Nanobodies in cytokine­mediated immunotherapy and immunoimaging (Review).

Cytokines are the main regulators of innate and adaptive immunity, mediating communications between the cells of the immune system and regulating biological functions, including cell motility, differentiation, growth and apoptosis. Cytokines and cytokine receptors have been used in the treatment of tumors and autoimmune diseases, and to intervene in cytokine storms. Indeed, the use of monoclonal antibodies to block cytokine­receptor interactions, as well as antibody­cytokine fusion proteins has exhibited immense potential for the treatment of tumors and autoimmune diseases. Compared with these traditional types of antibodies, nanobodies not only maintain a high affinity and specificity, but also have the advantages of high thermal stability, a high capacity for chemical manipulation, low immunogenicity, good tissue permeability, rapid clearance and economic production. Thus, nanobodies have extensive potential for use in the diagnosis and treatment of cytokine­related diseases. The present review summarizes the application of nanobodies in cytokine­mediated immunotherapy and immunoimaging.

Dendritic cells modified by tumor associated antigen SMP30 have enhanced antitumor effect against mouse hepatocarcinoma cells in vitro and in vivo.

OBJECTIVES: Tumor immunotherapy based on dendritic cells (DC) is one of the most promising approaches to treat cancers. This therapy uses an immunogenic tumor antigen to present it to T cells. Senescence marker protein 30 (SMP30) is identified as a tumor associated antigen (TAA) with high immunogenicity and specificity for hepatocellular carcinoma (HCC). DCs are the most potent antigen presenting cells, and can be transduced with tumor antigens to enhance antitumor immune response. The purpose of this study was to investigate the antitumor effect of DCs transduced with a recombinant lentiviral vector (LV-SMP30) expressing SMP30. METHODS: A recombinant lentiviral vector (LV-SMP30) expressing SMP30 was constructed and transduced into DCs. The expression of SMP30 was detected by western blot. Mouse bone marrow-derived DCs were divided into four groups: LV-SMP30 group (transduced with LV-SMP30), Protein group (co-cultured with SMP30 protein), LV group (transduced with the empty vector) and Untreated group (the normal DCs). The effect of LV-SMP30 on DCs was detected through surface markers (CD123, CD11c, CD80 and CD86) and cytokine production. The activation and proliferation of CD3+CD8+ T cells were detected by CCK-8 kit. Flow cytometry was used to detect CD3+CD8+ T cell-mediated cytotoxicity. After construction of a mouse subcutaneous xenograft model, the volume and growth of tumors in different groups were observed. The changes in serum immune indexes in the treated groups were compared with those in the control group. RESULTS: The LV-SMP30 recombinant was constructed and transduced into DCs successfully, and LV-SMP30-transduced DCs stably expressed SMP30. The percentages of expression in the LV-SMP30 and Protein groups were significantly higher than those in the LV or Untreated groups (P<0.05). Meanwhile, after the DCs were cultured for 72 hours, the levels of IL-2, IL-6, IL-12, and IFN-γ were significantly higher in the LV-SMP30 and Protein groups than in the LV group or Untreated group (P<0.05). After the DCs were continuously cultured for one week, however, the cytokine levels in the LV-SMP30 group were significantly higher than those in the Protein group (P<0.05). In addition, CD3+CD8+ T cell proliferation and activation levels were substantially higher in the LV-SMP30 and Protein groups than in the LV or Untreated groups (P<0.05). Furthermore, as the ratio of effectors/target cells increasing in the LV-SMP30 group, CD3+CD8+ T cell-mediated cytotoxicity in H22 cells became higher (0:1, 10:1; 20:1; 40:1, respectively). In comparison to the control group, the cytotoxicity of the LV-SMP30 group was considerably increased at the ratios of 10:1, 20:1 and 40:1 (P<0.05). However, in the case of Hep1-6 cells, there was no significant difference in CD3+CD8+ T cell-mediated cytotoxicity among the groups. In addition, when compared with other groups, the mice in the LV-SMP30 group showed the most volume reduction, the slowest tumor growth, and the highest level of IL-2 and IFN-γ (P<0.05). CONCLUSION: DCs transduced with LV-SMP30 can dramatically enhance specific CD3+CD8+ T cell immune responses against mouse hepatocarcinoma cells in vitro and in vivo. These findings lend significant support to the development of the DC-based SMP30 antigen vaccine for hepatocarcinoma immunotherapy.

