Phagocytic NADPH oxidase and its role in chronic granulomatous disease

Case-control and cohort studies support the hypothesis that postmenopausal oestrogen-replacement therapy reduces the risk of atherosclerotic disease manifestations. The evidence for a cardioprotective effect of such a therapy is, however, incomplete because randomized prospective studies are missing. Because it may be almost impossible to conduct placebo-controlled trials in the future, other study designs will be needed to minimize selection bias. Further work is required to define the optimal dose and administration schedule of oestrogen and to determine whether addition of progestogens alters the beneficial effect of oestrogen on the cardiovascular system. Such studies may also provide mechanistic insight into the interaction between lipoprotein metabolism and haemostasis and its relation to the atherosclerotic disease process.     

Histo-blood group p: biosynthesis of globoseries glycolipids in EBV-transformed B cell lines

PURPOSE: In vitro and in vivo investigations were performed to evaluate the Irie retrievable inferior vena caval (IVC) filter. MATERIALS AND METHODS: The clot capturing performance of the Irie and five other IVC filters were assessed in both horizontal and vertical orientations within a pulsed-flow circuit with 240 clot challenges for each filter. Subjective comparisons of the flow disturbance characteristics of the Irie and three other filters were also performed. In vivo studies consisted of 13 Irie filter insertions and eight attempted retrievals in 11 pigs. Histologic evaluation of the IVC was performed with the Irie filter in situ and following retrieval. RESULTS: In vitro testing demonstrated the clot capturing capability and flow disturbance characteristics of the Irie filter to be similar to those of other IVC filters. Filter deployment problems occurred during three of the 13 insertions. Six of the eight retrieval procedures were successful; four filters were retrieved 1 month after insertion. Follow-up cavography demonstrated two tilted filters and three caval perforations. CONCLUSION: The performance of the Irie filter is similar to that of other currently available IVC filters. The filter can be retrieved after neointimal incorporation of the struts into the IVC wall.     

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.     

The isolation and analysis of lymphoblastoid cell lines from patients with xeroderma pigmentosum and progeria

Lymphoblastoid cell lines from patients with xeroderma pigmentosum (2 forms) and progeria (unusual form) were established using transformation of peripheral blood lymphocytes by Epstein--Barr virus. The influence of different UV doses on cell vitality, proliferation and cell cycle progression was studied by means of flow cytometry. The cell vitality was determined after incubation of cells with etidium bromide and FDA. We used cytograms with two logarithmic signals (log green/log red) to discriminate the cell cycle status. Cell cultures were used with density of 500,000 cells per 1 ml, previously synchronized at G-phase by the incubation in a medium with low serum content. The effect of UV irradiation was followed during 72 h. Among four analysed cell lines only line XP2SP demonstrated enhanced UV sensitivity, expressed by decreasing of the amount of living cells after the UV dose of 2.5 J/m2 and higher. The cell cycle studies showed that cells were blocked in S-phase and simultaneously the amount of apoptotic cells with both reduced DNA content and ability to bind FDA was seen increased. Similar events were observed in the control line only after the dose of 20 J/m2 and higher.     

The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes

Normal human polymorphonuclear leukocytes (PMN) placed in anaerobic chambers reaching pO2's of less than 5 mm Hg fail to generate O2-, iodinate ingested particles, and stimulate glucose-1-14C oxidation through the hexose monophosphate shunt. The observation that anaerobic cells are incapable of generating O2- or reducing nitroblue tetrazolium (NBT) to formazan supports the idea that NBT reduction in phagocytizing PMN is due exclusively to oxygen-dependent O2- generating oxidase which is deficient in chronic granulomatous disease leukocytes, despite their hyperphagocytic capacity.     

Hypomethylation of classical satellite DNA and chromosome instability in lymphoblastoid cell lines

To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.     

