76 research outputs found
The chimeric antibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather than via its neutralizing activity
Podoplanin (PDPN/Aggrus/T1α) binds to C-type lectin-like receptor-2 (CLEC-2) and induces platelet aggregation. PDPN is associated with malignant progression, tumor metastasis, and poor prognosis in several types of cancer. Although many anti-human PDPN (hPDPN) monoclonal antibodies (mAbs), such as D2-40 and NZ-1, have been established, these epitopes are limited to the platelet aggregation-stimulating (PLAG) domain (amino acids 29-54) of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-7, which is more sensitive than D2-40 and NZ-1, using the Cancer-specific mAb (CasMab) method. The epitope of LpMab-7 was shown to be entirely different from that of NZ-1, a neutralizing mAb against the PLAG domain according to an inhibition assay and lectin microarray analysis. In the present study, we produced a mouse-human chimeric anti-hPDPN mAb, chLpMab-7. ChLpMab-7 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Furthermore, chLpMab-7 inhibited the growth of hPDPN-expressing tumors in vivo. Although chLpMab-7 recognizes a non-PLAG domain of hPDPN, it suppressed the hematogenous metastasis of hPDPN-expressing tumors. These results indicated that chLpMab-7 suppressed tumor development and hematogenous metastasis in a neutralization-independent manner. In conclusion, hPDPN shows promise as a target in the development of a novel antibody-based therapy
Chimeric Anti-PDPN Antibody ChLpMab-2
Human podoplanin (hPDPN ), a platelet aggregationâinducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to Câtype lectinâlike receptor 2 (CLEC â2). The overexpression of hPDPN is involved in invasion and metastasis. AntiâhPDPN monoclonal antibodies (mAbs) such as NZ â1 have shown antitumor and antimetastatic activities by binding to the platelet aggregationâstimulating (PLAG ) domain of hPDPN . Recently, we developed a novel mouse antiâhPDPN mAb, LpMabâ2, using the cancerâspecific mAb (CasMab) technology. In this study we developed chLpMabâ2, a humanâmouse chimeric antiâhPDPN antibody, derived from LpMabâ2. chLpMabâ2 was produced using fucosyltransferase 8âknockout (KO ) Chinese hamster ovary (CHO )âS cell lines. By flow cytometry, chLpMabâ2 reacted with hPDPN âexpressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMabâ2 exhibited high antibodyâdependent cellular cytotoxicity (ADCC ) against PDPN âexpressing cells, despite its low complementâdependent cytotoxicity. Furthermore, treatment with chLpMabâ2 abolished tumor growth in xenograft models of CHO /hPDPN , indicating that chLpMabâ2 suppressed tumor development via ADCC . In conclusion, chLpMabâ2 could be useful as a novel antibodyâbased therapy against hPDPN âexpressing tumors
Hydrophobicity of Residue 128 of the Stress-Inducible Sigma Factor RpoS Is Critical for Its Activity
RpoS is a key stress-inducible sigma factor that regulates stress resistance genes in Escherichia coli, such as the katE gene encoding catalase HPII and the glg genes encoding glycogen synthesis proteins. Monitoring RpoS activity can provide information on the stress sensitivity of E. coli isolates in clinical settings because the RpoS in these isolates is often mutated. In the present study, we found a novel, missense point mutation at RpoS residue 128 in a clinical Shiga toxin-producing E. coli (STEC) isolate. This mutation caused RpoS dysfunction and increased stress sensitivity. A mutant rpoS was cloned from a clinical STEC that is vulnerable to cold temperature and oxidative stresses. Mutant RpoS protein expression was detected in the clinical isolate, and this RpoS was non-functional according to HPII activity and glycogen levels, which are positively regulated by RpoS and thus are used as indicators for RpoS function. A reporter assay with ÎČ-galactosidase indicated that the dysfunction occurred at the transcriptional level of genes regulated by RpoS. Furthermore, substitution analysis indicated that the hydrophobicity of the amino acid at residue 128 was critical for RpoS activity; the simulation analysis indicated that the amino acids of RNA polymerase (RNAP) that interact with RpoS residue 128 are hydrophobic, suggesting that this hydrophobic interaction is critical for RpoS activity. In addition, substitution of Ile128 to Pro128 abolished RpoS activity, possibly as a result of disruption of the secondsary structure around residue 128, indicating that the structure is also a crucial factor for RpoS activity. These results indicate that only one point mutation at a hydrophobic residue of the complex formed during transcription leads to a critical change in RpoS regulation. Moreover, we found that Ile128 is widely conserved among various bacteria: several bacterial strains have Met128 or Leu128, which are hydrophobic residues, and these strains had similar or higher RpoS activity than that observed with Ile128 in this study. These data indicate that the hydrophobicity of the amino acid at residue 128 is critical for RpoS activity and is consequently important for bacterial survival. Taken together, these findings may contribute to a deeper understanding of protein functional mechanisms and bacterial stress responses
A Case of Malignant Melanoma Diagnosed during Pregnancy who Underwent a Premature Cesarean Section
Observation of Clinical Course with Light-cured Fluoride Releasing Composite Resin UniFil^[â!R] F
Four Cases of Atopic Dermatitis Complicated by Sjogren's Syndrome: Link between Dry Skin and Autoimmune Anhidrosis
We report four adult cases of atopic dermatitis (AD) complicated by Sjogren's syndrome (SS). The patients fulfilled diagnostic criteria for AD and SS. All cases showed persistent itchy dry skin and eczematous lesions complicated by sicca symptoms including dry eyes and dry mouth with moderate joint pain. One case manifested annular erythema and another manifested widespread discoid erythema. To investigate the underlying cause of dry skin in these cases, sweating function was evaluated using a quantitative sudomotor axon reflex test (QSART) in which the axon reflex is stimulated by acetylcholine iontophoresis. The sweating latency time was significantly prolonged in eczematous skin of AD and AD/SS compared to normal controls. Axon reflex (AXR) sweat volume was also significantly reduced in AD (normal and eczematous skin) and AD/SS (normal and eczema) compared to normal control. In contrast, the direct sweat volume of lesional or non-lesional AD skin induced by direct stimulation with acetylcholine was only slightly reduced compared to that in normal controls, but not in SS and lesional skin of AD/SS patients. These results suggest that the impaired sweat response in AD is attributable to an abnormal sudomotor axon reflex, which is accelerated and modulated when complicated by SS resulting in dry skin in the present cases
Design of a liquid cell toward three-dimensional imaging of unidirectionally-aligned particles in solution using X-ray free-electron lasers
X-ray free-electron lasers (XFELs) opened up a possibility for molecular-scale single particle imaging (SPI) without the need for crystallization. In SPI experiments, the orientation of each particle has to be determined from the measured diffraction pattern. Preparing unidirectionally-aligned particles can facilitate the determination of the sample orientation. Here, we show the design principles of a liquid cell for three-dimensional imaging of unidirectionally-aligned particles in solution with XFELs. The liquid cell was designed so that neither incident X-rays nor diffracted X-rays are blocked by the substrate of the liquid cell even at high tilt angles. As a feasibility evaluation, we performed coherent diffraction measurements using the cells with a 1 mm focused XFEL beam. We successfully measured coherent diffraction patterns of a nano-fabricated metal pattern at 701 tilt angle and obtained the reconstructed image by applying iterative phase retrieval. The liquid cell will be usefully applied to molecular-scale SPI by using more tightly focused XFELs. In particular, imaging of membrane proteins embedded in lipid membranes is expected to have an enormous impact on life science and medicine
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