Human anp

LPS from Escherichia coli 0111:B4 was formulated as a 10 mg ml⁻¹ solution in water and stored in −80 °C. In BALB/C mice, LPS was injected intraperitoneally at a lethal dose of 3.5 mg kg⁻¹. This lethal dose was found to cause 70–90% death rate and be optimal for demonstrating the protective effects of ANP and MTR. In experiments with catecholamine pumps that were implanted a day before, a sublethal dose of LPS with 15–35% death rate was optimized in BALB/C mice. In Th^+/+ and Th^ΔLysM mice with C57Bl/6 background, a lethal dose of LPS was optimized at 5 mg kg⁻¹. Human ANP (Sigma) was dissolved in saline, loaded in mini-osmotic pumps (ALZET) with a release rate of 12 μg per day and implanted subcutaneously in the back of mice 12 h before the LPS injection. Mice implanted with pumps loaded with saline served as controls. MTR was freshly dissolved in PBS and injected intraperitoneally at the indicated doses for three days before the LPS treatment. One hour before the LPS injection, MTR was injected into the lower abdomen contralateral to the side of LPS injection. The control groups were injected with PBS. For the following 3 days, MTR was injected intraperitoneally at reduced indicated doses. Hydration of mice was supported by daily subcutaneous injection of 0.5 ml saline.

The colon cancer cell line CT26 was injected subcutaneously into the right flank of 6–8-week-old female BALB/C mice as described previously^{6 (link)}. Tumour sizes were measured with a caliper and calculated as (L × W × H)/2. When tumours reached 600–900 mm³ after about two weeks, 12 × 10⁶ spores of C. novyi-NT or ANP-C. novyi-NT at 3 × 10⁶ per μl were injected intratumourally into four central parts of the tumour with a 32G Hamilton syringe needle. The bacteria typically germinated in the tumours within 24 h, turning them necrotic. Hydration of the mice was supported by daily subcutaneous injections of 500 μl saline. Human ANP (Sigma) was dissolved in saline, loaded in mini-osmotic pumps (ALZET) with a release rate of 12 μg per day and implanted subcutaneously in the back of mice 12 h before the spore injection. Pumps loaded with saline served as controls. MTR was dissolved in PBS and injected intraperitoneally at 60 mg kg⁻¹ per day for three days before the C. novyi injection to deplete catecholamines in storage. Two hours after the spore injection, 60 mg kg⁻¹ of MTR was injected intraperitoneally. For each of the next three days, intraperitoneal injections of MTR at 30 mg kg⁻¹ were administered. Control groups were injected with PBS at the same time points.