Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

Fate of the capping agent of biologically produced gold nanoparticles and adsorption of enzymes onto their surface.

Enzymotherapy based on DNase I or RNase A has often been suggested as an optional strategy for cancer treatment. The efficacy of such procedures is limited e.g. by a short half-time of the enzymes or a low rate of their internalization. The use of nanoparticles, such as gold nanoparticles (AuNPs), helps to overcome these limits. Specifically, biologically produced AuNPs represent an interesting variant here due to naturally occurring capping agents (CA) on their surface. The composition of the CA depends on the producing microorganism. CAs are responsible for the stabilization of the nanoparticles, and promote the direct linking of targeting and therapeutic molecules. This study provided proof of enzyme adsorption onto gold nanoparticles and digestion efficacy of AuNPs-adsorbed enzymes. We employed Fusarium oxysporum extract to produce AuNPs. These nanoparticles were round or polygonal with a size of about 5 nm, negative surface charge of about - 33 mV, and maximum absorption peak at 530 nm. After the adsorption of DNAse I, RNase A, or Proteinase K onto the AuNPs surface, the nanoparticles exhibited shifts in surface charge (values between - 22 and - 13 mV) and maximum absorption peak (values between 513 and 534 nm). The ability of AuNP-enzyme complexes to digest different targets was compared to enzymes alone. We found a remarkable degradation of ssDNA, and dsDNA by AuNP-DNAse I, and a modest degradation of ssRNA by AuNP-RNase A. The presence of particular enzymes on the AuNP surface was proved by liquid chromatography-mass spectrometry (LC-MS). Using SDS-PAGE electrophoresis, we detected a remarkable digestion of collagen type I and fibrinogen by AuNP-proteinase K complexes. We concluded that the biologically produced AuNPs directly bound DNase I, RNase A, and proteinase K while preserving their ability to digest specific targets. Therefore, according to our results, AuNPs can be used as effective enzyme carriers and the AuNP-enzyme conjugates can be effective tools for enzymotherapy.

Investigation of Protein Corona Formed around Biologically Produced Gold Nanoparticles.

Although there are several research articles on the detection and characterization of protein corona on the surface of various nanoparticles, there are no detailed studies on the formation, detection, and characterization of protein corona on the surface of biologically produced gold nanoparticles (AuNPs). AuNPs were prepared from Fusarium oxysporum at two different temperatures and characterized by spectrophotometry, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDS). The zeta potential of AuNPs was determined using a Zetasizer. AuNPs were incubated with 3 different concentrations of mouse plasma, and the hard protein corona was detected first by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then by electrospray liquid chromatography-mass spectrometry (LC-MS). The profiles were compared to AuNPs alone that served as control. The results showed that round and oval AuNPs with sizes below 50 nm were produced at both temperatures. The AuNPs were stable after the formation of the protein corona and had sizes larger than 86 nm, and their zeta potential remained negative. We found that capping agents in the control samples contained small peptides/amino acids but almost no protein(s). After hard protein corona formation, we identified plasma proteins present on the surface of AuNPs. The identified plasma proteins may contribute to the AuNPs being shielded from phagocytizing immune cells, which makes the AuNPs a promising candidate for in vivo drug delivery. The protein corona on the surface of biologically produced AuNPs differed depending on the capping agents of the individual AuNP samples and the plasma concentration.

Coated nanodiamonds interact with tubulin beta-III negative cells of adult brain tissue.

Fluorescent nanodiamonds (NDs) coated with therapeutics and cell-targeting structures serve as effective tools for drug delivery. However, NDs circulating in blood can eventually interact with the blood-brain barrier, resulting in undesired pathology. Here, we aimed to detect interaction between NDs and adult brain tissue. First, we cultured neuronal tissue with ND ex vivo and studied cell prosperity, regeneration, cytokine secretion, and nanodiamond uptake. Then, we applied NDs systemically into C57BL/6 animals and assessed accumulation of nanodiamonds in brain tissue and cytokine response. We found that only non-neuronal cells internalized coated nanodiamonds and responded by excretion of interleukin-6 and interferon-γ. Cells of neuronal origin expressing tubulin beta-III did not internalize any NDs. Once we applied coated NDs intravenously, we found no presence of NDs in the adult cortex but observed transient release of interleukin-1α. We conclude that specialized adult neuronal cells do not internalize plain or coated NDs. However, coated nanodiamonds interact with non-neuronal cells present within the cortex tissue. Moreover, the coated NDs do not cross the blood-brain barrier but they interact with adjacent barrier cells and trigger a temporary cytokine response. This study represents the first report concerning interaction of NDs with adult brain tissue.

