Population-based study of a free rubella-specific antibody testing and immunization campaign in Chiba city in response to the 2018–2019 nationwide rubella outbreak in Japan

Japan has not been able to eliminate rubella; as a result, the large rubella epidemic has occurred. Considering the complicated history of the vaccine policy in Japan, some susceptible populations became infected with rubella, resulting in an outbreak. We conducted a large serosurveillance against rubella in Chiba city after initiating free rubella-specific antibody testing and an immunization campaign during 2018–2019. The total number of rubella specific antibody tests that was conducted in the nationwide campaign and Chiba city original campaign was 8277 and 6104, respectively. The proportion of participants with an antibody titer of ≤1:16 using the hemagglutination inhibition (HI) test was higher in those in their 20–30s. On the contrary, the proportion of participants with an antibody titer of <1:8 using the HI test was higher in men in their 40–50s. This discrepancy possibly reflects the complicated history of the vaccine policy. The number of participants in the nationwide immunization campaign in this city was 1517, whereas that in the Chiba city campaign was 3607. The Chiba city campaign was effective against women in their 20–30s (child-bearing generation); however, the nationwide campaign was not sufficiently effective against men in their 40–50s because many workers were did not visit medical facilities to receive the measles–rubella vaccine

CD271 Defines a Stem Cell-Like Population in Hypopharyngeal Cancer

<div><p>Cancer stem cells contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. Here, we report that the CD271⁺ population sorted from xenotransplanted HPCs possesses an enhanced tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271⁺ cells contained both CD271⁺ and CD271⁻ cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271⁺ cells localized to a perivascular niche near CD34⁺ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, <i>Nanog</i>, and <i>matrix metalloproteinases (MMPs)</i>, which are implicated in cancer invasion, were significantly up-regulated in the CD271⁺ compared to the CD271<b>⁻</b> cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Taken together, our findings indicate that CD271 is a novel marker for HPC stem-like cells and for HPC prognosis.</p></div

Tumor initiation capability of CD271⁺ cells vs, CD271⁻ cells.

<p>Tumor initiation capability of CD271⁺ cells vs, CD271⁻ cells.</p

CD271 expression in HPC clinical specimens and progression of HPC patients.

<p>(A) Representative results of IHC analyses for CD271 in clinical specimens obtained from 83 HPC patients by surgery or biopsy. Immunopositivity appears brown. Specimens with more than 50% positive cells in the entire tumor were classified as “strong” (top), and less than 50%, as “moderate-to-weak” (bottom). (B) Kaplan-Meier analysis for the disease-specific survival rate (3 years) of the “strong” and “moderate-to-weak” groups. *Statistically significant.</p

In vivo Chemoresistance of CD271⁺ cells.

<p>(A) HPCM1- transplanted mice were treated with 7.5 mg/kg of CDDP for a week, then the generated tumors were analyzed by IHC for CD271. Boxed areas are linked to their respective high-magnification images by horizontal arrows. Scale bar: 100 µm. (B) FACS analyses for CD271 in HPCM1 cells derived from mice with or without CDDP treatment. (C) Expression of <i>ABCC2</i>, <i>ABCB5</i>, and <i>ABCG2</i> in CD271⁺ and CD271⁻ cells analyzed by real-time RT-PCR. Transcript levels were normalized to those of <i>GAPDH</i>, and the fold increase of each gene expression levels in CD271⁺ cells versus CD271⁻ cells are shown. Values are the mean±SD of triplicate experiments.</p

In vivo tumorigenicity and differentiation capacity of CD271⁺ cells.

<p>(A) Representative tumors in mice into which the indicated number of cells were transplanted. Red arrowheads indicate CD271⁺ cell injection sites (right side), and blue arrowheads indicate CD271⁻ cell injection sites (left side). Circles and dashed circles indicate transplantation locations resulting in success and failure of tumor formation, respectively. (B) Flow cytometry analysis of the sorted cells (96.8∼99.5% CD271⁺ or CD271⁻ cells). (C) Thirty CD271⁺ cells (right side) and CD271⁻ cells (left side) were transplanted into a mouse, and the generated tumors were resected. Tumor growth was plotted using the average value. (D) Flow cytometry analysis of the CD271 expression of tumor cells resulting from the injection of CD271⁺ cells. Tumor sections were stained with hematoxylin and eosin (H&E). Scale bar: 100 µm.</p

Expression profile of Nanog and MMPs in CD271⁺ and CD271⁻ cells.

<p><i>Nanog</i> expression (A) and <i>MMP1</i>, <i>MMP2</i>, and <i>MMP10</i> expression (B) in CD271⁺ and CD271⁻ cells derived from the three HPC lines were analyzed by real-time RT-PCR. The transcript levels were normalized to those for <i>GAPDH</i>, and the fold change in the MMP expression level in CD271⁺ cells versus CD271⁻ cells was calculated for each sample. Values are the mean±SD of triplicate experiments.</p

Distribution of CD271⁺ cells in HPC.

<p>(A) Cells derived from three HPC xenotransplanted lines were stained for CD271 and analyzed by FACS. (B) Tumor tissues dissected from mice transplanted with the three HPC lines were analyzed for CD271 expression by IHC. Immunopositivity appears brown (a, b, and c). High-magnification images are linked to their respective boxed areas by arrows. Scale bar: 100 µm. Red line indicates the border of the tumor and stroma (d). Red line indicates the border of a CD271⁺ cell cluster and CD271⁻ cells, and red and blue arrowheads show CD271⁺ cells and CD271⁻ cells, respectively (e). (C) Representative results of IHC for CD271 in clinical specimens. Normal mucosa (a), carcinoma <i>in situ</i>. (b). Arrowheads indicate an invasive front, and strongly positive CD271 expression (c, d). (D) IHC for CD34 with New Fuchsin substrate (a), and double staining for CD34 (New Fuchsin) and CD271 (DAB) (b-e). CD34 immunopositivity appears red. Insets show high-magnification images of the boxed areas. Red arrowheads indicate CD34-positive microvessels, and brown arrowheads indicate CD271⁺ cells. (E) Sphere-forming cells of HPCM1, and elevated CD271 expression in the FACS analysis. Scale bar: 25 µm. (F) IHC for the double staining of Ki-67 (New Fuchsin) and CD271 (DAB). Ki-67 immunopositivity appears red in the nucleus. Red arrowheads indicate Ki-67-positive cells. Scale bar: 100 µm.</p

Gene expression of CD271 and Nanog in clinical specimens of HPC.

<p>(A) Kaplan-Meier analysis for the relapse-free survival rate according to the <i>CD271</i> mRNA level, in tumor tissues derived from 28 HPC patients by surgery. Real-time RT-PCR analyses were conducted, normalized to the expression of <i>GAPDH</i>, and the fold-change in <i>CD271</i> expression levels in the tumor versus normal mucosa was calculated. The cut-off value for the <i>CD271</i>-expression index was 1.5. *Statistically significant. (B) <i>Nanog</i> expression was examined by real-time RT-PCR. Cut-off value for the <i>Nanog</i> expression index was 1.5. *Statistically significant. Closed circles, CD271 high; open circles, CD271 low.</p