Apoptosis-derived membrane vesicles drive the cGAS-STING pathway and enhance type I IFN production in systemic lupus erythematosus.

OBJECTIVE: Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS-STING pathway in the IFN-I-producing cascade driven by SLE serum. METHODS: We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction. RESULTS: IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells. CONCLUSIONS: AdMVs in SLE serum induce IFN-I production through activation of the cGAS-STING pathway. Thus, blockade of the cGAS-STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

The Brucella melitensis M5-90ΔmanB live vaccine candidate is safer than M5-90 and confers protection against wild-type challenge in BALB/c mice.

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (strain M5-90 or others) have several drawbacks. The first is their residual virulence for animals and humans and the second is their inability to differentiate natural infection from that caused by vaccination. In the present study, Brucella melitensis M5-90 manB mutant (M5-90ΔmanB) was generated to overcome these drawbacks. M5-90ΔmanB showed significantly reduced survival in macrophages and mice, and induced strong protective immunity in BALB/c mice. It elicited anti-Brucella-specific IgG1 and IgG2a subtype responses and induced the secretion of gamma interferon (IFN-γ) and interleukin-4(IL-4). Results of immune assays showed, M5-90ΔmanB immunization induced the secretion of IFN-γ in goats, and serum samples from goats inoculated with M5-90ΔmanB were negative by Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Further, the ManB antigen also allows serological assays differentiate infections caused by wild strains from infections by vaccination. These results show that M5-90ΔmanB is a suitable attenuated vaccine candidate against virulent Brucella melitensis 16 M (16 M) infection.

Intracellular pH regulation in leukocytes: mechanisms and functional significance.

The regulation of the intracellular pH (pHi) of phagocytic cells is critical to their function and viability. The acidic nature of the abscesses to which these cells migrate and the burden of acid that they generate in the activated state tend to perturb the pHi outside of the physiological range. Failure to maintain pHi homeostasis results in decreased cellular enzyme activity, cellular migration, and microbicidal function. Several pHi regulatory mechanisms, including the sodium-proton exchanger, proton conductive pathways, and a vacuolar type H(+)-ATPase exist in leukocytes and play an important role in preventing deviations in the pHi away from the physiological range. The last of these mechanisms is central to the regulation of cytoplasmic pH, and is modulated by mediator molecules in the inflammatory mileu.

Enzymatic imbalance in peripheral blood mononuclear cells isolated from individuals with anti-HIV antibodies.

Plasma membrane-bound 5'-nucleotidase (5'-NT), gamma-glutamyltransferase (gamma-GT) and soluble deoxynucleotidyltransferase (TdT) were studied in peripheral blood cells (PBMN) of 35 individuals, 26 male and 9 female, with circulating anti-HIV antibodies. Twenty-six were drug abusers, 2 were drug abusers and homosexuals and 4 were homosexuals. Three did not fall into any risk group. The surface immunologic phenotype of cells stained with the fluorescent monoclonal antibodies Leu 5, Leu 3, Leu 2, Leu 12, Leu M3, Leu M1, anti-CALLA and anti-HLA-DR was delineated by flow cytometry. While the gamma-GT activity did not change, the lymphocyte 5'-NT activity was significantly less than normal in anti-HIV positive individuals and in anti-HIV negative drug abusers. TdT activity was detectable in 14 anti-HIV positive patients (40%), who did not have clinical AIDS. Of 8 patients with AIDS, 3 had a low level of TdT activity but 5 had cells completely devoid of TdT and 5'-NT activity. 5'-nucleotidase activity and the frequency of Leu 2 suppressor antigen bearing cells were the only independent variables that correlated with AIDS incidence.

Galactose-1-phosphate uridyl transferase in density-fractionated erythrocytes. Studies of normal and mutant enzymes.

Galactose-1-phosphate uridyl transferase (GALT), the deficient enzyme in classical galactosemia, was studied by Percoll-gradient age-fractionation of erythrocytes. For normal GALT, a rapid and substantial decrease in GALT activity and loss of most of two isozymes was found to occur in the reticulocyte fractions. The loss of activity was then followed by relative stabilization of both GALT-specific activity and microheterogeneity in mature and aging erythrocytes. When applied to the study of mutant GALT from galactosemic patients, the Percoll-gradient fractionation method permitted detection in the reticulocyte-enriched fractions of up to 5% of normal GALT-specific activity and an isoelectric focusing pattern essentially the same as that of normal GALT. Percoll-gradient fractionation of erythrocytes offers a simple and direct method to study characteristics of GALT activity and microheterogeneity in normal and galactosemic human erythrocytes.