GP96 and SMP30 Protein Priming of Dendritic Cell Vaccination Induces a More Potent CTL Response against Hepatoma.

Heat-shock protein (HSP) GP96 is a well-known adjuvant in immunotherapy. It belongs to the HSP90 family. Our previous study demonstrated that DC pulsed with recombinant senescence marker protein 30 (SMP30) could induce cytotoxic T lymphocytes (CTLs) against liver cancer cells in vitro. In this study, SMP30 and GP96 were subcloned into lentiviruses and transfected into DCs from healthy donors. We included six groups: the GP96-SMP30 group, GP96 group, SMP30 group, DC group, empty vector control group, and hepatoma extracted protein group. We used ELISA to detect cytokines and flow cytometry to assess CD80 and CD86 on DCs and the effect of CTLs. Our vector design was considered successful and further studied. In the SMP30 group, DC expresses more CCR7 and CD86 than the control group; in the SMP30+GP96 group, DC express more CCR7, CD86, and CD80 than the control group. Transfected DCs secreted more TNF-α and interferon-ß and induced more CTLs than control DCs. SMP30 + GP96 effectively stimulated the proliferation of T cells compared with control treatment (P < 0.01). We detected the cytokines TNF-α, TNF-ß, IL-12, and IFN (α, ß, and γ) via ELISA (Figure 5) and verified the killing effect via FCM. Four E : T ratios (0 : 1, 10 : 1, 20 : 1, and 40 : 1) were tested. The higher the ratio was, the better the effects were. We successfully constructed a liver cancer model and tested the CTL effect in each group. The GP96 + SMP30 group showed a better effect than the other groups. GP96 and SMP30 can stimulate DCs together and produce more potent antitumor effects. Our research may provide a new efficient way to improve the therapeutic effect of DC vaccines in liver cancer.

Study on the Applicability of Reservoir Fractal Characterization in Middle-High Rank Coals with NMR: Implications for Pore-Fracture Structure Evolution within the Coalification Process.

In order to evaluate the applicability of the pore-fracture structure fractal characterizations in coal reservoirs and confirm the internal relationships between the porosity, permeability, coal metamorphic grade, and pore-fracture structure, the pore-fracture features of 21 middle-high rank coal samples from Anhe, Jiaozuo, and Huaibei coalfields in northern China were investigated using a low-field nuclear magnetic resonance (NMR). All the coal samples are characterized by low moisture content (M ad), low and medium ash yield (A ad), and high vitrinite (V) in coal maceral. The adsorption space fractal dimension (D A) is positively correlated with the Langmuir volume (V L) under the three-peak transverse relaxation time (T 2) spectrum. The fractal dimension of all effective T 2 points under saturated water (D NMR) is positively correlated with V L and the adsorption pore volume, but negatively correlated with the volume ratio of seepage pores and fractures. The free flow space fractal dimension (D M) is negatively correlated with the porosity of full saturated water (ΦF) and the porosity of movable water (ΦM). There is a negative correlation between ΦF and the seepage space fractal dimension (D S) in the coal samples with one-peak and two-peak T2 spectra, but a positive correlation can be found with the three-peak T2 spectrum. Therefore, it is necessary to consider the types of T2 spectral peak as a prerequisite to analyze the correlations between pore-fracture parameters and NMR fractal dimensions. With the increase of coal rank, the adsorption pore content, ΦF, and bulk volume immovable (BVI) fraction first increase and then decrease, whereas the seepage pore content, fracture development, bulk volume movable (BVM) fraction, and BVM/BVI first decrease and then increase. The inflection points of these changes correspond to the maximum vitrinite reflectance (R o,max) at 2.6-2.8%, which would be attributed to the third coalification jump. Generally, D A is the fractal dimension representing the coal pore surface, and D S and D M are closely related to the pore structure. Furthermore, D NMR not only represents the roughness of the pore surface but also the complexity of the pore structure.

