Plasma gelsolin accumulates in macrophage nodules in brains of simian immunodeficiency virus infected rhesus macaques.

Plasma gelsolin (pGSN), an isoform 1, is secreted by various types of cells in the central nervous system (CNS) and periphery, but not by the liver. pGSN circulates in blood and cerebrospinal fluid (CSF); however, its concentration in CSF is approximately twenty times lower than in plasma. It has been shown that several types of cells such as oligodendrocytes, neurons, and/or astrocytes contribute to the overall pool of pGSN in the CNS. Further, it has been postulated that pGSN plays multiple roles during microbial infection and modulates inflammatory responses; however, the exact mechanism of regulation is not known. We previously showed that levels of pGSN in CSF of individuals with advanced neurocognitive impairment due to HIV infection of the brain are decreased. Here, we show that macrophages express significant amounts of pGSN in response to HIV infection in vitro. Using immunohistochemistry of simian immunodeficiency virus infected rhesus monkey brains, we show that increased levels of pGSN are present in macrophage nodules creating locally a high level of this protein within the brain. This may not be reflected by the overall decreased level in the distinct CSF compartment.

Mass spectrometric characterization of gelsolin isoforms.

Gelsolin is the most widely expressed member of the actin capping and severing family of proteins. There are two isoforms of gelsolin: isoform 1, a secretory (plasma) protein that is 51 amino acids longer than isoform 2, a cytosolic protein, at the N-terminus; the first 27 amino acids is a signal sequence. Both isoforms are coded by a single gene and differ as a result of alternative initiation site/splicing. The level of gelsolin in the blood and cerebrospinal fluid (CSF) is altered in many diseases including amyloidoses and other neurodegenerative disorders. Although quantitative analysis of gelsolin has been reported, lack of suitable antibodies makes it impossible to differentiate these two isoforms by immunodetection techniques and no other technique is available. Therefore, ambiguity exists whether gelsolin present in circulation is isoform 1 or also isoform 2 released from lysed cells. We report in this communication a mass spectrometric approach to identify isoform 1 of gelsolin immunopurified from human plasma and CSF. Recombinant isoform 1 was used as reference.

Proteomic analyses of monocyte-derived macrophages infected with human immunodeficiency virus type 1 primary isolates from Hispanic women with and without cognitive impairment.

The signature for human immunodeficiency virus type 1 (HIV-1) neurovirulence remains a subject of intense debate. Macrophage viral tropism is one prerequisite but others, including virus-induced alterations in innate and adaptive immunity, remain under investigation. HIV-1-infected mononuclear phagocytes (MPs; perivascular macrophages and microglia) secrete toxins that affect neurons. The authors hypothesize that neurovirulent HIV-1 variants affect the MP proteome by inducing a signature of neurotoxic proteins and thus affect cognitive function. To test this hypothesis, HIV-1 isolates obtained from peripheral blood of women with normal cognition (NC) were compared to isolates obtained from women with cognitive impairment (CI) and to the laboratory adapted SF162, a spinal fluid R5 isolate from a patient with HIV-1-associated dementia. HIV-1 isolates were used to infect monocyte-derived macrophages (MDMs) and infection monitored by secreted HIV-1 p24 by enzyme-linked immunosorbent assay (ELISA). Cell lysates of uninfected and HIV-1-infected MDMs at 14 days post infection were fractionated by cationic exchange chromatography and analyzed by surface enhanced laser desorption ionization time of flight (SELDI-TOF) using generalized estimating equations statistics. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and identified by tandem mass spectrometry. Levels of viral replication were similar amongst the HIV-1 isolates, although higher levels were obtained from one viral strain obtained from a patient with CI. Significant differences were found in protein profiles between virus-infected MDMs with NC, CI, and SF162 isolates (adjusted P value after multiple testing corrections, or q value <.10). The authors identified 6 unique proteins in NC, 7 in SF162, and 20 in CI. Three proteins were common to SF162 and CI strains. The MDM proteins linked to infection with CI strains were related to apoptosis, chemotaxis, inflammation, and redox metabolism. These findings support the hypothesis that the macrophage proteome differ when infected with viral isolates of women with and without CI.

Proteomic analyses associate cystatin B with restricted HIV-1 replication in placental macrophages.

