Application and prospect of microfluidic devices for rapid assay of cell activities in the tumor microenvironment.

The global impact of cancer on human health has raised significant concern. In this context, the tumor microenvironment (TME) plays a pivotal role in the tumorigenesis and malignant progression. In order to enhance the accuracy and efficacy of therapeutic outcomes, there is an imminent requirement for in vitro models that can accurately replicate the intricate characteristics and constituents of TME. Microfluidic devices exhibit notable advantages in investigating the progression and treatment of tumors and have the potential to become a novel methodology for evaluating immune cell activities in TME and assist clinicians in assessing the prognosis of patients. In addition, it shows great advantages compared to traditional cell experiments. Therefore, the review first outlines the applications and advantages of microfluidic chips in facilitating tumor cell culture, constructing TME and investigating immune cell activities. Second, the roles of microfluidic devices in the analysis of circulating tumor cells, tumor prognosis, and drug screening have also been mentioned. Moreover, a forward-looking perspective is discussed, anticipating the widespread clinical adoption of microfluidic devices in the future.

Follistatin Is a Novel Chemoattractant for Migration and Invasion of Placental Trophoblasts of Mice.

Follistatin (FST) as a gonadal protein is central to the establishment and maintenance of pregnancy. Trophoblasts' migration and invasion into the endometrium are critical events in placental development. This study aimed to elucidate the role of FST in the migration and invasion of placental trophoblasts of mice. We found that FST increased the vitality and proliferation of primary cultured trophoblasts of embryonic day 8.5 (E8.5) mice and promoted wound healing of trophoblasts. Moreover, FST significantly induced migration of trophoblasts in a microfluidic device and increased the number of invasive trophoblasts by Matrigel-coated transwell invasion assay. Being treated with FST, the adhesion of trophoblasts was inhibited, but intracellular calcium flux of trophoblasts was increased. Western blotting results showed that FST had no significant effects on the level of p-Smad3 or the ratio of p-Smad3/Smad3 in trophoblasts. Interestingly, FST elevated the level of p-JNK; the ratio of p-JNK/JNK; and expression of migration-related proteins N-cadherin, vimentin, ezrin and MMP2 in trophoblasts. Additionally, the migration of trophoblasts and expression of N-cadherin, vimentin, and MMP2 in trophoblasts induced by FST were attenuated by JNK inhibitor AS601245. These findings suggest that the elevated FST in pregnancy may act as a chemokine to induce trophoblast migration and invasion through the enhanced JNK signaling to maintain trophoblast function and promote placental development.

Short-Term Instantaneous Prophylaxis and Efficient Treatment Against SARS-CoV-2 in hACE2 Mice Conferred by an Intranasal Nanobody (Nb22).

Current COVID-19 vaccines need to take at least one month to complete inoculation and then become effective. Around 51% of the global population is still not fully vaccinated. Instantaneous protection is an unmet need among those who are not fully vaccinated. In addition, breakthrough infections caused by SARS-CoV-2 are widely reported. All these highlight the unmet needing for short-term instantaneous prophylaxis (STIP) in the communities where SARS-CoV-2 is circulating. Previously, we reported nanobodies isolated from an alpaca immunized with the spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its variants. Herein, we found that Nb22, among our previously reported nanobodies, exhibited ultrapotent neutralization against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM). Furthermore, the crystal structural analysis revealed that the binding of Nb22 to WH01 and Delta RBDs both effectively blocked the binding of RBD to hACE2. Additionally, intranasal Nb22 exhibited protection against SARS-CoV-2 Delta variant in the post-exposure prophylaxis (PEP) and pre-exposure prophylaxis (PrEP). Of note, intranasal Nb22 also demonstrated high efficacy against SARS-CoV-2 Delta variant in STIP for seven days administered by single dose and exhibited long-lasting retention in the respiratory system for at least one month administered by four doses, providing a strategy of instantaneous short-term prophylaxis against SARS-CoV-2. Thus, ultrahigh potency, long-lasting retention in the respiratory system and stability at room-temperature make the intranasal or inhaled Nb22 to be a potential therapeutic or STIP agent against SARS-CoV-2.

A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration.

The dramatically expanding coronavirus disease 2019 (COVID-19) needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterize several receptor binding domain (RBD)-specific Nbs isolated from an Nb library derived from an alpaca immunized with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S); among them, three Nbs exhibit picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and circulating SARS-CoV-2 variants. To improve their efficacy, various configurations of Nbs are engineered. Nb15-NbH-Nb15, a trimer constituted of three Nbs, is constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibits single-digit ng/ml neutralization potency against the wild-type and Delta variants of SARS-CoV-2 with a long half-life in vivo. In addition, we show that intranasal administration of Nb15-NbH-Nb15 provides effective protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both the prevention and treatment of SARS-CoV-2 through respiratory administration.