Safety and effectiveness of long-term interferon gamma therapy in patients with chronic granulomatous disease

In chronic granulomatous disease (CGD), diminished or absent neutrophil NADPH oxidase function leads to recurrent pyogenic infections and granuloma formation. In a recent randomized, placebo-controlled trail, short-term prophylactic use of recombinant human interferon gamma (rIFN-gamma 1b) reduced the risk of serious infection in CGD patients by 67%, The current study evaluated the safety and effectiveness of long-term rIFN-gamma therapy in CGD patients. Patients were treated three times weekly with rIFN-gamma and evaluated semiannually. Serious infections (requiring hospitalization and parenteral antibiotic therapy), adverse clinical events, and measures of growth and development were noted. Thirty patients were evaluated for 12 months. The total average duration of rIFN-gamma therapy was 2.5 years. Three patients developed a total of four serious infections (0.13 infections per patient year). This rate compare favorably with rates of 1.10 and 0.38 infections per patient year found in the placebo and rIFN-gamma groups, respectively, during a previous study. Common adverse events were fever (23%), diarrhea (13%), and flu-like illness (13%). No serious adverse event was attributable to rIFN-gamma therapy and no obvious effects on growth and development were observed. rIFN-gamma is a safe and effective adjunctive therapy for reducing the frequency and severity of serious infections in CGD patients.     

Genitourinary involvement in chronic granulomatous disease of childhood

A second case of symptomatic renal involvement of chronic granulomatous disease of childhood is reported, and all previous cases of documented genitourinary lesions in this disease are reviewed. Although frequent infections of other organ systems are a well recognized part of chronic granulomatous disease, involvement of the urinary tract has been rarely reported. The nature of the few cases described suggests that extensive insidious destruction occurs before urinary tract involvement is clinically suspected.     

Protein kinases potentially capable of catalyzing the phosphorylation of p47-phox in normal neutrophils and neutrophils of patients with chronic granulomatous disease

A procedure for uncovering novel protein kinases was used to search for enzymes in neutrophils that may catalyze the phosphorylation of the 47-Kd subunit of the NADPH oxidase system (p47-phox). This component of the oxidase can undergo phosphorylation on multiple sites. The method is based on the ability of renatured kinases to recognize exogenous substrates fixed in gels. We report that neutrophils contain several uncharacterized protein kinases that catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297 through 331 of p47-phox. Some of these enzymes are strongly activated on stimulation of the cells with phorbol 12-myristate 13-acetate (PMA). The results indicate that the phosphorylation of p47-phox in neutrophils may be more complicated than previously appreciated and may involve multiple protein kinases. In addition, we have examined both the renaturable protein kinases and the properties of protein kinase C (PKC) in neutrophils from patients with chronic granulomatous disease (CGD) who are deficient in cytochrome b558. Previous studies have shown that these cells exhibit incomplete phosphorylation of p47-phox on stimulation. In this study, we were unable to detect any alterations in the renaturable protein kinases or PKC in CGD neutrophils that could explain these defects in the phosphorylation of p47-phox.     

X-Linked chronic granulomatous disease: mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.     

Transcriptional analysis of Marek''s disease virus (MDV) genes in MDV-transformed lymphoblastoid cell lines without MDV-activated cells

Prevention of the occurrence of diabetes-specific vascular complications is the final aim of clinical islet transplantation. Pancreatic islets isolated from adult pigs may be a suitable tissue source to transplant a large number of type 1 diabetic patients. Acute cellular rejection may be finally overcome by clinically applicable protocols for tolerance induction. However, primary nonfunction of the graft, as regularly observed in the porcine islet-to-rat xenotransplantation model, may be an additional problem. In this paper, species-specific inflammatory and immunological mechanisms are discussed which prevent early porcine islet graft function in rats but not in mice.     

Platelet activation in patients with sickle cell disease

Vascular occlusion and vasculopathy underlie much of the morbidity in patients with sickle cell anaemia. Platelets may play a role in this vasculopathy. Samples from 43 patients with sickle cell disease (SCD) were examined for evidence of platelet activation using fluorescent-labelled monoclonal antibodies and flow cytometry. There was increased expression of activation-dependent antigens on the platelets from patients with SCD compared to those from both Caucasian and African-American controls. In addition, SCD patients had increased levels of platelet microparticles. Platelets are activated in patients with sickle cell disease. The contribution of platelet activation to sickle cell pathophysiology is under active investigation in our laboratories.     

