition, double-immunofluorescent labeling for PHA-L and substance P was performed in a few cases, since substance P immunoreactivity is present in fibers in the medial LCN and also in cell bodies in lamina I; however, very few spinocervical fibers displayed immunoreactivity for both antigens. These observations indicate that the medial part of the LCN receives input from lamina I neurons, and probably from lamina III-V neurons as well, at cervical and lumbar spinal levels. The lamina I input to the medial LCN provides a basis for the small population of nociceptive neurons that differentiate the medial LCN. The lamina I input could also be responsible for the general inhibition of lateral LCN neurons by wide-field noxious stimulation, via activation of GABAergic interneurons in the medial LCN.(ABSTRACT TRUNCATED AT 400 WORDS)


Multiple neurotransmitters in the tuberomammillary nucleus: comparison of rat, mouse, and guinea pig. Tuberomammillary neurons in the posterior hypothalamus are the sole source of neuronal histamine in adult mammalian brain. In the rat, these cells are reported to contain immunoreactivity for gamma-aminobutyric acid (GABA) and several neuropeptides. We compared the presence of these substances in the tuberomammillary cells of the rat, mouse, and guinea pig. In all three species, all histamine-immunoreactive neuronal cell bodies were positive for GABA. This suggests that GABAergic transmission may be important in tuberomammillary function. No cell bodies immunoreactive for thyrotropin releasing hormone (TRH) were found in the guinea pig or mouse tuberomammillary area. In contrast, about 14% of the histamine-immunoreactive tuberomammillary cells in the rat were TRH-positive. These cells were small or medium-sized and were located only in the medial part of the tuberomammillary complex. An antibody against porcine galanin stained about 45% of the tuberomammillary cell bodies in the rat and about 28% in the mouse, but none in the guinea pig. A large proportion of the cells in the rat and mouse, but none in the guinea pig, were positive for met-enkephalin-arg-phe. In contrast, all histamine-containing tuberomammillary cells in the guinea pig, but none in the rat or mouse, were immunoreactive for met-enkephalin. This may indicate a different expression of proenkephalin-derived peptides in the tuberomammillary neurons in these species. Some substance P-immunoreactive cell bodies were located in the tuberomammillary area in all three species. However, only 3% of the histamine-immunoreactive cell bodies in the rat and mouse but none in the guinea pig were substance P-positive. The neurochemical properties of the tuberomammillary nucleus that exhibited species commonality deserve to be studied neurochemically and electrophysiologically in order to determine the functional relevance of coexisting transmitters in this nucleus.


Topographic organization of subcortical projections to the anterior thalamic nuclei in the rat. Subcortical projections to the anterior thalamic nuclei were studied in the rat, with special reference to projections from the mammillary nuclei, by retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. The medial mammillary nucleus (MM) projects predominantly ipsilaterally to the entire anterior thalamic nuclei, whereas the lateral mammillary nucleus projects bilaterally to the anterodorsal nucleus (AD) of the anterior thalamic nuclei. A topographic relationship was recognized between the MM and the anterior thalamic nuclei. The dorsal region of the pars mediana of the MM projects to the interanteromedial nucleus (IAM), whereas the ventral region projects to the rostral part of the anteromedial nucleus (AM). The dorsal and the ventral regions of the pars medialis project to the dorsomedial part of the AM at its caudal and rostral levels, respectively. The dorsomedial region of the pars lateralis projects to the ventral AM. The ventrolateral region of the pars lateralis projects to the ventral part of the anteroventral nucleus (AV) in such a manner that rostral cells project rostrally and caudal cells project caudally. The pars basalis projects predominantly ipsilaterally to the dorsolateral AV and bilaterally to the AD. The rostrolateral region of the pars posterior projects to the lateral AV, whereas the medial and the caudal regions of the pars posterior project to the dorsomedial AV. The rostrodorsal part of the nucleus reticularis thalami was found to project to the anterior thalamic nuclei; cells located rostrally in this part project to the IAM and AM, whereas cells located caudodorsally project to the AV and AD. The laterodorsal tegmental nucleus projects predominantly ipsilaterally to the AV, especially to its dorsolateral part. The present study demonstrates that subdivisions of the subcortical structures are connected to the subnuclei of the anterior thalamic nuclei, with a clear-cut topography arranged in the dorsoventral and the rostrocaudal dimensions.


Nerve growth factor receptor-immunoreactive neurons within the developing human cortex. A monoclonal antibody recognizing the p75 receptor for nerve growth factor (NGF) was used to assess the immunohistochemical expression of NGF receptors within the developing human neo-, limbic, and paralimbic cortices as well as the hippocampal complex. Between embryonic weeks 16 and 26, a transient population of neurons located within the upper and lower subplate zones of the neo-, limbic, and paralimbic cortices expressed the receptor for NGF. In contrast, NGF receptor-immunoreactive neurons were only observed in the upper subplate zone of the entorhinal cortex at embryonic week 40 (term), a staining pattern not observed in a 5-year-old specimen. The expression of NGF receptor-immunoreactive neurons within the upper subplate zone between embryonic weeks 16 and 40 was characterized by a dense band of immunoreactive neurons and neuropil. These neurons were bipolar with basal and apically directed neurites. NGF receptor-immunoreactive neurons were also scattered throughout the lower subplate zone and underlying white matter between embryonic weeks 19 and 26. These neurons were multipolar, with less apically directed neurites. NGF receptor-immunoreactive subplate neurons displayed a topographic distribution with the heaviest concentration found within limbic and paralimbic cortices as well as association neocortex. In contrast, light to moderate NGF receptor-immunoreactivity was seen in sensory-motor cortex. Within the hippocampal complex, only a few lightly stained NGF receptor-immunoreactive neurons were seen within the fimbria, hilar region of the dentate gyrus, and subiculum. The expression of NGF receptor-immunoreactivity increased within the subplate zone of the pre- and parasubiculum culminating in intense entorhinal cortex staining. As the entorhinal cortex merged with the developing inferior temporal association cortex, there was a marked reduction in staining intensity. In contrast to those in the subplate zone, neurons within the germinal zone and cortical plate were NGF receptor immunonegative at all times examined. The presence of NGF receptors in the subplate zone suggests that neurotrophins such as NGF play an important role in the transient viability of these neurons as well as in the guidance of cortical afferent inputs into topographically organized regions of the cerebral cortex.