Mononuclear phagocytes (MP; monocytes, tissue macrophages, and dendritic cells) are reservoirs, vehicles of dissemination, and targets for persistent HIV infection. However, not all MP population equally support viral growth. Such differential replication is typified by the greater ability of placental macrophages (PM), as compared to blood borne monocyte-derived macrophages (MDM), to restrict viral replication. Since cytosolic protein patterns can differentiate macrophage subtypes, we used a proteomics approach consisting of surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF), tandem mass spectrometry, and Western blots to identify differences between the uninfected and HIV-infected PM and MDM protein profiles linked to viral growth. We performed proteome analysis of PM in the molecular range of 5-20kDa. We found that a SELDI-TOF protein peak with an m/z of 11,100, which was significantly lower in uninfected and HIV-infected PM than in MDM, was identified as cystatin B (CSTB). Studies of siRNA against CSTB treatment in MDM associated its expression with HIV replication. These data demonstrate that the low molecular weight placental macrophage cytosolic proteins are differentially expressed in HIV-infected PM and MDM and identify a potential role for CSTB in HIV replication. This work also serves to elucidate a mechanism by which the placenta protects the fetus from HIV transmission.

Recombinant human tumor antigen MUC1 expressed in insect cells: structure and immunogenicity.

MUC1, a member of the mucin family of molecules, is a transmembrane glycoprotein abundantly expressed on human ductal epithelial cells and tumors originating from those cells. MUC1 expressed by malignant cells is aberrantly O-glycosylated. Differences in O-glycosylation of the tandem repeat region of MUC1 make tumor and normal forms of this antigen immunologically distinct. The tumor-specific glycoform is, therefore, expected to be a good target for immunotherapy and a good immunogen for generation of antitumor immune responses. We have generated a renewable source of this glycoform by expressing MUC1 cDNA in Sf-9 insect cells using a baculovirus vector. This form of MUC1 (BV-MUC1) is O-glycosylated at a very low level, approximately 0.3% (w/w), and this is not due to the lack of appropriate glycosylotransferases in insect cells. Peptidyl GalNAc-transferases isolated from Sf-9 cells were able to glycosylate in vitro a synthetic MUC1 peptide as efficiently as the transferases isolated from human milk. Neither preparation of peptidyl GalNAc-transferases, however, was able to glycosylate BV-MUC1. This underglycosylated recombinant MUC1 mimics underglycosylated MUC1 on human tumor cells and could serve as an immunogen to stimulate responses that would recognize MUC1 on tumor cells. To test this we immunized mice with Sf-9 cells expressing BV-MUC1. Sera from immunized mice recognized MUC1 on human tumor cells. We also generated MUC1-specific T cells that proliferated in response to synthetic MUC1 peptide.

A new strategy for tumor antigen discovery based on in vitro priming of naive T cells with dendritic cells.

We describe a method for discovery of new tumor antigens that uses dendritic cells (DCs) as antigen-presenting cells to prime autologous naive CD4+ and CD8+ T cells from healthy donors against tumor proteins and peptides. For the identification of HLA class I-restricted tumor antigens, peptides were extracted from tumor HLA class I molecules, fractionated by reverse phase-high performance liquid chromatography, and loaded onto in vitro-generated DCs to prime naïve CD8+ T cells. Our results show that we were able to prime naive CD8+ T cells in vitro to several peptide fractions and generate specificity for the tumor. Electrospray ionization mass spectrometry was used to confirm that these fractions contained peptides derived from MHC class I molecules, and the primed CD8+ T cells were used to further analyze the immunostimulatory peptide fractions. For the identification of HLA class II-restricted tumor antigens, we fractionated tumor protein extracts using reverse phase-high performance liquid chromatography and loaded individual fractions onto DCs to prime naive CD4+ T cells. Our results show that we were also able to prime naive CD4+ T cells to several protein fractions and generate specificity for the tumor. These results illustrate the potential of this method to identify new immunostimulatory MHC class I- and class II-restricted tumor antigens.

Biochemical characterization of the soluble form of tumor antigen MUC1 isolated from sera and ascites fluid of breast and pancreatic cancer patients.

Transmembrane glycoprotein tumor antigen MUC1 that is overexpressed on pancreatic and breast tumor cells can be found in large amounts in soluble form in serum and ascites fluid. MUC1 has been identified as a target of human antitumor antibody and CTL responses that are generated in the absence of helper T cells. The soluble form of MUC1 should support generation of helper T cells, but we have found recently that this form, although effectively endocytosed by dendritic cells, remains trapped in early endosomes and is not trafficked to antigen-processing compartments. The exact biochemical structure of this form of MUC1 has not been elucidated to date, and it is thus not clear what structural characteristics may be responsible for its retention in early endosomes. We have purified soluble MUC1 from ascites fluid of breast/pancreatic cancer patients (ASC-MUC1) and quantitated O-linked carbohydrates. We have altered ASC-MUC1 by enzymatic treatment: trypsin or clostripain digestion, desialylation, and further in vitro glycosylation. We have found that desialylated ASC-MUC1 was further glycosylated by peptidyl N-acetylgalactosamine transferases and was not when sialic acid was present. These alterations created new forms of ASC-MUC1 that might be handled more efficiently by antigen-presenting cells to generate better tumor-specific immunity and used to identify structures that are directly involved in retention of this antigen in early endosomes.

The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells.