Potent Neutralizing Humanized Antibody With Topical Therapeutic Potential Against HPV18-Related Cervical Cancer.

Cervical cancer caused by human papillomavirus (HPV) infections is the fourth most common cancer in women worldwide. Current prophylactic HPV vaccines have achieved promising success in preventing HPV infection. However, still 570,000 new cases were reported in 2018. The current primary treatment for the patient with cervical cancer is either surgery or chemoradiotherapy. Cervical cancer still lacks standard medical therapy. HPV18 induced cervical cancer has the worst prognosis and high mortality compared to other HPV infections. The development of HPV18 related with cervical malignancy requires the persistent infection of cervical-vaginal epithelium by HPV18 subtype, which can take years to transform the epithelium. This period of repeated infection provides a window for therapeutic intervention. Neutralizing antibodies formulated as topical agents that inhibit HPV18 infection should reduce the chance of cervical malignancy. We previously demonstrated that potent neutralizing anti-sera against HPV18 infection were induced by HPV18 viral like particle (VLP) generated in mammalian cells. We, therefore, isolated two potent neutralizing antibodies, 2A12 and 8H4, from over 3,810 hybridomas prepared from mice immunized with HPV18 VLP. 2A12 and 8H4 exhibited excellent potency, with 50% virus-inhibitory concentrations (IC50) of 0.4 and 0.9 ng/ml, respectively. Furthermore, 2A12 and 8H4 recognized distinct and non-overlapping quaternary epitopes and bound specifically with HPV18. Humanized 2A12 (Hu2A12) retained comparable neutralizing activity against HPV18 infection in various acidic pH settings and in hydrogel formulation with IC50 values of 0.04 to 0.77 ng/ml, indicating that Hu2A12 will be a promising candidate for clinical development as a topical vaginal biopharmaceutical agent against HPV18 infection.

Synthesis of a series of validoxylamine A esters and their biological activities.

BACKGROUND: The worldwide reduction in food production due to pests and diseases is still an important challenge facing today. Validoxylamine A (VAA) is a natural polyhydroxyl compound derived from validamycin, acting as an efficient trehalase inhibitor with insecticidal and antifungal activities. To extend the application and discover green pesticide, a series of ester derivatives were prepared based on VAA as a lead compound. Their biological activities were investigated against three typically agricultural disease, Rhizoctonia solani, Sclerotinia sclerotiorum and Aphis craccivora. RESULTS: This study involved 30 novel validoxylamine A fatty acid esters (VAFAEs) synthesized by Novozym 435 and they were characterized with high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and proton nuclear magnetic resonance (1 H-NMR). Of these 30 derivatives, most compounds showed improved antifungal activity, and 12 novel compounds showed improved insecticidal activity. When reacted with pentadecanoic acid, compound 14 showed the highest inhibitory activity against R. solani [median effective concentration (EC50 ) 0.01 µmol L-1 ], while the EC50 value of VAA was 34.99 µmol L-1 . Furthermore, 21 novel VAFAEs showed higher inhibitory activity against S. sclerotiorum. Validoxylamine A oleic acid ester, compound 21, exhibited the highest insecticidal activity against A. craccivora [median lethal concentration (LC50 ) 39.63 µmol L-1 ], while the LC50 value of Pymetrozine was 50.45 µmol L-1 , a commercialized pesticide against A. craccivora. CONCLUSION: Combining our results, esterification of VAA by introducing different acyl donors was beneficial for the development of new eco-friendly drugs in the field of pesticides.

AAV-mediated in vivo CAR gene therapy for targeting human T-cell leukemia.

Chimeric antigen receptor (CAR) T-cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T-cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV) vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NOD.Cg-Prkdcscid Il2rgem26/Nju tumor mouse model of human T-cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo-generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ß processes of traditional CAR T-cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.

A single-domain antibody inhibits SFTSV and mitigates virus-induced pathogenesis in vivo.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel tick-borne bunyavirus that recently emerged in East Asian countries. SFTS is characterized by high fever, thrombocytopenia, leukopenia, multiorgan failure, and hemorrhage with case fatality rates of 6.3% to 30%. Neither antivirals nor vaccines are available at present. We previously demonstrated that neutralizing antibodies specific for SFTSV glycoprotein (Gn) played a vital role in the survival of patients with SFTS. Nanobodies from camels present unique properties, such as thermostability, high affinity, and low immunogenicity. In the current study, mammalian expressed SFTSV Gn was used to immunize a camel, and functional nanobodies were isolated from the B cell nanobody library constructed from the immunized animal. Clone SNB02 was selected for in-depth analysis for its inhibition of SFTSV replication both in vitro and in vivo. We showed that SNB02 potently inhibited SFTSV infection and prevented thrombocytopenia in a humanized mouse model and is a potential candidate for therapeutics.

