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Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.

Malaria infection is initiated when an infected anopheline mosquito injects sporozoites during a bloodmeal. After injection, sporozoites enter the circulation and rapidly invade hepatocytes [1], where they develop into exoerythrocytic forms (EEF). There is a large body of evidence (reviewed in [2]) suggesting that sporozoite arrest in the liver is mediated by the binding of the parasite’s major surface protein, the circumsporozoite protein (CS; [3]), to heparan sulfate proteoglycans (HSPGs) on the hepatocyte surface. CS binding to HSPGs is mediated by region II-plus, a highly conserved stretch of amino acids, containing a motif of basic and hydrophobic residues, which binds to the negatively-charged sulfate moieties of HSPG glycosaminoglycan (GAG) chains. Physiologic ligands for these hepatic HSPGs include chylomicron and very low density lipoprotein remnants [4]. Like Plasmodium sporozoites, these lipoprotein remnants are rapidly cleared from the circulation by the liver. Clearance of these lipoproteins is mediated by apolipoprotein (apo) E which, like CS, binds to HSPGs. We have previously shown, that remnant lipoproteins can competitively inhibit sporozoite invasion of HepG2 cells in vitro and can decrease sporozoite infectivity in vivo, suggesting that sporozoites and remnant lipoproteins compete for the same HSPG binding sites at the cell surface [5]. After their sequestration from the plasma, by hepatic HSPGs [6], remnant lipoproteins are internalized by one of two receptors found on the hepatocyte surface, the LDL receptor (LDLR) and the LDL receptor-related protein (LRP) [7– * Corresponding author. Tel.: +1-212-263-6818; fax: +1212-263-8116. E-mail address: photini.sinnis@med.nyu.edu (P. Sinnis)

Introduction: Several cytokines (including TNF-alpha, IL-6 and VEGF) are reported to play a critical role in the pathogenesis of CLL cells. Targeting the tumor microenvironment through modulation of the cytokine milieu thus is a potential way to treat CLL. In 2 separate clinical studies we investigated the role of IMiDs (thalidomide and lenalidomide) either in combination with standard chemotherapy (thalidomide + fludarabine, FT) or single agent (lenalidomide, L) in pts with previously untreated or relapsed/refractory CLL, respectively. In these studies we observed tumor flare reaction not previously reported. In this report we describe the clinical characteristics of this side effect, its management and correlation with changes in serum cytokine levels. Flare Reaction (FR): CLL pts treated with IMiDs were noted to have tender increase in lymph nodes size starting within 24–48 hours of initiating therapy with or without erythema and rash. Some patients also increased their total white cell as well as absolute lymphocyte count (ALC). Results: A total of 48 pts (20 on FT and 28 on lenalidomide) are collectively treated on 2 clinical trials. Median age was------, -----had limited stage (0-II) and ----- has advance Rai stage (III, IV). ------(%) on the FT and 22 (78.5%) on the L developed a FR, which was noted as early as 24 hours after initiation of therapy (median 48 hours). Most pts resolved FR by day 14. A comparison of serum cytokine level at day 0 and 7 was done in 10 pts (9 with FR) treated with L. An increase in TNF-a (n=4), MIG (n=6), IP-10 (n=8), IL-8 (n=7) and IL-10 (n=6) was noted at day 7. Management: FR was treated with ibuprofen 400 mg q 6 hr with addition of oral morphine (or agents of equivalent potency) for improved pain control. Conclusion: FR is a new side effect noted in CLL pts treated with IMiDs that is fairly well managed by anti-inflammatory agent along with aggressive pain control. Higher incidence is noted with L. There seems to be no correlation of FR with clinical response to therapy. Identification of the FR is important to avoid confusion with disease progression. An increase in levels of inflammatory cytokine was noted in all pts treated with L which may explain the underlying mechanism of this phenomenon.

Using polyclonal and monoclonal antibodies to human recombinant IL-2 (rIL-2), we developed a sensitive radioimmunoassay (RIA) for quantitation of human IL-2. In this assay, microtitration plates pre-coated with an anti-rIL-2 monoclonal antibody (35H10), recognizing residues 59-72 of human IL-2, are incubated with serial dilutions of test samples. Captured IL-2 is quantitated by adding an affinity-purified rabbit anti-rIL-2 antibody followed by an 125I-labeled goat anti-rabbit IgG. Antibodies to chemically synthesized IL-2 peptides could replace the polyspecific rabbit anti-rIL-2 antibody as the second specific reagent in the assay. This configuration was more sensitive than others tested, approaching the level of detection of the conventional IL-2 bioassay, thus allowing detection of as little as 100-200 pg IL-2. Serum or plasma fluids, however, inhibited the assay, reducing its sensitivity by approximately 5-fold. This RIA correlated well with the conventional bioassay in measuring IL-2 levels in sera from IL-2-treated patients. This, and similar, RIAs may serve as a useful adjunct or alternative to the conventional IL-2 bioassay in detecting and quantitating human IL-2 in culture supernatants and clinical samples.

We describe a new approach to the analysis of the structural polymorphism of the DR beta, DQ alpha, and DQ beta polypeptide chains of human histocompatibility class II antigens. In comparison to conventional two-dimensional gel studies, this method provides sharper definition of the protein bands and side-by-side comparisons within the same gel, thereby permitting the detection of minor differences in the isoelectric points of the protein chains. Using this methodology we have analyzed the IEF polymorphism and the variability in the number of the DR beta chains encoded by different DR haplotypes. Twenty DR beta chain variants, which include the products of no less than two separate DR beta loci, have been thus far identified. Alleles at one of these loci are assumed to code for DR beta chains carrying the DR alloespecificities DR1, DR2, DR3, DR4, DR5, DRw6, DR7, and DR8. Alleles at a second DR beta locus encode DR beta chains that may be shared by serologically DR-different haplotypes and carry supertypic serologic specificities (i.e., DRw52 and DRw53). We also demonstrate here that the structural polymorphisms of the DQ alpha and DQ beta chains are more extensive than previously thought, report the characterization of 14 DQ beta variants, and define their relationship to the previously described DQw serologic specificities. In addition, we describe the class II haplotype associations observed for the different DR and DQ variants characterized.

The human class II HLA-DP molecules are heterodimers of a 34–36,000 d polypeptide (DPα-chain) noncovalently associated to a 28–29,000 d polypeptide (DPβ-chain) with both the DPα and DPβ chains being encoded by genes located in the most centromeric region of the human major histocompatibility complex (MHC) (1–3). DP molecules, as the other class II molecules DR and DQ, can act as restriction elements for T-cell recognition of foreign antigens (4) and represent important histocompatibility antigens. Although the genetic polymorphism of the DP molecules can be shown by the use of serologic and cellular reagents (5,6), the precise biochemical characterization of the different polymorphic variants has been made difficult by the low levels of expression of these molecules and by their extensive microheterogeneity, particularly that of the DPβ chain. We report here on results of the biochemical analysis of the DPα and DPβ products in 137 individuals from 25 multiplex insulin-dependent diabetes mellitus (IDDM) families which allowed for the direct observation by segregation of the DP molecules encoded by 94 haplotypes. Two DPα and 7 DPβ isoelectric focusing (IEF) variants were characterized. Haplotypic associations between the DP IEF-variants and those of the DR and DQ loci were encountered which suggest a way to discriminate between high and low IDDM risk among otherwise indistinguishable HLA haplotypes.