Ahmi Ben-Yehudah

Tryptase activates peripheral blood mononuclear cells causing the synthesis and release of TNF-α, IL-6 and IL-1β: possible relevance to multiple sclerosis

Abstract Presence of mast cells and an increase in the concentration of their products has been reported in multiple sclerosis (MS) plaques. The most abundant secretory mediator of the human mast cell is the tetrameric protease tryptase. We demonstrate that tryptase can activate peripheral mononuclear cells (PBMCs), isolated from healthy donors as well as MS patients for the release of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β. Cytokine secretion was significantly higher in secondary progressive (SP) MS patients and healthy control (HC) individuals than in relapsing–remitting (RR) patients. Our findings suggest that tryptase is, most probably, an important mediator of inflammation in MS.

Targeted cancer therapy with gonadotropin-releasing hormone chimeric proteins.

Tumor-associated antigens (TAAs) have been identified mainly to determine cancer prognosis. In the past few years, TAAs have been used in the development of treatment modalities such as tumor vaccination. This review describes an additional application of TAAs: as a target for specific antitumor treatment. Since TAAs are overexpressed on the tumor cell surface, they can be targeted to deliver drugs directly to cancer cells. One such delivery system exploits chimeric proteins. Chimeric proteins are a class of targeted molecules designed to recognize and specifically destroy cells that overexpress specific receptors. These molecules, designed and constructed by gene fusion techniques, comprise both cell-targeting and cell-killing moieties. The authors’ laboratory has developed a number of chimeric proteins using gonadotropin-releasing hormone (GnRH) as the targeting moiety. These chimeras recognize a GnRH binding site that is expressed on adenocarcinoma cells. GnRH was fused to a large number of killing moieties, including bacterial and human proapoptotic proteins. All GnRH-based chimeric proteins selectively killed adenocarcinoma cells both in vitro and in vivo. Utilizing chimeric proteins for targeted therapy represents a new and exciting therapeutic modality for the treatment of cancer in humans.

Mechanism of action of interleukin-2 (IL-2)-Bax, an apoptosis-inducing chimaeric protein targeted against cells expressing the IL-2 receptor.

The chimaeric protein interleukin-2 (IL-2)-Bax was designed to target and kill specific cell populations expressing the IL-2 receptor. However, it is not well understood how IL-2-Bax causes target cells to die. In the present study, we investigated the pathway of apoptosis evoked by IL-2-Bax and the possible involvement of endogenous Bax in this process. We report here that, upon internalization of IL-2-Bax into target cells, it is localized first mainly in the nucleus, and only later is it translocated to the mitochondria. Similarly, endogenous Bax is also partially localized in the nucleus, and accumulates mainly in this compartment soon after physiological triggering of apoptosis. Despite the fact that Bax has no nuclear localization sequence, our data suggest that Bax has one or more physiological roles and/or substrates within the nucleus. Indeed, a dramatic repression of nuclear Tax protein expression was induced following treatment of HUT-102 cells with IL-2-Bax, similar to what occurs following serum deprivation of these cells. Unexpectedly, induction of apoptosis using IL-2-Bax was preceded by enhanced expression of newly synthesized Bax protein and suppression of Bcl-2. This imbalance between the pro- and anti-apoptotic genes was associated with p53 induction, although IL-2-Bax activity was also evident in cells lacking p53 expression. By studying the mechanism of action of IL-2-Bax, we were able to follow the intrinsic events and their cascade that culminates in cell death. We have shown that the ability of IL-2-Bax to affect the intracellular apoptotic machinery within the target cells, and to cause the cells to die, uses a mechanism similar to that induced following a normal apoptotic signal.

I.V. Administration of L‐GNRH‐PE66 efficiently inhibits growth of colon adenocarcinoma xenografts in nude mice

When developing new anti‐cancer therapeutic treatments, it is crucial to find the correct route of administration and timetable for treatment. Recently, we constructed the L‐GnRH‐PE66 chimeric protein, which can target and kill adenocarcinoma cells both in vitro and in vivo. We examined the ability of the L‐GnRH‐PE66 chimeric protein to inhibit tumor growth in colon carcinoma xenografted nude mice, using different routes of administration and various timetables of treatment. In addition, we examined the ability of the chimeric protein to inhibit tumor growth of large tumors that resemble those encountered in human patients in the clinical setting. We found that an i.v. dose of 12.5 μg given every 48 hr was the most efficacious in inhibiting tumor growth. Tumors treated with this concentration of the chimeric protein were 4.4 times smaller in volume and 3.4 times smaller in weight than those in the control groups. This protocol of L‐GnRH‐PE66 treatment is an improvement on our previously suggested treatment for adenocarcinoma in humans. An i.v. injection every 48 hr is effective, less toxic and less painful. Our results further support the use of L‐GnRH‐PE66 as an effective treatment for adenocarcinoma in humans.

Linker-based GnRH-PE chimeric proteins inhibit cancer growth in nude mice

Since the number of cancer-related deaths has not decreased in recent years, major efforts are being made to find new drugs for cancer treatment. In this report we introduce the gonadotropin releasing hormone-Pseudomonas exotoxin (GnRH-PE) based chimeric proteins L-GnRH-PE66 and L-GnRH-PE40. These proteins are composed of a GnRH moiety attached to modified forms ofPseudomonas exotoxin via a polylinker (gly4ser)2. The chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 have the ability to target and kill adenocarcinoma cell linesin vitro, whereas non-adenocarcinoma cell lines are not affected. We demonstrate that L-GnRH-PE66 and L-GnRH-PE40 efficiently inhibit cancer growth. Nude mice were injected subcutaneously with the SW-48 adenocarcinoma cell line to produce xenograft tumours. When the tumours were established and visible, the animals were injected with chimeric proteins for 10 days. At the end of this period, a reduction of up to 3-fold in tumor size was obtained in the treated mice, as compared with the control group, which received equivalent amounts of GnRH; the difference was even greater 13 days after termination of treatment. Thus, the chimeric proteins L-GnRH-PE66 and L-GnRH-PE40 are promising candidates for treatment of a variety of adenocarcinomas and their use in humans should be considered.