Biomedical subjects
Publications and source records attributed to G D Johnson.
For more than a century follicles have been recognized as a site of intense cell proliferation and cell death. At last the significance of this activity is beginning to emerge: antigen-driven B-cell proliferation, somatic mutation, positive and negative selection, and memory and plasma cell development all appear to take place within the follicle.
Germinal centers develop in follicles during TD antibody responses in the first 3 wk following each immunization. In primary responses to protein-based antigens, T-cell help is limiting, follicles develop towards the end of the 1st wk from immunization and the size of the follicular response in relatively small. When T-cell help is provided the primary B-cell response in follicles is much larger, B cells start to proliferate in follicles within a few hours of immunization and reach peak size 3-4 d later. Available evidence suggests that virgin B cells that colonize follicles to form germinal centers must first be activated by antigen outside follicles, probably in T zones. Memory B cells also proliferate in follicles and they can do so without first being activated outside follicles. Germinal center formation consists of an initial phase of exponential proliferation of B cells within the follicular dendritic cell network. After a single immunization the follicular response is oligoclonal and on average only 3 cells colonize each follicle. In responses to hapten-protein in rats primed previously with the carrier protein these 3 cells increase to around 10(4) cells in 3 d with a cell cycle time of about 6 h. At the end of the period of exponential growth of B blasts, the classical structure of germinal centers emerges. The B blasts become centroblasts in the dark zone of the germinal center which develops at that pole of the FDC network nearer the T zones. The centroblasts are still in rapid cell cycle but do not more than sustain their numbers. The rest of their progeny move to the heart of the FDC network where they come out of cell cycle as centrocytes. Evidence is cited which indicates that somatic mutation occurs in the Ig V-region genes of centroblasts and that centrocytes are selected on the basis of their ability to respond to antigen held on FDC. Centrocytes not receiving this antigen-dependent signal kill themselves by apoptosis. Centrocytes positively selected by interaction with antigen on FDC receive further signals which induce the cells to differentiate to become either plasma cells or memory B cells. The nature of some of these differentiation signals is described. It is shown that proliferation, selection and differentiation occur within germinal centers in distinct micro-environments.(ABSTRACT TRUNCATED AT 400 WORDS)
Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.
B cells undergo selection within germinal centers on the basis of their capacity to be activated by antigen held on follicular dendritic cells. Isolated germinal center B cells in culture kill themselves by apoptosis but this is prevented if their receptors for antigen are cross-linked. In this study it is confirmed that almost all germinal center B cells, unlike other B cells, do not express the 25-kDa protein encoded by the bcl-2 oncogene. Cross-linking the surface Ig of isolated germinal center cells causes them to express bcl-2 protein. Two other stimuli which inhibit the entry of germinal center cells to apoptosis result in the expression of bcl-2 protein. These stimuli are: (a) CD40 antibody and (b) recombinant 25-kDa fragment of the CD23 protein plus recombinant interleukin 1 alpha. Respectively, these induce germinal center cells to differentiate to resting B cells or plasmablasts. Dual-fluorescence studies on small lymphocytes confirm the presence of bcl-2 protein in mitochondria but show that this is also present in other extra-nuclear areas. Burkitt lymphoma cells have a phenotype which indicates that they are neoplastic cells of germinal center origin. The expression of bcl-2 protein by Burkitt lymphoma lines was also studied. Burkitt lines which retain the phenotype of fresh Burkitt lymphoma cells can be induced to enter apoptosis on culture with the Ca2+ ionophore ionomycin. These cells were found not to express bcl-2 protein. By contrast, Burkitt lines which have drifted towards a lymphoblastoid cell line phenotype and are resistant to the induction of apoptosis express high levels of the bcl-2 protein. The findings support the concept that the susceptibility of germinal center cells to entering apoptosis is associated with their lack of expression of bcl-2 protein. Aberrant expression of bcl-2 protein by some neoplastic germinal center cells may allow survival in situations where their normal counterparts die.