Novel versatile 3D bio-scaffold made of natural biocompatible hagfish exudate for tissue growth and organoid modeling.

Hagfish exudate is a natural biological macromolecule made of keratin intermediate filament protein skeins and mucin vesicles. Here, we successfully examined this remarkable biomaterial as a substrate for three-dimensional (3D) cell culturing purposes. After the sterilization with chloroform vapor, Dulbecco's modified eagle medium was mixed with the exudate to rupture the vesicles and skeins; a highly soft, adherent, fibrous and biocompatible hydrogel was formed. A variety of cells, including Hela-FUCCI, NMuMG-FUCCI, 10T1/2 and C2C12, was cultured on the hagfish exudate. A remarkable 3D growth by ~2.5 folds after day 3, ~5 folds after day 5, ~10 folds after day 7 and ~15 folds after day 14 were seen compared to day one of culturing in the hagfish exudate scaffold. In addition, the phase contrast, fluorescent and confocal microscopy observations confirmed the organoid shape formation within the three-week culture. The viability of cells was almost 100% indicating the great in vitro and in vivo potential of this exceptional biomaterial with no cytotoxic effect.

Why nanodiamond carriers manage to overcome drug resistance in cancer.

Nanodiamonds represent an attractive potential carrier for anticancer drugs. The main advantages of nanodiamond particles with respect to medical applications are their high compatibility with non-cancerous cells, feasible surface decoration with therapeutic and cancer-cell targeting molecules, and their relatively low manufacturing cost. Additionally, nanodiamond carriers significantly increase treatment efficacy of the loaded drug, so anticancer drugs execute more effectively at a lower dose. Subsequently, lower drug dose results in less extensive side effects. The carriers decorated with a targeting molecule accumulate primarily in the tumor tissue, and those nanodiamond particles impair efflux of the drug from cancer cells. Therapeutic approaches considering nanodiamond carriers were already tested in vitro, as well as in vivo. Now, researchers focus particularly on the possible side effects of nanodiamond carriers applied systemically in vivo. The behavior of nanodiamond carriers depends heavily on their surface coatings, so each therapeutic complex must be evaluated separately. Generally, it seems that site-specific application of nanodiamond carriers is a rather safe therapeutic approach, but intravenous application needs further study. The benefits of nanodiamond carriers are remarkable and represent a potent approach to overcome the drug resistance of many cancers.

Knockdown of microRNA-135b in Mammary Carcinoma by Targeted Nanodiamonds: Potentials and Pitfalls of In Vivo Applications.

Nanodiamonds (ND) serve as RNA carriers with potential for in vivo application. ND coatings and their administration strategy significantly change their fate, toxicity, and effectivity within a multicellular system. Our goal was to develop multiple ND coating for effective RNA delivery in vivo. Our final complex (NDA135b) consisted of ND, polymer, antisense RNA, and transferrin. We aimed (i) to assess if a tumor-specific coating promotes NDA135b tumor accumulation and effective inhibition of oncogenic microRNA-135b and (ii) to outline off-targets and immune cell interactions. First, we tested NDA135b toxicity and effectivity in tumorospheres co-cultured with immune cells ex vivo. We found NDA135b to target tumor cells, but it binds also to granulocytes. Then, we followed with NDA135b intravenous and intratumoral applications in tumor-bearing animals in vivo. Application of NDA135b in vivo led to the effective knockdown of microRNA-135b in tumor tissue regardless administration. Only intravenous application resulted in NDA135b circulation in peripheral blood and urine and the decreased granularity of splenocytes. Our data show that localized intratumoral application of NDA135b represents a suitable and safe approach for in vivo application of nanodiamond-based constructs. Systemic intravenous application led to an interaction of NDA135b with bio-interface, and needs further examination regarding its safety.

