A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells.

Human epithelial growth factor receptor-2 (HER2) plays an oncogenic role in numerous tumors, including breast, gastric, and various other solid tumors. While anti-HER2 therapies are approved for the treatment of HER2-positive tumors, a necessity persists for creating novel HER2-targeted agents to resolve therapeutic resistance. Utilizing a synthetic nanobody library and affinity maturation, our study identified four anti-HER2 nanobodies that exhibited high affinity and specificity. These nanobodies recognized three distinct epitopes of HER2-ECD. Additionally, we constructed VHH-Fc and discovered that they facilitated superior internalization and showed moderate growth inhibition. Compared to the combination of trastuzumab and pertuzumab, the VHH-Fc combos or their combination with trastuzumab demonstrated greater or comparable antitumor activity in both ligand-independent and ligand-driven tumors. Most remarkably, A9B5-Fc, which targeted domain I of HER2-ECD, displayed significantly enhanced trastuzumab-synergistic antitumor efficacy compared to pertuzumab under trastuzumab-resistant conditions. Our findings offer anti-HER2 nanobodies with high affinity and non-overlapping epitope recognition. The novel nanobody-based HER2-targeted antibody, A9B5-Fc, binding to HER2-ECD I, mediates promising receptor internalization. It possesses the potential to serve as a potent synergistic partner with trastuzumab, contributing to overcoming acquired resistance.

miR­3613­3p/MAP3K2/p38/caspase­3 pathway regulates the heat­stress­induced apoptosis of endothelial cells.

Previous studies have identified microRNA (miRNA/miR)­3613­3p as a heat stress (HS)­related miRNA in endothelial cells that can lead to apoptosis. However, the mechanism underlying the miR­3613­3p­mediated apoptosis of HS­exposed endothelial cells remains unclear. In the present study, western blot analysis and reverse transcription­quantitative PCR were used to determine protein and miRNA expression levels, respectively. Annexin V­fluorescein isothiocyanate/propidium iodide staining, caspase­3 activity measurements and DNA fragmentation assays were performed to detect apoptosis. To evaluate whether mitogen­activated protein kinase kinase kinase 2 (MAP3K2) was a direct target of miR­3613­3p, a luciferase reporter assay was performed. In addition, transient transfection was used to carry out loss­ and gain­of­function experiments. The results revealed that miR­3613­3p expression was reduced in human umbilical vein endothelial cells (HUVECs) following HS, which led to apoptosis. Mechanistically, following HS, a decrease in miR­3613­3p binding to the 3'­untranslated region of MAP3K2 directly upregulated its expression, and the downstream p38 and caspase­3 pathways, thereby leading to apoptosis. Taken together, the results of the present study demonstrated that HS suppressed miR­3613­3p expression, which activated the MAP3K2/p38/caspase­3 pathway, leading to the apoptosis of HUVECs. In conclusion, the miR­3613­3p/MAP3K2/p38/caspase­3 pathway may serve an indispensable role in regulating the progression of apoptosis, indicating a regulatory role of miR­3613­3p in the pathophysiology of HS­exposed endothelial cells.

Analysis of Syk/PECAM-1 signaling pathway in low shear stress induced atherosclerosis based on ultrasound imaging.

BACKGROUND AND OBJECTIVE: Low shear stress (LSS) has been demonstrated to be involved in function of vascular endothelial cells. Here we tested the hypothesis that activation of Syk played an important in LSS-induced atherosclerosis via PECAM-1 signaling pathway. METHODS: In vitro, primary human umbilical vein endothelial cells (HUVECs) were stimulated with parallel plate flow chamber system for 12h under normal shear stress (NSS, 15dyne/cm2), LSS (5dyne/cm2) and high shear stress (HSS, 25dyne/cm2), respectively, followed by inflammatory response analysis. In vivo, animal models of rat fed atherogenic diet were treated with LSS stimulation by constricting abdominal aorta with a blunted needle (0.6mm in diameter). The spatial distribution of WSS of blood vessels was generated by WSS quantitative analysis software through color Doppler flow imaging with a high-frequency small animal ultrasound system. Small molecule R406, a well-demonstrated Syk inhibitor, was applied to animals as well as HUVEC cells. RESULTS: In vivo, comparison with the control group was performed, the mean value of WSS distribution of blood vessels was lower in LSS model rat. LSS promoted expression of phosphorylated PECAM-1 (p-PECAM-1) and Syk in LSS model rats. Compared with control group, endothelial cells of the abdominal aorta become less elongated and more polygonal in LSS group, and had a slender shape in LSS with R406 group. In vitro, LSS increased the expression of p-PECAM-1, Syk and NF-κB in HUVECs. Inhibition of Syk attenuated LSS-induced inflammatory response. CONCLUSIONS: Activation of Syk resulted in LSS-induced inflammatory response via PECAM-1 signaling pathway both in vitro and in vivo. Syk might be involved in morphological changes of ECs under the influence of LSS.