The surgical management of patients with slow-growing benign temporal bone neoplasms has been criticized because of its significant morbidity and mortality compared with results after radiation therapy, but long-term control by irradiation remains unproved. Long-term surgical results have not been studied previously. One hundred twenty-nine skull base operations were performed in 126 patients at the Otology Group, Nashville, Tenn., from January 1970 through May 1987. Fifty-eight patients responded to questionnaires focusing on recovery from loss of cranial nerves. All patients regained some degree of facial function (class V or better), no alimentary tubes or tracheotomies were in use, and no patients had debilitating aspiration. Long-term compensation from the cranial nerve deficits of lateral skull base surgery can be expected in most patients and should not be used as an argument for irradiation in patients with a long life expectancy at time of diagnosis.
Bovine herpesvirus-1 (BHV-1) was detected in Musca autumnalis De Geer after feeding on virus suspensions in bovine albumin or sucrose. Virus was detected for 48 h in flies fed an albumin suspension, with the highest titers recovered in crops (1.0 x 10(4) TCID50/ml) and whole-body (5 x 10(4) TCID50/ml/fly) specimens immediately after feeding; the level of virus declined rapidly thereafter. Virus titers in flies fed sucrose suspensions were 5 x 10(4) TCID50/ml in excised crops and 5 x 10(5) TCID50/ml in whole-body specimens; virus was not detected in flies 72 h after feeding. Four Hereford calves exposed for 4 h to BHV-1 fed flies did not develop any clinical symptoms related to BHV-1 transmission (i.e., elevated temperatures, ocular/nasal discharges, and labored breathing). Serum neutralization and plaque reduction tests did not show antibody production in calves exposed to BHV-1 infected flies. While we demonstrated that face flies retained BHV-1 for a short period of time, it does not appear that M. autumnalis is involved in either the mechanical or biological transmission of BHV-1.
It has been suggested that immunological mechanisms involving lymphocyte-mediated damage are important in the characteristic bile-duct damage that occurs in primary biliary cirrhosis and primary sclerosing cholangitis. Because adhesion is necessary for the interaction of lymphocytes with their target structures, we have studied the expression of intercellular adhesion molecule 1, a ligand for the leukocyte adhesion receptor lymphocyte function-associated antigen 1 in the liver of patients with primary biliary cirrhosis and primary sclerosing cholangitis. Strong expression of intercellular adhesion molecule 1 was seen on interlobular bile ducts and proliferating bile ductules in both conditions. In primary biliary cirrhosis, medium-sized ducts, which are spared by the disease, were negative. Minimal bile-duct staining was seen in conditions in which bile-duct damage is not a major feature, such as nonbiliary cirrhosis and acute liver diseases. In patients with cirrhosis from any cause, strong expression of intercellular adhesion molecule 1 was detected on the periseptal hepatocytes adjacent to new connective tissue. The intensity of immunohistochemical staining was recorded using a semiquantitative visual scoring system that was subsequently validated quantitatively by confocal laser scanning microscopy. The expression/induction of intercellular adhesion molecule 1 on bile ducts may be important in the pathogenesis of bile-duct damage in primary biliary cirrhosis and primary sclerosing cholangitis and is further evidence to support an immune pathogenesis in these two conditions. Furthermore, the induction of intercellular adhesion molecule 1 on hepatocytes may be an important factor in the liver-cell damage and fibrosis that occur during the development of cirrhosis.