The structure of the diencephalic prepacemaker nucleus revisited: light microscopic and ultrastructural studies. The prepacemaker nucleus (PPn), a bilateral cluster of neurons at the boundary of diencephalon and mesencephalon, controls frequency modulations of the electric organ discharge in weakly electric knifefish (Eigenmannia sp.). Previous light microscopic studies employing retrograde labelling with horseradish peroxidase suggested that the PPn is restricted to a small area, located approximately 400 microns laterally from the third ventricle and fusing at its medial edge with the thalamic central posterior nucleus (CP). In the present investigation we used Phaseolus vulgaris-leucoagglutinin and cholera toxin as highly sensitive markers. In contrast to the previous studies, these experiments yielded a large number of labelled cells not only in the region of the traditionally defined PPn but also in an area reaching far into the CP. Since the PPn has been defined by retrograde labelling rather than by topographic criteria, this result questions the traditional separation between PPn and CP. Such a notion is in agreement with observations of Nissl-stained sections at the light microscopic level and with a quantitative analysis of several morphological characteristics of the cell bodies in the PPn and CP at the ultrastructural level. Both sets of experiments failed to find differences between the two nuclei. Furthermore, autoradiographic studies have shown that, even in adulthood, cells are continuously born within the ventricular zone of the CP, and at least some of these newborn cells differentiate into CP cells and migrate laterally towards the PPn. Therefore, we postulate that CP and PPn form one large complex, with the medial CP providing precursors of neurons in the lateral CP and PPn.


A multiterminal stretch receptor, chordotonal organ, and hair plate at the wing-hinge of Manduca sexta: unravelling the mystery of the noctuid moth ear B cell. The present study aims to shed light on the evolutionary origin of the B cell, a sensory element of unknown function in the noctuid moth ear. Peripheral projections of the metathoracic nerve IIIN1b1, homologue of the noctuid moth tympanic nerve, are described in the atympanate moth Manduca sexta on the basis of dissections with the aid of Janus Green B, and intracellular tracer dyes Lucifer yellow and cobalt lysine. A large multiterminal (Type II) neurone, attaching to membranous cuticle ventral to the hind wing axillary cord, was discovered. This cell appears to be homologous to the B cell in the noctuid moth ear. Recordings from the IIIN1b1 nerve in M. sexta reveal a continuous train of large, uniform spikes, presumed to originate from the multiterminal cell. This unit increases its rate of firing in response to hind wing elevation, suggesting that it functions as a stretch receptor monitoring wing movements during flight. Also identified in the tympanic nerve homologue, and closely associated with the multiterminal cell, were a chordotonal organ and hair plate. The chordotonal organ consists of a proximal scolopidial region and a distal strand that attaches to the sclerotized epimeron slightly medial to the multiterminal cell. This simple chordotonal organ, having three uniterminal (Type I) sensory cells, is homologous to the auditory cells of the noctuid moth ear. The significance of these receptors as proprioceptors in M. sexta, and as evolutionary precursors to the noctuid moth ear, is discussed.


Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus. To investigate synaptic mechanisms in taste buds and collect information about synaptic transmission in these sensory organs, we have examined taste buds of the mudpuppy, Necturus maculosus for the presence of neurotransmitters and neuromodulators. Immunocytochemical staining at the light microscopic level revealed the presence of serotonin-like and cholecystokinin-like (CCK) immunoreactivity in basal cells in the taste bud. Nerve fibers innervating taste buds were immunoreactive for vasoactive intestinal peptide-like (VIP), substance P-like, and calcitonin gene-related peptide-like (CGRP) or compounds closely related to these substances. Immunoreactivity for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the taste cells and nerve fibers was absent. These data suggest that serotonin, CCK, VIP, substance P, and CGRP are involved in synaptic transmission or neuromodulation in the peripheral organs of taste. No evidence was found for cholinergic or adrenergic mechanisms on the basis of the absence of immunocytochemical staining for key enzymes involved in these two transmitter systems.


Topography of ganglion cells in the dog and wolf retina. The topographical distribution of retinal ganglion cells in seven breeds of dog (Canis lupus f. familiaris) and in the wolf (Canis lupus) was studied in retinal wholemounts stained with cresyl violet or with a reduced silver method. A prominent feature of all wolf retinae was a pronounced "visual streak" of high ganglion cell density, extending from the central area far into both temporal and nasal retina. By contrast, either a pronounced or a moderate visual streak was found in dog retinae. It is hypothesized that a pronounced streak is an archetypal feature of Canis lupus, and that the moderate streak in some dogs is a corollary of breeding during domestication. Irrespective of the differences in streak form and retinal area, the estimated total number of ganglion cells was about 200,000 cells in the wolf and 115,000 in the dog. Ganglion cell density maxima in the central area of the wolf were about 12,000-14,000/mm2, and in the dog they ranged from 6,400/mm2 to 14,400/mm2. This implies individual differences in visual acuity. Alpha ganglion cells constituted 3-14% of all ganglion cells in the dog and 1-18% in the wolf, depending on retinal location. A distinct feature of all dogs and wolves was the absence of alpha cells in a substantial region of temporal peripheral retina. This has not been found in any other mammalian species and suggests corresponding functional deficits.


Pathological changes in the reproductive tract of male rhesus monkeys associated with age and simian AIDS. The pathological changes associated with ageing and simian immunodeficiency virus (SIV) infection in groups of immature, adult and ageing Rhesus monkeys were studied. Eighty three per cent (5 of 6) of uninfected ageing animals had hyperplasia of the prostate, 33 per cent (2 of 6) had mild prostatitis and in 66 per cent (4 of 6) there were calcified concretions in the seminal vesicles. The testes were normal and showed active spermatogenesis. In the SIV-infected animals, two types of lesion occurred; the most common, in 81 per cent (18 of 22 monkeys), was the presence of focal lymphoid infiltrations in the epididymis, prostate or seminal vesicles. The other was hypospermatogenesis (23 per cent, 4 of 17) with degeneration of seminiferous tubules. Immunocytochemical staining demonstrated that the lymphoid masses contained approximately equal numbers of B and T lymphocytes, but the majority of diffusely scattered cells were T lymphocytes. Staining for SIV antigen identified small numbers of positive lymphocytes and macrophages in all tissues.