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.

Presentation of MUC1 tumor antigen by class I MHC and CTL function correlate with the glycosylation state of the protein taken Up by dendritic cells.

We previously reported that the glycosylated MUC1 tumor antigen circulating as soluble protein in patients' serum is not processed by dendritic cells and does not elicit MHC-Class II-restricted T helper responses in vitro. In contrast, a long synthetic peptide from the MUC1 tandem repeat region is presented by Class II molecules, resulting in the initiation of T helper cell responses. Here we addressed the ability of dendritic cells to present various glycosylated or not glycosylated forms of MUC1 by MHC Class I. We found that three different forms of MUC1, ranging from glycosylated and underglycosylated protein to unglycosylated synthetic peptide, were able to elicit MUC1-specific, Class-I-restricted CTL responses. The efficiency of processing and the resulting strength of CTL activity were inversely correlated with the degree of glycosylation of the antigen. Furthermore, the more efficiently processed 100mer peptide primed a broader repertoire of CTL than the glycosylated protein.

Naturally processed class II epitope from the tumor antigen MUC1 primes human CD4+ T cells.

Epithelial cell mucin MUC1 is expressed on adenocarcinomas in an underglycosylated form that serves as a tumor antigen in breast, pancreatic, ovarian, and other tumors. Two predominant MUC1-specific immune responses are found in patients: CD8+ CTLs, which recognize tandemly repeated epitopes on the MUC1 protein core, and IgM antibodies. There have been no reports to date of MUC1-specific CD4+ T-helper cells in cancer patients. We show here that MUC1-specific CD4+ T cells are neither deleted nor tolerized and that CD4+ T cell responses can be generated when an appropriate soluble form of MUC1 is used. Naive CD4+ T cells from healthy donors were primed in vitro to a synthetic MUC1 peptide of 100 amino acids, representing five unglycosylated tandem repeats, presented by dendritic cells. They produced IFN-gamma and had moderate cytolytic activity. We identified one core peptide sequence, PGSTAPPAHGVT, that elicits this response when it is presented by HLA-DR3.

Intercellular and intracellular events following the MHC-unrestricted TCR recognition of a tumor-specific peptide epitope on the epithelial antigen MUC1.

We examined the functional and molecular parameters involved in direct TCR recognition of a tumor-specific peptide epitope on the tumor Ag MUC1. This peptide epitope is tandemly repeated and recognized on the native molecule rather than processed and bound to the MHC. Even though the TCR was not MHC restricted, intercellular interactions found to facilitate this recognition included intercellular adhesion molecule-1/LFA-1, LFA-3/CD2, and class I/CD8. Intracellular parameters of MHC-unrestricted CTL activation were examined to compare the recognition of the MUC1 epitope presented on synthetic microspheres, with the recognition of the native epitope in the context of other molecules on the target cells. The epitope on microspheres induced a transient influx of Ca2+ that was not accompanied by detectable tyrosine phosphorylation of the zeta-associated protein ZAP-70, whereas recognition of MUC1 epitopes on tumor cells caused a sustained Ca2+ influx and ZAP-70 phosphorylation. The transient influx of Ca2+ was not sufficient to cause translocation of the nuclear factor of activated T cells (NF-AT) into the nucleus or CTL proliferation. In contrast, recognition of the MUC1 epitope on tumor cells resulted in full activation of the CTL, nuclear translocation of NF-AT, and proliferation. MHC-unrestricted TCR triggering, therefore, involves similar intercellular and intracellular events that participate in the conventional, MHC-restricted Ag recognition. Direct recognition of the MUC1 peptide epitope by the TCR in the absence of presentation by the MHC induces a partial signal that is completed by further interactions of other receptor/ligand pairs on the surface of the CTL and their target cells.

Screening of anti-MUC1 antibodies for reactivity with native (ascites) and recombinant (baculovirus) MUC1 and for blocking MUC1 specific cytotoxic T-lymphocytes.

We report the results of a panel of 56 monoclonal antibodies submitted to the ISOBM TD-4 Workshop for the study of the epithelial MUC1 mucin. We used three forms of the MUC1 as antigen. One form of mucin was the native, highly glycosylated MUC1 isolated from the ascites of breast or pancreatic cancer patients. Two other forms of the mucin were recombinantly expressed in a baculovirus expression system as either fully glycosylated, or underglycosylated. Based on the results of Western blot analysis we were able to group the antibodies into 7 clusters depending on their recognition of the MUC1 forms tested. We then selected several antibodies, representatives of each cluster, to test for the ability to block MUC1-specific cytotoxic T-lymphocyte (CTL) function. We found that antibodies ISOBM-163 and ISOBM-147 blocked cytotoxicity of MUC1-specific CTL against two tumor targets in a concentration-dependent manner.

