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.

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.

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.

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.