Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

Background

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy.

Design and Methods

We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d⁺ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice.

Results

The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d⁺ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d⁺ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules.

Conclusions

Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d⁺ hematologic malignancies.     

Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.     

The natural killer cell cytotoxic function is modulated by HIV-1 accessory proteins

Natural killer (NK) cells' major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu's ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.     

Expression and clinical significance of CD73 and hypoxia-inducible factor-1α in gastric carcinoma

AIM: To investigate the expression of CD73 and hypoxia-inducible factor-1α (HIF-1α) in human gastric carcinoma, and explore their clinical significance and prognostic value. METHODS: CD73 and HIF-1α expressions were detected by immunohistochemistry in consecutive sections of tissue samples from 68 gastric carcinoma patients. The peritumor tissues 2 cm away from the tumor were obtained and served as controls. The presence of CD73 and HIF-1α was analyzed by immunohis-tochemistry using the Envision technique. RESULTS: CD73 and HIF-1α expressions in gastric carcinoma were significantly higher than those in gastric mucosal tissues as control (P < 0.001) and showed a close correlation (Spearman r = 0.390, P = 0.001). Overexpression of CD73 was positively correlated with differentiation of tumor (P = 0.000), histopathology (P = 0.041), depth of invasion (P < 0.001), nodal status (P = 0.003), metastasis (P = 0.013), and the American Joint Committee on Cancer (AJCC) stage (P < 0.001). High expression of HIF-1α was positively correlated with tumor diameter (P = 0.031), depth of invasion (P = 0.022), and AJCC stage (P = 0.035). The overall survival rate was low in the patients with high expression of CD73 (P < 0.001). Moreover, CD73+/HIF-1α+ patients had the worst prognosis (P < 0.001). CD73 expression was proven to be an independent predictor for patients with gastric carcinoma by both multivariate Cox regression analysis (P = 0.021) and receiver operating characteristic curves (P = 0.001).CONCLUSION: CD73 expression correlates closely with HIF-1α expression in gastric carcinoma. CD73 could be an independent prognostic indicator for gastric carcinoma.     

聚乙二醇干扰素α-2b治疗慢性乙型肝炎患者外周血T淋巴细胞亚群和血清细胞因子水平变化*

目的 研究聚乙二醇干扰素α-2b治疗慢性乙型肝炎患者外周血T淋巴细胞亚群和血清细胞因子水平的变化。 方法 2014年1月~2016年1月我院收治的184例慢性乙型肝炎患者,92例接受聚乙二醇干扰素α-2b联合恩替卡韦治疗48 w,另92例只接受恩替卡韦治疗。使用流式细胞仪检测外周血T淋巴细胞亚群,采用放射免疫法检测血清IL-6、INF-ɑ、IL-4,采用酶联免疫吸附法检测血清IL-17、TGF-β1和HBV标记物,采用荧光定量PCR法检测血清HBV DNA、核转录因子RORγt、Foxp3、IL-17mRNA。 结果 在停药随访24 w,联合组与恩替卡韦组血清HBV DNA阴转率分别为86.96%和84.78%（P>0.05）;联合组血清HBeAg阴转率为28.89%（13/45）,与恩替卡韦组的15.22%（7/46）比,无显著性差异（P>0.05）;联合组血清ALT水平为（34.6±11.6） U/L,显著低于恩替卡韦组【（64.6±20.5） U/L,P<0.05】;联合组外周血CD3+、CD4+细胞和CD4+/CD8+比值分别为（75.6±14.5）%、（42.7±10.3）%和（1.4±0.6）,显著高于恩替卡韦组【（66.8±14.4）%、（36.7±8.5）%和（1.0±0.5）,P<0.05】,CD8+细胞百分比为（29.3±7.3） %,显著低于恩替卡韦组【（34.8±8.5） %,P<0.05】,两组NK细胞百分比比较无显著性差异（P>0.05）;治疗前两组血清IL-6、IL-17、IL-4、INF-ɑ、TGF-β1水平比较无显著性差异（P>0.05）,治疗后联合组血清IL-6水平为（6.8±1.2）pg/ml,显著高于恩替卡韦组【（3.5±0.8） pg/ml,P<0.05】,IL-17水平为（0.7±0.3） pg/ml,显著低于恩替卡韦组【（2.8±0.9） pg/ml,P<0.05】,IL-4水平为（1.4±0.5）pg/ml,显著低于恩替卡韦组【（3.8±1.5）pg/ml,P<0.05】,INF-ɑ水平为（4.0±1.3） pg/ml,显著高于恩替卡韦组【（2.6±0.9）pg/ml,P<0.05】,两组血清TGF-β1水平比较无显著性差异（P>0.05）;治疗前两组血清Foxp3、IL-17和RORγt mRNA水平比较无显著性差异（P>0.05）,治疗后联合组血清RORγt水平为（0.86±0.31）,显著低于恩替卡韦组【（1.56±0.43）,P>0.05】,而两组血清Foxp3和IL-17mRNA水平比较无显著性差异（P>0.05）。 结论 聚乙二醇干扰素α-2b能通过调控细胞因子和核转录因子水平,从多个环节调控慢性乙型肝炎患者免疫功能,发挥抗病毒作用。     

Skint-1 is a highly specific, unique selecting component for epidermal T cells

αβ T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1(+) dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1(+) dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.     

CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

OBJECTIVES—To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.
METHODS—Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.
RESULTS—HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1.
CONCLUSION—IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

Keywords: synovial lining cells; hyaluronan, interleukin 1β; transforming growth factor β1     

Construction, expression and characterization of human interferon α2b-（G4S）n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNc(2b and To(1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris.METHODS: Using PCR and molecular clone techniques,the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1(n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex^TM 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex^TM 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins‘ function in vivo.     

肝泡状棘球蚴组织中HIF-1α、VEGFA的表达及对血管生成的作用

目的 探讨HIF-1α、VEGFA在沙鼠肝泡状棘球蚴组织的表达及血管生成过程中的作用。方法 126只沙鼠随机分成空白组(6只)、假手术组(60只)、模型组(60只),采用开腹直视肝脏穿刺法建立泡状棘球蚴动物模型,假手术组接种同体积的PBS,术后第3 d、7 d、14 d、28 d、42 d、56 d、70 d、84 d、98 d、112 d随机取6只沙鼠,取临近病变的边缘区组织及正常肝组织。采用HE观察病理改变;qRT-PCR、原位杂交、免疫组化检测HIF-1α、VEGFA表达,CD34标记微血管进行MVD计数。结果 HE染色根据病理特点将病程分为早期(14 d内)、中期(14 d~56 d)、晚期(56 d后)。模型组泡状棘球蚴组织随感染时间不同,其HIF-1αmRNA随感染时间呈动态改变,术后第14 d其表达量高于空白组、低于假手术组(F=82.732,P< 0.001);术后第42 d、112 d其相对表达量均高于空白组与假手术组(χ²=11.536,χ²=15.189,P< 0.01);模型组术后第14 d VEGFA mRNA相对表达量低于空白组及假手术组(χ²=15.174,P< 0.01);术后第42 d、112 d,VEGFA mRNA表达量均高于空白组与假手术组(χ²=15.158,χ²=15.158,P< 0.01)。模型组术后第14 d、42 d、112 d HIF-1α表达均高于空白组、假手术组(χ²=8.627,χ²=9.000,F=15.690,P< 0.01);模型组术后第14 d、42 d VEGFA表达高于空白组、假手术组(F=11.250, F=70.059,P< 0.001);模型组术后第14 d、42 d、112 d,其MVD-CD34均高于空白组、假手术组(χ²=12.517,P< 0.01,χ²=13.157,P< 0.01;χ²=13.220,P< 0.01)。结论 沙鼠感染肝泡状棘球蚴中期,病变边缘区HIF-1α、VEGFA表达均升高,同时伴有大量微血管生成,可能存在HIF-1α转录因子激活并上调VEGFA的表达,促进肝泡状棘球蚴组织边缘区的血管新生。;     

Advances in refractory ulcerative colitis treatment: A new therapeutic target,Annexin A2

Medical treatment has progressed significantly over the past decade towards achieving and maintaining clinical remission in patients with refractory ulcerative colitis(UC). Proposed mediators of inflammation in UC include pro-inflammatory cytokines such as tumor necrosis factor-α(TNF-α) and interleukin-2, and the cellsurface adhesive molecule integrin α4β7. Conventional therapeutics for active UC include 5-aminosalicylic acid, corticosteroids and purine analogues(azathioprine and 6-mercaptopurine). Patients who fail to respond to conventional therapy are treated with agents such as the calicineurin inhibitors cyclosporine and tacrolimus, the TNF-α inhibitors infliximab or adalimumab, or a neutralizing antibody(vedolizumab) directed against integrin α4β7. These therapeutic agents are of benefit for patients with refractory UC, but are not universally effective. Our recent research on TNF-α shedding demonstrated that inhibition of annexin(ANX) A2 may be a new therapeutic strategy for the prevention of TNF-α shedding during inflammatory bowel disease(IBD) inflammation. In this review, we provide an overview of therapeutic treatments that are effective and currently available for UC patients, as well as some that are likely to be available in the near future. We also propose the potential of ANX A2 as a new molecular target for IBD treatment.     