Upregulating KTN1 promotes Hepatocellular Carcinoma progression.

Background: Hepatocellular carcinoma (HCC) presents a common malignant tumor worldwide. Although kinectin 1 (KTN1) is the most frequently identified antigen in HCC tissues, the detailed roles of KTN1 in HCC remain unknown. This study seeks to clarify the expression status and clinical value of KTN1 in HCC and to explore the complicated biological functions of KTN1 and its underlying mechanisms. Methods: In-house reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of KTN1 in HCC tissues. External gene microarrays and RNA-sequencing datasets were downloaded to confirm the expression patterns of KTN1. The prognostic ability of KTN1 in HCC was assessed by a Kaplan-Meier curve and a hazard ratio forest plot. The CRISPR/Cas9 gene-editing system was used to knock out KTN1 in Huh7 cells, which was verified by PCR-Sanger sequencing and western blotting. Assays of cell migration, invasion, viability, cell cycle, and apoptosis were conducted to explore the biological functions. RNA sequencing was performed to quantitatively analyze the functional deregulation in KTN1-knockout cells compared to Huh7-wild-type cells. Upregulated genes that co-expressed with KTN1 were identified from HCC tissues and were functionally annotated. Results: KTN1 expression was increased in HCC tissues (standardized mean difference [SMD] = 0.20 [0.04, 0.37]). High KTN1 expression was significantly correlated with poorer prognosis of HCC patients, and KTN1 may be an independent risk factor for HCC (pooled HRs = 1.31 [1.05, 1.64]). After KTN1-knockout, the viability, migration, and invasion ability of HCC cells were inhibited. The proportion of HCC cells in the G0-G1 phases increased after KTN1 knockout, which also elevated the apoptosis rates in HCC cells. Several cascades, including innate immune response, chemical carcinogenesis, and positive regulation of transcription by RNA polymerase II, were dramatically changed after KTN1 knockout. KTN1 primarily participated in the cell cycle, DNA replication, and microRNAs in cancer pathways in HCC tissues. Conclusion: Upregulation of KTN1 served as a promising prognosticator in HCC patients. KTN1 promotes the occurrence and deterioration of HCC by mediating cell survival, migration, invasion, cell cycle activation, and apoptotic inhibition. KTN1 may be a therapeutic target in HCC patients.

Impact of blood-brain barrier disruption on newly diagnosed neuromyelitis optica spectrum disorder symptoms and prognosis.

BACKGROUND: Blood-brain barrier (BBB) disruption and ensuing immune activation are central to the pathogenesis of central nervous system (CNS) inflammatory diseases. However, the influence of BBB permeability on the clinical signs and prognosis of newly diagnosed neuromyelitis optica spectrum disorder (NMOSD) has not been examined. We investigate the relationships between BBB permeability as showed by the albumin quotient (qalb) and clinical features of NMOSD. METHODS: Demographic and clinical data of 46 patients, including peripheral blood (PB) measures (serum albumin concentration and total leukocyte, neutrophil, total lymphocyte, CD4+ T cell, and CD8+ T cell counts, complement C3 and C4 concentrations, AQP4-IgG titer),autoimmune antibody titers (ANA/SSA/SSB/Ro-52), and cerebrospinal fluid (CSF) parameters (total leukocyte count, total protein and albumin concentrations, AQP4-IgG titer), were compared between qalb(BBB permeability) increased and normal groups. Complete measures were not obtained from 9 patients, but all other measures were included in the analysis. RESULTS: According to the calculated qalb, 15 patients with albumin quotient (qalb) > (4 + age/15) × 10-3 were assigned to the qalb increased (high BBB permeability) group (33%) and the remainder to the qalb normal group. Compared to the qalb normal group, the qalb increased group exhibited significantly lower serum albumin (P=0.001) and CD4+ T cell count (P=0.044), CD8+ T cell count (P=0.014), and total T lymphocyte count (P=0.016). The qalb increased group proved higher CSF albumin, total protein, leukocyte count, and IgG titer (all P=0.000). Optic neuritis and optic nerve abnormalities on magnetic resonance images were also more frequent in the qalb increased group (P=0.037 and 0.038, respectively). Patients in the qalb increased group showed significantly poorer treatment response as indicated by the lower post-treatment change in Expanded Disability Status Scale (EDSS) score compared to the qalb normal group. CONCLUSIONS: BBB permeability is strongly associated with the clinical features and treatment response of newly diagnosed NMOSD. The qalb is a potentially valuable indicator of disease severity and an index to guide personalized treatment.

