EBV abortive lytic cycle promotes nasopharyngeal carcinoma progression through recruiting monocytes and regulating their directed differentiation.

Epstein-Barr virus (EBV) is associated with several types of human cancer including nasopharyngeal carcinoma (NPC). The activation of EBV to the lytic cycle has been observed in advanced NPC and is believed to contribute to late-stage NPC development. However, how EBV lytic cycle promotes NPC progression remains elusive. Analysis of clinical NPC samples indicated that EBV reactivation and immunosuppression were found in advanced NPC samples, as well as abnormal angiogenesis and invasiveness. To investigate the role of the EBV lytic cycle in tumor development, we established a system that consists of two NPC cell lines, respectively, in EBV abortive lytic cycle and latency. In a comparative analysis using this system, we found that the NPC cell line in EBV abortive lytic cycle exhibited the superior chemotactic capacity to recruit monocytes and polarized their differentiation toward tumor-associated macrophage (TAM)-like phenotype and away from DCs, compared to EBV-negative or EBV-latency NPC cells. EBV-encoded transcription activator ZTA is responsible for regulating monocyte chemotaxis and TAM phenotype by up-regulating the expression of GM-CSF, IL-8, and GRO-α. As a result, TAM induced by EBV abortive lytic cycle promotes NPC angiogenesis, invasion, and migration. Overall, this study elucidated the role of the EBV lytic life cycle in the late development of NPC and revealed a mechanism underlying the ZTA-mediated establishment of the tumor microenvironment (TME) that promotes NPC late-stage progression.

Circulating cell-free DNA fragmentation is a stepwise and conserved process linked to apoptosis.

BACKGROUND: Circulating cell-free DNA (cfDNA) is a pool of short DNA fragments mainly released from apoptotic hematopoietic cells. Nevertheless, the precise physiological process governing the DNA fragmentation and molecular profile of cfDNA remains obscure. To dissect the DNA fragmentation process, we use a human leukemia cell line HL60 undergoing apoptosis to analyze the size distribution of DNA fragments by shallow whole-genome sequencing (sWGS). Meanwhile, we also scrutinize the size profile of plasma cfDNA in 901 healthy human subjects and 38 dogs, as well as 438 patients with six common cancer types by sWGS. RESULTS: Distinct size distribution profiles were observed in the HL60 cell pellet and supernatant, suggesting fragmentation is a stepwise process. Meanwhile, C-end preference was seen in both intracellular and extracellular cfDNA fragments. Moreover, the cfDNA profiles are characteristic and conserved across mammals. Compared with healthy subjects, distinct cfDNA profiles with a higher proportion of short fragments and lower C-end preference were found in cancer patients. CONCLUSIONS: Our study provides new insight into fragmentomics of circulating cfDNA processing, which will be useful for early diagnosis of cancer and surveillance during cancer progression.

Castor1 overexpression regulates microglia M1/M2 polarization via inhibiting mTOR pathway.

Microglia are resident immune cells in the brain and are closely associated with central nervous system inflammation and neurodegenerative diseases. It is known that mammalian target of rapamycin (mTOR) pathway plays an important role in the polarization of microglia. Castor1 has been identified as the cytosolic arginine sensor for the mTOR complex 1 (mTORC1) pathway, but the role of Castor1 in microglial polarization is still unknown. The purpose of this study was to explore the regulatory effect of Castor1 on microglial polarization and the underlying mechanism. The results demonstrated that Castor1 expression was significantly decreased in lipopolysaccharides (LPS) and interferon (IFN)-γ treated microglia. Castor1 overexpression inhibited the microglia M1 polarization by reducing the expression of M1 related markers. However, the expression of M2-related genes was promoted when Castor1 was overexpressed in IL-4 treated microglia. Mechanistically, Castor1 overexpression inhibited the activation of mTOR signaling pathway. In addition, after treatment with the mTOR activator MHY1485, the inhibitory effect of Castor1 overexpression on M1 polarization was attenuated, indicating that the regulation effects of Castor1 on M1 polarization was dependent on its inhibition of mTOR pathway. We propose that Castor1-mTOR signaling pathway could be considered as a potential target for treatment and intervention of central nervous system-related diseases by regulating microglia polarization.

