Intensive Immunofluorescence Staining Methods for Low Expression Protein: Detection of Intestinal Stem Cell Marker LGR5

Leucine-rich repeat-containing G-protein coupled receptor 5, or LGR5, is a molecule that recognizes stem cells in multiple organs and also in colon cancer. Previously, we have developed monoclonal antibodies specific to LGR5 protein that can be used for immunofluorescence staining, but because a very low level of LGR5 protein is expressed, the visualization technique needed to be enhanced. To develop procedures to detect LGR5 protein in various specimens by immunofluorescence staining, we evaluated the Alexa-labeled streptavidin biotin (LSAB), the Qdot, and the tyramide methods. The detection sensitivity was highest in the tyramide method followed by the Qdot method, whereas subcellular localization of the protein was most clear in the Qdot method, because the Qdot method gave a high S/N ratio that could show a low background. Thus, the tyramide method is superior to the Q-dot method for intensifying the signal of a low expression protein, and the Qdot method is superior to the tyramide method for identifying the subcellular localization of the target protein. The results of the present study will be helpful in providing more insight into the pathophysiological roles of LGR5-positive cancer stem cells and in developing therapeutic approaches for targeting cancer stem cells.     

Development of an Immunochromatographic Assay Kit Using Fluorescent Silica Nanoparticles for Rapid Diagnosis of Acanthamoeba Keratitis

We developed an immunochromatographic assay kit that uses fluorescent silica nanoparticles bound to anti-Acanthamoeba antibodies (fluorescent immunochromatographic assay [FICGA]) and evaluated its efficacy for the detection of Acanthamoeba and diagnosis of Acanthamoeba keratitis (AK). The sensitivity of the FICGA kit was evaluated using samples of Acanthamoeba trophozoites and cysts diluted to various concentrations. A conventional immunochromatographic assay kit with latex labels (LICGA) was also evaluated to determine its sensitivity in detecting Acanthamoeba trophozoites. To check for cross-reactivity, the FICGA was performed by using samples of other common causative pathogens of infectious keratitis, such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans. Corneal scrapings from patients with suspected AK were tested with the FICGA kit to detect the presence of Acanthamoeba, and the results were compared with those of real-time PCR. The FICGA kit detected organisms at concentrations as low as 5 trophozoites or 40 cysts per sample. There were no cross-reactivities with other pathogens. The FICGA was approximately 20 times more sensitive than the LICGA for the detection of Acanthamoeba trophozoites. The FICGA kit yielded positive results for all 10 patients, which corresponded well with the real-time PCR results. The FICGA kit demonstrated high sensitivity for the detection of Acanthamoeba and may be useful for the diagnosis of AK.     

The short-term outcomes of robotic sphincter-preserving surgery for rectal cancer: comparison with open and laparoscopic surgery using a propensity score analysis

Purpose

The aim of this study is to clarify the short-term outcomes of robotic sphincter-preserving surgery for rectal cancer in a retrospective study.

Methods

The short-term outcomes of robotic sphincter-preserving surgery (n?=?130) were retrospectively compared to open (n?=?234) and laparoscopic surgery (n?=?318) by a propensity score analysis.

Results

Robotic surgery was performed more frequently for patients with lower rectal cancer (55%) than open (30%, p?<?0.0001) or laparoscopic surgery (36%, p?<?0.0001). None of the robotic surgery cases were converted to open surgery. After propensity score matching, robotic surgery was found to be associated with a longer operation time (342 vs. 230 min, p?<?0.0001) and less blood loss (7 vs. 420 mL, p?<?0.0001) than open surgery. The overall complication rate of robotic surgery was lower than that of open surgery (13 vs. 28%, p?=?0.032). Robotic surgery was associated with a lower incidence of surgical site infections (SSIs) than laparoscopic surgery (0 vs. 7%, p?=?0.028). There were no cases of anastomotic leakage after robotic surgery. The circumferential resection margin was involved in 0.8% of the patients who underwent robotic surgery; the incidence did not differ among the treatment groups.

Conclusions

Although robotic surgery for rectal cancer was associated with a longer operation time, it was associated with a very low incidence of SSIs. The degree of safety was comparable to both open and laparoscopic surgery.

     

A Cold Agglutinin: Om

An IgM kappa cold agglutinin reacted with cord blood as well as with adult red blood cells (RBC). The agglutinin reacted strongly with papainized and neuraminidase (RDE)-treated RBC. It resembled anti-Me cold agglutinin in these serological findings. However, the activity of this agglutinin against RBC was markedly decreased in the presence of human milk. The data suggest that the agglutinin is directed against a new glycolipid antigen common to human RBC, termed Om antigen.     

Determinants of participation in prostate cancer screening: A simple analytical framework to account for healthy‐user bias

In Japan at present, fecal occult blood testing (FOBT) is recommended for cancer screening while routine population‐based prostate‐specific antigen (PSA) screening is not. In future it may be necessary to increase participation in the former and decrease it in the latter. Our objectives were to explore determinants of PSA‐screening participation while simultaneously taking into account factors associated with FOBT. Data were gathered from a cross‐sectional study conducted with random sampling of 6191 adults in Osaka city in 2011. Of 3244 subjects (return rate 52.4%), 936 men aged 40–64 years were analyzed using log‐binomial regression to explore factors related to PSA‐screening participation within 1 year. Only responders for cancer screening, defined as men who participated in either FOBT or PSA‐testing, were used as main study subjects. Men who were older (prevalence ratio [PR] [95% confidence interval (CI)] = 2.17 [1.43, 3.28] for 60–64 years compared with 40–49 years), had technical or junior college education (PR [95% CI] = 1.76 [1.19, 2.59] compared with men with high school or less) and followed doctors' recommendations (PR [95% CI] = 1.50 [1.00, 2.26]) were significantly more likely to have PSA‐screening after multiple variable adjustment among cancer‐screening responders. Attenuation in PR of hypothesized common factors was observed among cancer‐screening responders compared with the usual approach (among total subjects). Using the analytical framework to account for healthy‐user bias, we found three factors related to participation in PSA‐screening with attenuated association of common factors. This approach may provide a more sophisticated interpretation of participation in various screenings with different levels of recommendation.