Functional studies of Drosophila zinc transporters reveal the mechanism for zinc excretion in Malpighian tubules.

BACKGROUND: Zinc is an essential metal involved in many physiological processes. Previous work has identified a set of zinc transporters involved in Drosophila dietary zinc absorption. However, zinc excretion and reabsorption, the other two important processes to maintain zinc homeostasis, are not as well understood. In this work, we screened all the potential zinc transporter Zip (SLC39) and ZnT (SLC30) members for their likely roles in zinc excretion in Malpighian tubules, an insect organ functionally analogous to mammalian kidneys. RESULTS: Zip71B (CG10006, most homologous to hZIP5), in addition to the previously characterized ZnT35C (CG3994), was identified as being critical in zinc excretion. Tubule-specific knockdown of Zip71B/dZip5 reduces zinc accumulation in the tubules, but increases zinc levels in the body, resulting in survival defect under zinc excess conditions. Zip71B/dZip5 is localized to the plasma membrane at the basolateral side of the tubules, and is functionally epistatic to the apically localized ZnT35C in regulating the tubule zinc homeostasis. Our results indicate that Zip71B/dZip5 is involved in zinc import into the tubular cells from the circulation, and ZnT35C in turn effluxes the tubular zinc out. Notably, mammalian ZIP5, which is expressed in the kidney, functions analogously to Zip71B/dZip5 in the fly while hZIP4 cannot complement the loss of Zip71B/dZip5 function. Furthermore, Zip71B/dZip5 expression is regulated by zinc so that, in response to toxic levels of zinc, the tubules can increase zinc efflux capability. We also characterized the role of dZnT1 (CG17723) in zinc reabsorption in Malpighian tubules. Finally, using a tubule calcification model, we were able to show that knockdown of Zip71B/dZip5 or ZnT35C was able to mitigate stone formation, consistent with their roles in tubular zinc homeostasis. CONCLUSIONS: Our results start to sketch out a relatively complete picture of the zinc excretion process in Drosophila Malpighian tubules, and may provide a reference for relevant mammalian studies.

Lateralization of gene expression in the honeybee brain during olfactory learning.

In the last decade, it has been demonstrated that brain functional asymmetry occurs not only in vertebrates but also in invertebrates. However, the mechanisms underlying functional asymmetry remain unclear. In the present study, we trained honeybees of the same parentage and age, on the proboscis extension reflex (PER) paradigm with only one antenna in use. The comparisons of gene expression between the left and right hemispheres were carried out using high throughput sequencing. Our research revealed that gene expression in the honeybee brain is also asymmetric, with more genes having higher expression in the right hemisphere than the left hemisphere. Our studies show that during olfactory learning, the left hemisphere is more responsible for long term memory and the right hemisphere is more responsible for the learning and short term memory.

Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana).

BACKGROUND: Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager). RESULTS: Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs. CONCLUSIONS: In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

The integrative analysis of microRNA and mRNA expression in Apis mellifera following maze-based visual pattern learning.

