Complement Activation-Related Pathophysiological Changes in Anesthetized Rats: Activator-Dependent Variations of Symptoms and Mediators of Pseudoallergy.

Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.

Single cell measurement of calpain activity in neutrophils reveals link to cytosolic Ca2+ elevation and individual phagocytotic events.

It has been proposed that Ca2+ activation of calpain-1 is important for the rapid cell shape changes which accompany phagocytosis. In this paper, we use a fluorogenic calpain substrate, (CBZ-Ala Ala)2 R110, and find that there was a low calpain activity measureable in resting (ie without intentional activation) neutrophils, but that it was accelerated by an elevation of cytosolic free Ca2+ (ionomycin -induced) and inhibited by calpeptin (an established calpain-1 inhibitor). The fluorescence signal was sufficiently bright for detection in individual neutrophils that enabled the quantification of dynamic changes in calpain activity to be related to elevations in cytosolic Ca2+ within individual neutrophils. It was found that during phagocytosis of C3bi-opsonised zymosan particles, calpain activity was elevated incrementally, each step increase corresponding to the phagocytosis of an individual particle. The sub-cellular source of the fluorescent product of calpain activity was the phagocytic site itself and originated at the phagocytic cup. It was thus concluded that calpain was activated locally during the formation of the phagocytic cup. These data were consistent with central role of Ca2+ activated calpain activation in controlling phagocytosis.

Development of a Quantitative Assay for the Characterization of Human Collectin-11 (CL-11, CL-K1).

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement with diverse functions spanning from host defense to embryonic development. CL-11 is found in the circulation in heterocomplexes with the homologous collectin-10 (CL-10). Abnormal CL-11 plasma levels are associated with the presence of disseminated intravascular coagulation, urinary schistosomiasis, and congenital disorders. Although there has been a marked development in the characterization of CL-11 there is still a scarcity of clinical tools for its analysis. Thus, we generated monoclonal antibodies and developed a quantitative ELISA to measure CL-11 in the circulation. The antibodies were screened against recombinant CL-11 and validated by ELISA and immunoprecipitation of serum and plasma. The best candidates were pairwise compared to develop a quantitative ELISA. The assay was validated regarding its sensitivity, reproducibility, and dilution linearity, demonstrating a satisfactory variability over a working range of 0.29-18.75 ng/ml. The mean plasma concentration of CL-11 in healthy controls was determined to be 289.4 ng/ml (range 143.2-459.4 ng/ml), highly correlated to the levels of CL/10/11 complexes (r = 0.729). Plasma CL-11 and CL-10/11 co-migrated in size exclusion chromatography as two major complexes of ~400 and >600 kDa. Furthermore, we observed a significant decrease at admission in CL-11 plasma levels in patients admitted to intensive care with systemic inflammatory response syndrome. By using the in-house antibodies and recombinant CL-11, we found that CL-11 can bind to zymosan independently of calcium by a separate site from the carbohydrate-binding region. Finally, we showed that CL-11/MASP-2 complexes trigger C4b deposition on zymosan. In conclusion, we have developed a specific and sensitive ELISA to investigate the ever-expanding roles of CL-11 in health and disease and shown a novel interaction between CL-11 and zymosan.

Heterogeneity of hypochlorous acid production in individual neutrophil phagosomes revealed by a rhodamine-based probe.

The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in ∼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.

Effect of zymosan and poly (I:C) adjuvants on responses to microneedle immunization coated with whole inactivated influenza vaccine.

Microneedles are the micrometer size devices used for the delivery of vaccines and biotherapeutics. In order to increase the vaccine efficacy and reduce the antigen dose, there is a significant need to find some adjuvants for the microneedle vaccination. In this study, zymosan, which is the cell wall preparation of Saccharomyces cerevisiae, or poly (I:C) was coated on a microneedle with inactivated influenza virus, and then immunized into BALB/c mouse to determine the immunogenicity, protection and synergetic effect between two adjuvants. As a result, the group administered with zymosan and vaccine antigen showed significantly stronger IgG response, HI titer and IgG subtypes without any adverse effects, compared to the group immunized with the vaccine antigen alone. Also, there were enhanced cellular immune responses in the group received adjuvant with vaccine antigen. In addition, they showed superior protection and lung viral reduction against lethal viral challenge. Taken together, this study confirms that zymosan can be used as an immunostimulant for microneedle vaccination.

Structures and recognition modes of toll-like receptors.

Toll-like receptors (TLRs) recognize common structural patterns in diverse microbial molecules and play central roles in the innate immune response. The structures of extracellular domains and their ligand complexes of several TLRs have been determined by X-ray crystallography. Here, we discuss recent advances on structures and activation mechanisms of TLRs. Despite the differences in interaction areas of ligand with TLRs, the extracellular domains of TLRs all adopt horseshoe-shaped structures and the overall M-shape of the TLR-ligand complexes is strikingly similar. The structural rearrangement information of TLRs sheds new light on their ligand-recognition and -activation mechanisms. Proteins 2016; 85:3-9. © 2016 Wiley Periodicals, Inc.

