Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C44Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3-10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3-10 and HSC-3 was 1.6 × 10-7 M and 1.7 × 10-7 M, respectively. Furthermore, C44Mab-18 detected CD44v3-10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

EMab-300 Detects Mouse Epidermal Growth Factor Receptor-Expressing Cancer Cell Lines in Flow Cytometry.

Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling, which is an important hallmark of cancer. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. Therefore, sensitive mAbs against mouse EGFR (mEGFR) should be established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa), reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells, using flow cytometry. The kinetic analysis using flow cytometry indicated that the KD of EMab-300 for CHO/mEGFR and NMuMG was 4.3 × 10-8 M and 1.9 × 10-8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

A Novel Anti-CD44 Variant 3 Monoclonal Antibody C44Mab-6 Was Established for Multiple Applications.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3-10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3-10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3-10, COLO205, and HSC-3 was 1.5 × 10-9 M, 6.3 × 10-9 M, and 1.9 × 10-9 M, respectively. C44Mab-6 could detect the CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.

Trismus after partial maxillectomy and radiotherapy: Free flap reconstruction versus prosthetic obturation.

OBJECTIVE: Partial maxillectomy and postoperative radiotherapy are both risk factors associated with trismus. This retrospective study aimed to evaluate the incidence and severity of trismus in patients following partial maxillectomy with or without postoperative radiotherapy and to compare free flap reconstruction and prosthetic obturation. METHODS: A retrospective review of 40 oral cancer patients who underwent partial maxillectomy with or without postoperative radiotherapy was performed. Maximum interincisal distance recorded at least 6 months after surgery was classified according to a revised subjective-objective management-analytical (SOMA) scale and compared between the free flap reconstruction group (n = 12) and the prosthetic obturation group (n = 28). RESULTS: Trismus was observed in 16/40 (40%) patients, and severe trismus was observed in 4/40 (10%) patients. Although no significant difference in trismus grade was observed between the free flap reconstruction and prosthetic obturation groups, both severe trismus and radiation-induced osteonecrosis were only seen in the prosthetic obturation group with postoperative radiotherapy. CONCLUSION: Free flap reconstruction was preferable to prosthetic obturation to avoid severe trismus and radiation-induced osteonecrosis in patients who underwent both partial maxillectomy and postoperative radiotherapy.

High diagnostic accuracy for lymph node metastasis of oral squamous cell carcinoma using PET/CT with a silicon photomultiplier.

OBJECTIVE: The higher sensitivity of the new-generation positron emission tomography/computed tomography (PET/CT) with silicon photomultiplier (SiPM) may increase false-positive rates in detecting metastatic lymph nodes (LNs). This study aimed to clarify the usefulness of the SiPM PET scanner in diagnosing LN metastasis of oral squamous cell carcinoma (SCC). METHODS: We retrospectively reviewed consecutive F-18 fluorodeoxyglucose PET/CT images of 39 SCC patients using SiPM PET and 31 SCC patients using non-SiPM PET. We measured the maximum standardized uptake value (SUVmax) of the LNs on PET images and maximum short-axis diameter on transverse CT images. RESULTS: The sensitivity and specificity of SiPM PET were 86.2% and 95.6%, respectively (cut-off SUVmax, 4.6). The area under the curve (AUC) of SiPM PET (0.977; 95% confidence interval [CI], 0.958-0.995) was significantly higher than that of non-SiPM PET (0.825; 95% CI 0.717-0.934) (P < 0.01). In a size-limited analysis of diameter, the AUC of SiPM PET (≥ 0.96 for all diameters) was significantly higher than that of non-SiPM PET (tended to decrease as the LN diameter decreased) for the diagnosis of LN metastasis by SUVmax. CONCLUSION: SiPM PET had higher diagnostic accuracy for LN metastasis of oral SCC than non-SiPM PET, even for small LN metastasis without increasing false-positives.

Area-Efficient Post-Processing Circuits for Physically Unclonable Function with 2-Mpixel CMOS Image Sensor.

In order to realize image information security starting from the data source, challenge-response (CR) device authentication, based on a Physically Unclonable Function (PUF) with a 2 Mpixel CMOS image sensor (CIS), is studied, in which variation of the transistor in the pixel array is utilized. As each CR pair can be used only once to make the CIS PUF resistant to the modeling attack, CR authentication with CIS can be carried out 4050 times, with basic post-processing to generate the PUF ID. If a larger number of authentications is required, advanced post-processing using Lehmer encoding can be utilized to carry out authentication 14,858 times. According to the PUF performance evaluation, the authentication error rate is less than 0.001 ppm. Furthermore, the area overhead of the CIS chip for the basic and advanced post-processing is only 1% and 2%, respectively, based on a Verilog HDL model circuit design.

Chromobacterium haemolyticum Pneumonia Associated with Near-Drowning and River Water, Japan.

We report a severe case of Chromobacterium haemolyticum pneumonia associated with near-drowning and detail the investigation of the pathogen and river water. Our genomic and environmental investigation demonstrated that river water in a temperate region can be a source of C. haemolyticum causing human infections.

An Over 90 dB Intra-Scene Single-Exposure Dynamic Range CMOS Image Sensor Using a 3.0 µm Triple-Gain Pixel Fabricated in a Standard BSI Process.

To respond to the high demand for high dynamic range imaging suitable for moving objects with few artifacts, we have developed a single-exposure dynamic range image sensor by introducing a triple-gain pixel and a low noise dual-gain readout circuit. The developed 3 µm pixel is capable of having three conversion gains. Introducing a new split-pinned photodiode structure, linear full well reaches 40 ke-. Readout noise under the highest pixel gain condition is 1 e- with a low noise readout circuit. Merging two signals, one with high pixel gain and high analog gain, and the other with low pixel gain and low analog gain, a single exposure dynamic rage (SEHDR) signal is obtained. Using this technology, a 1/2.7", 2M-pixel CMOS image sensor has been developed and characterized. The image sensor also employs an on-chip linearization function, yielding a 16-bit linear signal at 60 fps, and an intra-scene dynamic range of higher than 90 dB was successfully demonstrated. This SEHDR approach inherently mitigates the artifacts from moving objects or time-varying light sources that can appear in the multiple exposure high dynamic range (MEHDR) approach.