Label‐free multiphoton excitation imaging as a promising diagnostic tool for breast cancer

Histopathological diagnosis is the ultimate method of attaining the final diagnosis; however, the observation range is limited to the two‐dimensional plane, and it requires thin slicing of the tissue, which limits diagnostic information. To seek solutions for these problems, we proposed a novel imaging‐based histopathological examination. We used the multiphoton excitation microscopy (MPM) technique to establish a method for visualizing unfixed/unstained human breast tissues. Under near‐infrared ray excitation, fresh human breast tissues emitted fluorescent signals with three major peaks, which enabled visualizing the breast tissue morphology without any fixation or dye staining. Our study using human breast tissue samples from 32 patients indicated that experienced pathologists can estimate normal or cancerous lesions using only these MPM images with a kappa coefficient of 1.0. Moreover, we developed an image classification algorithm with artificial intelligence that enabled us to automatically define cancer cells in small areas with a high sensitivity of ≥0.942. Taken together, label‐free MPM imaging is a promising method for the real‐time automatic diagnosis of breast cancer.     

Miller Fisher Syndrome Following Vaccination against SARS-CoV-2

After BNT162b2 messenger ribonucleic acid (mRNA) coronavirus disease 2019 (COVID-19) vaccination, a 30-year-old man developed bilateral lateral gaze palsy, diplopia, absent tendon reflexes, and ataxic gait. Serum anti-GQ1b and anti-GT1a immunoglobulin G (IgG) antibodies were strongly positive. Based on those findings, he was diagnosed with Miller Fisher syndrome (MFS). Intravenous immunoglobulin therapy was administered, and his symptoms fully recovered within approximately 3 months. To the best of our knowledge, this is the first report to describe the development of MFS after COVID-19 mRNA vaccination.     

Expression of parathyroid hormone/parathyroid hormone-related peptide receptor 1 in normal and diseased bladder detrusor muscles: a clinico-pathological study

Background

To investigate the expression of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor 1 (PTH1R) in clinical specimens of normal and diseased bladders. PTHrP is a unique stretch-induced endogenous detrusor relaxant that functions via PTH1R. We hypothesized that suppression of this axis could be involved in the pathogenesis of bladder disease.

Methods

PTH1R expression in clinical samples was examined by immunohistochemistry. Normal kidney tissue from a patient with renal cancer and bladder specimens from patients undergoing ureteral reimplantation for vesicoureteral reflux or partial cystectomy for urachal cyst were examined as normal control organs. These were compared with 13 diseased bladder specimens from patients undergoing bladder augmentation. The augmentation patients ranged from 8 to 31 years old (median 15 years), including 9 males and 4 females. Seven patients had spinal disorders, 3 had posterior urethral valves and 3 non-neurogenic neurogenic bladders (Hinman syndrome).

Results

Renal tubules, detrusor muscle and blood vessels in normal control bladders stained positive for PTH1R. According to preoperative urodynamic studies of augmentation patients, the median percent bladder capacity compared with the age-standard was 43.6% (range 1.5–86.6%), median intravesical pressure at maximal capacity was 30 cmH₂O (range 10–107 cmH₂O), and median compliance was 3.93 ml/cmH₂O (range 0.05–30.3 ml/cmH₂O). Detrusor overactivity was observed in five cases (38.5%). All augmented bladders showed negative stainings in PTH1R expression in the detrusor tissue, but positive staining of blood vessels in majority of the cases.

Conclusions

Downregulation of PTH1R may be involved in the pathogenesis of human end-stage bladder disease requiring augmentation.     

Negative regulation of cell motile and invasive activities by lysophosphatidic acid receptor-3 in colon cancer HCT116 cells

Lysophosphatidic acid (LPA) mediates a wide range of biological responses with G protein-coupled transmembrane receptors (LPA receptors). So far, at least six types of LPA receptors (LPA receptor-1 (LPA₁) to LPA₆) have been identified. Recently, it has been reported that LPA₃ indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA₃ on cell migration ability of colon cancer cells, we generated LPA receptor-3 (LPAR3) knockdown (HCT-sh3-3) cells from HCT116 and measured cell motile and invasion activities. In motility assay with a cell culture insert, HCT-sh3-3 cells showed significantly high cell motile activity, compared with control cells. For invasion assay, the filter was coated with Matrigel. The invasive activity of HCT-sh3-3 cells was significantly higher than that of control cells. Furthermore, we also examined the effects of LPAR3 knockdown on the interaction between colon cancer cells and endothelial F-2 cells. When F-2 cells were cultured with serum-free DMEM containing a supernatant from HCT-sh3-3 cells, the cell growth rate and migration activity of F-2 cells were significantly stimulated, associating with the elevated expressions of vascular endothelial growth factor (VEGF)-A and VEGF-C genes in HCT-sh3-3 cells. These results suggest that LPA₃ may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells.     

