Electroporation of synthetic oligodeoxynucleotides: a novel technique for ex vivo bone marrow purging

Recent data suggest that tumor cells contaminating reinfused bone marrow may contribute to relapse in patients undergoing autologous bone marrow transplantation. Purging strategies that are able to remove these contaminating tumor cells need to be developed. This study describes how electroporation (EP) can be used to improve intracellular delivery of synthetic antisense oligodeoxynucleotides (ODNs), thereby enhancing their ability to suppress a target protein. Antisense ODNs that were introduced into cells by EP led to immediate suppression of targeted c-myc protein; this was associated with rapid cell death in the diffuse histiocytic lymphoma, U937; Burkitt's lymphoma, ST486; breast carcinoma, MCF-7; and Ewing's sarcoma, CHP-100, cell lines. Electroporation was found to have little or no detrimental effect on cells responsible for murine hematopoietic long-term reconstitution as determined from in vivo competitive repopulation studies. Using human c- myc-directed antisense ODNs as a model for the application of this approach to bone marrow purging, selective killing of human lymphoma U937 cells relative to normal human bone marrow cells was shown in cell mixing studies. In vivo studies were performed in which a survival advantage was shown for athymic mice that were inoculated with antisense-treated U937 cells as opposed to control cells. These studies suggest that EP of bone marrow may be of use in enhancing intracellular delivery of a variety of molecular/pharmaceutical agents. Taken together, these data suggest that the use of electroporation to enhance delivery of antisense ODNs is a promising new approach towards ex vivo bone marrow purging.     

Hepatitis B virus DNA in the serum of Sardinian blood donors negative for the hepatitis B surface antigen

The high endemicity of hepatitis B virus (HBV) infection and liver disease in Sardinia led us to assess the occurrence of HBV DNA in 1,411 sera of two selected groups of hepatitis B surface antigen (HBsAg)- negative blood donors: 793 with abnormal serum alanine aminotransferase (ALT) and 618 with normal serum ALT values (determined during routine testing of their blood donation). HBV DNA sequences were detected by dot-blot hybridization in 68 of 793 subjects (9%) with abnormal ALT but only in three of 618 subjects (0.5%) with normal ALT. HBV-core antibody (anti-HBc) was detected in 338 of 793 subjects (43%) with abnormal ALT as well as in 125 of 618 subjects (20.2%) with normal ALT. Among the 71 subjects positive for serum HBV DNA, 22 (31%) were positive for anti- HBc, while 49 (69%) were negative for all serologic markers of HBV infection. Thus, a high frequency of anti-HBc in apparently healthy HBsAg-negative individuals and a high prevalence of serum HBV DNA in the absence of immunologic markers of HBV infection suggest the existence of genetic variants of HBV that may be responsible for some of the presumed NANB hepatitis encountered in Sardinia and possibly other areas of high endemicity for HBV.     

Early detection of antibodies against rDNA-produced HIV proteins with a flow cytometric assay

There is evidence that some human immunodeficiency virus (HIV)-infected individuals have prolonged periods of seronegativity. A flow cytometric immunoreactive bead (IRB) assay is described for quantitative, simultaneous, and early detection of antibodies to HIV. Polystyrene beads of four diameters, each size coated with a different HIV recombinant DNA-produced protein (p24, p31, gp41, or gp120), bound anti- HIV antibodies detected with fluorescent antiglobulin. The IRB assay was performed on a panel of blood donor samples, many giving consistently false-positive enzyme immunoassay (EIA) and indeterminant Western blot (WB) results. The IRB assay proved as sensitive and more specific than currently licensed EIA and WB tests. Results on serial samples from eight HIV-infected individuals indicated that quantitation of anti-p24 by IRB assay may be useful in monitoring disease progression. Sequential pre- and post-EIA seroconversion sera from 35 HIV-infected homosexual men were tested by the IRB assay using IgM- and IgG-specific fluorescent probes. All 35 cases were IRB assay positive for at least one rDNA-p either before (17 of 35, 49%) or at the time of EIA positivity. Eleven cases (31%) initially had only IgM anti-HIV, primarily to gp41 (17%). In two individuals, the IgM response was detected at least 18 months before EIA seroconversion. The IRB assay is a widely applicable analytic procedure, potentially useful in pretransfusion anti-HIV screening of blood.     