Regulation of platelet granule exocytosis by S-nitrosylation

Nitric oxide (NO) regulates platelet activation by cGMP-dependent mechanisms and by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules, releasing mediators that regulate interactions between platelets, leukocytes, and endothelial cells. Exocytosis is mediated in part by N-ethylmaleimide-sensitive factor (NSF), an ATPase that disassembles complexes of soluble NSF attachment protein receptors. We now demonstrate that NO inhibits exocytosis of dense granules, lysosomal granules, and alpha-granules from human platelets by S-nitrosylation of NSF. Platelets lacking endothelial NO synthase show increased rolling on venules, increased thrombosis in arterioles, and increased exocytosis in vivo. Regulation of exocytosis is thus a mechanism by which NO regulates thrombosis.     

Estrogen receptor alpha and imprinting of the neonatal mouse ventral prostate by estrogen

Exposure to estrogen in the neonatal period affects prostatic growth and leads to an increased incidence of prostatic intraepithelial neoplasia in later life. The effects of neonatal estrogen are clearly dependent on estrogen receptor (ER) alpha because they do not occur in ERalpha-knockout mice. Because ERalpha is expressed in the stroma, but not in the epithelium, of the adult ventral prostate, the concept of indirect estrogen effects through stromal signaling has been proposed. Here, we show that during the first 4 weeks of life, there are profound and rapid changes in the ER profile in the mouse ventral prostate. ERalpha is abundant in the stroma during week 1, but by week 2 it is exclusively epithelial, and then by week 4, ERalpha is lost and ERbeta is dominant in the prostatic epithelium. The presence of ERalpha is associated with a high proliferation index, and ERbeta is associated with quiescence. Branching morphogenesis was altered in ERalpha-/-, but not in ERbeta-/-, mice. We conclude that imprinting and branching morphogenesis of the ventral prostate are mediated by estrogen acting directly on epithelial and stromal ERalpha during the first 2 weeks of life.     

Regulation of memory CD4 T-cell pool size and function by natural killer T cells in vivo

To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4⁺ T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naïve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-γ expression by memory Th2 cells. These IFN-γ–producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.     

Bone Marrow Cells Ameliorate Liver Fibrosis and Express Albumin after Transplantation in CCl4 -Induced Fibrotic Liver

Background/Aim:

We investigated the effect of bone marrow-derived stem cell (BMSC) transplantation on carbon tetrachloride (CCl₄)-induced liver fibrosis.

Patients and Methods:

BMSCs of green fluorescent protein (GFP) mice were transplanted into 4-week CCl₄ -treated C57BL/6 mice directly to the liver, and the mice were treated for 4 more weeks with CCl₄ (total, 8 weeks). After sacrificing the animals, quantitative data of percentage fibrosis area and the number of cells expressing albumin was obtained. One-way analysis of variance was applied to calculate the significance of the data.

Results:

GFP expressing cells clearly indicated migrated BMSCs with strong expression of albumin after 28 days post-transplantation shown by anti-albumin antibody. Double fluorescent immunohistochemistry showed reduced expression of αSMA on GFP-positive cells. Four weeks after BMSC transplantation, mice had significantly reduced liver fibrosis as compared with that of mice treated with CCl₄ assessed by Sirius red staining.

Conclusion:

Mice with BMSC transplantation with continuous CCl₄ injection had reduced liver fibrosis and a significantly improved expression of albumin compared with mice treated with CCl₄ alone. These findings strengthen the concept of cellular therapy in liver fibrosis.     

Inhibition of amyloid precursor protein processing by beta-secretase through site-directed antibodies

Amyloid-beta peptide (AbetaP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of beta- and gamma-secretases. The beta-secretase APP cleaving enzyme (BACE), which generates the N terminus of AbetaP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AbetaP and, subsequently, plaque formation. The search for specific inhibitors of beta-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP-BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP-BACE interaction at the cell surface may affect amyloid-beta production. We present here a unique approach to inhibit AbetaP production by means of antibodies against the beta-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the beta-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AbetaP formation in these cells.     

Control of feeding behavior in C. elegans by human G protein-coupled receptors permits screening for agonist-expressing bacteria

G protein-coupled receptors (GPCRs) have a key role in many biological processes and are important drug targets for many human diseases. Therefore, understanding the molecular interactions between GPCRs and their ligands would improve drug design. Here, we describe an approach that allows the rapid identification of functional agonists expressed in bacteria. Transgenic Caenorhabditis elegans expressing the human chemokine receptor 5 (CCR5) in nociceptive neurons show avoidance behavior on encounter with the ligand MIP-1α and avoid feeding on Escherichia coli expressing MIP-1α compared with control bacteria. This system allows a simple activity screen, based on the distribution of transgenic worms in a binary food-choice assay, without a requirement for protein purification or tagging. By using this approach, a library of 68 MIP-1α variants was screened, and 13 critical agonist residues involved in CCR5 activation were identified, four of which (T8, A9, N22, and A25) have not been described previously, to our knowledge. Identified residues were subsequently validated in receptor binding assays and by calcium flux assays in mammalian cells. This approach serves not only for structure/function studies as demonstrated, but may be used to facilitate the discovery of agonists within bacterial libraries.