Objective:To discuss the effect of long non-coding RNA(lncRNA)DUXAP8 in exosomes(Exo)derived from the lung cancer A549 cells on the growth and immune escape of the lung cancer cells,and to clarify the mechanism.Methods:The human lung cancer cell line A549 was cultured,and its exosomes were extracted and identified.The A549 cells were treated with PKH67-labeled Exo to observe the uptake of Exo by A549 cells.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression level of lncRNA DUXAP8 in A549 cells before and after Exo treatment.The A549 cells were divided into control group(no treatment),Exo group(A549 cells treated with Exo),Exo+sh-NC group(A549 cells treated with Exo and then transfected with sh-NC),and Exo+sh-DUXAP8 group(A549 cells treated with Exo and then transfected with sh-DUXAP8).RT-qPCR method was used to detect the expression level of lncRNA DUXAP8 in A549 cells in various groups;colony formation assay was used to detect the colony formation abilities of the A549 cells in various groups;5-ethynyl-2'-deoxyuridine(EdU)staining method was used to detect the proliferation abilities of the A549 cells in various groups.After co-culturing A549 cells in various groups with human peripheral blood lymphocytes,flow cytometry was used to detect the percentages of activated CD8+T lymphocytes in the human peripheral blood lymphocytes in various groups;3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method was used to detect the killing rates of human peripheral blood lymphocytes on the A549 cells in various groups.Results:The diameter of Exo vesicles was 50-150 nm,and the exosome-specific marker proteins cluster of differentiation 63(CD63),cluster of differentiation 9(CD9),tumor susceptibility gene 101(TSG101),and heat shock protein 70(HSP70)were positively expressed,indicating successful exosome extraction.A549 cells efficiently took up PKH67-labeled Exo.The RT-PCR results showed that compared with A549 cells cultured alone,the expression level of lncRNA DUXAP8 in the A549 cells was increased after treatment with Exo derived from A549 cells(P<0.05).compared with control group,the expression level of lncRNA DUXAP8 in the A549 cells in Exo group was increased(P<0.05);compared with Exo group,the expression level of lncRNA DUXAP8 in the A549 cells in Exo+sh-DUXAP8 group was decreased(P<0.05),while there were no significant difference in the expression level of IncRNA DUXAP8 in the cells in Exo+sh-NC group(P>0.05).The colony formation assay results showed that compared with control group,the number of colony formation of the A549 cells in Exo group was increased(P<0.05);compared with Exo group,the number of colony formation of the A549 cells in Exo+sh-DUXAP8 group was decreased(P<0.05),while there was no significant difference in the number of colony formation of the A549 cells in Exo+sh-NC group(P>0.05).The EdU staining results showed that compared with control group,the EdU-positive rate of the A549 cells in Exo group was increased(P<0.05);compared with Exo group,the EdU-positive rate in A549 cells in Exo+sh-DUXAP8 group was decreased(P<0.05),while there was no significant difference in the EDU-positive rate in the cells in Exo+sh-NC group(P>0.05).The flow cytometry results showed that compared with control group,the percentage of activated CD8+T lymphocytes in the human peripheral blood lymphocytes in Exo group was decreased(P<0.05);compared with Exo group,the percentage of activated CD8+T lymphocytes in the human peripheral blood lymphocytes in Exo+sh-DUXAP8 group was increased(P<0.05),while there was no significant difference in the percentage of activated CD8+T lymphaytes in Exo+sh-NC group(P>0.05).The MTT assay results showed that compared with control group,the killing rate of human peripheral blood lymphocytes on the A549 cells in Exo group was decreased(P<0.05);compared with Exo group,the killing rate of human peripheral blood lymphocytes on A549 cells in Exo+sh-DUXAP8 group was increased(P<0.05),while no significant difference was observed in Exo+sh-NC group(P>0.05).Conclusion:The lncRNA DUXAP8 in exosomes derived from the lung cancer A549 cells promotes the proliferation of lung cancer cells and tumor immune escape.