The induction and characterization of monoclonal antibodies specific to GP of Ebola virus.

The Ebola virus is highly infectious and characterized by hemorrhagic fever, headache, and so on with a high mortality rate. Currently, there are neither therapeutic drugs or vaccines against the Ebola virus nor fast diagnostic methods for the detection of Ebola virus infection. This study reported the induction and isolation of two monoclonal antibodies that specifically recognized the glycoprotein (GP) and secreted glycoprotein (sGP) of the Ebola virus. Plasmids encoding either GP or sGP were constructed and immunized BALB/c mice, accordingly purified sGP was boosted. The antisera were analyzed for binding activity against sGP protein in enzyme-linked immunosorbent assay (ELISA) and neutralization activity in a pseudotyped virus neutralization assay. A number of reactive clones were isolated and two monoclonal antibodies T231 and T242 were identified to react with both GP and sGP. Western blot and ELISA assays showed that the monoclonal antibodies could react with GP and sGP, respectively. Moreover, they could recognize Ebola pseudovirus by cellular immunochemistry assay. We labeled the monoclonal antibody T231 with biotin and analyzed the competitiveness of the two antibodies by the ELISA test. The results showed that the binding epitopes of the two monoclonal antibodies to sGP were partially overlapped. In summary, two GP-specific mAbs were identified, which will be used to detect the Ebola virus or investigate GP.

Efficient chiral 1H NMR analysis of indoloquinazoline alkaloids phaitanthrin A, cephalanthrin-A and their analogues with a chiral phosphoric acid.

A chiral phosphoric acid promoted enantioselective NMR recognition and determination of indoloquinazoline alkaloids phaitanthrin A, cephalanthrin-A and their analogues was described, which conveniently reveals their optical purities with high accuracy. Besides, pyrazine type tertiary alcohols, cyclic amino alcohols and diamines can also be well resolved under optimal conditions. Importantly, this methodology was further employed in the direct analysis of reaction mixtures of amino acid metal salt catalyzed asymmetric synthesis of phaitanthrin A, providing access to the optimized reaction conditions in high efficiency.

Platelets selectively enhance lymphocyte adhesion on subendothelial matrix under arterial flow conditions.

Platelet adhesion at sites of cardiovascular injury may facilitate leukocyte deposition. We asked if and how platelets enhance lymphocyte adhesion on different subendothelial matrix protein (SEMP)-coated surface at arterial shear stress. Hirudinised whole blood was subjected to an arterial shear rate (500 s(-1)) in a Cone and Plate(let) analyser (CPA) for 5 minutes using plates coated with bovine serum albumin (BSA), collagen, fibrinogen, von Willebrand factor (vWF), or fibronectin. Platelet and lymphocyte adhesion were monitored by CPA and flow cytometry. Exposure of blood to collagen, fibrinogen, and vWF-coated surfaces induced platelet activation. The most marked effect was seen with collagen-coating, which markedly enhanced the adhesion of all lymphocyte subpopulations compared to BSA-coating. Fibrinogen-coating supported both T and NK cell adhesion, while vWF-coated surface only enhanced NK cell deposition. In contrast, fibronectin enhanced neither platelet activation nor lymphocyte adhesion. Moreover, platelets preferentially facilitated adhesion of large CD4(+) and CD8(+) T cells and NK cells, and of small B cells. Enhanced cell adhesion of larger lymphocytes was associated with elevated platelet conjugation and higher lymphocyte expression of PSGL-1, Mac-1, and CD40L. The enhancement of lymphocyte adhesion was totally platelet-dependent, and was abolished in platelet-depleted blood. Moreover, blockade of the platelet adhesion molecules P-selectin, GPIIb/IIIa, and CD40L attenuated platelet-dependent lymphocyte deposition. In conclusion, platelets support lymphocyte adhesion on SEMP-coated surfaces under arterial shear. The enhancement is selective for large T and NK cells and small B cells.

Platelets regulate CD4⁺ T-cell differentiation via multiple chemokines in humans.