Patterns of adenosine deaminase, ecto-5'-nucleotidase, poly(A)polymerase and surface light chain expression in chronic lymphocytic leukemias.

The levels of activity of three enzymes have been measured in the circulating malignant lymphocytes of 47 patients with B chronic lymphocytic leukemia (CLL). These were the purine degradative enzymes, adenosine deaminase (ADA) and ecto-5'-nucleotidase (5'NT) and the enzyme responsible for the polyadenylation of mRNA, poly(A) polymerase. The patterns of activity of the above enzymes and the expression of surface immunoglobulin light chains were examined. A heterogeneity in the specific activity of the enzymes was observed which could not be attributed to variations of the percentage of B lymphocytes. A positive correlation was found between ADA and poly(A)polymerase activity (r = 0.383, p less than 0.01). Furthermore, the expression of immunoglobulin light chain phenotype was inversely related to 5'NT specific activity; CLL cases in which less than 20% of the cells expressed lambda chain phenotype, presented 5'NT specific activity of 16.7 +/- 3.3 (S.E.) nmol/h/10(6) cells, whereas in CLL cases with more than 20% of the cells expressing this phenotype the enzyme specific activity was 4.8 +/- 1.6 (S.E.) nmol/h/10(6) cells (p less than 0.02). These findings suggest that the simultaneous determination of enzymatic activities and immunological markers, might be useful in defining subsets in CLL and the subsequent clinical treatment.

Gonadal failure in young women and galactose-1-phosphate uridyl transferase activity.

The present study allows the conclusion that low Gal-1-P transferase activity resulting from GtD/gt heterozygosity is not an major cause of early ovarian failure. However, larger groups of patients need to be examined to fully exclude an association between these rare conditions.

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes.

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.

Idiopathic presenile cataract formation and galactosaemia.

Five hundred patients undergoing cataract surgery were prospectively examined, and 46 Caucasian patients were found to have strictly idiopathic cataracts severe enough to warrant surgery on or before age 55. In a masked fashion we determined the activity of galactokinase (GK) and galactose-1-phosphate uridyl transferase (GPUT) in these patients as well as on 53 age matched controls. With respect to GK no cataract patient had an enzyme level of less than 2 standard deviations below the control mean. However, 3 of 45 (6.7%) patients in the cataract group had a GPUT level less than 2 standard deviations below the mean for controls, and were presumably heterozygotes for this enzyme. In comparison with the expected population rate of 0.8% this is highly significant (p = 0.006). Abnormalities in galactose pathway enzymes may therefore predispose to development of presenile cataracts. In affected people there is a possibility of treating these patients clinically by dietary restriction of dairy products or by using aldose reductase inhibitors to prevent or reverse cataract formation.

Relation between the activity of poly(A)polymerase and the levels of protein synthesis in the lymphocytes of patients with chronic lymphocytic leukemia.

The activity levels of the enzyme poly(A)polymerase and the levels of protein synthesis primed by endogenous messenger RNA (mRNA) as well as polyuridylic acid, poly(U) directed polyphenylalanine synthesis, were determined in lymphocytic extracts from 17 patients with chronic lymphocytic leukemia of the B cell type. The enzyme activity values have not been found to correlate with the poly(U)-protein synthesis, whereas a positive linear correlation has been established between the activity levels of poly(A)polymerase and the endogenous mRNA-primed protein synthesis (r = 0.735, p less than 0.01). This difference between exogenously and endogenously primed protein synthesis in concern with poly(A)polymerase is discussed.

Poly(A) polymerase activity in murine serum. Elevation in animals with proliferative changes.

Sera of normal rats contain polynucleotide adenylyltransferase [poly(A) polymerase] activity. The enzymatic activity has been optimized with regard to primer concentration, ion requirements, kinetics, and protein. Results based on inclusion of inhibitors in the assay system show that the enzyme is poly(A) polymerase. High levels of the enzymatic activity were prevalent in sera of (a) BUF/SimfBR rats bearing sc transplanted hepatomas; (b) Sprague-Dawley rats with hepatoma cells grown in ascites; (c) partially hepatectomized Sprague-Dawley rats; and (d) MRL/lpr mice, which are in a massive lymphoproliferative autoimmune state.