Marjolin's ulcer: immunohistochemical study of 17 cases and comparison with common squamous cell carcinoma and basal cell carcinoma. Formalin-fixed, paraffin-embedded biopsy specimens of 17 cases of squamous cell carcinoma of Marjolin's ulcer (SCC-MU), 6 cases of common SCC (SCC), and 5 cases of basal cell carcinoma (BCC) were stained with three monoclonal antikeratin antibodies (CAM 5.2, MAK-6, and MA-903), a monoclonal antivimentin antibody (V9), and a polyclonal anticarcinoembryonic antigen antiserum (A115). Neoplastic cells of SCC-MU, SCC, and BCC showed consistently negative staining for CAM 5.2. A wide range of reactivity, from negative to diffuse strong positivity, among neoplastic cells of SCC-MU and SCC was noted with MAK-6. Alternatively, neoplastic cells of SCC-MU, SCC, and BCC consistently showed diffuse moderate to strong reactivity with MA-903. These findings imply that SCC-MU has largely high-molecular-weight keratins. They also showed a wide range of reactivity with V9. However, neoplastic cells of five of the six SCC and five cases of BCC were negative for V9. These findings suggest that neoplastic cells of SCC-MU contain vimentin in higher frequency than in the more usual SCC.


Spindle cell neoplasms coexpressing cytokeratin and vimentin (metaplastic squamous cell carcinoma). Spindle cell squamous carcinoma (SCSC) and atypical fibroxanthoma (AFX) are both spindle cell neoplasms (SCN) that usually arise in areas of solar or ionizing radiation of elderly patients. Both lesions have a similar biologic behavior. In addition, the morphologic and ultrastructural similarities found in AFX and the spindle cell component of SCSC, have led some investigators to conclude that these tumors have a similar cell of origin. We studied 15 SCNs with no evidence of epithelial origin and no morphologic epithelial component, that showed immunohistochemical and ultrastructural evidence that would support metaplastic changes of a squamous cell carcinoma to a neoplasm with mesenchymal characteristics.


Rat palatal histology related to denture-like appliances. Two designs of palatal denture-like appliance were fitted to rats. Tooth- and tissue-borne appliances caused changes in epithelial thickness and keratin thickness, but these varied with anatomical site and type of appliance. A single inoculation of Candida albicans at the time of insertion of the appliance failed to establish infection consistently. Debris accumulation under the appliances was a problem which will restrict long-term studies with this model.


MRI of temporal lobe pleomorphic xanthoastrocytoma. Pleomorphic xanthoastrocytomas (PXA) are classically supratentorial, peripherally located, well-circumscribed, partially cystic neoplasms, which can enhance on CT following the administration of intravenous contrast agents. Focal calcification may also be seen. Although the CT appearance has been described, we report the MR findings in two cases of histologically documented temporal lobe PXA. The two well-circumscribed lesions were predominantly cystic and both contained a Gd-DTPA enhancing mural nodule. The latter was isointense with gray matter on T1-weighted images and hyperintense on T2-weighted scans. Minimal surrounding edema was present. Histologically, PXA may be confused with glioblastoma multiforme (GBM) due to the pronounced cellular pleomorphism. Because of their potentially more indolent behavior compared with GBM, it is important to recognize the gross morphologic characteristics of this rare tumor on MR. The MR pattern of a cystic lesion with enhancing mural nodule is characteristic of PXA, but not diagnostic, and other lower grade gliomas such as ganglioglioma and pilocytic astrocytoma need to be considered. The MR and CT appearance of PXA can provide critical information for the pathologist, especially when only a small amount of tissue is obtained for histologic evaluation.


A technique for three-dimensional microleakage assessment using tooth sections. Microleakage is commonly assessed using restored teeth, sectioned through the midline of the restoration. It is impossible to determine if this is representative of the leakage throughout the whole tooth. This pilot study examined the feasibility of calculating areas and volumes of leakage using serial sections of restored teeth which had been subjected to dye immersion and image analysis. Using the sections, perspex models of the teeth were constructed to present the pattern of the dye leakage into dentine. This appears to be a viable technique for the three-dimensional assessment of microleakage.


Medical humanities in nursing: thought provoking? Medical humanities is an innovative way of learning. Discussing literary texts of nursing practice has been used to help students analyse attitudes, values and ethics; it has also been used to help practitioners review and reflect on their own experience and philosophy of nursing. In nursing education, it has been used to explore difficult issues in a safe environment. The value of this approach in nursing education and practice is that it can encourage reflection, promote self-awareness and stimulate debate on difficult issues: for example, death and dying, power and institutionalization (of patients and staff) and pain. This paper gives a detailed worked example of how a literary text can be used in this way, the aim being to provide a resource which readers can then use with a group of students or colleagues. Finally, the authors explore the question of where medical humanities might have a place in the curriculum: as a lecture/tutorial in a course (e.g. Ethics), as a module in the curriculum, as a method of teaching nursing subjects (e.g. communication skills), as a discussion group (outside the curriculum), as a study guide, using literary texts alongside nursing text books. Any of these strategies can be a powerful vehicle for preserving the 'human factor' in both nursing education and continuing professional development.


Effects of acute and chronic treatment with fluvoxamine on extracellular and platelet serotonin in the blood of major depressive patients. Relationship to clinical improvement. The effects of the treatment with fluvoxamine (FVX) on platelet and plasma serotonin (5-HT) have been examined in eleven drug-free major depressive patients. Acute FVX was without effect, whereas the repeated oral treatment (100-150 mg daily, 12 weeks) reduced platelet 5-HT (-89%, P less than 0.001) and plasma 5-HT (-60%, P less than 0.02). Patients who responded to the treatment at 6 weeks (Hamilton score less than or equal to 10) had significantly lower (-39%, P less than 0.02) pretreatment values of platelet 5-HT than the rest. This suggests that 'low 5-HT' patients may have a more rapid improvement after fluvoxamine. Platelet 5-HT and HDRS correlated significantly along the treatment (r = 0.679, P less than 0.01). These data demonstrate a marked action of fluvoxamine as 5-HT uptake inhibitor at therapeutic doses and confirm that this mechanism is relevant for its efficacy as antidepressant drug.