Psychological stress induces depressive-like behavior associated with bone marrow-derived monocyte infiltration into the hippocampus independent of blood-brain barrier disruption.

BACKGROUND: Psychological stress is one of the most important factors that trigger emotional disorders, such as depression and anxiety. Emerging evidence suggests that neuroinflammation exacerbated by bidirectional communication between the peripheral immune system and the central nervous system facilitates abnormal psychiatric symptoms. This study aimed to investigate the hippocampal migration of bone marrow (BM)-derived monocytes and its role in regulating depressive-like behaviors using the chronic psychological stress (CPS) mouse model. More importantly, whether the central migration of these peripheral BM-derived cells depend on the disruption of the blood-brain barrier (BBB) was also investigated. METHODS AND FINDINGS: Green fluorescent protein-positive (GFP+) BM chimeric mice were used to distinguish BM-derived monocytes within the brain. A CPS mouse model was established to explore the effect of CPS on hippocampal migration of BM-derived monocytes and its role in the regulation of depressive-like behaviors. The results revealed that BM-derived GFP+ cells accumulated in the hippocampus and differentiated into microglia-like cells after exposure to CPS. Interestingly, this migration was not associated with BBB disruption. Furthermore, treatment with C-C chemokine receptor 2 (CCR2) antagonist (RS102895) suppressed the recruitment of BM-derived monocytes to the hippocampus and alleviated depressive-like symptoms. CONCLUSION: These findings indicate that monocyte recruitment to the hippocampus in response to psychological stress may represent a novel cellular mechanism that contributes to the development of depression.

Automatic system for high-throughput and high-sensitivity diagnosis of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had severe consequences for health and the global economy. To control the transmission, there is an urgent demand for early diagnosis and treatment in the general population. In the present study, an automatic system for SARS-CoV-2 diagnosis is designed and built to deliver high specification, high sensitivity, and high throughput with minimal workforce involvement. The system, set up with cross-priming amplification (CPA) rather than conventional reverse transcription-polymerase chain reaction (RT-PCR), was evaluated using more than 1000 real-world samples for direct comparison. This fully automated robotic system performed SARS-CoV-2 nucleic acid-based diagnosis with 192 samples in under 180 min at 100 copies per reaction in a "specimen in data out" manner. This throughput translates to a daily screening capacity of 800-1000 in an assembly-line manner with limited workforce involvement. The sensitivity of this device could be further improved using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based assay, which opens the door to mixed samples, potentially include SARS-CoV-2 variants screening in extensively scaled testing for fighting COVID-19.

Advanced glycation end products reduce macrophage-mediated killing of Staphylococcus aureus by ARL8 upregulation and inhibition of autolysosome formation.

Staphylococcus aureus, a pathogen most frequently found in diabetic foot ulcer infection, was recently suggested as an intracellular pathogen. Autophagy in professional phagocytes like macrophages allows selective destruction of intracellular pathogens, and its dysfunction can increase the survival of internalized pathogens, causing infections to worsen and spread. Previous works have shown that S. aureus infections in diabetes appeared more severe and invasive, and coincided with the suppressed autophagy in dermal tissues of diabetic rat, but the exact mechanisms are unclear. Here, we demonstrated that accumulation of advanced glycation end products (AGEs) contributed to the diminished autophagy-mediated clearance of S. aureus in the macrophages differentiated from PMA-treated human monocytic cell line THP-1. Importantly, infected macrophages showed increased S. aureus containing autophagosome, but the subsequent fusion of S. aureus containing autophagosome and lysosome was suppressed in AGEs-pretreated cells, suggesting AGEs blocked the autophagic flux and enabled S. aureus survival and escape. At the molecular level, elevated lysosomal ARL8 expression in AGEs-treated macrophages was required for AGEs-mediated inhibition of autophagosome-lysosome fusion. Silencing ARL8 in AGEs-treated macrophages restored autophagic flux and increased S. aureus clearance. Our results therefore demonstrate a new mechanism, in which AGEs accelerate S. aureus immune evasion in macrophages by ARL8-dependent suppression of autophagosome-lysosome fusion and bactericidal capability.

Low prevalence of antibodies against SARS-CoV-2 among voluntary blood donors in Guangzhou, China.

