Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay

Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells

Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E₂ (PGE₂) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE₂ production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE₂, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE₂, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS

Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.     

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets

The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either T. brucei CaaX protease when examined in the context of the yeast a-factor reporter but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that T. brucei Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases and that a subset of these compounds disrupt T. brucei Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both T. brucei CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences T. brucei CaaX protease specificity.Certain isoprenylated proteins are synthesized as precursors having a highly degenerate C-terminal tetrapeptide CaaX motif (C, cysteine; a, aliphatic amino acid; X, one of several amino acids). This motif typically directs three posttranslational modifications that include covalent attachment of an isoprenoid lipid to the cysteine residue, followed by endoproteolytic removal of the terminal three residues (i.e., aaX), and lastly, carboxyl methyl esterification of the farnesylated cysteine (49, 50). Relevant examples of proteins subject to the above modifications, also referred to as CaaX proteins, include the Ras and Ras-related GTPases, Gγ subunits, prelamin A, members of the Hsp40 family of chaperones, and fungal mating pheromones.Isoprenylation of CaaX proteins is performed by either the farnesyltransferase (FTase) or the geranylgeranyl transferase I (GGTase I). The particular isoprenoid attached, C15 farnesyl or C20 geranylgeranyl, respectively, depends in part on the sequence of the CaaX motif (8, 26, 31). Proteolysis of isoprenylated intermediates is carried out by the otherwise unrelated Rce1p (Ras converting enzyme 1) and Ste24p (sterile mutant 24) enzymes, collectively referred to as CaaX proteases, which are integral membrane proteins residing within the endoplasmic reticulum (3, 40, 45). Studies to elucidate the specificities of the CaaX proteases have often involved reporters designed from biological substrates (e.g., Ras GTPases) (2, 3, 16, 21, 22, 24, 34). Although these studies suggest that isoprenylated CaaX tetrapeptides alone are sufficient for recognition as a substrate, insufficient evidence exists to assert whether this sequence contains all of the necessary information for substrate specificity. Reporters are typically cleaved by either Rce1p or Ste24p. The Saccharomyces cerevisiae a-factor mating pheromone is a rather unusual biological reporter since it is cleaved by both yeast CaaX proteases. Orthologs of the CaaX proteases from humans, worms, and plants can also cleave a-factor when heterologously expressed in yeast, thereby making a-factor a convenient reporter for comparative analyses of CaaX protease activities (3, 5, 6, 36). Where evaluated using the a-factor reporter, Rce1p and Ste24p display partially overlapping target specificity, and this is an expected property of CaaX proteases in all eukaryotic systems (5, 6, 36, 47). Unlike the isoprenylation and proteolysis steps, carboxyl methyl esterification exclusively relies on a single enzyme, the isoprenylcysteine carboxyl methyltransferase (ICMT) (23, 50). A farnesylated cysteine appears to be the sole recognition determinant of the endoplasmic reticulum-localized ICMT (10, 23, 38).Disruption of the posttranslational modifications associated with CaaX proteins is often perceived as an anticancer strategy because of the prominent role of CaaX proteins in cellular transformation (i.e., the Ras GTPases) (49). To date, the most advanced drug discovery efforts have focused on farnesyltransferase inhibitors (FTIs) (9, 53). Inhibitors of the CaaX proteases and ICMT are also being developed (1, 11, 28, 37, 39, 48). Disrupting CaaX protein modifications has therapeutic application to other diseases as well. The relief of prelamin A toxicity by FTIs is a well-documented example (51). Accumulation of the farnesylated but unproteolysed precursor of lamin A results in a progeroid phenotype in individuals lacking ZmpSte24 proteolytic activity. The treatment of parasitic disease is another area under investigation (13). A number of FTIs have been developed that inhibit protozoan FTases, and in vivo testing is a continued effort (15, 32). Although research is less advanced with respect to CaaX protease and ICMT inhibitors, RNA interference experiments on the bloodstream form of Trypanosoma brucei indicate that CaaX processing enzymes are required for viability and proliferation of the parasite (20).In the present study, we evaluated the enzymatic properties of the trypanosomal CaaX proteases. We establish through the use of a variety of in vivo and in vitro assays that T. brucei Rce1 and T. brucei Ste24 are active when heterologously expressed in S. cerevisiae and have partially overlapping substrate specificities. The assays rely on various reporters, specifically the yeast a-factor mating pheromone, a K-Ras4B-based fluorogenic peptide, a green fluorescent protein (GFP)-GTPase fusion, and a T. brucei Hsp40 protein. All but the GTPase reporter could be effectively cleaved by both T. brucei CaaX proteases. We also demonstrate that the trypanosomal CaaX proteases can be targeted for inhibition by small molecules both in vitro and when heterologously expressed in yeast, suggesting that the trypanosomal CaaX proteases may be attractive drug targets for pharmacological inhibition.