The reason for the close association between primary biliary cirrhosis and the appearance of antibodies that recognize the E2 component of pyruvate dehydrogenase complex is not understood. The distribution of the three pyruvate dehydrogenase complex subunits was examined in the liver and lymph nodes of patients with primary biliary cirrhosis, patients with other liver diseases and normal subjects by immunohistochemistry using affinity-purified antibodies. Intensity of staining was assessed semiquantitatively and validated by scanning laser confocal microscopy. In primary biliary cirrhosis tissue, the E2 staining pattern did not parallel the reported distribution of mitochondria. E2 staining in biliary epithelial cells was consistently stronger than in hepatocytes. In primary biliary cirrhotic liver, staining of biliary epithelium was significantly stronger than in normal or other liver disease controls; many bile ducts in primary biliary cirrhotic liver demonstrated very high intensity, diffuse distribution of stain. No differences in staining intensity were seen between perivenular hepatocytes in primary biliary cirrhotic liver and those in controls; periportal hepatocytes in primary biliary cirrhotic liver were, however, more intensely stained than perivenular cells. In primary biliary cirrhotic portal lymph nodes, a subset of macrophages showed high-intensity, diffuse distribution of stain. By contrast, staining with antibodies to E1 and E3 (other components of pyruvate dehydrogenase complex) produced uniform-intensity, mitochondrial distribution both in primary biliary cirrhosis and control tissue. The increased intensity of E2 in primary biliary cirrhotic tissue could be explained in terms of abnormal metabolism of E2 by biliary epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
A small number of children have been identified as having an interruption in their communicative progress known as Landau-Kleffner syndrome, acquired epileptic aphasia, or aphasia with convulsive disorder. Although presenting symptoms have differed among the cases reported, a progressive or acute language loss and inattentiveness to auditory stimuli are the most common manifestations. Typically, these children begin developing language normally and then, for no apparent reason, language progress is disrupted. This disruption is accompanied by the onset of seizure activity and/or abnormal electroencephalographic (EEG) findings. While this disorder appears to be relatively uncommon, its frequency is questionable due to its unfamiliarity among the audiology and otology communities and, thus, it is subject to the likelihood of misdiagnosis. A case of acquired epileptic aphasia is described herein. A team diagnostic and management approach, which can include audiology, otology, psychology, neurology, and speech-language pathology is recommended for such cases. Earlier identification of this debilitating disorder is needed in order to secure appropriate intervention and reestablish communication systems for these children.
We have observed that the red reaction product of alkaline phosphatase immuno-conjugates and certain substrate preparations produces a brilliant red fluorescence that is visible by fluorescence microscopy using both fluorescein and rhodamine filter combinations. This provides a level of sensitivity greater than that obtained with other commonly used red fluorochromes or by inspection of the reaction product under bright field illumination. Of particular value, the reaction product is unaffected by the denaturing conditions required for the detection of incorporated nuclear BrdU with FITC conjugated anti-BrdU antibody and provides a simple and robust method for the simultaneous detection of cell proliferation and cell surface markers.
The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using L-alanyl-beta-naphthylamide as substrate Vmax of the reaction was shown to be pH independent over the range of 5.5-9.0, while Km exhibited a pKa of 7.7. This latter value corresponds to the pKa of the amino group of the substrate. Using X-Ala and X-Leu to examine the specificity of the P1 site it was found that Arg and Lys exhibit the highest affinity, followed by Met, Val, Leu, Trp, and Phe, which bind congruent to 5- to 20-fold less well. Although Km varied more than 20-fold within this series, Vmax showed considerably less variation. Significantly weaker binding was observed with a P1 Gly, Ala, Ser, or Pro with no binding detectable with a P1 Glu. The presence of a P'1 Leu compared to P'1 Ala results in an approximate 10-fold decrease in Km with little change in Vmax. The effect of varying P'1 residues was examined with the series Leu-X. In this case basic and hydrophobic amino acids, with the exception of Val, all exhibit nearly the same Km. The binding of Arg-Arg and Lys-Lys showed the same Km as obtained for Arg-Leu or Lys-Leu, respectively. When Leu-Ser-Phe was compared to Leu-Ser the P'2 residue led to a 100-fold decrease in Km and slightly less than a 5-fold increase in Vmax. In contrast the addition of a P'2 Met to Leu-Trp results in only a 3-fold decrease in Km and a 3-fold increase in Vmax. The results indicate a preference for a basic or hydrophobic residue in the P1 and P'1 sites and indicate subsite-subsite interactions which primarily affect binding.
Following the observation of Bonnefoy et al. (J. Exp. Med. 1988. 167:57), that the low-affinity IgE receptor (CD23) on B lymphocytes can be coupled (with the use of chemical cross-linking reagents) to major histocompatibility complex (MHC) class II DR molecules, we now report that ligands binding within the lectin-homology region of CD23 prevent B cells from stimulating allogeneic mixed lymphocyte responses. Ligands capable of blocking mixed lymphocyte responses include the anti-CD23 antibodies MHM6 and EBVCS 4 but not EBVCS 1 and 5. IgE itself, and small peptides representing sequences within the CH3 domain of IgE. The detailed topographical relationship between CD23 and MHC class II on the B lymphocyte surface was examined using dual immuno-fluorescence labeling of cells and direct visualization of the staining by confocal laser scanning microscopy. On transformed B lymphoblasts, the two antigens were seen to co-localize in discrete patches; on normal B cells which had been cultured for 2 days with interleukin 4, CD23 and MHC class II converged at a single pole which exhibited a tendency to pseudopod formation and provided a focus for homotypic cell-cell interactions. The possibility that CD23 could serve as a co-stimulatory-adhesion molecule in antigen presentation by B lymphocytes is discussed with special reference to a potential role in the regulation of IgE synthesis.