Since the first case of COVID-19 reported in late December of 2019 in Wuhan, China, the SARS-CoV-2 virus has caused approximately 20 million infections and 732 thousand deaths around the world by 11 August 2020. Although the pathogen generally infects the respiratory system, whether it is present in the bloodstream and whether it poses a threat to the blood supply during the period of the outbreak is of serious public concern. In this study, we used enzyme-linked immunosorbent assay (ELISA) to screen total antibodies against SARS-CoV-2 in 2199 blood donors, who had donated blood at the Guangzhou Blood Center during the epidemic. The Ig-reactive samples were further characterized for IgA, IgG, and IgM subtypes by ELISA and viral nucleic acid by real-time polymerase chain reaction. Among the 2199 plasma samples, seven were reactive under total antibodies' screening. Further testing revealed that none of them had detectable viral nucleic acid or IgM antibody, but two samples contained IgA and IgG. The IgG antibody titers of both positive samples were 1:16 and 1:4, respectively. Our results indicated a low prevalence of past SARS-CoV-2 infection in our blood donors, as none of the tests were positive for viral nucleic acid and only 2 out of 2199 (0.09%) of samples were positive for IgG and IgA. There would be a limited necessity for the implementation of such testing in blood screening in a COVID-19 low-risk area.

IL-6 and IL-10 gene polymorphisms and cirrhosis of liver risk from a comprehensive analysis.

BACKGROUND: Different inflammatory and immune cytokines play a key role in the development of cirrhosis of liver (CL). To investigate the association between interleukin-6,10 (IL-6,10) genes polymorphisms and CL risk through comparison of the allele and genotype distribution frequencies by meta-analysis. METHODS: A literature search covered with the PubMed, Embase, Cochrane Library, Web of Science, Google Scholar, SinoMed (CNKI and Wanfang) through 20th April, 2021. Odds ratios (OR) and 95% confidence intervals (CI) were used to assess the strength of associations. RESULTS: After a comprehensive search, three common polymorphisms (rs1800872, rs1800871, rs1800896) in IL-10 gene were selected, and three common polymorphisms (rs1800795, rs1800796, rs1800797) in IL-6 gene were also identified. The important finding was that IL-10 rs1800872 was a risk factor for CL development. For example, there has a significantly increased relationship between rs1800872 polymorphism and CL both in the whole group (OR: 1.30, 95%CI: 1.01-1.67 in heterozygote model), Asian population (OR: 1.40, 95%CI: 1.03-1.88 in heterozygote model) and hospital-based source of control (OR: 1.40, 95%CI: 1.01-1.96 in dominant model). In addition, significant association was found between rs1800896 and primary biliary cirrhosis subtype disease (OR: 1.30, 95%CI: 1.01-1.68 in allelic contrast model). No association was observed in all three polymorphisms in IL-6 gene. CONCLUSION: Our present study suggests that the IL-10 rs1800872 and rs1800896 polymorphisms is potentially associated with the risk of CL susceptibility.

Methods used for the measurement of blood-brain barrier integrity.

The blood-brain barrier (BBB) comprises the interface between blood, brain and cerebrospinal fluid. Its primary function, which is mainly carried out by tight junctions, is to stabilize the tightly controlled microenvironment of the brain. To study the development and maintenance of the BBB, as well as various roles their intrinsic mechanisms that play in neurological disorders, suitable measurements are required to demonstrate integrity and functional changes at the interfaces between the blood and brain tissue. Markers and plasma proteins with different molecular weight (MW) are used to measure the permeability of BBB. In addition, the expression changes of tight-junction proteins form the basic structure of BBB, and imaging modalities are available to study the disruption of BBB. In the present review, above mentioned methods are depicted in details, together with the pros and cons as well as the differences between these methods, which maybe benefit research studies focused on the detection of BBB breakdown.

Extensive serum cytokine analysis in patients with prostate cancer.

