The oncolytic peptide LTX-315 triggers necrotic cell death

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.     

The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.     

Spirochete round bodies Syphilis, Lyme disease & AIDS: Resurgence of “the great imitator”?

We advocate investigation of spirochete cyclical symbioses (e.g.,Borrelia sp.,Leptospira sp., Treponema sp.) given the newly established verification of a developmental history in these gram-negative motile helical eubacteria, both in pure culture and in mammals. Symbiotic spirochetes can be compared to free-living relatives for their levels of integration (behavioral, metabolic, gene product or genetic levels), Detailed research that correlates life histories of symbiotic spirochetes to changes in the immune system of associated vertebrates is sorely needed. Genome analyses show that in necrotrophic symbioses (Borrelia andTreponema sp.) of humans and other primates, integration of the bionts occurs at the gene product and genetic level. Spirochete round bodies (also called cysts, L-forms and sphaeroplasts) can be induced by many types of unfavorable conditions (e.g., threats of starvation, desiccation, oxidation, penicillin and other antibiotics). Reversion to familiar helical, motile active swimmers by placement of pure cultures into favorable environments in some cases can be controlled. These observations are supported by a European literature, especially Russian, apparently unknown to American medicine and medical research.     

Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus

Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune‐dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species‐dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA‐dependent secretory pathway. This led to a general inhibition of protein secretion by PDT‐treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin‐based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro‐apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.     

Therapeutic vaccination against a murine lymphoma by intratumoral injection of a cationic anticancer peptide

Cationic antimicrobial peptides (CAPs) exhibit promising anticancer activities. In the present study, we have examined the in vivo antitumoral effects of a 9-mer peptide, LTX-302, which is derived from the CAP bovine lactoferricin (LfcinB). A20 B cell lymphomas of BALB/c origin were established by subcutaneous inoculation in syngeneic mice. Intratumoral LTX-302 injection resulted in tumor necrosis and infiltration of inflammatory cells followed by complete regression of the tumors in the majority of the animals. This effect was T cell dependent, since the intervention was inefficient in nude mice. Successfully treated mice were protected against rechallenge with A20 cells, but not against Meth A sarcoma cells. Tumor resistance could be adoptively transferred with spleen cells from LTX-302-treated mice. Resistance was abrogated by depletion of T lymphocytes, or either the CD4⁺ or CD8⁺ T cell subsets. Taken together, these data suggest that LTX-302 treatment induced long-term, specific cellular immunity against the A20 lymphoma and that both CD4⁺ and CD8⁺ T cells were required. Thus, intratumoral administration of lytic peptide might, in addition to providing local tumor control, confer a novel strategy for therapeutic vaccination against cancer.     

Structural requirements for furin-induced cleavage and activation of Shiga toxin

Shiga toxin has a protease-sensitive site in the disulfide loop region of the A-chain. Cleavage of this site by furin is essential for rapid intoxication of cells by Shiga toxin. We have here investigated whether in addition to the Arg-X-X-Arg sequence, there are other structural requirements in the disulfide loop region for furin cleavage. A toxin mutant (Shiga-2D toxin) still containing the consensus motif for cleavage by furin, but lacking ten amino acids in the disulfide loop, was generated. Trypsin was able to cleave Shiga-2D toxin in vitro, demonstrating that the protease-sensitive region is intact. However, Shiga-2D toxin was not efficiently cleaved by furin either in vitro or in vivo. Furthermore, unless it was precleaved with trypsin, Shiga-2D toxin was much less toxic than wild type Shiga toxin in LoVo cells expressing functional furin. In contrast, LoVo/neo cells lacking functional furin were unable to activate both wild type Shiga toxin and Shiga-2D toxin. In conclusion, an extended loop structure is required for furin-induced cleavage of Shiga toxin.     

Daylength and temperature during seed production interactively affect adaptive performance of Picea abies progenies

Adaptive traits in Picea abies (Norway spruce) progenies are influenced by the maternal temperatures during seed production. Here, we have extended these studies by testing the effects of maternal photoperiod and temperature on phenology and frost hardiness on progenies. Using eight phytotron rooms, seeds from three unrelated crosses were made in an environmental 2 x 2 factorial combination of long and short days and high and low temperatures. The progenies were then forced to cease growth rapidly at the end of the first growing season. An interactive memory effect was expressed the second growth season. Progenies from high temperature and short days, and from low temperatures and long days, started growth later in spring, ceased shoot growth later in summer, grew taller and were less frost hardy in the autumn than their full siblings from low temperatures and short days, and from high temperatures and long days. Norway spruce has developed a memory mechanism, regulating adaptive plasticity by photoperiod and temperature, which could counteract harmful effects of a rapidly changing climate.     

Complete regression and systemic protective immune responses obtained in B16 melanomas after treatment with LTX-315

Malignant melanoma is the most aggressive and deadliest form of skin cancer due to its highly metastatic potential, which calls for new and improved therapies. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising potential as novel anticancer agents. Structure–activity relationship studies on the CAP bovine lactoferricin allowed us to de novo design short chemically modified lytic anticancer peptides. In the present study, we investigated the in vivo antitumor effects of LTX-315 against intradermally established B16 melanomas in syngeneic mice. Intratumoral administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by complete regression of the tumor in the majority of the animals. LTX-315 induced the release of danger-associated molecular pattern molecules such as the high mobility group box-1 protein in vitro and the subsequent upregulation of proinflammatory cytokines such as interleukin (IL) 1β, IL6 and IL18 in vivo. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells both intradermally and intravenously. Together, our data indicate that intratumoral treatment with LTX-315 can provide local tumor control followed by protective immune responses and has potential as a new immunotherapeutic agent.     

Antimicrobial activity of short arginine- and tryptophan-rich peptides.

Highly antimicrobial active arginine- and tryptophan-rich peptides were synthesized ranging in size from 11 to five amino acid residues in order to elucidate the main structural requirement for such short antimicrobial peptides. The amino acid sequences of the peptides were based on previous studies of longer bovine and murine lactoferricin derivatives. Most of the peptides showed strong inhibitory action against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacterium Staphylococcus aureus. For the most active derivatives, the minimal inhibitory concentration values observed for the Gram-negative bacteria were 5 microg/ml (3.5 microM), whereas it was 2.5 microg/ml (1.5 microM) for the Gram-positive bacterium. It was essential for the antimicrobial activity that the peptides contained a minimum of three tryptophan and three arginine residues, and carried a free N-terminal amino group and an amidated C-terminal end. Furthermore, a minimum sequence size of seven amino acid residues was required for a high antimicrobial activity against Pseudomonas aeruginosa. The insertion of additional arginine and tryptophan residues into the peptides resulted only in small variations in the antimicrobial activity, whereas replacement of a tryptophan residue with tyrosine in the hepta- and hexapeptides resulted in reduced antimicrobial activity, especially against the Gram-negative bacteria. The peptides were non-haemolytic, making them highly potent as prospective antibiotic agents.