A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.
In appropriately selected patients, glomus tumors of the head and neck are best treated surgically. Unresectability is not a factor in therapeutic planning for local disease control. Existing techniques and exposures for tumor removal can be reliably applied to these paragangliomas, with acceptable morbidity and mortality. A team approach to this problem is mandatory.
Using a simian virus 40/adenovirus 12 (Ad12)-recombinant virus, the Ad12 early region 1B (E1B) 19K protein has been produced at high levels after infection of Cos 1 cells. Expression of the 19K polypeptide reaches a maximum at about 48 h post-infection, declining at later times as host cell death occurs. Using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis, we have shown that the Ad12 E1B protein is a major species following infection of Cos 1 cells with recombinant virus. Two forms of 19K polypeptide can be distinguished following isoelectric focusing. Using subcellular fractionation of infected cells, it was found that a similar distribution of 19K protein occurred after recombinant virus and Ad12 infection, with the polypeptide being most abundant in nuclear and membranous fractions. Similarly, as in Ad12-infected cells, a certain proportion of the protein is located on the outside surface of the cell after recombinant viral infection. Immunohistochemical studies suggest that, at early times post-infection, the E1B 19K protein is located in the nuclear membrane, the Golgi apparatus and the endoplasmic reticulum. At later times, it can be seen to have spread to the cytoplasm as well as to the other organelles. These results are discussed in relation to the known functions of the 19K E1B protein.
The regulatory mechanisms that monitor the size of the peripheral B cell pool and determine cell death or survival are poorly understood. In rodents B lymphopoiesis is maintained at a high rate throughout adult life, and under resting conditions there is little recruitment into the long-lived peripheral pool; it therefore follows that most newly formed B lymphocytes have a very short lifespan. The maturation stages of B lymphopoiesis in humans and in experimental mammals appear to be similar. We have determined the phenotype of sIgM- and sIgD-expressing cells from normal adult human bone-marrow and peripheral blood by dual immunofluorescence with an extensive panel of monoclonal antibodies representative of major B cell clusters, in order to identify antigenic differences that may play a regulatory role. Antibodies of the CD21, CD22 and CD9 clusters, the unclustered restricted B antibody 7-F-7 and anti-IgD were reactive with different proportions of sIgM+ cells in blood and bone marrow; 29.5% (range 5-60%) of sIgM+ cells in marrow were sIgD- and most of these cells were also CD21- and CD22-, thus defining a unique marrow population. However, newly formed and mature re-circulating cells comprising the sIgM+sIgD+ population could not be distinguished by the panel of antibodies.
Dual fluorescence analysis with a single-laser fluorescence-activated cell sorter is dependent on the use of two fluorochromes with similar excitation wavelengths but different emission wavelengths. The dye pair fluorescein and R-phycoerythrin (RPE) have been widely employed for this purpose and interaction between the two dyes has not been observed. Here evidence is presented to show that at high concentrations RPE can completely quench the fluorescein signal in dual fluorescence analysis of human tonsil lymphocytes labelled with pairs of monoclonal antibodies. Reduction in the fluorescein signal correlated with the intensity of red (RPE) staining. This phenomenon can seriously compromise interpretation of dual immunofluorescence carried out on a single laser instrument and can best be avoided by careful analysis of single colour controls.
The histopathologic findings for a patient with acute facial paralysis caused by herpes zoster oticus who obtained no return of active facial function after 1 year are presented. All imaging studies were nondiagnostic. Biopsy of the labyrinthine segment was performed. Histopathologic analysis showed a sharp line of demarcation between sclerotic nerve proximal to and necrotic nerve distal to the meatal foramen area of the fallopian canal. This finding is consistent with observations that the lesion producing Bell's palsy and herpes zoster oticus usually is situated at the meatal foramen.