Cutting Edge Issues in Autoimmune Gastritis

Autoimmune gastritis is the outcome of a pathological CD4 T cell-mediated autoimmune response directed against the gastric H/K-ATPase. Silent initially, the gastric lesion becomes manifest in humans by the development of megaloblastic pernicious anemia arising from vitamin B12 deficiency. Cutting edge issues in this disease relate to its epidemiology, immunogenetics, a role for Helicobacter pylori as an infective trigger through molecular mimicry, its immunopathogenesis, associated organ-specific autoimmune diseases, laboratory diagnosis, and approaches to curative therapy.     

Bronchoscopic management of airway obstruction in pediatric endobronchial tuberculosis

The present report describes a case of severe airway obstruction caused by endobronchial tuberculosis in an 11-year-old girl who was successfully treated by bronchoscopic balloon dilation. This case illustrates the insidious presentation and the increasingly important role of bronchoscopic intervention in the management of endobronchial tuberculosis. In addition, a brief literature review of the condition in the pediatric age group is included.     

Endogenous protein phosphorylation by resting and activated human neutrophils

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen- dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl- phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.     

Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme- linked differential antibody immunosorbent assay

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF- deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF- deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time- dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.     

Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate

Proteases can complicate the characterization of proteins from cells, especially human polymorphonuclear leukocytes (PMN), which contain abundant neutral proteases. We tested the ability of agents to inhibit proteolysis, with special reference to the subunit polypeptides of the contractile proteins actin, myosin, and actin-binding protein (ABP). Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N- ethylmaleimide, alone or in combinations, failed to prevent extensive proteolysis of the PMN proteins during solubilization of cells with dodecyl sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin inhibitor similarly could not prevent proteolysis during homogenization of cells in cold isosomolar sucrose. Treatment of PMN with greater than or equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or homogenization markedly inhibited proteolysis. PMSF and DFP were equally effective in inhibiting proteolysis in PMN extracts, suggesting that the efficacy of DFP may result from its permeation of intact cells and granules before barriers are disrupted by detergents or homogenization. Treatment of PMN with DFP under conditions inhibiting proteolysis did not affect their rate of phagocytosis. We recommend the use of DFP in future studies correlating functions and protein structure of PMN.     

Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.     

Phenotyping of malignant hematopoietic cells

Summary 1255 cases of leukemia-lymphoma were tested between 1972 and 1984 by multiple marker analysis. Routine leukemia phenotyping was performed using standard morphological and cytochemical techniques in combination with clinical and histo-pathological information; the main emphasis was put on immunological surface marker analysis using erythrocyte rosette assays, TdT and a large panel of poly- and monoclonal antibody tests. The 1255 cases were divided into these major types and subtypes: 349 cases of ALL and related immature T- and Burkitt-lymphomas (cALL, pre B-ALL, B-ALL and Burkitt-lymphomas, T-ALL and immature, mostly leukemic T-lymphomas, Null-ALL), 454 cases of mature T- and B-cell malignancies (T-CLL, mycosis fungoides, Sezary-syndrome, T-lymphomas, B-CLL, hairy cell leukemia, multiple myeloma, B-lymphomas), 263 cases of acute myeloid leukemias (AML, AMMoL/AMoL), 182 cases of chronic myeloid leukemias (CML in chronic phase, CMoL, CML in blast crisis), 6 cases of erythroleukemia and 1 case of megakaryoblastic leukemia. A simplified classification scheme which has been used in our laboratories is presented. Phenotyping is of diagnostic, prognostic and therapeutic relevance, most evidently for patients with ALL. Routine leukemia phenotyping should be performed with highly standardized techniques and reagents and by combining information from several fields in the multiple marker analysis. New areas of leukemia research might become very useful for the routine procedure of phenotyping.Abbreviations ALL acute lymphoblastic leukemia - AML acute myeloblastic leukemia - AMMoL acute myelomonoblastic leukemia - AMoL acute monoblastic leukemia - cALL common ALL - CLL chronic lymphocytic leukemia - CML chronic myelocytic leukemia - CML-BC CML in blastic crisis - CMoL chronic monocytic leukemia