Prostate cancer (CaP) is a common male malignancy. Using prostate specific antigen (PSA) and prostate cancer antigen 3 (PCA3) in the diagnosis of prostate cancer, sensitivity and specificity still require improvement. Additional targets are urgently needed for the diagnosis, prognosis, and prediction of therapeutic response, leading to better treatments in order to reduce the mortality of CaP. Here, we utilized a solid-phase antibody array, which can simultaneously detect 200 proteins, for the screening of novel blood-based biomarkers. The proteins differentially expressed in the pathogenesis of CaP were further analyzed using bioinformatics methods. The identified targets were further validated by the enzyme-linked immunosorbent assay (ELISA). A total of 38 proteins were identified with significantly differential levels in CaP serum compared to healthy control serum, including 21 up-regulated and 17 down-regulated cytokines. ELISA result showed that validated six ones of these differential cytokines were significantly differential between CaP and control, consistent with the antibody array result. The protein-protein interaction (PPI) analysis for these differentially expressed cytokines showed the top five cytokines interacting with most other cytokines were insulin, SDF-1a, CD40L, IL-18 and NCAM-1, suggesting these five targets are important in the pathogenesis of CaP, and more sensitive for the early diagnosis and prognosis of CaP. Targeting these cytokines may be more effective therapies against CaP. Among these differentially expressed cytokines, it was found that AR, BTC, IL-1 F8, IL-31, Marapsin, b-NGF, EDA-A2, MCP-3, MCP-4, MIP-3a, PIGF, and TECK decreased, while Fas, Flt-3L, and NCAM-1 increased in CaP when compared to the controls. Taken together, those 38 differentially expressed cytokines may service as novel serum biomarkers for CaP, which will be further validated with more clinical samples.

Extensive serum biomarker analysis in patients with non-small-cell lung carcinoma.

Lung cancer is a common malignant disease, nearly 2.09 million new patients occurred last year. Approximately 85% of the patients are classified as non-small-cell lung cancer (NSCLC). It is therefore important to identify new diagnostic and prognostic biomarkers for the early detection of this disease. The presented study identifies biomarkers in the serum of NSCLC patients. The expression of 274 cytokines was measured by a novel antibody array methodology and ELISA was applied to validate the array results. The levels of MIP-1 α, IL-8, MIP-1 ß, Resistin, GDF-15, HGF, CA125, FLRG, VCAM-1, DKK-3, sTNF-R1, CTACK, Acrp30, CXCL-16 and LYVE-1 were significantly higher in serum from NSCLC patients, while the level of TIMP-2 and IGFBP-6 were lower. More importantly, the validation supported the result of the antibody array. The result of the antibody array indicates that these cytokines might be novel auxiliary biomarkers in the diagnosis and prognosis of NSCLC.

Extensive serum biomarker analysis in patients with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a fast-growing cancer characterized by high occurrences of nodal and distant metastases and poor prognosis. It is therefore important to identify new serum biomarkers for the early diagnosis and prognostic prediction of this disease. The present study identifies biomarkers in NPC patient serum using a solid-phase antibody array detecting the expression profiles of 174 cytokines in a single experiment. ELISA was performed to validate the array results. The levels of TIMP-2, SELL, CCL24, MMP-1, MMP-3, IGF-I and IL-8 were significantly higher in serum from NPC patients, while the levels of MSP-alpha and HCC-4 were lower. Furthermore, the validation results were identical to those obtained from the antibody array. These results indicate that these cytokines might serve as novel biomarkers for the diagnosis and prognostic prediction of NPC.

Identification and characterization of Orf virus 050 protein proteolysis.

The Orf virus 050 (ORFV050) gene is located in the core region of the ORFV genome. It is similar to Vaccinia virus (VV) Copenhagen L4R, and encodes the DNA-binding virion core protein VP8, which has structures similar to the VV P25K core protein and may undergo similar proteolytic processing during virus assembly. Three conserved Ala-Gly-X motifs at putative cleavage sites were identified in ORFV050. To investigate the proteolysis of ORFV050 and its participation in viral assembly, full-length and site-directed mutant ORFV050 recombinant proteins were constructed and expressed. Two distinct protein bands of 28.5 and 25 kDa were detected in the infected cells using anti-ORFV050 polyclonal antiserum. A potential cleavage site was identified at amino acids 30-32 of ORFV050. Mutation of AG/A to (R) in ORFV050 abolished the process of proteolysis. ORFV050 is a late gene synthesized during viral replication in the host cytoplasm. According to these results, we conclude that ORFV050 undergoes proteolysis and plays an important role in viral assembly.

