Regression of line-10 hepatocellular carcinomas following treatment with water-soluble,microbial extracts combined with trehalose or arabinose mycolates

Summary Intratumor injections of the aqueous phase of phenol-water extracts of Re mutant Salmonella typhimurium (Re glycolipid) in combination with trehalose dimycolate at dose levels of 150 to 15 g were consistently and highly effective (65–93%) in producing regression of line-10 tumors in strain-2 guinea pigs. We observed that the rate of regression was more rapid than that seen after treatment with cell walls from Mycobacterium bovis strain Bacillus of Calmette and Guèrin (BCG). Arabinose mycolate could be substituted for trehalose dimycolate in the Re glycolipid-mycolate mixture without appreciably compromising antitumor activity, providing that the level of arabinose mycolate was not reduced below 15 g. In addition to the Re glycolipid preparation, similarly prepared aqueous extracts from Mycobacterium bovis strain BCG and strain AN5 in combination with trehalose dimycolate also possessed tumor-regressive activity. The activity of these last extracts was reduced when the arabinose mycolate was substituted for the trehalose dimycolate. The aqueous extract of a rickettsia, Coxiella burnetii, in combination with either trehalose dimycolate or arabinose mycolate was also active (50 and 80% tumor regression rates, respectively). Intracutaneous administration of Re glycolipid or aqueous extracts from BCG in combination with trehalose or arabinose mycolates did not produce life-threatening, clinical signs of toxicity in young mice. If additional toxicity studies demonstrate that adverse side effects can be satisfactorily controlled, these watersoluble extracts may prove beneficial in the treatment of spontaneous tumors of humans and other animals.     

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

Summary We made a comparative study of the in vivo binding of immunoglobulins (Ig) to a polyoma virus-induced ascitic tumor propagated in syngeneic or allogeneic mice. The Ig coat was found to appear more rapidly and to be denser in H 2-incompatible than in H 2-compatible mice. This suggests that antibodies were fixed specifically on strong normal transplantation antigens (H-2) recognized as non-self by allogeneic mice. Experiments with mice in which immunosuppression had been achieved by means of X-irradiation confirmed that the Ig fixed on SEWA cells are actively bound antibodies. The only mice that could fix Ig on tumor cells were those that had been specifically immunized against cell surface antigens shared by SEWA cells before irradiation, while mice hyperimmunized against nonrelated antigens could not.In partial fulfilment of doctorate thesis requirements     

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Endotoxic glycolipid extracted from the heptose-less mutant of $\text{Salmonella typhimurium}$ was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen’s persistence.     

Synthetic Studies on Formamidopyrimidines Related to Clofarabine

Degradation of clofarabine (3) in 0.9% saline solution at 100°C afforded three degradation products which were determined to be formamidopyrimidines 4–6.Compounds 4 and 5 were assigned as C_1′ anomers on the basis of one-dimensional and two-dimensional NMR experiments, whereas 6 was found to be the formamidopyrimidine lacking the sugar moiety. An improved procedure for the synthesis of formamidopyrimidines was developed, wherein benzoylated clofarabine (11) was treated with allyl chloroformate, followed by deprotection of the alloc group with catalytic Pd(PPh₃)₄ and dimedone. A synthesis of compound 6 from 4 is also described.