The honeybee (Apis mellifera) is a social insect with strong sensory capacity and diverse behavioral repertoire and is recognized as a good model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the changes in microRNA (miRNA) and messenger RNA (mRNA) following maze-based visual learning using next-generation small RNA sequencing and Solexa/lllumina Digital Gene Expression tag profiling (DGE). For small RNA sequencing, we obtained 13 367 770 and 13 132 655 clean tags from the maze and control groups, respectively. A total of 40 differentially expressed known miRNAs were detected between these two samples, and all of them were up-regulated in the maze group compared to the control group. For DGE, 5 681 320 and 5 939 855 clean tags were detected from the maze and control groups, respectively. There were a total of 388 differentially expressed genes between these two samples, with 45 genes up-regulated and 343 genes down-regulated in the maze group, compared to the control group. Additionally, the expression levels of 10 differentially expressed genes were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and the expression trends of eight of them were consistent with the DGE result, although the degree of change was lower in amplitude. The integrative analysis of miRNA and mRNA expression showed that, among the 40 differentially expressed known miRNAs and 388 differentially expressed genes, 60 pairs of miRNA/mRNA were identified as co-expressed in our present study. These results suggest that both miRNA and mRNA may play a pivotal role in the process of learning and memory in honeybees. Our sequencing data provide comprehensive miRNA and gene expression information for maze-based visual learning, which will facilitate understanding of the molecular mechanisms of honeybee learning and memory.

Functional studies of Drosophila zinc transporters reveal the mechanism for dietary zinc absorption and regulation.

BACKGROUND: Zinc is key to the function of many proteins, but the process of dietary zinc absorption is not well clarified. Current knowledge about dietary zinc absorption is fragmented, and mostly derives from incomplete mammalian studies. To gain a comprehensive picture of this process, we systematically characterized all zinc transporters (that is, the Zip and ZnT family members) for their possible roles in dietary zinc absorption in a genetically amenable model organism, Drosophila melanogaster. RESULTS: A set of plasma membrane-resident zinc transporters was identified to be responsible for absorbing zinc from the lumen into the enterocyte and the subsequent exit of zinc to the circulation. dZip1 and dZip2, two functionally overlapping zinc importers, are responsible for absorbing zinc from the lumen into the enterocyte. Exit of zinc to the circulation is mediated through another two functionally overlapping zinc exporters, dZnT1, and its homolog CG5130 (dZnT77C). Somewhat surprisingly, it appears that the array of intracellular ZnT proteins, including the Golgi-resident dZnT7, is not directly involved in dietary zinc absorption. By modulating zinc status in different parts of the body, we found that regulation of dietary zinc absorption, in contrast to that of iron, is unresponsive to bodily needs or zinc status outside the gut. The zinc transporters that are involved in dietary zinc absorption, including the importers dZip1 and dZip2, and the exporter dZnT1, are respectively regulated at the RNA and protein levels by zinc in the enterocyte. CONCLUSIONS: Our study using the model organism Drosophila thus starts to reveal a comprehensive sketch of dietary zinc absorption and its regulatory control, a process that is still incompletely understood in mammalian organisms. The knowledge gained will act as a reference for future mammalian studies, and also enable an appreciation of this important process from an evolutionary perspective.

Gene expression analysis following olfactory learning in Apis mellifera.

The honeybee has a strong learning and memory ability, and is recognized as the best model organism for studying the neurobiological basis of learning and memory. In this study, we analyzed the gene expression difference following proboscis extension response-based olfactory learning in the A. mellifera using a tag-based digital gene expression (DGE) method. We obtained about 5.71 and 5.65 million clean tags from the trained group and untrained group, respectively. A total of 259 differentially expressed genes were detected between these two samples, with 30 genes up-regulated and 229 genes down-regulated in trained group compared to the untrained group. These results suggest that bees tend to actively suppress some genes instead of activating previously silent genes after olfactory learning. Our DGE data provide comprehensive gene expression information for olfactory learning, which will facilitate our understanding of the molecular mechanism of honey bee learning and memory.

Comparison of learning and memory of Apis cerana and Apis mellifera.

The honeybee is an excellent model organism for research on learning and memory among invertebrates. Learning and memory in honeybees has intrigued neuroscientists and entomologists in the last few decades, but attention has focused almost solely on the Western honeybee, Apis mellifera. In contrast, there have been few studies on learning and memory in the Eastern honeybee, Apis cerana. Here we report comparative behavioral data of color and grating learning and memory for A. cerana and A. mellifera in China, gathered using a Y-maze apparatus. We show for the first time that the learning and memory performance of A. cerana is significantly better on both color and grating patterns than that of A. mellifera. This study provides the first evidence of a learning and memory difference between A. cerana and A. mellifera under controlled conditions, and it is an important basis for the further study of the mechanism of learning and memory in honeybees.