Objective To investigate the regulatory effects and molecular mechanisms of PUS3 on GBM cell malignant behaviors(proliferation,apoptosis,invasion)in vitro,providing potential therapeutic targets for GBM.Methods The expression of PUS3 in GBM was analyzed using the GEPIA2 database.Kaplan-Meier survival analysis evaluated the survival difference between PUS3 high-and low-expression patients.qRT-PCR and Western blot were performed to detect PUS3 expression in normal glial cells(HEB)and GBM cell lines(U87,LN229,U251,T98G).PUS3-stably overexpressing GBM cell lines were constructed.Colony formation,CCK-8 assay,and flow cytometry were used to assess proliferation and apoptosis.Transwell assay evaluated cell invasion.Immunohis-tochemistry(IHC)detected PUS3 expression in GBM patient tissues.Western blot analyzed tumor-related pathway proteins after PUS3 overexpression.Results The expression level of PUS3 is elevated in GBM patient tissues,and the mRNA and protein expression levels in GBM cells are significantly higher than those in HEB cells(P<0.01).After overexpression of PUS3,the proliferation ability of GBM cells was enhanced(P<0.05),the apoptosis rate decreased(P<0.01),and the number of invasive cells increased(P<0.001).Mechanistically,overexpression of PUS3 significantly activates the AKT pathway,and the use of AKT inhibitors in PUS3 overexpressing cells can reverse the pro cancer effect.Conclusion PUS3 is highly expressed in glioblastoma(GBM)and promotes tumor cell proliferation,invasion,and apoptosis inhibition by activating the AKT pathway,suggesting its potential as a therapeutic target for GBM treatment.

Objective To investigate the regulatory effects and molecular mechanisms of PUS3 on GBM cell malignant behaviors(proliferation,apoptosis,invasion)in vitro,providing potential therapeutic targets for GBM.Methods The expression of PUS3 in GBM was analyzed using the GEPIA2 database.Kaplan-Meier survival analysis evaluated the survival difference between PUS3 high-and low-expression patients.qRT-PCR and Western blot were performed to detect PUS3 expression in normal glial cells(HEB)and GBM cell lines(U87,LN229,U251,T98G).PUS3-stably overexpressing GBM cell lines were constructed.Colony formation,CCK-8 assay,and flow cytometry were used to assess proliferation and apoptosis.Transwell assay evaluated cell invasion.Immunohis-tochemistry(IHC)detected PUS3 expression in GBM patient tissues.Western blot analyzed tumor-related pathway proteins after PUS3 overexpression.Results The expression level of PUS3 is elevated in GBM patient tissues,and the mRNA and protein expression levels in GBM cells are significantly higher than those in HEB cells(P<0.01).After overexpression of PUS3,the proliferation ability of GBM cells was enhanced(P<0.05),the apoptosis rate decreased(P<0.01),and the number of invasive cells increased(P<0.001).Mechanistically,overexpression of PUS3 significantly activates the AKT pathway,and the use of AKT inhibitors in PUS3 overexpressing cells can reverse the pro cancer effect.Conclusion PUS3 is highly expressed in glioblastoma(GBM)and promotes tumor cell proliferation,invasion,and apoptosis inhibition by activating the AKT pathway,suggesting its potential as a therapeutic target for GBM treatment.

Objectives:To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms.Methods:Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT).Results:Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated ( r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 ( r=-0.167, P=0.044), the level of serum CEA ( r=-0.061, P=0.032), and the level of serum ALT (r=-0.147, P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 ( r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT ( r=0.164, P=0.029). Conclusion:Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

Objectives:To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms.Methods:Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT).Results:Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated ( r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 ( r=-0.167, P=0.044), the level of serum CEA ( r=-0.061, P=0.032), and the level of serum ALT (r=-0.147, P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 ( r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT ( r=0.164, P=0.029). Conclusion:Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

TROP-2, a tumor-associated antigen, has been implicated in the progression of various epithelial tumors. Due to its favorable expression profile, TROP-2 has emerged as a promising target for antibody-drug conjugates (ADCs) based anti-tumor therapies. Although ADCs have shown efficacy in cancer treatment, their application in solid tumors is hindered by their high molecular weight, poor tumor penetration, and release of cytotoxic molecules. Therefore, a recombinant immunotoxin was developed based on a shark-derived variable domain of immunoglobulin new antigen receptor (VNAR) antibody. VNARs are only one-tenth the size of IgG antibodies and possess remarkable tissue penetration capabilities and high stability. In this study, a shark VNAR phage display library was created, leading to the identification of shark VNAR-5G8 that targets TROP-2. VNAR-5G8 exhibited a high affinity and cellular internalization ability towards cells expressing high levels of TROP-2. Epitope analysis revealed that VNAR-5G8 recognizes a hidden epitope consisting of CRD and TY-1 on TROP-2. Subsequently, VNAR-5G8 was fused with a truncated form of Pseudomonas exotoxin (PE38) to create the recombinant immunotoxin (5G8-PE38), which exhibited significant anti-tumor activity in vitro and in vivo. Overall, this study highlights the promise of 5G8-PE38 as a valuable candidate for cancer therapy.