Atherosclerosis is an inflammatory and thrombotic disease. Both platelets and lymphocytes play important roles in atherogenesis. However, information on their interaction is limited. We therefore studied how platelets regulate CD4+ T cell activation and differentiation. Human CD4+ T cells and autologous platelets were co-cultured. Platelets concentration-dependently enhanced anti-CD3/CD28-induced IFNγ production by CD4+ T cells, but attenuated their proliferation. Abrogation of heterotypic cell-cell contact partially reversed the enhancement, and supernatant from activated platelets partially mimicked the enhancement, suggesting that platelets exert their effects via both soluble mediators and direct cell-cell contact. Platelets enhanced the production of IL-10 and cytokines characteristic for type 1 T helper (TH1) (IFNγ/TNFα) and TH17 (IL-17) cells, but influenced TH2 cytokines (IL-4/IL-5) little. The cytokine responses were accompanied by enhanced TH1/TH17/TReg differentiation. Using neutralising antibodies and recombinant PF4, RANTES, and TGFß, we found that platelet-derived PF4 and RANTES enhanced both pro- and anti-inflammatory cytokine production, whilst recombinant TGFß enhanced IL-10 but not TNFα production. In conclusion, platelets enhance the differentiation and cytokine production of anti-CD3/CD28-stimulated CD4+ T cells via both multiple chemokines and direct cell-cell contact. Our study provides new insights into the cross-talk between thrombosis and adaptive immunity, and indicates that platelets can enhance T-effector cell development.

Distinct platelet packaging, release, and surface expression of proangiogenic and antiangiogenic factors on different platelet stimuli.

The present study characterized platelet secretion and surface expression of proangiogenic stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) and antiangiogenic PF4 and endostatin on activation. The angiogenic factors presented in randomly distributed granules in resting platelets, which were peripherized on activation. Confocal and immunogold electron microscopy demonstrated that SDF-1α/CXCL12 and PF4/CXCL4 mostly present in different granules. Platelet activation induced marked SDF-1α and endostatin but mild PF4 or no VEGF surface expression. PAR1-activating peptide (PAR1-AP), adenosine diphosphate (via P2Y1/P2Y12), and glycoprotein VI-targeting collagen-related peptide induced massive SDF-1α and VEGF but modest PF4 or no endostatin release. In contrast, PAR4-AP triggered marked PF4 and sole endostatin release but limited SDF-1α or VEGF secretion. Distinct platelet release of SDF-1α and endostatin involved different engagements of intracellular signaling pathways. In conclusion, different platelet stimuli evoke distinct secretion and surface expression of proangiogenic and antiangiogenic factors. PAR1, adenosine diphosphate, and glycoprotein VI stimulation favors proangiogenic, whereas PAR4 promotes antiangiogenic, factor release.

Platelets enhance lymphocyte adhesion and infiltration into arterial thrombus.

Lymphocytes are present in atherosclerotic lesion. We hypothesise that platelets may facilitate lymphocyte infiltration into the arterial wall. Reconstituted human blood or whole blood was perfused through a collagen-coated parallel-plate flow chamber at different shear rates. Adhered platelets markedly enhanced lymphocyte adhesion that increased lymphocyte deposition from 10 ± 3 cells/mm2 of platelet-depleted blood to 38 ± 11 cells/mm2 of platelet-containing blood at the arterial shear rate of 500 s-1. Platelet-dependent lymphocyte adhesion was inhibited by P-selectin, CD40L, and GPIIb/IIIa-blocking agents, suggesting the involvement of multiple adhesion molecules in this heterotypic interaction. Lymphocyte deposition was more marked among T cells, and seen in both small and large cells. B and natural killer cell adhesion was, however, mainly seen in small cells. Platelet-conjugation facilitated lymphocyte adhesion, as suggested by the selective deposition of platelet-conjugated lymphocytes. In a mouse model of arterial thrombosis, FeCl3-induced thrombus formation markedly enhanced lymphocyte adhesion and infiltration into platelet thrombi, which was abolished by GPIIb/IIIa inhibition. In conclusion, platelets support lymphocyte adhesion under arterial flow conditions, which is selective among T cells and involves multiple adhesion molecules. Our data imply that platelets may facilitate the recruitment of circulating lymphocytes at the arterial injured sites.

[Function of TM4SF-integrins complexes in regulating cancer metastasis].

Distant metastasis is the main cause of cancer death. Tetraspanins (transmembrane 4 superfamily, TM4SF) is capable of forming transmembrane complexes with integrin family participating in cell adhesion, migration and tumor metastasis. This review elucidates the structure of tetraspanins and its function in regulating metastasis as form of multimolecular transmembrane complexes with integrin.