Fluorescent Nanodiamonds are Efficient, Easy-to-Use Cyto-Compatible Vehicles for Monitored Delivery of Non-Coding Regulatory RNAs.

MicroRNAs are short molecules of RNA regulating most cellular processes via the mechanism of RNA interference. Their dysregulation leads to a disease burden, making them important therapeutic targets. For the successful development of a therapeutic device, the uptake of a functionalized carrier by live cells and the sufficient release of effector therapeutic molecules are limiting factors. Here for the first time, the inhibition of oncogenic microRNA-21 in CT-26 colon cancer cells is achieved, using an advanced nanosystem consisting of fluorescent nanodiamond and antisense RNA. Stable nanocomplexes efficiently deliver antisense RNA into cell cytoplasm, encouraging further study of microRNA-21 function in target cells. Engaging the fluorescent nanoparticle enables monitoring of transfection and release of the antisense RNA load into cell cytoplasm. Importantly, the internalized antisense RNA effectively destroys target microRNA-21 in CT-26 cancer cells. The absence of oncogenic microRNA-21 liberates tumor suppressor genes Pdcd4 and Timp3 from silencing, and results in a decrease of cell invasion and migration, and in the induction of apoptotic cell death. This study uses a nanodiamond-based imaging and delivery system, and shows that the multidimensional performance of the presented device makes nanodiamond-based complexes promising therapeutic devices.

Interaction of colon cancer cells with glycoconjugates triggers complex changes in gene expression, glucose transporters and cell invasion.

Glycan metabolism balance is critical for cell prosperity, and macromolecule glycosylation is essential for cell communication, signaling and survival. Thus, glycotherapy may be a potential cancer treatment. The aim of the present study was to determine whether combined synthetic glycoconjugates (GCs) induce changes in gene expression that alter the survival of colon cancer cells. The current study evaluated the effect of the GCs N­acetyl­D­glucosamine modified polyamidoamine dendrimer and calix[4]arene scaffold on cancer cell proliferation, apoptosis, invasion and sensitivity to immune cell­mediated killing. Using reverse transcription­quantitative polymerase chain reaction, the expression of genes involved in the aforementioned processes was measured. It was determined that GCs reduce the expression of the glucosaminyltransferases Mgat3 and Mgat5 responsible for surface glycosylation and employed components of the Wnt signaling pathway Wnt2B and Wnt9B. In addition, the calix[4]arene­based GC reduced cell colony formation; this was accompanied by the downregulation of the metalloproteinase Mmp3. By contrast, the dendrimer­based GC affected the expression of the glucose transporter components Sglt1 and Egfr1. Therefore, to the best of our knowledge, the present study is the first to reveal that N­acetyl­D­glucosamine­dendrimer/calix[4]arene GCs alter mRNA expression in a comprehensive way, resulting in the reduced malignant phenotype of the colon cancer cell line HT­29.

Prognostic Significance of TMPRSS2-ERG Fusion Gene in Prostate Cancer.