Background: Excessive growth of keratinocytes is the critical event in the etiology of psoriasis.However, the underlying molecular mechanism of psoriatic keratinocyte hyperproliferation is still unclear. Objective: This study aimed to figure out the potential contributory role of S-phase kinase-associated protein 2 (SKP2) in promoting the hyperproliferation of keratinocytes in psoriasis. Methods: We analyzed microarray data (GSE41662) to investigate the gene expression of SKP2in psoriatic lesion skins compared with their adjacent non-lesional skin. Then, we further confirmed the mRNA and protein expression of SKP2 in human psoriatic skin tissues, imiquimod (IMQ)-induced psoriatic mice back skins and tumor necrosis factor α (TNF-α), interleukin (IL)-17A and IL-6-stimulated keratinocytes by using real-time quantitative polymerase chain reaction and western blot (WB). Furthermore, we explored the potential pathogenic role and its underlying cellular mechanism of SKP2 in promoting keratinocytes hyperproliferation through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle detection, 5-ethynyl-2′-deoxyuridine staining and WB. Finally, we determined whether inhibition of SKP2 can effectively alleviate the keratinocytes hyperproliferation in vivo. Results: We identified that SKP2 is aberrantly upregulated in the psoriatic lesion skin and cytokines-stimulated keratinocytes. Moreover, upregulated SKP2 augments cytokines-induced keratinocytes hyperproliferation. Mechanistically, enhanced SKP2 increased the S phase ratio through inhibiting Cyclin-Dependent Kinase Inhibitor p27 (P27 Kip1) expression. Correspondingly, suppression of SKP2 with SMIP004 can significantly ease the epidermis hyperplasia in vivo. Conclusion Our results suggest that elevated SKP2 can empower keratinocytes proliferation and psoriasis-like epidermis hyperplasia via downregulation of P27 Kip1. Therefore, targeting SKP2-P27 Kip1 axis might be a promising therapeutic strategy for the treatment of psoriasis in future.

Glioma is a primary brain tumor with high incidence rate. High-grade gliomas (HGG) are those with the highest degree of malignancy and the lowest degree of survival. Surgical resection and postoperative adjuvant chemoradiotherapy are often used in clinical treatment, so accurate segmentation of tumor-related areas is of great significance for the treatment of patients. In order to improve the segmentation accuracy of HGG, this paper proposes a multi-modal glioma semantic segmentation network with multi-scale feature extraction and multi-attention fusion mechanism. The main contributions are, (1) Multi-scale residual structures were used to extract features from multi-modal gliomas magnetic resonance imaging (MRI); (2) Two types of attention modules were used for features aggregating in channel and spatial; (3) In order to improve the segmentation performance of the whole network, the branch classifier was constructed using ensemble learning strategy to adjust and correct the classification results of the backbone classifier. The experimental results showed that the Dice coefficient values of the proposed segmentation method in this article were 0.909 7, 0.877 3 and 0.839 6 for whole tumor, tumor core and enhanced tumor respectively, and the segmentation results had good boundary continuity in the three-dimensional direction. Therefore, the proposed semantic segmentation network has good segmentation performance for high-grade gliomas lesions.
Attention ; Glioma/diagnostic imaging* ; Humans ; Magnetic Resonance Imaging/methods* ; Semantics