Accuracy of in-vivo assessment of prostatic volume by MRI and transrectal ultrasonography. More accurate noninvasive estimation of prostate size is important in therapeutic trials for benign prostatic hyperplasia. The accuracy of MRI and transrectal ultrasound (TRUS) in assessing prostate weight was evaluated in 48 patients who underwent radical prostatectomy for stage A or B cancer. The volume derived from the wet weight of the freshly excised specimen was used as a reference. We compared that volume with volume estimates derived from the three-axis linear dimension measurement by MRI and TRUS using a tissue density of 1.05 g/cc and the standard formula for an ellipsoid object. Prostate and seminal vesicle volumes were also computed by contouring T2-weighted 5 mm thick contiguous MR images using a semiautomatic edge detection program and pixel summation. Three-axis volume MRI method versus volume from wet weight has slightly less scatter than TRUS three-axis method (r = 0.85 vs r = 0.81). Contoured MR volume method has the least scatter r = 0.93, statistically better than the linear axis method. Contoured MRI volumetric analysis appears superior to linear MRI or TRUS methods in estimating true prostate volume.


Transcatheter umbrella closure of valvular and paravalvular leaks. Our aim was to adapt the technique of transcatheter umbrella closure of intracardiac defects for closure of valvular and paravalvular defects. The double-umbrella device developed by Rashkind and Cuaso has been safely and effectively delivered across a host of intracardiac defects, but transcatheter closure of valvular and paravalvular leaks has not been reported. Between February 1987 and September 1990, eight patients who were believed to be poor operative candidates were taken to the catheterization laboratory for transcatheter double-umbrella closure of a valvular or a paravalvular leak. Four patients had a paravalvular leak around a prosthetic aortic valve. The other four patients had a valvular leak: one patient with a regurgitant native aortic valve after a Stansel procedure and three patients with a regurgitant porcine valve in a left ventricular apex to descending aorta conduit. Placement of a double-umbrella device was attempted in seven of the eight patients and was successful in all seven. Device placement was not attempted in one patient because of the crescentic shape of his defect. Two patients required two devices for each closure; the other five required only one device each. Angiography, performed on six patients after device closure, demonstrated that three patients had a completely occluded defect, two had trivial residual flow and one patient had mild residual flow through the device. All significant complications occurred in one patient who had hemolysis and oliguria that resolved when the initial umbrella was replaced by a larger device. In addition, two devices migrated to the patient's pulmonary arteries but were retrieved in the catheterization laboratory without difficulty. No other early or late complications occurred in 21 to 50 months of follow-up. Of the four patients with a paravalvular leak, the one who did not receive a device died at operation, one patient died at operation for an associated defect (in the operating room the umbrella was found securely in place across the paraaortic defect) and two patients are clinically well at home after 21 and 32 months, respectively. Of the four patients with closure of a valvular leak, one patient remains well at home 50 months later, one patient died at operation for associated defects and two patients had additional successful surgical treatment and remain well 29 months after device placement. Transcatheter umbrella closure appears to be a reasonable alternative for closure of a valvular or paravalvular leak in patients who are poor operative candidates.


Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite). The activation of CD4+ T lymphocytes, which play a central role in allergic inflammation, depends on the recognition of allergen-derived peptides in association with major histocompatibility complex class II gene products. In this report we demonstrate, at a clonal level, that a component of the T-cell repertoire reactive with Dermatophagoides spp. (house dust mite) in atopic individuals, is restricted by HLA-DP class II molecules. This supports the recent results emerging from genetic epidemiologic studies that indicate positive associations between the HLA-DP phenotype and immune responsiveness to a variety of common allergens. Our findings also reveal that the T cells restricted by HLA-DP recognize a species-specific epitope located in the group I allergen of Dermatophagoides pteronyssinus (residues 101-119). Furthermore, we report that the pretreatment of the T cells restricted by HLA-DP with the Der p I peptide renders them nonresponsive to an immunogenic challenge with house dust mite allergen, and the loss of antigen-dependent proliferation is associated with downregulation of membrane expression of the T-cell antigen receptor. The ability to functionally inactivate T cells restricted by HLA-DP, as well as those that recognize allergen in association with HLA-DR class II molecules, suggests that desensitization with allergen-derived peptides may have therapeutic potential in the management of allergic diseases irrespective of their HLA class II association.


A survey of the vision assessment of the developmentally disabled and multi-handicapped in University Affiliated Programs (UAPs). In 1989 we conducted a survey to assess the availability of vision assessments (screening and complete eye/vision examinations) in University Affiliated Programs for Persons with Developmental Disabilities (UAPs). Analysis of the results suggests that although the UAPs are continuing to provide some services for eye/vision care, only 58 percent of these centers have facilities for the screening of vision problems. Ninety-six percent of the respondents, however, feel that vision screening is important. The developmentally disabled and multi-handicapped child is at high risk for vision/eye problems. Unless this difference between service availability and the perceived importance of vision services is addressed, there is an increased risk that the child may not reach his/her full potential. The UAPs need to increase the availability of eye/vision care within the UAPs and to expand training to providers in the community to deal with the developmentally disabled and multi-handicapped.


Acute and subacute toxicity of 7.5% hypertonic saline/6% dextran-70 (HSD) in dogs. 1. Serum immunoglobulin and complement responses. Clinical use of modern dextran solutions has been limited by concerns of anaphylactoid reactions. To assess the short-term antigenic response to 7.5% hypertonic saline in 6% Dextran-70 (HSD), sera were obtained from dogs involved in the acute and subacute toxicology testing of HSD and its individual components, and analyzed for IgG, IgM and C3 complement. In separate studies, beagles were infused i.v. with a single dose of HSD or its components at 20 ml kg-1 (the maximum tolerated dose; MTD), or the MTD daily for 14 days, and serum was obtained prior to and at various times after infusion up to 14 days. In both studies, despite serum dextran concentrations exceeding 2000 mg dl-1, no induction of IgG, IgM or C3 complement concentrations were observed. In addition, serum IgG immunoelectrophoretic patterns were of normal curvature, position and intensity; the immunoprecipitin bands were not displaced, bowed, inhibited or thicker than the normal preinfusion immunoelectrophoretograms. The data suggest that single or multiple HSD i.v. injections, as much as five times the proposed therapeutic level for the treatment of hypovolemia, evoked no increase in antibody titers in dogs. Therefore, therapeutic use of HSD in the treatment of hemorrhagic shock should not be associated with widespread concomitant allergic complications.