BACKGROUND/AIM: Current research of prostate cancer (PCa) offers a promising way of identifying patients with adverse prognosis who do benefit from radical treatment that can affect quality of life as resections are associated with numerous side-effects. The aim of our study was to evaluate the relationship of TMPRSS2-ERG fusion gene status, tumor tissue prostate-specific antigen (PSA), prostate cancer antigen 3 (PCA3), miR-23b, miR-26a and miR-221 expression levels in combination with preoperative serum PSA level to the risk of PCa recurrence after radical prostatectomy. PATIENTS AND METHODS: The study group consisted of 108 patients who underwent radical prostatectomy. PSA was measured in peripheral blood collected preoperativelly. The expression of TMPRSS2-ERG transcript and the expression of miR-23b, miR-26a and miR-221 in formalin-fixed, paraffin-embedded (FFPE) tumor tissues was analyzed by reverse transcription (RT) real-time polymerase chain reaction (PCR). RESULTS: Significantly shorter time to recurrence was observed in patients with high expression of TMPRSS2-ERG (p=0.0020). High levels of preoperative PSA (>10.0 ng/ml) proved to be marker of shorter time to recurrence (p=0.0153). The most promising marker of the risk of recurrence after radical prostatectomy was a combination of high level of preoperative serum PSA and high expression of TMPRSS2-ERG fusion transcript in tumor tissue (p=0.0001). CONCLUSION: A combination of high preoperative serum PSA and high expression of TMPRSS2-ERG could be promising in distinguishing those tumors that are aggressive and life-threatening.

Re-evaluation of the involvement of NK cells and C-type lectin-like NK receptors in modulation of immune responses by multivalent GlcNAc-terminated oligosaccharides.

Recognition of glycosylation patterns is one of the basic features of innate immunity. Ability of C-type lectin-like receptors such as NKR-P1 to bind saccharide moieties has become recently a controversial issue. In the present study, binding assay with soluble fluorescently labeled recombinant rat NKR-P1A and mouse NKR-P1C proteins revealed apparently no affinity to the various neoglycoproteins. Lack of functional linkage between NKR-P1 and previously described saccharide binder was supported by the fact, that synthetic N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P) did not change gene expression of NKR-P1 isoforms in C57BL/6 and BALB/c mice divergent in the NK gene complex (both in vitro and in vivo). Surprisingly, N-acetyl-D-glucosamine-coated tetrabranched polyamido-amine dendrimer specifically binds to NKT cells and macrophages but not to NK cells (consistently with changes in cytokine patterns). Despite the fact that GN8P has been tested as an immunomodulator in anti-cancer treatment animal models for many years, surprisingly no changes in cytokine profiles in serum relevant to anti-cancer responses using B16F10 and CT26 harboring mouse strains C57BL/6 and BALB/c are observed. Our results indicate possible indirect involvement of NK cells in GN8P mediated immune responses.

N-acetyl-D-glucosamine-coated polyamidoamine dendrimer promotes tumor-specific B cell responses via natural killer cell activation.

N-acetyl-D-glucosamine-coated polyamidoamine dendrimer (GN8P), exerting high binding affinity to rodent recombinant NKR-P1A and NKR-P1C activating proteins, was shown previously to delay the development of rat colorectal carcinoma as well as mouse B16F10 melanoma, and to potentiate antigen-specific antibody formation in healthy C57BL/6 mice via NK cell stimulation. In this study, we investigated whether GN8P also modulates tumor-specific B cell responses. Serum anti-B16F10 melanoma IgG levels, IgG2a mRNA expression, antibody dependent cell-mediated cytotoxicity (ADCC), and counts of plasma as well as antigen presenting B cells were evaluated in tumor-bearing C57BL/6 mice treated with GN8P and in respective controls. To reveal the mechanism of GN8P effects, the synthesis of interferon-gamma (IFN-γ) and interleukin-4 (IL-4), cytokines involved in regulation of immunoglobulin class switch, was determined. The GN8P treatment significantly elevated IgG, and particularly IgG2a, response against B16F10 melanoma, which led to augmented ADCC reaction. The significant increase in production of IFN-γ, which is known to support IgG2a secretion, was observed solely in NK1.1 expressing cell populations, predominantly in NK cells. Moreover, GN8P raised the number of plasma cells, and promoted antigen presenting capacity of I-A/I-E-positive B lymphocytes by up-regulation of their CD80 and CD86 co-stimulatory molecule expression. These results indicate that GN8P-induced enhancement of tumor-specific antibody formation is triggered by NK cell activation, and contributes to complexity of anticancer immune response involving lectin-saccharide interaction.

Glycosylation regulates NK cell-mediated effector function through PI3K pathway.