The use of Zymosan A and bacteria anchored to tumor cells for effective cancer immunotherapy: B16-F10 murine melanoma model.

The idea of using killed microorganisms or their parts for a stimulation of immunity in the cancer immunotherapy is very old, but the question of interactions and binding of these preparations to tumor cells has not been addressed so far. The attachment of Zymosan A and both Gram-positive and Gram-negative bacteria to tumor cells was tested in in vivo experiments. This binding was accomplished by charge interactions, anchoring based on hydrophobic chains and covalent bonds and proved to be crucial for a strong immunotherapeutic effect. The establishment of conditions for simultaneous stimulation of both Toll-like and phagocytic receptors led to very strong synergy. It resulted in tumor shrinkage and its temporary or permanent elimination. The role of neutrophils in cancer immunotherapy was demonstrated and the mechanism of their action (frustrated phagocytosis) was proposed. Finally, therapeutic approaches applicable for safe human cancer immunotherapy are discussed. Heat killed Mycobacterium tuberculosis covalently attached to tumor cells seems to be promising tool for this therapy.

Binding activities of non-ß-glucan glycoclusters to dectin-1 and exploration of their binding site.

Dectin-1, which specifically recognizes ß-(1,3)-glucans, plays an important role in innate immune responses. For the first time, in this study we found that a series of non-ß-glucan glycoclusters can bind to dectin-1 by means of surface plasmon resonance (SPR) assay. Hexavalent lactoside Ju-6 showed the strongest affinity property (KD=1.6 µM). Interestingly, a continuous binding-dissociation experiment on SPR showed that Ju-6 and Laminarin binding to dectin-1 are independent of each other. Moreover, RT-PCR assay showed that Ju-6 cannot up-regulate cytokine gene expression or inhibit the promoting effect caused by Zymosan (a long-chain ß-glucan). These results indicated that there might be a possible new carbohydrate binding site on dectin-1.

Effects of Toll-like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis.

In this study we compared the effects of the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS), flagellin, the synthetic bacterial triacylated lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), Polyinosinic:polycytidylic acid (Poly I:C), and macrophage-activating lipopeptide (MALP-2), which are TLR4, TLR5, TLR1/2, TLR3, and TLR2/6 agonists, respectively, on cell morphology and phagocytosis of zymosan particles, derived from Saccharomyces cerevisiae, and rich in fungal PAMPs including beta-glucan, mannose, and chitin. LPS, Pam3CSK4, and MALP-2 induced an activated macrophage phenotype and enhanced zymosan phagocytosis. In contrast, flagellin and Poly I:C, respectively, had little effect on cell morphology and phagocytosis. We examined the role of scavenger receptor A (SR-A) on zymosan phagocytosis. Cells cultured in medium alone expressed SR-A, and LPS induced further expression of the receptor. We also observed inhibitory effects of scavenger receptor antagonists fucoidan, dextran sulphate, and Polyinosinic (Poly I), respectively, on zymosan phagocytosis of cells in medium alone and those pre-treated with LPS. We conclude that exposure to specific TLR ligands impacts both cellular morphology and phagocytic capacity, and that scavenger receptors contribute to zymosan ingestion as well as LPS-induced augmentation of phagocytosis.

MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

Comparison of complement activation-related pseudoallergy in miniature and domestic pigs: foundation of a validatable immune toxicity model.

Complement activation-related pseudoallergy (CARPA) is an acute adverse immune reaction caused by many nanomedicines. There is a regulatory need for a sensitive and standardizable in vivo predictive assay. While domestic pigs are a sensitive animal model, miniature pigs are favored in toxicological studies yet their utility as a CARPA model has not yet been explored. Herein, we used liposomal doxorubicin and amphotericin B (Doxil/Caelyx and AmBisome), Cremophor EL and zymosan as CARPA triggers to induce reactions in miniature and domestic pigs, and compared the hemodynamic, hematological, biochemical, and skin alterations. The changes observed after administration of the test agents were very similar in both pig strains, suggesting that miniature pigs are a sensitive, reproducible, and, hence, validatable animal model for CARPA regulatory testing. FROM THE CLINICAL EDITOR: With the advances in nanomedicine research, many new agents are now tested for use in clinical setting. Nonetheless, complement activation-related pseudoallergy (CARPA) is a well known phenomenon which can be caused by nanoparticles. In this study, the authors looked at and compared the use of domestic pigs versus miniature pigs as experimental animals for toxicological studies. Their findings confirmed the possible use of miniature pigs for regulatory testing.

