Micro-carrier-test: evaluating disinfectants for HIV.

To determine the effect of disinfectants against viruses in vitro, I devised the Micro-Carrier-Test of dry-fixed virus-infected cells. Human immunodeficiency virus type 1-infected Molt-4 cells (1 x 10(5) cells in 5 microliters of 10% fetal bovine serum) were dry-fixed at the bottom of each well of a 96-well flat-bottomed microtiter plate for 120 minutes at room temperature. Disinfectants were added and allowed to remain for designated times and the wells were washed three times with PBS. Uninfected Molt-4 cells (1 x 10(4) cells/well) were inoculated and cultured for 4 weeks. The culture supernatant was harvested to measure reverse transcriptase activity by non-radioisotopic reverse transcriptase assay every week. Residual cytotoxicity of the disinfectant was determined by cytotoxicity assay. To evaluate the new method, the virucidal efficacy of several well-known disinfectants was reevaluated. Dose- and time-dependent effects of the disinfectants were determined. The minimal effective concentrations after 5 minutes of contact were 20% ethanol, 0.01% glutaraldehyde and 0.1% sodium hypochlorite. These results are almost the same as those reported previously, but there are some discrepancies. The differences between the present and previous protocols are discussed. This Micro-Carrier-Test promises to be useful in the screening of disinfectants.

A micro-suspension-test for evaluation of disinfectants against human immunodeficiency virus.

We devised a micro-suspension-test to evaluate disinfectants against human immunodeficiency virus type-1 (HIV-1) and confirmed its reliability. Suspensions of persistently HIV-1-infected Molt-4 cells were used as targets of disinfectants and residual infectivity was measured by an infectivity assay: after cocultivation with uninfected Molt-4 cells reverse transcriptase activity (RTA) in the supernatant and giant cell formation (GCF) were monitored. Our new infectivity assay consists of a short-term assay, that is RTA and GCF monitoring on the second day of co-culture, and a long-term assay, that is RTA monitoring up to the 28th day of co-culture. The sensitivity of the short-term assay was 1 x 10(3) infected cells and that of the long-term assay 1 x 10(1) infected cells. All the chemical disinfectants examined in this study showed dose- and time-dependent inactivation of HIV-1. By 5-minute contact with ethanol, glutaraldehyde, formalin, sodium hypochlorite and povidone-iodine, HIV-1 was effectively inactivated at concentrations of 20, 0.01, 5, 0.05 and 0.1%, respectively. Since the micro-suspension-test is easy and sensitive, we recommend it as a method for evaluating disinfectants against HIV-1.

1-Hydroxyethylidene-1,1-bisphosphonate decreases the postovariectomy enhanced interleukin 1 secretion from peritoneal macrophages in adult rats.

Bisphosphonates are potent inhibitors of bone resorption. In previous studies, we have shown that ovariectomy accelerates bone resorption and 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) inhibits ovariectomy-accelerated bone resorption in female Wistar adult rats. As interleukin 1 (IL-1) stimulates bone resorption in vitro and in vivo, we have investigated the effects of ovariectomy and HEBP administered in vivo on IL-1 secretion from peritoneal macrophages in adult rats. Ovariectomy or sham surgery were performed in female Wistar rats at 40 weeks of age. Ovariectomized and sham-operated rats were administered with HEBP (10 mg) or saline, 10 times in total, from 43 to 46 weeks of age. Paraffin oil-induced peritoneal macrophages at 46 weeks of age were cultured for 24 hours. Lipopolysaccharide (LPS)-stimulated peritoneal macrophages from ovariectomized rats secreted more IL-1 than sham-operated rats. HEBP decreased LPS-stimulated IL-1 secretion from peritoneal macrophages in ovariectomized rats, but not in sham-operated rats. In vivo administration of HEBP decreased IL-1 secretion only in postovariectomy hyperresorptive states. These results suggest that alterations in LPS-stimulated IL-1 secretion from oil-induced peritoneal macrophages may be responsible, at least in part, for the postovariectomy acceleration in bone resorption and its inhibition by HEBP.

Effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on interleukin 1 production by macrophages.

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the in vitro functions of guinea pig macrophages were studied. A high dose (1 mg/ml) of EHDP inhibited interleukin 1 (IL 1) production by oil-induced peritoneal macrophages stimulated with muramyl dipeptide (MDP), lipopolysaccharide (LPS), phorbol myristic acetate (PMA), heat-aggregated IgG2 or calcium ionophore A23187. On the other hand, low doses (less than 0.125 mg/ml) of EHDP augmented the MDP induced IL 1 production by macrophages. This biphasic effect was also observed when macrophages were exposed to EHDP at 37 C for 24 hr and then stimulated with IL 1 inducers. Superoxide anion generation induced by formyl peptide or PMA was not affected by preincubation of the macrophages with doses of EHDP up to 1 mg/ml. Adherence and spreading of macrophages was inhibited by EHDP in a dose dependent manner without affecting cell viability. These results demonstrated that EHDP acted on macrophages directly and modulated IL 1 production in vitro.