Overexpression of arginine transporter CAT-1 is associated with accumulation of L-arginine and cell growth in human colorectal cancer tissue.

We previously showed that L-arginine (Arg) accumulates in colorectal cancer tissues. The aim of this study was to investigate the mechanism by which Arg accumulates and determine its biological significance. The concentration of Arg and Citrulline (Cit) in sera and tumor tissues from colorectal cancer (CRC) patients was analyzed by high-performance liquid chromatography (HPLC). The expression of Arg transporters was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis of tissue microarray. We also transfected the colon cancer cell line HCT-116 with siRNA specific for the Arg transporter CAT-1 and measured the induction of apoptosis by flow cytometry and cell proliferation by MTT assay. Consistent with our previous results, serum Arg and Cit concentrations in colorectal cancer patients were significantly lower than those in normal volunteers, while Arg and Cit concentrations in colorectal cancer tissues were significantly higher than in matched adjacent normal colon tissues. Quantitative RT-PCR showed that the CAT-1 gene was highly overexpressed in 70.5% of colorectal cancer tissue samples relative to adjacent normal colon tissues in all 122 patients with colorectal cancer. Immunohistochemical analysis of tissue microarray confirmed that the expression of CAT-1 was higher in all 25 colorectal cancer tissues tested. CAT-1 siRNA significantly induced apoptosis of HCT-116 cells and subsequently inhibited cell growth by 20-50%. Our findings indicate that accumulation of L-Arg and Cit and cell growth in colorectal cancer tissues is associated with over-expression of the Arg transporter gene CAT-1. Our results may be useful for the development of molecular diagnostic tools and targeted therapy for colorectal cancer.

Covert laparoscopic cholecystectomy:a new minimally invasive technique.

To further improve our developed transumbilical endoscopic surgery (TUES), we developed a completely covert laparoscopic cholecystectomy (LC). Twelve cases of LC were recruited for this new approach. First, a 10-mm trocar was placed above the umbilicus for inserting the laparoscope. Two 5-mm trocars were then placed near the right and left ends of the superior margin of the suprapubic hair. After the 5-mm 30° laparoscope was shifted to the left suprapubic trocar, the harmonic scalper, electric hook, and grasper were inserted either through the 10-mm umbilical trocar or through the right suprapubic trocar. All gallbladders were successfully removed without intraoperative complications. The mean operating time was 28.5 ± 5.7 min (range 20-45 min). All patients felt well after surgery and did not need postoperative analgesia. They resumed free oral intake 6h after the procedure. All patients were satisfied with the appearance of the incisions, which were completely hidden in the umbilicus and suprapubic hair. The approach we developed has overcome both external instrument interference around the umbilicus and the loss of triangulation in the operative field. It is relatively simpler than a typical TUES and offers better cosmetic results.

Syk protein tyrosine kinase involves PECAM-1 signaling through tandem immunotyrosine inhibitory motifs in human THP-1 macrophages.

Although recent evidence supports a functional relationship between platelet endothelial cell adhesion molecule (PECAM-1) and Syk tyrosine kinase, little is known about the interaction of Syk with PECAM-1. We report that down-regulation of Syk inhibits the spreading of human THP-1 macrophage cells. Moreover, our data indicate that Syk binds PECAM-1 through its immune tyrosine-based inhibitory motif (ITIM), and dual phosphorylation of the ITIM domain of PECAM-1 leads to activation of Syk. Our results indicate that the distance between the phosphotyrosines could be up to 22 amino acids in length, depending on the conformational flexibility, and that the dual ITIM tyrosine motifs of PECAM-1 facilitate immunoreceptor tyrosine-based activation motif-like signaling. The preferential binding of PECAM-1 to Src homology region 2 domain-containing phosphatase-2 or Syk may depend on their relative affinities, and could provide a mechanism by which signal transduction from PECAM-1 is internally regulated by both positive and negative signaling enzymes.

Gas-free single-port transumbilical laparoscopic cholecystolithotomy: preliminary report on eight cases.