Aberrant glycosylation, which impairs recognition capability of NK cells or modifies recognition pattern of target cells, is associated with cancer. Synthetic glycoconjugates (GCs), which modulate cell glycosylation, increase the sensitivity of tumor cells to therapy or boost anti-cancer immune response. In the current study, we employed N-acetyl-D-glucosamine-calix[4]arene (GN4C) as a modulator of cell glycosylation of NK cells represented by the NK-92 cell line and fresh human NK cells. For the first time, we have demonstrated that calix[4]arene-based GC down-regulated the expression of glycosyltransferases MGAT3 and MGAT5 in NK-92 and fresh NK cells. GN4C increased the susceptibility of tumor cells to cytotoxicity by purified fresh NK cells or NK-92 cells. This functional activation of NK cells and the NK-92 cell line correlated with an increased expression of NKG2D mRNA. In the NK-92 cell line, GN4C induced the synthesis of IL-2, IFN-gamma and tumor necrosis factor-alpha as well. Cellular signaling triggered by GN4C engaged PI3-kinase/ERK but not phospholipase C-gamma/JNK pathways. Simultaneously, in transformed NK-92 cells, GN4C reduced the rate of proliferation and down-regulated the c-MYC, EGF-receptor 1 and REL-A molecules. In conclusion, the modulation of glycosyltransferases MGAT3 and MGAT5 by synthetic GN4C correlated with the improvement of NK cell effector functions and the augmentation of tumor cells sensitivity to NK cell-mediated cytotoxicity.

Colorectal carcinoma: Importance of colonic environment for anti-cancer response and systemic immunity.

The intestinal environment is considered to play an important role both in colorectal tumor development and in the evolution and modulation of mucosal immunity. Studies in animals reared in germ-free (GF, without any intestinal microflora) versus conventional (CV, with regular microflora in bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and anti-cancer immune responses in situ. The lower incidence of colon cancers and better immunological parameters in GF animals versus CV ones after chemically-induced carcinogenesis raises questions about specific characteristics of the immunological networks in each respective condition. Different levels of tolerance/regulatory mechanisms in the GF versus CV animals may influence the development of immune responses not only at the level of mucosal, but also at the systemic, immunity. We hypothesize that GF animals can better recognize and respond to evolving neoplasias in the bowel as a consequence of their less-tolerogenic immunity (i.e., due to their more limited exposure to antigens to become tolerated against at the intestinal level). In this paper, we review the role of bacteria in modulating gut environment and mucosal immunity, their importance in cancer development, and aspects of immune regulation (both at local and systemic level) that can be modified by bacterial microflora. Lastly, the use of GF animals in comparison with conventionally-raised animals is proposed as a suitable and potent model for understanding the inflammatory network and its effect on cancer immunity especially during colorectal cancer development.

Acquired genetic and functional alterations associated with transforming growth factor beta type I resistance in Hep3B human hepatocellular carcinoma cell line.

During the neoplastic process tumour cells frequently acquire resistance to the antiproliferative signals of transforming growth factor-beta (TGF-beta). Here we examined a human hepatocellular carcinoma cell line (Hep3B-TS) sensitive to TGF-beta signalling, and a derivative line (Hep3B-TR) rendered resistant to TGF-beta by stepwise exposure to TGF-beta(1). Comprehensive molecular cytogenetic analysis revealed that the acquisition of TGF-beta-resistance by Hep3B-TR cells was due to loss of TGF-beta receptor 2 (TGFbetaRII) gene. As demonstrated by spectral karyotyping and array-based comparative genomic hybridization, and in difference to Hep3B-TS cells, which have three rearranged and two normal copies of chromosome 3 that harbour the TGFbetaRII gene, Hep3B-TR cells have four rearranged and one apparently normal chromosome 3, which nonetheless underwent a critical microdeletion at the site of TGFbetaRII gene. Gene expression analysis using an oligonucleotide microarray of 21,397 genes showed that Hep3B-TR differentially expressed 307 genes, out of which 197 and 110 were up- and down-regulated, respectively, compared to Hep3B-TS. Six of differentially expressed genes were identified as downstream targets of the tumour necrosis factor (TNF) gene, suggesting that loss of TGFbetaRII triggered activation of the TNF pathway known to be regulated by TGF-beta(1) network. On the functional level, the TGF-beta-resistant Hep3B-TR cells displayed significantly enhanced capacity for anchorage independent growth and cell migration in vitro, and also increased tumorigenicity in vivo and in vitro and in vivo tumorigenicity compared with parental sensitive cells.

