Proteomic analyses of different human tumour-derived chaperone-rich cell lysate (CRCL) anti-cancer vaccines reveal antigen content and strong similarities amongst the vaccines along with a basis for CRCL's unique structure: CRCL vaccine proteome leads to unique structure.

PURPOSE: The aim of this paper was to compare protein content of chaperone-rich cell lysate (CRCL) anti-cancer vaccines prepared from human tumours of different histological origins to evaluate the uniformity of their protein content. MATERIALS AND METHODS: Clinical grade CRCL was prepared under Good Manufacturing Practice (GMP) conditions from surgically resected human tumours (colorectal cancer, glioblastoma, non-small cell lung cancer, ovarian cancer). Protein samples were separated by SDS-PAGE and slices cut from gels for protease digestion followed by mass spectrometry analysis. Proteins were identified, and the content assessed by gene ontogeny/networking programmatic computation. CRCL preparations were also analysed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). RESULTS: We identified between 200 and 550 proteins in the various CRCL preparations. Gene ontogeny analysis indicated that the vaccines showed clear relationships, despite different tumour origins. A total of 95 proteins were common to all the CRCLs. Networking analyses implicated heat shock proteins in antigen processing pathways, and showed connections to the cytoskeletal network. We found that CRCL vaccines showed a particulate structure by NTA, and TEM revealed an extended fence-like structural network in CRCL, with regions that were microns in size. CONCLUSIONS: We conclude that it is feasible to prepare and characterise CRCL from a variety of different tissue sources; a substantial portion of the protein content is identical among the different CRCLs, while the overall compositions also suggest high overlaps in functional categories. The protein content indicates the presence of antigens and implies a potential structure, which we believe may play a role in CRCL's ability to stimulate innate antigen presenting cell activation.

Prolonged remission of advanced bronchoalveolar adenocarcinoma in a dog treated with autologous, tumour-derived chaperone-rich cell lysate (CRCL) vaccine.

PURPOSE: This paper presents the treatment of a 12-year-old female spayed Great Dane who presented with vestibular signs (ataxia, nystagmus, hind end collapse). Thoracic radiographs revealed a discrete pulmonary nodule in the right cranial lung lobe. Ultrasound-guided fine needle aspirate detected primary bronchoalveolar adenocarcinoma, verified via computed tomography, with a second smaller nodule discovered in the right cranial lung lobe. MATERIALS AND METHODS: A lateral thoracotomy with right cranial lung lobectomy was performed. Histopathological analysis of the nodules and an excised lymph node identified grade III bronchoalveolar adenocarcinoma with vascular infiltration and lymph node metastasis - a grim diagnosis with a reported median survival time of 6-27 days. A 10-g sample of the tumour was processed into a chaperone-rich cell lysate (CRCL) vaccine, which was administered weekly to the patient. Imiquimod - a Toll-like receptor 7 (TLR7) agonist - was applied topically for the first 12 treatments to stimulate local Langerhans cells. A single injection of bacillus Calmette-Guerin (BCG) was administered for additional immune stimulation at week 30 of treatment. RESULTS: The dog remained stable and in otherwise good health until diffuse relapse occurred 44 weeks after the initial treatment; following gastrointestinal bleeding, the dog was euthanised 50+ weeks post diagnosis. CONCLUSION: To the authors' knowledge, this is the first report of significantly prolonged survival following a diagnosis of grade III/stage III bronchoalveolar adenocarcinoma in a canine patient. This case report suggests that CRCL vaccine combined with topical imiquimod is a safe, effective treatment for canine tumours.

Targeting of prion-infected lymphoid cells to the central nervous system accelerates prion infection.