Clinical evaluation of dengue RNA, NS1, and IgM for diagnosis of dengue in Southern China.

In 2014, a large outbreak of dengue occurred in Guangzhou, China. This outbreak prompted us to evaluate NS1 and RNA for the early diagnosis of acute dengue infection, in addition to the combination with IgM antibody. We aimed to find the differences of three assays about dengue diagnosis. This study was an evaluation of diagnosis test. Based on WHO criteria 2009, dengue RNA, NS1, and IgM/IgG were detected from 294 patients (180 dengue patients, 114 non-dengue patients) by three diagnostic kits made in China. The χ(2) test, sensitivity, and specificity were used in statistical analysis. The ratios of dengue patients with low platelet counts (<100 × 10(9) /L 32.2%) or white blood cell counts (<4.0 × 10(9) /L 58.9%) were significantly higher compared to non-dengue patients (P < 0.05). Dengue NS1 was shown sensitive (93.9%) for diagnostic use. RNA had a better performance with 98.1% of sensitivity from day 1 to day 4 after illness onset. IgM performed better at day 5 or more with 74.0% of sensitivity. The diagnostic rate using a combination of RNA and IgM was 97.8% and 96.7% using NS1 and IgM. A patient with low platelet and white blood cell counts needs additional tests for dengue during an epidemic. RNA and NS1 were most valuable for early diagnosis of dengue, whereas IgM was best suited as a supplementary method for patients at day 5 or more after illness onset.

Declined Natural Killer Cells Emerging in Women with Unexplained Recurrent Spontaneous Abortion and Further Reducing after Medical Therapy.

BACKGROUND: As focus grows on reproduction, the issue of Recurrent Spontaneous Abortion (RSA), especially for unexplained reasons (URSA), is grabbing more and more attention in gynecological immunology. We investigated the changes of peripheral lymphocyte subsets focusing on whether they had some relationship with development of URSA. METHODS: The percentage and absolute count of lymphocyte subsets (T cells, Th cells, Ts cells, B cells, NK cells) were simultaneously evaluated by flow cytometry in URSA patients (n = 48) and healthy controls (HC, n = 22). RESULTS: Significantly lower percentage and absolute counts of NKT cells and NK cells were observed in URSA compared to the HC. After medical therapy, an obviously increase was shown in the percentage of both T cells and B cells, whereas it presented a reduction in the percentage of NK cells. CONCLUSIONS: The flow cytometry test in T, B, NK cells is a method available to identify URSA patients from healthy women and to provide reference guides for clinical therapy.

Detection of Human Epididymis Protein 4 (HE4) in Human Serum Samples Using a Specific Monoclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay (ELISA).

BACKGROUND: The human epididymis protein 4 (HE4) may have high specificity in the detection of malignant diseases, making the development of an immunoassay for HE4 essential. METHODS: In our study, a fusion gene was constructed encoded with the HE4 protein. This protein was then produced in the bacterial cells (Escherichia coli) and used to immunize mice in order to eventually generate hybridomas specific to HE4. The hybridoma supernatants were then screened, and four positive anti-HE4 cell lines were selected. These cell lines produce monoclonal antibodies against HE4 epitopes, as demonstrated in the Western blot as well as by direct enzyme-linked immunosorbent assay (ELISA). Using the developed antibodies, we successfully identified several good antibody pairs from the hybridomas, which allowed for the development of a sandwich ELISA to measure HE4 levels. By using the HE4 ELISA, we measured HE4 levels of 60 clinical human serum samples. RESULTS: Compared with the Food and Drug Administration (FDA) approved kit (Roche), our results showed a strong positive correlation to those of the FDA-approved kit. CONCLUSIONS: In summary, highly sensitive antibody pairs were screened against HE4, and a sandwich ELISA was developed as an accurate analytical tool for the detection of HE4 in human serum, which could be especially valuable for diagnosing ovarian carcinomas.