Pulsatile release of histamine in the hypothalamus of conscious rats. The pattern of histamine release was investigated in the hypothalamus of the conscious, freely moving rat over 20 h. Under anaesthesia, a guide cannula was stereotaxically inserted into the posterior hypothalamus. In the conscious animal, the stylet of the guide cannula was replaced by a push-pull cannula, and the hypothalamus was superfused with artificial cerebrospinal fluid. Histamine was determined radioenzymatically in the superfusate which was continuously collected in time periods of 20 min. The release rate of histamine fluctuated according to an ultradian rhythm (frequency: 1 cycle per 83 min) and a circadian rhythm with the highest release rate of histamine between 11:00 p.m. and 1:00 a.m. The release rate of histamine during darkness was higher than that during the light period. The results demonstrate that, in the brain, neuronal histamine is released according to rhythms with various frequencies.


Presence and regional variation in peptide-containing nerves in the human ureter. The occurrence, distribution and regional variation of neurones immunoreactive for the neuropeptides, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and substance P (SP) were investigated in human ureters by indirect immunohistochemistry. In addition, immunoreactivities to tyrosine hydroxylase (TH), a marker of noradrenergic neurones and to protein gene product (PGP) 9.5, a general marker of neurones, were also studied. Neurones displaying PGP-, NPY-, VIP- and TH-like immunoreactivity (-LIR) provided a rich innervation to the smooth muscle and blood vessels of the ureter, where they formed dense muscular and perivascular nerve plexuses. In contrast, there was only a moderate to sparse innervation by SP and CGRP-LIR neurones, most of which were distributed to blood vessels and to the sub mucosal layer, and only rarely to smooth muscle bundles. No ENK-LIR was detected in this study. Nerve fibre bundle densities were estimated for each of the localized neurochemicals according to a method described. NPY-LIR nerve fibre bundles were found to account for 80% of the total nerve fibre bundles (i.e. PGP-LIR) in the ureter. On the other hand, TH-LIR and VIP-LIR nerve fibre bundles each accounted for 50% of the total ureteral innervation, whereas SP- and CGRP-LIR nerve fibre bundles each comprised 20% of the total innervation. The abundance and pattern of tissues innervated by these immunoreactive neurones is consistent with the view that some of these neuropeptide substances co-exist with other peptide substances and/or with other known neurotransmitters, such as noradrenaline or acetylcholine. A gradient of innervation was found to exist for all the neurochemicals demonstrated in the ureter, whereby the lower ureter receives a greater density of innervation than the upper ureter. This finding suggests the human ureter is primarily innervated by fibres arising from or via the lower pelvis, i.e. the pelvic plexus. It also supports the view that the lower ureter may perform an important physiological role, such as coordinating the tone of this region during bladder filling and emptying.


[Macular involvement in angioid streaks. Gronblad-Strandberg syndrome]. Between 1971 and 1988, we examined 116 eyes of 58 patients with Gronblad-Strandberg syndrome in private practice and in the Retinal Department of the Eye Clinic, Faculty of Medicine, Ankara University. There was initial macular involvement in 79% of cases with angioid streaks. The most frequently encountered lesion, found in 53% of the cases, was choroidal neovascularization. Macular involvement had become bilateral in 80% of the cases after 12.5 months median follow up. Thirteen of the 50 eyes with choroidal neovascularization were treated by argon or red krypton laser photocoagulation. Vision decreased during follow-up in 15% of the 13 eyes treated whereas it decreased in 32% of the 69 eyes unsuitable for laser treatment.


Breast cancer screening in older women: law and patient rights. Legal principles that apply to breast cancer in older women have been developed in judicial decisions related to other medical screening tests. There are no special legal rules for either mammography or older women, although older women seldom file malpractice suits. The general standard is that a screening test must be offered to any particular age group when it is considered "reasonably prudent" to do so, and this almost always means when the medical profession--usually speaking though its specialty boards--declares it the standard of care. The standard of care should be set by medical professionals, with open opportunity for public input, rather than by lawyers or risk managers. In actual practice, private regulation may not be sufficient to protect the public, and both state and federal regulation of mammography facilities now seems inevitable. Patients have the right to be fully informed prior to screening, the right to refuse screening, and the right to have full knowledge of the consequences of such refusal. Mammography is not a consumer good, but American women should be actively involved in determining issues of costs and benefits, as well as helping to develop the best strategies for counseling and informed consent.


Suicide risk and serotonin.

Suicide risk and serotonin. The accumulating research evidence for the psychobiological reconceptualization of suicidal behaviour is briefly reviewed, particularly regarding the serotonin hypothesis of suicide risk. The first suggestion of a link between suicide and serotonin came with the biochemical results from postmortem brain studies of suicide victims. Currently available research data suggest decreased brain stem levels of serotonin and 5-HIAA, altered distribution of presynaptic binding of the ligand 3H-imipramine to serotonin neurons and a region-specific increase in the number of postsynaptic 5HT2 receptors in the prefrontal cortex in suicide victims. Early CSF studies have indicated decreased CSF 5-HIAA in patients recently attempting suicide and follow-up studies of large samples of mood-disorder patients confirm the original hypothesis that low CSF 5-HIAA predicts suicide risk after attempted suicide. However, the clinical utility of these compelling biological correlates of suicide in the management of suicide risk in psychiatric patients will depend on the results of psychopharmacological treatment research targeting suicidal behaviour.