Objective:To evaluate the status and influencing factors of psychiatric comorbidities of patients with epilepsy (PWEs) in Hubei province during the outbreak of COVID-19.Methods:From February 23, 2020 to March 5, 2020, a network questionnaire survey (including demographic characteristics, seizures, Generalized Anxiety Disorder Scale-7 score, Patient Health Questionnaire-9 score, Insomnia Severity Index score) was conducted among 570 PWEs who visited the Epilepsy Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology during April 1, 2019 and January 20, 2020. SPSS 22.0 software was used for correlation analysis of sociodemographic characteristics, epilepsy related factors, perceived threat to the COVID-19 and psychiatric comorbidity (depression, anxiety and insomnia) of PWEs during the COVID-19 epidemic.Results:A total of 362 valid questionnaires were included for analysis (the response rate was 63.51%,362/570). Thirty-four (9.4%), forty-seven (13.0%) and seventy-one (19.6%) patients suffered from anxiety, depression and insomnia, respectively. Patients with seizure frequency ≥2 times/month before the epidemic ( OR=3.395,95% CI 1.561-7.384, P=0.002), poor subjective quality of life during the epidemic ( OR=10.753,95% CI 1.938-59.654, P=0.024), and moderate to severe worry about bad impact of the epidemic on epilepsy ( OR=3.077, 95% CI 1.382-6.853, P=0.006) were more likely to be anxious. Patients with poor subjective quality of life during the epidemic ( OR=6.188, 95% CI 1.317-29.079, P=0.021) were more likely to be depressed. Patients with COVID-19 related symptoms ( OR=3.609, 95% CI 1.674-7.778, P=0.001), children ( OR=3.090, 95% CI 1.759-5.431, P<0.001), seizure frequency ≥2 times/month before the epidemic ( OR=1.907, 95% CI 1.017-3.575, P=0.044), and moderate to severe worry about unanticipated seizures ( OR=2.555, 95% CI 1.370-4.764, P=0.003) were more likely to suffer from insomnia. Conclusions:During the COVID-19 epidemic, parts of PWEs suffered from anxiety, depression and insomnia. PWEs with poor subjective quality of life, high frequency of epileptic seizures before the epidemic, excessive worry about bad impact of the epidemic on epilepsy and excessive worry about unanticipated seizures were prone to anxiety, depression and insomnia.

Objective:To understand the agglutination characteristics of different subtypes of avian influenza viruses, we selected erythrocytes from different sources to find suitable erythrocytes for influenza environmental sample detection.Methods:Different subtypes of avian influenza viruses, which were isolated from environmental sample between 2009 and 2016 in China, were selected to do hemagglutination assay using 5 animal erythrocytes (chicken, turkey, guinea pig, horse, and sheep). Flow cytometry was used to detect expression level and type of sialic acid receptors of different erythrocytes, and the characteristics of the receptor binding domain (RBD) of the viral hemagglutinin protein were analyzed by amino acid sequence.Results:In this study, a total of 28 strains of avian influenza virus including 14 subtypes were detected. The result showed that all viruses could agglutinate with turkey and guinea pig erythrocytes and the rest three erythrocytes were unable to produce agglutination with some viruses; among them, one H9N2 virus (A/environment/Anhui/43762/2015) did not agglutinate with chicken erythrocytes, one H1N1 virus (A/environment/Shandong/76972/2014) and two H9N2 viruses (A/environment/Chongqing/79449/2014 and A/environment/Anhui/43762/2015) did not agglutinate with horse erythrocytes, two viruses of H9N2 (A/environment/Chongqing/79449/2014 and A/environment/Anhui/43762/2015) and two viruses of H13N8 (A/environment/Qinghai Lake/166/2012 and A/environment/Qinghai Lake/13/2012) did not agglutinate with sheep erythrocytes. The result of flow cytometry showed that two sialic acid receptors, α-2, 3 and α-2, 6, were detected on the surface of erythrocytes of turkey, chicken and guinea pig, but the expression ratios of the two receptors were different. Only the expression of α-2, 3 sialic acid receptors was detected in horse and sheep erythrocytes. Sequence analysis suggested that amino acid substitution in key regions of viral hemagglutinin protein RBD may be an important factor affecting the binding properties of different erythrocytes.Conclusions:Our result suggested that turkey and guinea pig erythrocytes are the most sensitive in the hemagglutination test. Receptor expression and type of erythrocytes from different sources can significantly affect the agglutination reaction of different subtypes of avian influenza virus, and the amino acid changes in key regions of RBD can also affect the result of agglutination reaction.