Recombinant epithelial cell mucin (MUC-1) expressed in baculovirus resembles antigenically tumor associated mucin, target for cancer immunotherapy.

Epithelial cell mucin encoded by the gene MUC-1, is expressed on several human adenocarcinomas in an aberrantly glycosylated form, and as such it has been identified as the target of human cellular as well as humoral responses. In order to harness this immunity to combat mucin-expressing tumors, various forms of this molecule, synthetic or highly purified, are being tested as possible cancer vaccines. We have expressed MUC-1 in baculovirus, and we report that the recombinant product has important similarities with the MUC-1 expressed on tumors, especially in regard to its aberrant glycosylation.

Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis.

Porphyromonas gingivalis, and organism implicated in the etiology and pathogenesis of human periodontal diseases, produces a variety of potent proteolytic enzymes, and it has been suggested that these enzymes play a direct role in the destruction of periodontal tissues. We now report that two cell-associated cysteine proteinases of P. gingivalis W12, with molecular masses of approximately 150 kDa (porphypain-1) and 120 kDa (porphypain-2), as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, have been separated and purified to apparent homogeneity. These proteinases appear to be SDS-stable conformational variants of a 180-kDa enzyme, and they are the largest cysteine proteinases yet purified from P. gingivalis. The purified proteinases hydrolyze fibrinogen, tosyl-Gly-L-Pro-L-Arg p-nitroanilide, and tosyl-Gly-L-Pro-L-Lys p-nitroanilide. While hydrolysis of both synthetic substrates by porphypain-1 and -2 requires activation by reducing agents, is inhibited by EDTA, and is stimulated in the presence of derivatives of glycine, the Arg-amidolytic activity is sensitive to leupeptin and H-D-tyrosyl-L-prolyl-L-arginyl chloromethyl ketone, whereas the Lys-amidolytic activity is sensitive to tosyl-L-lysyl chloromethyl ketone and insensitive to leupeptin. These data suggest that porphypains contain two types of active sites. These cell-associated P. gingivalis proteinases may contribute significantly and directly to periodontal tissue destruction.

Zymographic techniques for detection and characterization of microbial proteases.

We have presented a variety of zymographic techniques for identification and characterization of microbial proteases, using SDS-PAGE and PAGE in nondissociating gels. Techniques are described using copolymerized protein substrates, diffusable protein substrates, protein substrates incorporated into indicator gels, as well as synthetic esterase substrates. When a newly discovered protease is being characterized, it is advisable to try a variety of techniques, both to determine optimal conditions for enzyme detection and to characterize the protease. Zymography is a versatile two-stage technique involving protein separation by electrophoresis followed by detection of proteolytic activity. Each particular combination of protease separation and detection techniques had advantages and limitations. Protease separation by SDS-PAGE has as a limitation the fact that some proteases do not renature and hence cannot be detected following treatment with SDS. However, it has an advantage the fact that it allows estimation of the relative molecular weight of proteases. Protein separation using nondissociating PAGE is performed using much gentler protease inactivation conditions than those produced by treatment with SDS. Like SDS-PAGE, nondissociating PAGE permits detection of multiple forms of enzymes; however, a disadvantage is that it cannot be used to obtain molecular weight estimates of proteases. The main variable to control during development of zymograms is the length of time of incubations. Increasing incubation (development) time generally increases the sensitivity of protease detection; however, as the length of time of incubation increases so does the extent of diffusion of proteases and substrates. If incubations are prolonged, protease bands will diffuse, decreasing resolution. Additionally, zones of lysis produced by closely migrating proteolytically active species will merge, eliminating the possibility of detecting all proteolytic species in the sample. Zymographic techniques can be extremely useful in identification and characterization of microbial proteases. If a few properties of a protease are known, such as the pH range over which the enzyme is active, and whether it can renature after exposure to SDS, zymographic techniques can be specifically and readily adapted to optimize conditions for detection and assist in characterization of the enzyme.

Immunological response to a Staphylococcus aureus fibronectin-binding protein.

A protein (gal-FnBP), constructed by fusion of the genes encoding beta-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25% respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component.

Effect of milk on fibronectin and collagen type I binding to Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis.

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (Fn) [corrected], whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (Cn) [corrected] type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 125I-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 125I-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding.

Purification and partial characterization of two proteinases from Clostridium butyricum M 55.

Clostridium butyricum M55 proteinases were purified by application of a multistep procedure involving ethanol precipitation, DEAE cellulose chromatography and molecular sieving. The purified enzymes obtained were called proteinase I and proteinase II. They appeared to be homogeneous when examined by molecular sieving and polyacrylamide gel electrophoresis. The highly purified proteinases were studied for their physico-chemical properties. The influences of pH, temperature, ionic strength and amino acids composition were investigated. The effects of metal ions and of protein-structure-modifying agents support views suggesting the character of these enzymes.