Glycyrrhetinic acid inhibits contact hypersensitivity induced by trichophytin via dectin-1.

Trichophyton infection is highly prevalent and tends to be recurrent. Therefore, it is important to develop new therapeutic agents. Previously, we established a mouse model of Trichophyton-induced contact hypersensitivity (CHS) and demonstrated that dectin-1 was involved in inflammation induced by trichophytin, the Trichophyton antigen. Here, we used that model to investigate glycyrrhetinic acid (GA) from plants of the genus Glycyrrhiza as a potential anti-inflammatory agent against superficial mycoses. GA suppressed swelling and the expression of inflammatory cytokines, including macrophage inflammatory protein (MIP)-2, interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon (IFN)-γ mRNA. Anti-MIP-2 antibody suppressed trichophytin-induced inflammation, and antidectin-1 antibody suppressed zymosan-induced MIP-2 production in keratinocyte cells. These results suggest that MIP-2 is produced by dectin-1 activation and is involved in inflammation associated with CHS to trichophytin. GA also suppressed zymosan-induced MIP-2 and interleukin (IL)-8, production in mouse and human macrophages and keratinocytes. Furthermore, GA suppressed the phosphorylation of spleen tyrosine kinase (Syk) and inhibitor of nuclear factor-kappa B (IκBα) and the degradation of IκBα in zymosan-simulated RAW264.7 cells. The results of this study suggest that GA suppresses inflammation induced by trichophytin, partly by the downregulation of Syk phosphorylation.

[Peripheral blood cells luminol-dependent chemiluminescence at the different stages of atopic dermatitis].

UNLABELLED: Aim of this work was to record the luminol-dependent spontaneous and induced chemiluminescence at the different stages of atopic dermatitis. METHODS: Peripheral blood cells were obtained from adult patient with atopic dermatitis followed by the registration of luminol-dependent chemiluminescence on luminograph. Opsonized zymosan as well as yeasts Candida tropicalis have been used to induce the chemiluminescence. RESULTS: Spontaneous and induced chemiluminescence were slightly elevated at the mild atopic dermatitis but were decreased at the severe stage of disease. Statistically significant difference has been found between group with mild and severe atopic dermatitis, Skin contamination by yeasts Candida tropicalis causes the increased level of blood cells chemiluminescence at the first week of atopic relapse when the disease was mild. Severe stage of atopic dermatitis was coupled with statistically significant inhibition of both, spontaneous and induced chemiluminescence. CONCLUSIONS: Luminol-dependent chemiluminescence of peripheral blood cells from adult atopic dermatitis patients may be stimulated at the mild stage and suppressed at severe stage of atopic dermatitis.

Identification and characterization of Tube in the Chinese mitten crab Eriocheir sinensis.

As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but has not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E. sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was 2247-bp with a 1539-bp open reading frame (ORF) encoding a 512-amino acid protein. The protein contained a 116-residue death domain (DD) at its N-terminus and a 272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4 proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in the ovary and testis, and moderately expressed in the thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2 (November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (ß-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.

Effect of gold nanoparticles on production of reactive oxygen species by human peripheral blood leukocytes stimulated with opsonized zymosan.

We studied the effect of gold nanoparticles on ROS production by leukocytes. ROS production was detected by luminol-dependent chemiluminescence (LDCL) of human peripheral blood leukocytes stimulated with opsonized zymosan. Nanoparticle size was 5, 10 and 30 nm. Simultaneous addition of nanoparticles and opsonized zymosan showed that 5-nm nanoparticles inhibited LDCL intensity in comparison with the control, when LDCL recording was conducted in the presence of opsonized zymosan. Increasing nanoparticle size from 5 up to 30 nm enhanced LDCL intensity. Preincubation of gold nanoparticles with autologous blood plasma increased LDCL intensity. In the control (without gold nanoparticles), blood plasma produced no activating effect on LDCL. We found that the effect of gold nanoparticles on leukocyte LDCL depended on nanoparticle size: 10- and 30-nm nanoparticles inhibited LDCL intensity in comparison with the control (incubation in the absence of nanoparticles) irrespective of the duration of incubation, while 5-nm gold nanoparticles had no effect on LDCL intensity. Incubation of gold nanoparticles with autologous plasma increased LDCL intensity if nanoparticle size was 30 and 10 nm.

Elucidation of novel structural scaffold in rohu TLR2 and its binding site analysis with peptidoglycan, lipoteichoic acid and zymosan ligands, and downstream MyD88 adaptor protein.

Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)-ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16-19, LRR12-14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)-protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, α B-helix, α C-helix, and CD loop in TLR2-TIR and BB loop, α B-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling.

Effect of conglutinin on phagocytic activity of bovine granulocytes.