Epitope-selected monospecific antibodies to recombinant antigens from Toxoplasma gondii reacted with dense granules of tachyzoites. Epitope-selected monospecific antibodies were applied to investigate the localization of antigenic molecules in Toxoplasma gondii by immunoelectron microscopy. Eighty cDNA clones encoding antigenic polypeptides were immunoscreened from lambda gt11 expression library with T. gondii infected mouse sera. Twenty different clones with no crossreactivity were selected from eighty clones. Monospecific antibodies to antigens derived from respective cDNA clones extracted from infected mouse sera by the epitope selection method were used in Western blot analysis and immunoelectron microscopy. Eleven antigens were detected with epitope-selected antibodies in lysates of T. gondii tachyzoites. Five of the antigens with molecular weights of 60, 40, 35, 28, and 27 KD were localized in the dense granules. Monospecific antibodies purified by the epitope selection method were useful for investigating the localization of antigens without preparation of a monoclonal antibody from a hybridoma.


Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G. Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.


Monoclonal antibodies specific to quail embryo tissues: their epitopes in the developing quail embryo and their application to identification of quail cells in quail-chick chimeras. Quail-chick chimeras have been used extensively in the field of developmental biology. To detect quail cells more easily and to detect cellular processes of quail cells in quail-chick chimeras, we generated four monoclonal antibodies (MAb) specific to some quail tissues. MAb QCR1 recognizes blood vessels, blood cells, and cartilage cells, MAb QB1 recognizes quail blood vessels and blood cells, and MAb QB2 recognizes quail blood vessels, blood cells, and mesenchymal tissues. These antibodies bound to those tissues in 3-9-day quail embryos and did not bind to any tissues of 3-9-day chick embryos. MAb QSC1 is specific to the ventral half of spinal cord and thymus in 9-day quail embryo. No tissue in 9-day chick embryo reacted with this MAb. This antibody binds transiently to a small number of brain vesicle cells in developing chick embryo as well as in quail embryo. A preliminary application of two of these MAb, QCR1 and QSC1, on quail-chick chimeras of neural tube and somites is reported here.


B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes. In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.


Characteristics of murine monoclonal anti-CD4. Epitope recognition, idiotype expression, and variable region gene sequence. We have characterized a series of mouse monoclonal anti-CD4 and describe both their CD4 epitope recognition and Id expression. We also determined the V region gene sequences of these antibodies in an attempt to correlate epitope recognition and Id expression with V region sequence. All of these preparations recognize epitopes that cluster around the HIV gp120 binding site on the human CD4 molecule. However, we observed differences in epitope recognition among the anti-CD4 preparations, based on either competitive inhibition assays or functional assays, such as syncytium inhibition. Analysis of Id specificities using a polyclonal anti-Id generated against anti-Leu 3a indicated that five of the seven monoclonal anti-CD4 expressed a shared Id. Based on V region gene sequences, the V region kappa-chain (V[kappa]) from each of the seven antibodies was encoded by the V[kappa]21 gene family and expressed the J[kappa]4 gene segment. Those preparations that expressed the shared Id with anti-Leu 3a have virtually identical V[kappa] sequences, with a high degree of homology in the CDR. The VH region gene sequences of six of the seven antibodies also shared overall homology and appeared to be encoded by the J558 VH gene family. The seventh anti-CD4 VH region is encoded for by the VHGAM gene family. The majority of these antibodies used JH3 gene segment, although the JH2 and JH4 gene segments were also represented. In addition, several of these antibodies share a common sequence organization within their V-D-J joining regions that appears to involve N and P sequences to generate unique D segments. Together, these data suggest that differences in epitope recognition among the monoclonal anti-CD4 may reflect sequence variability primarily within the CDR3 region of both V[kappa] and VH. The basis for the detection of a shared Id most likely reflects the high degree of homology within the V[kappa] region sequences. In addition, these data, which are based on a limited analysis, suggest the possible restricted use of V region germ-line gene families in the secondary antibody response of BALB/c mice to specific epitopes on the human CD4 molecule.


Cytokines increase transporter in antigen processing-1 expression more rapidly than HLA class I expression in endothelial cells. Transporter in Ag processing-1 (TAP-1, previously called PSF-1 or Ring-4) is an MHC-encoded gene product that is required for efficient association of intracellular peptide Ag with nascent HLA class I H chain and beta 2-microglobulin, thereby permitting assembly and normal surface expression of the class I molecules. TAP-1 is thought to function as a component of a transmembrane pump, that transports cytoplasmically-derived peptides into the lumen of the endoplasmic reticulum where class I molecules assemble. Synthesis and expression of HLA class I molecules is increased in human endothelial cells by IFN-beta, IFN-gamma, and TNF. We report these same cytokines increase TAP-1 expression. As with class I, TAP-1 is also synergistically increased by combinations of TNF with IFN. Interestingly, cytokine-induced increases in TAP-1 mRNA are markedly more rapid than increases in class I mRNA. This rapid increase in TAP-1 mRNA is reflected in a rapid increase in TAP-1 protein. These results demonstrate that TAP-1 synthesis and class I synthesis are regulated in parallel. The rapidity of the cytokine response of TAP-1 compared to class I further suggests that the constitutive level of TAP-1 expression in endothelial cells is not sufficient to support inducible increases in class I expression.


Neuropeptides modulate human eosinophil chemotaxis. To investigate the role of neuropeptides in allergic inflammation, we examined the effect of peptides on eosinophil chemotaxis. Eosinophils were purified from the blood of allergic and normal subjects using a discontinuous Percoll density gradients. Chemotaxis was induced by platelet-activating factor (PAF) and leukotriene B4, and was assayed by a modified Boyden's chamber technique. Four neuropeptides were examined in this study: substance P (SP), neurokinin A, calcitonin gene-related peptide (CGRP), and cholecystokinin octapeptide. Peptides alone (10 nM to 10 microM) were not chemotactic for eosinophils. However, when eosinophils were pre-treated with peptides (100 nM) at 37 degrees C for 30 min, chemotactic response to PAF (10 nM) was significantly enhanced (p < 0.01) in allergic subjects; % control by SP, neurokinin A, CGRP and cholecystokinin octapeptide was 269 +/- 42, 243 +/- 32, 227 +/- 21, and 251 +/- 42, respectively (n = 8). Similar results were obtained in leukotriene B4-induced eosinophil chemotaxis. In contrast, no enhancement was observed in normal subjects. Potentiating effect of SP and CGRP on PAF-induced eosinophil chemotaxis in allergic subjects was significantly attenuated by SP antagonist [D-Pro2,D-Trp7,9]-SP and human CGRP (8-37) receptor antagonist, respectively. Neutral endopeptidase inhibitors (phosphoramidon, leupeptin, and bestatin) failed to significantly augment the PAF-induced eosinophil chemotaxis when the cells were pretreated with various peptides and neutral endopeptidase inhibitors. The C-terminal fragment of SP (SP6-11) had an effect similar to that of the intact SP molecule, whereas no potentiating effect by the N-terminal of SP (SP1-9) was observed. These results suggest that neuropeptides may play a significant role in eosinophil infiltration by priming cells in allergic inflammation.


Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present an immunodominant viral cytotoxic T lymphocyte epitope. For controlling infection of the mouse with the lymphocytic choriomeningitis (LCM) virus, CD8+ CTL are essential. In the infected BALB/c mouse the arising LCM virus-specific CTL are exclusively restricted by the class I MHC-encoded molecule L; K- or D-restricted antiviral CTL cannot be detected. Thus, the infected L-deficient BALB/c mutant C-H-2dm2 should not be capable of eliminating the virus. The experimental evidence proves the contrary, which is explained by K- and D-restricted CTL that this mouse generates. Why such cells remain undetectable in BALB/c mice is currently unexplained, because there is no lack of precursors and the corresponding virus Ag is presented. Despite the absence of lytic activity in vitro, other than the one associated with L, transfusion of day 8-immune spleen cells from BALB/c into infected C-H-2dm2 (L-deficient) mice results in accelerated virus elimination from the organs of the latter, which was manifest as soon as 8 h after cell transfer. Furthermore, lytic activity did not attain measurable levels in the recipients' spleens. Obviously, this infection can be terminated by CD8+ T lymphocytes even when these cells' lytic activity is below detectability.


Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis. Hepatitis C virus (HCV) is a major cause of post-transfusion and sporadic hepatitis worldwide, leading to chronic liver disease in at least 50% of infected individuals. The pathogenic mechanisms that result in chronic hepatitis are unknown. Lymphocytes are typically observed within the hepatic parenchyma, but the functional characteristics of these cells have not been defined. In this study, liver-infiltrating lymphocytes from two subjects with chronic HCV hepatitis were cloned at limiting dilution and tested for HCV-specific cytolytic activity using autologous target cells infected with vaccinia viruses expressing recombinant HCV Ag or sensitized with synthetic HCV peptides. In both subjects, HCV-specific, HLA class I-restricted CTL were identified that recognized epitopes in variable regions of either the envelope or nonstructural proteins. These results demonstrate the presence of HCV-specific CTL at the site of tissue damage in persons with chronic HCV hepatitis, and provide a means to evaluate the possible pathogenic role of these cells in HCV infection.


Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. Immune response of BALB/c mice to human immunodeficiency virus envelope glycoprotein. In vivo priming of CTL requires the association with MHC class I molecules of peptides derived from the processing of endogenously produced proteins. Immunization with exogenous proteins or peptides rarely induces MHC class I-restricted CTL unless they are associated with lipidic compounds. The capacity to induce CTL was compared in synthetic peptides and simple lipopeptides containing the Immunodominant MHC class I H-2Dd-restricted T-cell epitope of HIV-1 gp160. In contrast with free peptides in saline, lipopeptides induced strong primary CTL responses in vivo. These CTL were able to lyse cells infected with a recombinant vaccinia virus expressing the HIV-1 env gene. Priming of CTL was also successful when using 16-amino acid lipopeptides as 34-amino acid lipopeptides, suggesting that several epitopes might be included in a single construct. In vivo priming of CTL also requires CD4+ T cell help. We therefore searched for Th cell activation after priming with lipopeptides. Our results show that, as with CTL induction, Th cell activation with lipopeptides did not require mixing with adjuvant. In addition, lipopeptides were also efficient at stimulating antibody-mediated responses. Our results show that a single lipopeptidic construct can induce a total immune response, which is of importance in vaccine development.


B cell deficiency progresses with lineage maturation in nude.X-linked immunodeficient mice B cell deficiency progresses with lineage maturation. Previously we showed that unlike normal, nude, or X-linked immune deficient (xid) mice, nude.xid mice are deficient in bone marrow pre-B cell targets for Abelson murine leukemia virus transformation. We show that nude.xid bone marrow is deficient in both CD45(B220)+ and CD45(B220)- surface (s)IgM- progenitors that give rise to B cell colonies in Whitlock-Witte cultures. CD45(B220)+ precursors had normal differentiation potential in vitro. CD45(B220)- precursors differentiated into CD45(B220)+ cells at the same rate as normal controls, but acquired sIgM at a much slower rate. These results correlated with the observation that in nude.xid mice the severity of B lineage defects correlates with maturity: a profound (ninefold) deficit of sIgM+, CD45(B220)+ mature B cells, a fivefold deficit in the sIgM-, CD45(B220)+ precursors of short term B cell colonies (colonies forming within 4-5 days in Whitlock-Witte cultures), and a moderate (twofold) decrease in the frequency of sIgM-, CD45(B220)- (less mature) precursors of long term B cell colonies (colonies forming after 14 days of Whitlock-Witte culture. Thus the combination of the nude and xid mutations produces a deficiency in early B cell progenitors and the deficiency becomes more profound with further maturation. Therefore the lack of mature B cells is the result of a cascade effect. Inasmuch as bone marrow progenitors are affected, and these are the source of the vast majority of B cells, most B cells are affected by the xid mutation and the xid defect cannot be attributed to a loss of a fetal lineage of B cells. These results suggest that xid affected cells lack the capacity to progress efficiently through differentiation in the absence of an exogenous factor(s) that is dependent on the product of a normal allele at the nude locus. This product might be supplied in vivo by a T cell or T cell-dependent source and/or epithelial elements such as bone marrow stromal cells all of which are known to be affected by the nude mutation.