On the appearance of polyadenylate polymerase activity in blood serum of animals exposed to a short-term influence by N,N'-ethylenethiourea, a well known hepatic carcinogen: a preliminary report.

Daily intragastric administration of a carcinogen N,N'-ethylenethiourea (ETU) (85 mg/kg body wt, congruent to 0.1 DL50) leads to significant polyadenylate polymerase (PAP) activity in rat blood serum by the 10th day of experiment. A similar course of N,N'-ethyleneurea (EU) fails to affect this enzyme activity in the blood. Also, PAP activity is not registered in the blood serum of intact rats.

Development of a protocol for newborn screening for disorders of the galactose metabolic pathway.

The protocol evaluated in this paper employs an enzymatic assay of galactose metabolites, thin layer chromatography, and an assay of galactose-1-phosphate uridyl transferase on a single sample of blood collected routinely for newborn screening. Its effectiveness was tested by a retrospective study of known galactosemic blood samples, and also by a prospective study of 207,000 newborn samples from which 6 infants with severe transferase deficient galactosaemia and 2 infants with red cell epimerase deficiency were identified. The detection rate for severe transferase deficiency in the newborn population was 1:35,000. Advantages include low false positive rate, definitive diagnosis within 6 hours of sample receipt, and the use of technically simple and robust procedures. This protocol overcomes the difficulties encountered with previously described procedures.

Effects of hyperthermia and nicotinamide on DNA repair synthesis, ADP-ribosyl transferase activity, NAD+ and ATP pools, and cytotoxicity in gamma-irradiated human mononuclear leukocytes.

Effects of hyperthermia and nicotinamide on ADP-ribosyl transferase activity (ADPRT), unscheduled DNA synthesis (UDS), NAD+- and ATP-pools and cytotoxicity were investigated in gamma-irradiated human mononuclear leukocytes. A significant decrease in radiation-induced UDS after heat treatment for 45 min was found. Nicotinamide increased the UDS levels in irradiated cells, but no effect of hyperthermia on these increased UDS values was observed. In the presence of 2 mM nicotinamide radiation-induced ADPRT activity was reduced to about 50 per cent. However, hyperthermia for 45 min was found to have no effect on the enzyme activity for temperatures below 46 degrees C. Nicotinamide increased the NAD+ pool in unirradiated cells. Damaging the cells with gamma-radiation leads to a severe depletion of the NAD+ pool. The NAD+ pool is restored, however, if the cells repair for 5 h at 37 degrees C. When radiation-damaged cells were treated with hyperthermia, exogenously supplied nicotinamide could not be converted to NAD+ in sufficient amounts to prevent NAD+ depletion. These data indicate that the radiosensitizing effect of heat and nicotinamide could both be explained by effects on the enzyme ADPRT, i.e. nicotinamide by directly blocking the enzyme and hyperthermia by limiting the co-substrate (NAD+).

Tumor promoter phorbol-12-myristate-13-acetate induces poly ADP-ribosylation in human monocytes.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) induces rapid poly ADP-ribosylation and a drop in cellular NAD concentration in human monocytes. The antioxidants CuZn-superoxide dismutase, catalase, glutathione peroxidase and butylated-hydroxytoluene inhibit the reaction indicating that active oxygen species produced in the PMA-induced oxidative burst represent intermediates. The inhibitor of ADP-ribosyl-transferase, 3-amino-benzamide, inhibited poly ADP-ribosylation but did not prevent the drop in NAD-levels. PMA also causes the slow accumulation of DNA strand breaks in monocytes. The difference in the kinetics of poly ADP-ribosylation and DNA breakage argues against a simple relationship between the two reactions.

De novo tandem duplication 9p (p12----p24) with normal GALT activity in red cells.

A 3 month old boy with a tandem duplication 9p (p12----p24) is reported. Both clinical and dermatoglyphic features were consistent with those of the trisomy 9p syndrome. However, the red cell galactose-1-P uridyl transferase (GALT) activity was normal despite the presence of the duplicated segment 9p13.