Recurrent and nonrandom DNA copy number and chromosome alterations in Myc transgenic mouse model for hepatocellular carcinogenesis: implications for human disease.

Mouse models for hepatocellular carcinoma (HCC) provide an experimental ground for dissecting the genetic and biological complexities of human liver cancer and contribute to our ability to gain insights into the relevance of candidate cancer genes. We examined, using spectral karyotyping (SKY) and array-based CGH (aCGH), seven cell lines derived from HCC spontaneously developed in transgenic Myc mice (Myc), and four cell lines established from tumors induced in nude mice by inoculation with the original Myc cells (nuMyc). All the cell lines exhibited gain of material from chromosomes 5, 6, 8, 10, 11, 15, and 19 and DNA copy-number loss from chromosomes 2, 4, 7, 9, 12, 14, and X. In addition, several recurrent chromosome reorganizations were found, including del(3), t(3;8), del(4), t(4;11), t(6;5), del(7), del(8), del(9), t(10;14), del(11), and del(16). Chromosome breakpoints underlying rearrangements clustered in the regions previously identified as important for the early stages of Myc-induced hepatocarcinogenesis. The results strongly suggest the importance of recurrent breakage and loss of chromosomes 4, 9, and 14 and gain of chromosomes 15 and 19 in mouse liver neoplasia. Genomic changes observed in Myc HCC cell lines are also recurrent in HCC developed in other transgenic mouse models, in mouse spontaneous HCC and derivative cell lines, and in preneoplastic liver lesions induced with chemical carcinogens. Overall, the present results document selective, nonrandom genomic changes involving chromosomal regions homologous to those implicated in human HCC.

N-Acetyl-D-glucosamine-coated polyamidoamine dendrimer modulates antibody formation via natural killer cell activation.

N-Acetyl-D-glucosamine-coated polyamidoamine dendrimer (GlcNAc8) was shown previously to exhibit binding affinity to the rat recombinant NKR-P1 molecule (known in mice also as NK1.1) and to induce NK cell-mediated cytotoxicity. In this study, we investigated whether GlcNAc8 modulates antibody formation as activated NK cells were reported to participate in its regulation. C57BL/6 mice treated with GlcNAc8 and intact controls were immunized either with sheep red blood cells (SRBCs), 2,4-dinitrophenylated-lipopolysaccharide (DNP-LPS) or keyhole limpet hemocyanin (KLH) for evaluation of splenic antibody forming cell counts and serum immunoglobulin (Ig) levels. In vitro Ig formation was determined using supernatants of spleen mononuclear cells (SMCs) and CD49b or NK1.1-depleted SMC subpopulations. Serum antigen-specific IgG2a levels were also measured in DBA/2 and BALB/c mice (NK1.1-negative mouse strains on the basis of flow cytometric analysis) which possess different Nkr-p1c gene form than C57BL/6 ones. A significant increase in anti-SRBC IgG forming cells, serum levels of anti-KLH as well as anti-DNP IgG and IgG2a was observed after GlcNAc8 administration in C57BL/6 mice. IgM levels in supernatants of SMCs stimulated in vitro simultaneously with DNP-LPS and GlcNAc8 were significantly mounted compared with supernatants of SMCs primed with the antigen alone, but this enhancement was blocked after depletion of CD49b-positive or NK1.1-positive cells. In DBA/2 and BALB/c mice, GlcNAc8 influenced neither serum levels of anti-KLH nor anti-DNP IgG2a. These results indicate that GlcNAc8-induced upregulation of antibody formation is triggered by NK cell stimulation and depends on expressed NKR-P1 isoforms, particularly NKR-P1C.