Superantigen and HLA-DR ligation induce phospholipase-C gamma 1 activation in class II+ T cells. Bacterial enterotoxin superantigens bind directly to HLA class II molecules (HLA-DR) expressed on both APC and activated human T cells, and simultaneously bind to certain V beta chains of the TCR. In this report, we compared early T cell signaling events in human alloantigen-stimulated T cells when activated by HLA-DR ligation through antibody cross-linking or by direct enterotoxin superantigen binding. Both types of stimuli induced tyrosine phosphorylation of phosphatidylinositol-specific phospholipase C gamma 1 (PLC gamma 1) and an increase in intracellular calcium concentration; however, superantigen-induced signaling was stronger than class II ligation alone. Antibody-mediated ligation of HLA-DR with CD3 resulted in augmented PLC gamma 1 activation and increased calcium mobilization, consistent with a mechanism of superantigen activity through a combination of class II and CD3/Ti signals. In addition, down-modulation of CD3 receptors with antibody demonstrated that superantigen-induced signaling events were CD3-dependent. Superantigen signaling was also class II-dependent, in that resting T cells were not responsive to direct enterotoxin stimulation. To address how early signal transducing activity correlated with T cell responsiveness, alloantigen-primed T cells were activated with immobilized class II-specific mAb or soluble superantigen. Both HLA-DR mAb-stimulated T cells and enterotoxin-treated T cells proliferated strongly in response to co-stimulation by a combination of CD28 receptor engagement and PMA addition. In addition, superantigen-induced growth was induced by CD28 receptor ligation with antibody or the B7 counter-receptor expressed on Chinese hamster ovary cells. Taken together, these results indicate that class II molecules expressed on activated T cells are directly coupled to the PLC gamma 1 signal transduction pathway, and that coligation of HLA-DR with CD3 augments T cell signaling comparable to that induced by enterotoxin superantigen. Thus, we suggest that superantigen-induced early signaling responses in activated T cells may be due in part to class II transmembrane signals induced when HLA-DR and V beta are ligated in cis.


Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1. Decay-accelerating factor (DAF or CD55) is a 70-kDa glycosyl-phosphatidylinositol (GPI)-anchored protein that protects cells from complement-mediated lysis by either preventing the formation of or dissociating C3 convertases. Cross-linking of DAF on human peripheral T cells by polyclonal antibodies has previously been reported to lead to lymphocyte proliferation. Two mAb, both mapping to the third short consensus repeat region of DAF, were able to trigger proliferation of human peripheral T cells. To determine the role of the GPI anchor in cell activation, we transfected EL-4 murine thymoma cells with cDNA encoding either DAF or a transmembrane form of DAF (DAF-TM). The DAF-transfected cells were able to transduce late activation events as evidenced by IL-2 production, whereas DAF-TM transfected cells were unable to do so. The GPI-anchored DAF was able to transduce early activation events leading to the tyrosine phosphorylation of a 40-kDa protein and several proteins in the 85-95 kDa range--an event absent in DAF-TM-transfected cells. Furthermore, anti-DAF immunoprecipitates of DAF-transfected cells contain tyrosine kinase activity leading to the phosphorylation of 40-, 56-60-, and 85-kDa proteins, whereas anti-DAF immunoprecipitates of DAF-TM-transfected cells did not have an associated kinase activity. Both p56lck and p59fyn were associated with DAF in DAF-transfected EL-4 cells. In HeLa cells transfected with fyn, DAF associated with p59fyn. This complex of DAF with src family protein tyrosine kinases requires the GPI anchor and suggests a pathway for signaling through GPI-anchored membrane proteins.


Serologic cross-reactivities poorly reflect allelic relationships in the HLA-B12 and HLA-B21 groups. Dominant epitopes of the alpha 2 helix. Previous analysis has emphasized the correlation between primary structures of class I HLA molecules and their patterns of serologic cross-reactivity. Here we describe the structures of two serologic groups of HLA-B alleles for which this is not the case. HLA-B45, an allele associated with black populations, is serologically paired with B44 in the B12 group; its structure, however, is divergent from that of B44 but closely related to B50. The BN21 (B*4005) allele is associated with native Americans and is serologically grouped with B50 in the B21 group; its structure, however, is more closely related to alleles of the B40 group. The B44 and B45 serologically cross-reactive molecules differ at seven functional positions of the Ag recognition site; the B50 and BN21 molecules differ at four such residues. These differences are predicted to alter peptide presentation and be capable of eliciting strong alloreactive T cell responses. For these pairs of B12 and B21 Ag, serology appears dominated by epitopes formed by short sequences of the alpha 2 helix which have been shuffled by recombination between alleles. The implications of these results for HLA matching in transplantation are discussed.


IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors. The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.


Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood. The Fas Ag is a newly defined cell-surface molecule that may mediate apoptosis. The antibody against Fas Ag can induce the apoptotic cell death in cell lines expressing this Ag. PBL subpopulations at various ages were here examined for Fas expression by two-or three-color flow-cytometric analyses using anti-Fas mAb. It was found that Fas Ag was appreciably detected on a proportion of T and B cells, whereas its expression was absent for NK cells. For CD4+ and CD8+ T cells, Fas Ag was expressed preferentially on CD45RO+ (memory or previously activated) populations, but not on CD45RO- naive ones. TCR-gamma/delta+ T cells, especially their CD45RO+ subsets, also expressed Fas Ag. Expectably, neonatal T cell subpopulations, most of which had the naive (CD45RO-) phenotype, expressed little Fas Ag. Fas-expressing B cells dominated in surface(s) IgD- populations, but neonatal B cells as well as adult sIgD+ B cells had little Fas Ag. The Fas Ag was inducible after in vitro mitogenic stimulation of naive T and B cells from neonatal blood. These observations suggested that expression of Fas Ag on T and B cells in the peripheral blood might reflect their in vivo Ag-activated status. In contrast to Fas-expressing cultured cell lines, however, viability of in vitro stimulated T and B cells as well as freshly isolated CD45RO+ T cells was not significantly changed after the treatment with anti-Fas mAb, indicating that additional cellular conditions to Fas expression might be required for anti-Fas-induced cell death.


Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins. Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.