Spontaneous production of Epstein-Barr virus by B lymphoblastoid cell lines obtained from patients with Sj?gren's syndrome. Possible involvement of a novel strain of Epstein-Barr virus in disease pathogenesis

OBJECTIVE: To investigate the involvement of Epstein-Barr virus (EBV) in the pathogenesis of Sj?gren's syndrome (SS) and to examine whether the spontaneous production of EBV is unique to SS B cell lines. METHODS: B cell lines were established from peripheral blood mononuclear cells (PBMC) of patients with systemic lupus erythematosus, rheumatoid arthritis, and SS. The cord blood immortalization assay, flow cytometric analysis, and polymerase chain reaction (PCR) were used to detect EBV production by B cell lines. RESULTS: SS B cell lines produced EBV at a higher frequency, and in significantly larger amounts, than did other B cell lines. However, no correlation with the amount of EBV DNA in the genome of B cell lines was found. PCR analysis revealed that EBV with a B95-8--like U2 region was dominant in SS B cell lines. CONCLUSION: Spontaneous, massive production of EBV by B cell lines is unique to SS, and may contribute to the polyclonal B cell activation seen in this disease.     

Expression of human placental cell surface antigens on peripheral blood lymphocytes and lymphoblastoid cell lines

The expression of human placental cell surface antigens was examined in cells of lymphoid origin, including peripheral blood lymphocytes and cultured lymphoblastoid cells of bone marrow or thymus derivation. A select group of this defined set of surface antigens was detected on all three cell preparations. The most remarkable observation was the conspicuous absence of three subunits previously demonstrated to be present on all human cell surfaces examined to date. Antiserum directed against several placental components prevents adhesion and spreading of cells which grow attached to surfaces. These results suggest a role for these three glycoproteins in mediating cellular adhesion.     

Arcuate dermal erythema in a carrier of chronic granulomatous disease

A female carrier of chronic granulomatous disease developed arcuate, erythematous dermal plaques on her back and face. The lesions were clinically and histopathologically suggestive of Jessner benign lymphocytic infiltration of the skin. Some women with relatively fixed arcuate or annular, erythematous, dermal plaques may be carriers of chronic granulomatous disease.     

Prevalence, genetics and clinical presentation of chronic granulomatous disease in Sweden

To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty-one patients (belonging to 16 different families) were found, corresponding to a prevalence of approximately 1/450,000 individuals. The patients with X-linked disease, lacking a functional gp91phox protein (n = 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n = 7) or p67phox (n = 1), respectively. All unrelated patients with X-linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n = 3), missense (n = 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47phox-deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox-deficient patient displayed a heterozygous in-frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from pseudomonas cepacia infections. Patients with X-linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses (n = 111), followed by lymphadenitis (n = 82) and pneumonias (n = 73). Inflammatory bowel disease-like symptoms, mimicking Crohn's disease of the colon, was seen in three CGD patients.     

Differential expression of nucleoskeleton- and cytoskeleton-associated proteins in Burkitt lymphoma-derived and Epstein-Barr virus-immortalized lymphoblastoid cell lines