BACKGROUND: To explore the feasibility and safety of gas-free single-port transumbilical laparoscopic cholecystolithotomy. METHODS: An incision of 1.5-2.0 cm was made through all layers of the umbilicus, and a specially designed silicone plug with three 5-mm ports was inserted. The surgical space was created by lifting the right abdominal wall with an abdominal suspension set. A laparoscope, S-type dissector, grasper,electric needle, and needle-holder were used to perform a cholecystolithotomy. The procedure was performed in 8 patients with gall stones. RESULTS: All stones were successfully removed. No postoperative complications, such as bleeding or bile leakage, occurred. The operative time was 45-120 minutes (mean 77.5 ± 24). The mean length of hospital stay was 2 days, and no postoperartive analgesics were used. There were no visible scars on the abdominal wall. CONCLUSIONS: The gas-free single-port transumbilical laparoscopic approach was safe and feasible for cholecystolithotomy. This approach expands the applications of laparoendoscopic single-site surgery and avoids the use of highly concentrated CO(2) in the body and its potential side effects.

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia.

Simultaneous determination of l-citrulline and l-arginine in plasma by high performance liquid chromatography.

OBJECTIVE: To develop a method for simultaneously determining l-citrulline and L-arginine levels in plasma using RP-HPLC with ultraviolet detection. DESIGN AND METHODS: Plasma samples were deproteinized by trichloroacetic acid and heat. Phenyl-isothiocyanate (PITC) solution was used as derivatization reagent and a gradient elution was carried out. RESULTS: The linearity for L-citrulline and L-arginine ranged from 0 to at least 1000 micromol/L. R(2) values were above 0.9999 for both. LODs for L-citrulline and L-arginine were 0.0201 micromol/L and 0.0476 micromol/L, respectively, while LOQs were 0.240 micromol/L and 0.448 micromol/L, respectively. Intra- and inter-day CVs were less than 3.40% and 7.2%, respectively. The average recovery was from 86.22% to 118.9%. L-citrulline and L-arginine concentrations in healthy controls were 60.77+/-9.18 micromol/L and 58.19+/-16.43 micromol/L, respectively. CONCLUSION: This approach offers a reliable, efficient analytical platform for the simultaneous determination of citrulline and arginine levels in plasma.

Characterization of the purification and primary culture of adult canine myoblasts in vitro.

The aim of the present study was to explore a simple and efficient procedure to culture and purify adult canine myoblasts in vitro. Muscle from adult Beagle canines was isolated and harvested by mechanical decomposition and two-step enzyme digestion. The cells were then purified by a method involving differential adherent velocity and flow cytometry. The morphologic properties and growth states of the cells were observed, and the cell phenotype was characterized by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Approximately 98% of the adult canine muscle cells were positive for CD56 by flow cytometry. These cells expressed MyoD and myogenin as determined by RT-PCR, and desmin as determined by immunocytochemistry. Our method proved to be convenient and practical with a high probability of success. Studies of cell therapy using highly purified myoblasts may now be broadly applied to canine models of human muscle and non-muscle diseases.

Myoblast therapy: from bench to bedside.

Myoblasts are defined as stem cells containing skeletal muscle cell precursors. A decade of experimental work has revealed many properties of myoblasts, including the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity. Early phase clinical trials also showed that myoblast-based therapy is a promising approach for many intractable clinical conditions, including both muscle-related and non-muscle-related diseases. The potential application of myoblast therapy may be in the treatment of genetic muscle diseases, cardiomyocyte damaged heart diseases, and urinary incontinence. This review will provide an overview of myoblast biology, along with discussion of the potential application in clinical medicine. In addition, problems in current myoblast therapy and possible future improvements will be addressed.

[Application of flow cytometry to the evaluation of semen quality].

OBJECTIVE: To evaluate the role of flow cytometry in semen assessment. METHODS: Semen samples from 104 infertile male patients (as the case group) and 10 fertilized donors (as the control group) were analyzed for the volume of ejaculate and sperm concentration, motility and atypical morphology by computer-assisted semen analysis (CASA), the viability, chromatin structure and mitochondrial membrane potential (MMP) of the sperm stained by SYBR-14/PI, AO and JC-1 respectively, and assessed with flow cytometry. The results were analyzed through SAS software. RESULTS: U tests indicated that the semen from the infertile patients had not only lower concentration (U = 2.51, P = 0.0143), lower motility (U = 3.44, P = 0.001) and higher rate of atypical morphology (U = -5.88, P < 0.0001), but also lower viability (U = 4.72, P < 0.0001), MMP (U = -2.53, P = 0.0309), and chromatin integrity (alpha t: U = -3.82, P = 0.0003; SD alpha t: U = -3.98, P = 0.0001; COMP alpha t: U = -3.57, P = 0.0005). The multiple stepwise regression analysis revealed that sperm motility was positively correlated with sperm membrane integrity (t = 1.66, P = 0.1016), sperm MMP (t = 3.33, P = 0.0014) and sperm acrosome integrity (t = 3.24, P = 0.0019), while sperm MMP was negatively correlated with the rates of sperm neck and tail defects (t = -3.44, P = 0.001). CONCLUSION: Flow cytometry plays a significant role in the evaluation of the quality of human sperm, and can be adopted as a useful tool in the diagnosis and treatment of male infertility.