Low Frequency of Peripheral Lymphocyte in Chronic Hepatitis B Patients Predicts Poor Progression to Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and the second leading cause of cancer-related deaths worldwide. The poor prognosis of HCC is mainly because of its discovery at advanced stages. Because chronic hepatitis B (CHB) accounts for 50-80% HCC occurrence worldwide, and immunity is regarded as an emerging hallmark of cancer, we investigated the predictive role of peripheral immune cells in HCC incidence in CHB patients. METHODS: This investigation collected and analyzed data from 89 CHB patients, 94 primary HCC patients with hepatitis B virus (HBV), 81 primary HCC patients without HBV, 69 normal healthy patients, and 257 CHB patients with at least 3-year regular followup. RESULTS: The results demonstrated that CHB and primary HCC patients had different concentrations of lymphocytes, neutrophils, and monocytes in their peripheral circulation. Further study showed that the peripheral lymphocyte concentration was an independent prognostic factor for HCC incidence in CHB patients during the 3 years of followup. Finally, a predictive HCC incidence model with an AUROC (area under the receiver operating characteristic) of 0.832 was constructed based on the peripheral lymphocyte concentration, serum alpha-fetoprotein (AFP) concentration, and cirrhosis status of CHB patients. CONCLUSIONS: The peripheral lymphocyte concentration was an independent prognostic factor for HCC incidence in CHB patients, and a more accurate predictive model based on peripheral lymphocytes, serum AFP, and cirrhosis status was constructed.

Comparative genomic sequence analysis of Chinese orf virus strain NA1/11 with other parapoxviruses.

Orf virus (ORFV) is a typical member of the genus Parapoxvirus. The parapoxvirus genome consists of highly variable terminal regions and relatively conserved central regions with a high G + C content. In our previous study, a novel ORFV strain, NA1/11, was isolated from northeastern China. To fully characterize this strain, we sequenced the entire genome of NA1/11 and conducted a comparative analysis using multiple parapoxviruses. The genomic sequence of NA1/11 was found to consist of 137,080 nucleotides with a G + C content of 63.6 %, but it did not contain the terminal hairpin sequence. Alignment of ORFs from NA1/11 with NZ2, IA82 and SA00 revealed several highly variable ORFs, while the most evident ones are ORFs 001, 103, 109-110, 116 and 132. An odd phenomenon in the region of ORFs 118-120 is that the non-coding fragments are almost as long as the coding fragments. By comparative analysis of inverted terminal repeats, we identified one repeat motif and a long conserved fragment. By comparing the ITRs of SA00 with those of three other ORFVs, more clues were obtained about the correlation between ITR sequence and host adaption. Comparison of the NA1/11 genome with the sequences of other strains of ORFV revealed highly variable regions, thus providing new insights into the genetic diversity of ORFV.

Assessment of a One-Step Intracellular Staining in Th1, Th2 and Th17 Cells of Clinical Samples.

BACKGROUND: Flow cytometry is a potent tool to dissect the phenotypes and functions of cell subsets by measuring multiple parameters on a single-cell basis. However, intracellular staining may be time consuming and more steps, particularly in cytokines, could be problematic for its use in daily routine or in large cohort testing. Lately, a novel reagent has been developed to perform intracellular staining in one step. The objective of our study was thus to assess this new method in comparison with the reference technique by focusing on CD4+ T-cell subsets such as Th1, Th2, and Th17 cells in clinical samples. METHODS: Peripheral blood was collected from 10 children with aplastic anemia and 10 healthy volunteers and stained using the reference and one-step methods. Different subsets of CD4+ T-cells, which are defined as Th1, Th2 and Th17 cells, were investigated by flow cytometry. The repetitive experiment was designed to study intraassay precision. Correlations were studied using Pearson's correlation coefficient test. RESULTS: When comparing results obtained with the two techniques, no statistical differences between the percentages of Th1, Th2, and Th17 cells were observed. Besides, a nice correlation between percentages of Th1 cells obtained with the two different methods was identified in the global population (r: 0.777, p < 0.01). Likewise, percentages of Th2 cells (r: 0.875, p < 0.01), and Th17 cells (r: 0.886, p < 0.01) were strongly correlated between reference and one-step procedures. Importantly, flow cytometry staining obtained with the one-step method was very robust with a nice intra-assay precision and a better discriminative power and repeatability. CONCLUSIONS: With better staining quality and a shorter realization time, one-step intracellular staining may provide an efficient way for daily routine testing of Th1, Th2 and Th17 cells, as well as for further research.