Intracellular Tracking of Single-plasmid DNA Particles After Delivery by Electroporation

Electroporation is a physical method of transferring molecules into cells and tissues. It takes advantage of the transient permeabilization of the cell membrane induced by electric field pulses, which gives hydrophilic molecules access to the cytoplasm. This method offers high transfer efficiency for small molecules that freely diffuse through electrically permeabilized membranes. Larger molecules, such as plasmid DNA, face several barriers (plasma membrane, cytoplasmic crowding, and nuclear envelope), which reduce transfection efficiency and engender a complex mechanism of transfer. Our work provides insight into the way electrotransferred DNA crosses the cytoplasm to reach the nucleus. For this purpose, single-particle tracking experiments of fluorescently labeled DNA were performed. Investigations were focused on the involvement of the cytoskeleton using drugs disrupting or stabilizing actin and tubulin filaments as the two relevant cellular networks for particle transport. The analysis of 315 movies (~4,000 trajectories) reveals that DNA is actively transported through the cytoskeleton. The large number of events allows a statistical quantification of the DNA motion kinetics inside the cell. Disruption of both filament types reduces occurrence and velocities of active transport and displacements of DNA particles. Interestingly, stabilization of both networks does not enhance DNA transport.     

HMG-CoA reductase inhibitor cerivastatin prolonged rat cardiac allograft survival by blocking intercellular signals.

BACKGROUND: The development of atherosclerotic cardiovascular complications caused by hyperlipidemia is a common and serious problem for long-term survivors of organ transplantation. However, adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are involved in allograft rejection, possibly by providing costimulatory signals. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor cerivastatin has been shown to suppress ICAM-1 expression in acute inflammatory responses. METHODS: In this study, we evaluated the immunosuppressive effects of cerivastatin in rat cardiac allografts. The hearts of Fischer rats were transplanted heterotopically into Lewis rats. Cerivastatin (2 mg/kg) was administrated intraperitoneally to recipients for 7 consecutive days from the day before transplantation. RESULTS: Graft survival in the cerivastatin-treated group (n = 8) was significantly longer than in controls (n = 10) (24.6 +/- 2.2 days vs 10.2 +/- 1.3 days, p < 0.05). Mixed lymphocyte reaction (MLR) showed that on Day 8 after grafting, the proliferative response of alloreactive T cells against F344 alloantigen in cerivastatin-treated rats was significantly more suppressed than in Lewis rats. The Interleukin-2 concentration of supernatant in MLR cultures in the cerivastatin-treated group was lower than in the control group. Immunohistochemical analysis showed that the percentage of CD4-positive cells to infiltrating mononuclear cells was less prominent in the cerivastatin-treated group (9.8% +/- 2.2%) than in the control group (20.9% +/- 3.2%). CONCLUSIONS: The HMG-CoA reductase inhibitor cerivastatin effectively suppressed acute graft rejection, possibly by blocking intercellular signals via ICAM/LFA-1, and cerivastatin may be a candidate for treating patients with hyperlipidemia who undergo organ transplantation.     

Blocking L-type calcium channels enhances long-term depression induced by low-frequency stimulation at hippocampal CA1 synapses

Specific contributions of voltage-dependent calcium channels (VDCCs) to induction of long-term depression (LTD) have not been thoroughly elucidated. The present study examined roles of T- and L-type VDCCs in N-methyl-D-aspartate (NMDA) receptor-dependent LTD induced at several different levels of synaptic activation (0.5- to 10-Hz presynaptic stimulations) at Schaffer collateral-CA1 synapses in rat hippocampal slices. Blockade of T-type VDCCs with nickel ions failed to change LTD magnitude at all levels of stimulation. However, blockade of L-type VDCCs reduced LTD in response to stimulation at 1 and 2 Hz and, conversely, enhanced LTD at a lower frequency (0.5 Hz). The enhancement of 0.5-Hz LTD under L-type VDCC blockade was shown pharmacologically to depend on NMDA receptors (NMDARs) and intracellular Ca(2+) release. Calcium imaging revealed that contribution of L-type VDCC-mediated calcium influx to the total calcium increase was greater during 0.5-Hz stimulation than during 1.0-Hz stimulation. This finding, combined with the reported suppression of NMDARs mediated by L-type VDCCs, may be relevant to the present enhancement of 0.5-Hz LTD due to L-type VDCC blockade.     

Gender differences in 3D morphology and bony impingement of human hips

For the proper diagnosis or treatment of hip joint disorders caused by anatomical abnormalities, the normal hip joint morphology must be studied to understand its influence on the maximum range of motion (ROM) until bony impingement by focusing on gender differences. Acetabular and femoral morphologies were analyzed from 3D CT images of 106 normal hip joints from elderly men (n = 36 joints) and women (n = 70 joints), and measurements of ROM until bony impingement were made in four directions (flexion, extension, and external and internal rotation at 90° flexion) using surface models of the pelvis and femur reconstructed from the CT data. Gender differences were found not only in joint orientation, including anteversion and inclination of the acetabulum and femoral neck anteversion, but also in the shape around the joint, including the acetabular rim and the femoral neck. This ROM study also showed gender differences in all four standard directions. In conclusion, significant gender differences were observed in the acetabular and femoral morphology, which led to significant gender differences in ROM until bony impingement. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:333–339, 2011