Specific tolerance induction of allo-K(b)-skin grafts by FK506 in the CD8-depleted H-2(k) recipients required low amounts of K(b)-antigen.

MHC class I allo-grafts can be directly rejected by recipient CD8 T cells and be indirectly rejected by recipient CD4 T cells. Although the experimental results using the bm mutant and C57BL/6 mice indicated that CD4-mediated rejection of class I-disparate grafts is a relatively weak process and is expected to be more sensitive to additional exogenous immunosuppression, it is unclear that whether this mechanism can be used for inducing a specific tolerance of class I disparate grafts. In this study, we hypothesize that a short course of FK506 may induce a specific tolerance of class I-disparate skin grafts in the CD8-depleted recipients. K(b)-transgenic C3H mice, Tg.H-2 K(b)-1 and Tg.H-2 K(b)-2 mice that express high copies and low copies of K(b)-antigen respectively were used as donors. Wild type C3H mice (H-2(k)) in which either CD4 or CD8 T cells were depleted by administration of anti-CD4 or CD8 monoclonal antibody (mAb) were used as recipients. Results showed that FK506 promoted longer survival of allo-K(b) skin grafts in CD8-depleted C3H mice than in CD4-depleted C3H mice. Graft survival from Tg.H-2 K(b)-2 mice was significantly longer than Tg.H-2 K(b)-1 mice. A short course of FK506 induced long-term survival of skin grafts from Tg.H-2K(b)-2 mice, but not from Tg.H-2K(b)-1 mice in CD8-depleted C3H recipients, even after FK506 was stopped. These mice also accepted grafts of Tg.H-2K(b)-1 mice when challenged with skin grafts from Tg.H-2K(b)-1 mice, but promptly rejected third party skin grafts from BALB/c (H-2(d)) mice. T cells from K(b)-tolerant C3H mice did not respond to allo-K(b)-antigen in in vitro assays of mixed lymphocyte culture and cell-mediated cytotoxicity. In conclusion we found that a short course of FK506 treatment and low amounts of K(b)-antigen induced a K(b)-specific tolerance in the CD8-depleted recipients, and this tolerance maintained even after withdrawing the anti-CD8 mAb treatment.

Adoptive immunotherapy of advanced tumors with CD62 L-selectin(low) tumor-sensitized T lymphocytes following ex vivo hyperexpansion.

Tumor-draining lymph nodes (TDLN) contain sensitized T cells with the phenotype CD62 L-selectin(low) (CD62L(low)) that can be activated ex vivo with anti-CD3 mAb and IL-2 to acquire potent dose-dependent effector function manifested upon adoptive transfer to secondary tumor-bearing hosts. In this study advanced tumor models were used as a stringent comparison of efficacy for the CD62L(low) subset, comprising 5-7% of the TDLN cells, vs the total population of TDLN cells following culture in high dose IL-2 (100 U/ml). During the 9-day activation period the total number of CD8+ T cells increased 1500-fold, with equivalent proliferation in the CD62L(low) vs the total TDLN cell cultures. Adoptive transfer of activated CD62L(low) cells eliminated 14-day pulmonary metastases and cured 10-day s.c. tumors, whereas transfer of maximally tolerated numbers of total TDLN cells was not therapeutic. Despite their propagation in a high concentration of IL-2, the hyperexpanded CD62L(low) subset of TDLN cells functioned in vivo without exogenous IL-2, and CD8+ T cells demonstrated relative helper independence. Moreover, the anti-tumor response was specific for the sensitizing tumor, and long term memory was established. The facile enrichment of tumor-reactive TDLN T cells, based on the CD62L(low) phenotype, circumvents the need for prior knowledge of the relevant tumor Ags. Coupling the isolation of pre-effector T cells with rapid ex vivo expansion to >3 logs could overcome some of the shortcomings of active immunotherapy or in vivo cytokine treatment, where selective robust expansion of effector cells has been difficult to achieve.