Mouse monoclonal antibodies raised against nuclear bodies isolated from an EBV-immortalized lymphoblastoid cell line (LCL) known to contain several viral and cellular proteins (Jiang et al., Exp. Cell Res., 197: 314-318, 1991; Szekely et al., J. Gen. Virol., 76: 2423-2432, 1995; Szekely et al., J. Virol., 70: 2562-2568, 1996). Seventy six clones gave detectable immunofluorescence staining on LCLs. Five independent monoclonal antibodies detected a group of apparently novel, high M(r) (> 200,000) proteins that shared common features of subcellular distribution. In LCLs, these proteins were preferentially associated with vimentin filaments in the cytoplasm and with distinct nuclear foci. The appearance of the latter differed from the premyelocytic leukemia-associated protein, EBV nuclear antigen #5, and retinoblastoma-protein-positive bodies that were used for immunization. They seemed to be connected to the cytoplasmic filaments through thin fibrillar nuclear structures. In mitotic cells, these complex structures rearranged into a perichromosomal basket that was associated with vimentin filaments. The target proteins, operationally designated as proteins associated with nuclear dots and cytoplasmic filaments (pNDCFs), were not present in resting human B cells or were expressed at a low level. The level increased considerably after EBV infection or mitogenic stimulation by interleukin 4 and anti-CD40 antibodies. In Burkitt lymphoma (BL) type I lines phenotypically representative of the in vivo tumors, the pNDCFs were either absent or exclusively localized to the nucleus, usually to well-defined nuclear foci. EBV-positive type I BLs often shift to a more LCL-like (type III) phenotype during prolonged in vitro propagation. Type I cells express only EBV nuclear antigen 1 and the surface markers CD10 and CD77, whereas type III express all nine growth-associated EBV-encoded proteins and a gamut of B-cell activation markers. Most of the type III BL cell lines contained increased amounts of pNDCFs bound to cytoplasmic filaments, as seen in the LCLs. We propose that the expression of vimentin-associated pNDCFs should be included in the definition of type III BL phenotype.     

Desensitization in normal and neoplastic human thyroid cell lines

Because some papillary thyroid cancers continue to grow when thyroid-stimulating hormone (TSH) levels are suppressed, we questioned whether desensitization (i.e., a decreased cAMP response to repeat stimulation with TSH) occurs in normal and neoplastic thyroid tissue. If desensitization does occur, is it similar or different in these human thyroid cells? Normal and papillary thyroid cancer cells from the same patient were cultured as we have previously described. Normal and neoplastic thyroid tissues responded to TSH (0.01-10.0 mU/ml) by increasing cAMP production and growth in a dose-dependent manner. In normal cells there was an 11-fold mean increase in cAMP production at 4 hours, and all thyroid cultures responded. In neoplastic cells cAMP production increased from 1.5-fold to 3.0-fold with a mean 2.0-fold increase at 4 hours. In normal thyroid cells the cAMP response to a second TSH stimulus (desensitization) decreased up to 75% (range 25-75%), and desensitization occurred in all normal thyroid cell cultures. In neoplastic thyroid cells, however, the cAMP response to a second TSH stimulus decreased up to 17% (range 0-17%); and desensitization occurred in only two of the five neoplastic thyroid cell cultures. Thus when normal thyroid and neoplastic cells from the same patients were studied, greater desensitization occurred in the normal cells (75% vs. 17%). These studies document that there is greater desensitization in normal tissue than in neoplastic thyroid tissue, which may account for the increased growth of thyroid neoplasms in the presence of ever-changing low levels of TSH.     

Telomerase activation in normal B lymphocytes and non-Hodgkin's lymphomas

Activation of telomerase seems to be a prerequisite for immortalization and is found in permanent cell lines and most malignant tumors. Normal somatic cells are generally telomerase negative, except for bone marrow stem cells. Weak activity is also present in peripheral blood cells. In the present study strong telomerase activity was demonstrated in vivo in normal mature cells of the immune system, as well as in malignant lymphomas. Benign lymph nodes had lower telomerase activity than benign tonsils, which exhibited intermediate to high activity comparable with findings in malignant lymphomas. In benign tonsils the activity seemed to be restricted to germinal center B cells. In benign lymphoid tissues telomerase activity correlated with B-cell numbers and cell proliferation, but this was not observed in the lymphoma group. High-grade lymphomas exhibited higher levels of telomerase compared with low-grade cases. The data showed that in vivo activation of telomerase is a characteristic feature of germinal center B cells. Different signals for activation of telomerase are likely to exist, one of them being immune stimulation. The data suggest that telomerase activity in malignant lymphomas can be explained by an "induction and retention" model, ie, transformation occurs in a normal, mature B cell with reactivated telomerase, which is retained in the neoplastic clone.