Chloride transport in primary cultures of rabbit colonocytes at different stages of development

BACKGROUND & AIMS: Ontogeny of colonic Cl- transport and its regulation has been characterized inadequately. The aim of this report was to study developmental changes in Cl- transport in primary cultures of rabbit distal colonocytes. METHODS: Colonocytes from newborn (7-9 days old), weanling (25-28 days old), and adult (6 months old) rabbits were cultured for 24 hours on a collagen IV matrix, and Cl- transport was measured using the fluoroprobe 6-methoxyquinolyl acetoethyl ester. RESULTS: Cl- permeabilities were dependent on [Cl-]o with maximal rates (in millimoles per liter per second) at [Cl-]o = 75 mmol/L (newborns; 0.15 +/- 0.04; weanlings; 0.2 +/- 0.02; and adults, 0.32 +/- 0.06). Influx was inhibited significantly by the Cl- channel (50 mumol/L diphenylamine-2-carboxylate) and the Na(+)-K(+)- 2Cl- cotransport (10 mumol/L furosemide) inhibitors. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretagogues, prostaglandin E1 (1 mumol/L), forskolin (1 mumol/L), and 8-bromo-cAMP (100 mumol/L), and the protein kinase C activator, phorbol 12-13 dibutyrate (1 mumol/L), increased Cl- influx significantly in all groups with adults showing greatest stimulation. However, taurodeoxycholate (0.025-1 mmol/L) had an effect only in the adult and the guanosine 3',5'-cyclic monophosphate (cGMP) activators STa and 8-bromo-cGMP had no effect. CONCLUSIONS: Rabbit distal colonocytes possess inhibitor-sensitive Cl- permeabilities even in neonates. However, the ontogeny of their regulation depends on the secretagogue-signaling pathway. (Gastroenterology 1996 Dec;111(6):1541-50)     

IL-1beta induced changes in hypothalamic IL-1R1 and IL-1R2 mRNA expression in the rat

Interleukin-1 receptor (IL-1R1 and IL-1R2) mRNA expression was detected within the rat hypothalamus, a primary site of IL-1 action, using RT-PCR. Levels of expression were unchanged by cardiac saline-perfusion. However, intracerebroventricular (i.c.v.) administration of IL-1beta caused changes in receptor mRNA expression in non-perfused animals that were profoundly different to those observed in their saline-perfused counterparts. This study demonstrates the importance of perfusing tissue to remove blood cells when determining changes in IL-1 receptor mRNA expression.     

Skin and platelet alpha-synuclein as peripheral biomarkers of Parkinson's disease

Parkinson's disease (PD) is a heterogeneous disease that can be difficult to diagnose, and for which we have no simple effective biomarker. In this study we have investigated whether peripheral alpha-synuclein might represent a useful biomarker given that it has a central role in the pathogenesis of PD. We found that full length and truncated alpha-synuclein is present in platelets, but the amount is very variable and does not correlate with disease presence or severity. Furthermore, we show that alpha-synuclein can be detected by immunoblotting in some, but not all, human skin biopsies, but again its level does not correlate with disease presence or severity. We conclude that skin or platelet alpha-synuclein would not be an appropriate diagnostic biomarker for PD.     

Vagotomy attenuates the behavioural but not the pyrogenic effects of interleukin-1 in rats

Vagal afferent signals, have been implicated in cytokine mediated interactions between the periphery and the central nervous system. Studies in experimental animals have shown that cytokine induced activation of brain mediated responses to infection such as fever, sickness behaviour and pituitary-adrenal activation, are inhibited by subdiaphragmatic vagotomy. We have previously proposed that the peripheral signal to the brain in fever is of a humoral nature while others have suggested that either neural afferents or a mixture of both humoral and neural signals may be involved. The objective of the present study was to examine further the role of vagal transmission, in mediating the febrile response to a systemic injection of IL-1beta in rats and to compare this with changes in social exploration behaviour. Intraperitoneal injection of IL-1beta (1.0-30.0 microg/kg) inhibited social exploration in rats and this was attenuated in vagotomized animals. Injection of increasing concentrations of IL-1beta (0.1-1.0 microg/rat) induced significant (P<0.001) increases in core body temperature. However, in contrast to effects on social exploration, the increase in temperature was not inhibited by vagotomy at any of the doses used. These observations demonstrate a dissociation between the two brain mediated events, one of which is dependent on the integrity of the vagus nerve (social exploration) while the other (fever) is apparently generated by different mechanisms which may include circulating pyrogens.