BACKGROUND: Prions, composed of a misfolded protein designated PrP(Sc), are infectious agents causing fatal neurodegenerative diseases. We have shown previously that, following induction of experimental autoimmune encephalomyelitis, prion-infected mice succumb to disease significantly earlier than controls, concomitant with the deposition of PrP(Sc) aggregates in inflamed white matter areas. In the present work, we asked whether prion disease acceleration by experimental autoimmune encephalomyelitis results from infiltration of viable prion-infected immune cells into the central nervous system. METHODS: C57Bl/6 J mice underwent intraperitoneal inoculation with scrapie brain homogenates and were later induced with experimental autoimmune encephalomyelitis by inoculation of MOG(35-55) in complete Freund's adjuvant supplemented with pertussis toxin. Spleen and lymph node cells from the co-induced animals were reactivated and subsequently injected into naïve mice as viable cells or as cell homogenates. Control groups were infected with viable and homogenized scrapie immune cells only with complete Freund's adjuvant. Prion disease incubation times as well as levels and sites of PrP(Sc) deposition were next evaluated. RESULTS: We first show that acceleration of prion disease by experimental autoimmune encephalomyelitis requires the presence of high levels of spleen PrP(Sc). Next, we present evidence that mice infected with activated prion-experimental autoimmune encephalomyelitis viable cells succumb to prion disease considerably faster than do mice infected with equivalent cell extracts or other controls, concomitant with the deposition of PrP(Sc) aggregates in white matter areas in brains and spinal cords. CONCLUSIONS: Our results indicate that inflammatory targeting of viable prion-infected immune cells to the central nervous system accelerates prion disease propagation. We also show that in the absence of such targeting it is the load of PrP(Sc) in the inoculum that determines the infectivity titers for subsequent transmissions. Both of these conclusions have important clinical implications as related to the risk of prion disease contamination of blood products.

Chemically induced accumulation of GAGs delays PrP(Sc) clearance but prolongs prion disease incubation time.

Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals. The only identified component of the infectious prion is PrP(Sc), an aberrantly folded isoform of PrP(C). Glycosaminoglycans, which constitute the main receptor for prions on cells, play a complex role in the pathogenesis of prion diseases. For example, while agents inducing aberrant lysosomal accumulation of GAGs such as Tilorone and Quinacrine significantly reduced PrP(Sc) content in scrapie-infected cells, administration of Quinacrine to prion-infected subjects did not improve their clinical status. In this study, we investigated the association of PrP(Sc )with cells cultured with Tilorone. We found that while the initial incorporation of PrP(Sc) was similar in the treated and untreated cells, clearance of PrP(Sc) from the Tilorone-treated cells was significantly impaired. Interestingly, prolonged administration of Tilorone to mice prior to prion infection resulted in a significant delay in disease onset, concomitantly with in vivo accumulation of lysosomal GAGs. We hypothesize that GAGs may complex with newly incorporated PrP(Sc) in lysosomes and further stabilize the prion protein conformation. Over-stabilized PrP(Sc) molecules have been shown to comprise reduced converting activity.

Prion urine comprises a glycosaminoglycan-light chain IgG complex that can be stained by Congo red.

Light chain IgG, a known amyloidotic protein, is present in the urine of prion disease affected individuals in a protease resistant form. In addition, it was shown recently that prion urine samples comprise a significant excess of glycosaminoglycans. Since amyloidotic proteins and glycosaminoglycans are the major components of amyloid aggregates, a Congo red dot blot assay was developed for detection of Creutzfeldt-Jacob disease (CJD) in urine. This assay was also positive for about 10% of patients suffering from diseases such as Alzheimer disease, cerebrovascular attacks and multiple sclerosis, but negative for healthy controls. Both glycosaminoglycans and proteins such as light chain IgG were required for the binding of Congo red to the urine fractions, as shown by the fact that Proteinase K digestion of the samples either after guanidine or after choindrotinase abolished the Congo red signal from the CJD samples.

The metabolism of glycosaminoglycans is impaired in prion diseases.

It is well established that the conversion of PrP(C) to PrP(Sc) is the key event in prion disease biology. In addition, several lines of evidence suggest that glycosaminoglycans (GAGs) and in particular heparan sulfate (HS) may play a role in the PrP(C) to PrP(Sc) conversion process. It has been proposed that PrP(Sc) accumulation in prion diseases may induce aberrant activation of lysosomal activity, which has been shown to result in neurodegeneration in a number of diseases, especially lysosomal storage disorders. Among such diseases, only the ones resulting from defects in GAGs degradation are accompanied by secretion of large amounts of GAG metabolites in urine. In this work, we show that GAGs are secreted in the urine of prion-infected animals and humans, and surprisingly, also in the urine of mice ablated for the PrP gene. We hypothesize that both the presence of PrP(Sc) or the absence of PrP(C) may alter the metabolism of GAGs.