In the present study we investigated the effect of bovine conglutinin on the phagocytic activity of leukocytes. We measured both the chemotactic activity of conglutinin and its effect on the internalization of zymosan particles and E. coli by granulocytes. We also assessed the binding of conglutinin to various microorganisms isolated from clinical cases in cattle. We showed that conglutinin binds strongly to the surface of yeast cells and to mannan-rich zymosan particles, while weak binding was observed in the case of the bacterial strains tested, including those whose O antigen is composed of mannan. Conglutinin (1-10 microg/ml) neither acts as a chemotactic factor for peripheral blood leukocytes nor affects ingestion of E. coli by granulocytes. However, as flow cytometry based assay showed, conglutinin (0.1-1 microg/ml) increased ingestion of zymosan expressed as mean fluorescence intensity (MFI) of positive cells.

Engagement of Siglec-7 receptor induces a pro-inflammatory response selectively in monocytes.

Sialic acid binding immunoglobulin-like lectin-7 (Siglec-7) is a trans-membrane receptor carrying immunoreceptor tyrosine based inhibitory motifs (ITIMs) and delivering inhibitory signals upon ligation with sialylated glycans. This inhibitory function can be also targeted by several pathogens that have evolved to express sialic acids on their surface to escape host immune responses. Here, we demonstrate that cross-linking of Siglec-7 by a specific monoclonal antibody (mAb) induces a remarkably high production of IL-6, IL-1α, CCL4/MIP-1ß, IL-8 and TNF-α. Among the three immune cell subsets known to constitutively express Siglec-7, the production of these pro-inflammatory cytokines and chemokines selectively occurs in monocytes and not in Natural Killer or T lymphocytes. This Siglec-7-mediated activating function is associated with the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. The present study also shows that sialic acid-free Zymosan yeast particles are able to bind Siglec-7 on monocytes and that this interaction mimics the ability of the anti Siglec-7 mAb to induce the production of pro-inflammatory mediators. Indeed, blocking or silencing Siglec-7 in primary monocytes greatly reduced the production of inflammatory cytokines and chemokines in response to Zymosan, thus confirming that Siglec-7 participates in generating a monocyte-mediated inflammatory outcome following pathogen recognition. The presence of an activating form of Siglec-7 in monocytes provides the host with a new and alternative mechanism to encounter pathogens not expressing sialylated glycans.

Studies on the characteristic and activity of low-molecular fragments from zymosan.

Zymosan was hydrolysed with HCl and fractionated by ultrafiltration and dialysis to obtain water-soluble fragments A, B and C. Physical and chemical analyses showed that these fractions are composed primarily of glucose and have molecular weights of 8 kDa, 5 kDa and 2 kDa, respectively. A glycosidic linkage analysis indicated that they are mainly composed of ß-1,3-glucans. Fragment A, which has the highest molecular weight, contains approximately 30% ß-1,6-linked glucans, but fragment C is almost entirely composed of linear ß-1,3-glucan chains. The anti-chronic atrophic gastritis activity experiments showed that fragment A has significant activity, the activity of zymosan is quite low and the activities of fragments B and C are in between those of fragment A and zymosan.

Production of functional soluble Dectin-1 glycoprotein using an IRES-linked destabilized-dihydrofolate reductase expression vector.

Dectin-1 (CLEC7A) is a C-type lectin receptor that binds to ß-glucans found in fungal cell walls to act as a major pattern recognition receptor (PRR). Since ß-glucans epitope is not present in human cells, we are of the opinion that Dectin-1 can have therapeutic functions against fungal infections. We thus set out to produce a soluble extracellular domain of murine Dectin-1 (called sDectin-1) in sufficient titers to facilitate such studies in mouse models. Since sDectin-1 has previously been shown to be glycosylated, we chose to produce this protein using Chinese Hamster Ovary (CHO) cells, a mammalian host cell line suitable for the high-titer production of recombinant glycoproteins. To ensure a high titer production of sDectin-1 and minimize the effects of gene fragmentation, we constructed a mammalian expression vector with a PEST-destabilized dhfr amplifiable marker downstream of an attenuated IRES element, which was in turn downstream of the sDectin-1 gene and a CMV IE promoter. Stably transfected and MTX-amplified cell pools were generated using this vector, and maximum sDectin-1 titers of 246 mg/l and 598 mg/l were obtained in shake flask batch culture and bioreactor fed-batch culture respectively. The purified recombinant sDectin-1 was shown to be glycosylated. Protein functionality was also demonstrated by its ability to bind to zymosan particles and to the cell wall of Saccharomyces cerevisiae. We describe for the first time the use of an attenuated IRES-linked PEST-destabilized dhfr amplifiable marker for the production of recombinant proteins with stably amplified cell pools. With our process, we reached the highest reported titer for producing recombinant proteins smaller than 50 kDa in cell pools. sDectin-1 protein produced is glycosylated and functional. This vector design can thus be used efficiently for the high-titer production of functional recombinant proteins.