The role of Cathepsin S as a marker of prognosis and predictor of chemotherapy benefit in adjuvant CRC: a pilot study.

BACKGROUND: The aim of this pilot retrospective study was to investigate the immunohistochemical expression of Cathepsin S (CatS) in three cohorts of colorectal cancer (CRC) patients (n=560). METHODS: Prevalence and association with histopathological variables were assessed across all cohorts. Association with clinical outcomes was investigated in the Northern Ireland Adjuvant Chemotherapy Trial cohort (n=211), where stage II/III CRC patients were randomised between surgery-alone or surgery with adjuvant fluorouracil/folinic acid (FU/FA) treatment. RESULTS: Greater than 95% of tumours had detectable CatS expression with significantly increased staining in tumours compared with matched normal colon (P>0.001). Increasing CatS was associated with reduced recurrence-free survival (RFS; P=0.03) among patients treated with surgery alone. Adjuvant FU/FA significantly improved RFS (hazard ratio (HR), 0.33; 95% CI, 0.12-0.89) and overall survival (OS; HR, 0.25; 95% CI, 0.08-0.81) among 36 patients with high CatS. Treatment did not benefit the 66 patients with low CatS, with a RFS HR of 1.34 (95% CI, 0.60-3.19) and OS HR of 1.33 (95% CI, 0.56-3.15). Interaction between CatS and treatment status was significant for RFS (P=0.02) and OS (P=0.04) in a multivariate model adjusted for known prognostic markers. CONCLUSION: These results signify that CatS may be an important prognostic biomarker and predictive of response to adjuvant FU/FA in CRC.

Characterization of trafficking pathways and membrane genesis in malaria-infected erythrocytes.

The origin of membraneous structures in the cytoplasm of human erythrocytes infected with the malaria parasite, Plasmodium falciparum, was determined by confocal fluorescence imaging microscopy. When infectious merozoites invaded erythrocytes labeled with the fluorescent, lipophilic, non-exchangeable molecules DiIC16 or DiOC16, a ring of fluorescence was observed surrounding the internal parasite, indicating that the parasitophorous vacuolar membrane (PVM) is formed in part from the erythrocyte membrane. As the parasites matured, fluorescent vesicles were seen to be exported into the erythrocyte cytoplasm, beginning at 6 h post-invasion. During intraerythrocytic development, these dyes were transferred from the PVM to the parasite. When fluorescently labeled merozoites were released from these cells and invaded unlabeled erythrocytes, fluorescence was confined to the parasite throughout the entire erythrocytic cycle. Taken together, these results demonstrate that all vesicles/membranous compartments in the erythrocyte cytoplasm of parasitized erythrocytes (IRBC) contain membrane derived from the PVM. Based on this information, we define pathways that the parasite utilizes to export proteins and lipids to the host cell cytoplasm and surface membrane. When IRBC were labeled post-invasion with DiIC16 or DiOC16 and the parasites allowed to mature for one life cycle, the dyes were confined to the erythrocyte membrane, demonstrating that the host cell membrane of IRBC does not endocytose and there is no membrane exchange from the erythrocyte to the parasite. This investigation helps to resolve two long-standing controversies and provides new insights into the transport pathways that malaria parasites utilize during their development within host erythrocytes.

PfEMP3 and HRP1: co-expressed genes localized to chromosome 2 of Plasmodium falciparum.

A malarial protein, Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3), has been recently characterized as a high-molecular-mass component (approx. 315 kDa) localized to the erythrocyte membrane of knob-bearing (K+), cytoadherent (C+) mature stages of P. falciparum-parasitized erythrocytes (PE) [Pasloske et al., Mol. Biochem. Parasitol. 59 (1993) 59-72]. Knobless (K-), non-cytoadherent (C-) parasites of the same strain were shown to lack the PfEMP3 gene. In view of the biological importance of the knobby and cytoadherent phenotypes with regard to parasite virulence, we extended the analysis of PfEMP3 and its gene product to other K+/K- and C+/C- parasites. Previously, other studies have shown that the malarial protein, knob-associated histidine-rich protein 1 (HRP1), is also strongly correlated with knob expression. Here, we show that PfEMP3 and HRP1 were absent from all the K- parasites tested, including the Palo Alto (PA) K-C+ strain. This result demonstrates that PfEMP3 and HRP1 are not essential for cytoadherence. PfEMP3 was localized to chromosome 2 of the K+ parasites, within no more than 130 kb of HRP1, between the telomere and HRP1. Stage-specific analysis of the mRNA for HRP1 and PfEMP3 indicated maximal transcription of the genes in ring-stage parasites, with little or no mRNA present during the mature parasite stages. Analysis of PfEMP3 and HRP1 by immunofluorescence assay (IFA) revealed identical staining patterns of fixed PE at all stages of the asexual life cycle. Hence, PfEMP3 and HRP1 are adjacent to each other in chromosome 2, co-expressed temporally and their gene products co-localized to the PE membrane.

Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight host membrane-associated protein, PfEMP3.

The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+PE). Both DNA fragments are absent from K - PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+PE, but no hybridization with K - PE. Immune sera were elicited against the lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+PE but not with K - PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K - PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda 12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways.

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum.

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.