Sex differences and risk factors in recurrent ischemic stroke.

Introduction: Recurrent ischemic stroke (RIS) is associated with increased mortality and poor outcomes. Therefore, secondary prevention is critical for reducing the risk of recurrent stroke. Previous studies have found sex differences in risk factors in patients with first-ever stroke; however, the results have been inconsistent for recurrent stroke. Therefore, this study aimed to investigate whether there are significant sex differences in the clinical characteristics and risk factors for recurrent ischemic stroke. Methods: We retrospectively studied 787 patients with recurrent ischemic stroke after first-ever stroke confirmation using magnetic resonance imaging (MRI) after visiting a regional tertiary hospital between 2014 and 2020. Demographic characteristics, laboratory findings, and risk factors were compared between the male and female patients. In addition, multivariate logistic regression was performed to identify the independent factors associated with stroke recurrence in male patients. Results: Among the 787 patients, 466 (59.2%) were males. Males were younger than females (67.6 vs. 71.9 years). Females had higher rates of hypertension, diabetes mellitus, dyslipidemia, and overweight than those of males. However, the alcohol drinking and smoking rate were significantly higher in males than that in females. There were no statistically significant sex-based differences in the laboratory findings. Among males, hypertension, alcohol drinking, smoking and dyslipidemia was a significant risk factor for ischemic stroke recurrence. Conclusion: Hypertension and dyslipidemia were significant risk factors of recurrent ischemic stroke in both genders. Smoking and alcohol drinking were significant risk factors associated with ischemic stroke recurrence in males. Therefore, smoking cessation and alcohol abstinence are recommended after the first stroke to prevent recurrent ischemic stroke especially for males. Diabetes was a significant risk factor of ischemic stroke recurrence in females. More extensive studies are needed to understand the causal relationship of each factors with ischemic stroke recurrence according to sex differences and specification of preventive management is needed.

Endolymphatic Hydrops in Patients With Vestibular Migraine and Concurrent Meniere's Disease.

Objective: Intravenous contrast agent enhanced, high-resolution magnetic resonance imaging of the inner ear (iMRI) confirmed that patients with Menière's disease (MD) and vestibular migraine (VM) could present with endolymphatic hydrops (EH). The present study aimed to investigate EH characteristics and their interrelation to neurotologic testing in patients with VM, MD, or VM with concurrent MD (VM-MD). Methods: Sixty-two patients (45 females, aged 23-81 years) with definite or probable VM (n = 25, 19 definite), MD (n = 29, 17 definite), or showing characteristics of both diseases (n = 8) were included in this study. Diagnostic workup included neurotologic assessments including video-oculography (VOG) during caloric stimulation and head-impulse test (HIT), ocular and cervical vestibular evoked myogenic potentials (o/cVEMP), pure tone audiometry (PTA), as well as iMRI. EH's degree was assessed visually and via volumetric quantification using a probabilistic atlas-based segmentation of the bony labyrinth and volumetric local thresholding (VOLT). Results: Although a relevant number of VM patients reported varying auditory symptoms (13 of 25, 52.0%), EH in VM was only observed twice. In contrast, EH in VM-MD was prevalent (2/8, 25%) and in MD frequent [23/29, 79.3%; χ2(2) = 29.1, p < 0.001, φ = 0.7]. Location and laterality of EH and neurophysiological testing classifications were highly associated (Fisher exact test, p < 0.005). In MD, visual semi-quantitative grading and volumetric quantification correlated highly to each other (r S = 0.8, p < 0.005, two-sided) and to side differences in VOG during caloric irrigation (vestibular EH ipsilateral: r S = 0.6, p < 0.05, two-sided). In VM, correlations were less pronounced. VM-MD assumed an intermediate position between VM and MD. Conclusion: Cochlear and vestibular hydrops can occur in MD and VM patients with auditory symptoms; this suggests inner ear damage irrespective of the diagnosis of MD or VM. The EH grades often correlated with auditory symptoms such as hearing impairment and tinnitus. Further research is required to uncover whether migraine is one causative factor of EH or whether EH in VM patients with auditory symptoms suggests an additional pathology due to MD.

Functional study of acetaldehyde dehydrogenase 1 (ALDH1) in keratinocytes: microarray integrating bioinformatics approaches.

The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.Communicated by Ramaswamy H. Sarma.

A Knock-Down Cell-Based Study for the Functional Analysis of Chloride Intracellular Channel 1 (CLIC1): Integrated Proteomics and Microarray Study.

BACKGROUND: Previously, we detected that chloride intracellular channel 1 (CLIC1) was involved in the pathogenesis of atopic dermatitis (AD). OBJECTIVE: In this study, we aimed to use high-throughput screening (HTS) approaches to identify critical factors associated with the function of CLIC1 in knock-down cells. METHODS: We down-regulated CLIC1 in human A549 cells via siRNA and then conducted serial HTS studies, including proteomics integrated with a microarray and the implementation of bioinformatics algorithms. RESULTS: Together, these approaches identified several important proteins and genes associated with the function of CLIC1. These proteins and genes included tumor rejection antigen (gp96) 1, nucleophosmin, annexin I, keratin 1 and 10, FLNA protein, enolase 1, and metalloprotease 1, which were found using two-dimensional electrophoresis (2-DE) proteomics. Separately, NTNG1, SEMA5A, CLEC3A, GRPR, GNGT2, GRM5, GRM7, DNMT3B, CXCR5, CCL11, CD86, IL2, MNDA, TLR5, IL23R, DPP6, DLGAP1, CAT, GSTA1, GSTA2, GSTA5, CYP2E1, ADH1A, ESR1, ARRDC3, A1F1, CCL5, CASP8, DNTT, SQSTM1, PCYT1A, and SLCO4C1 were found using a DNA microarray integrated with PPI mapping. CONCLUSION: CCL11 is thought to be a particularly critical gene among the candidate genes detected in this study. By integrating the datasets and utilizing the strengths of HTS, we obtained new insights into the functional role of CLIC1, including the use of CLIC1-associated applications in the treatment of human diseases such as AD.

Behavior of Muscle-Derived Stem Cells on Silica Nanostructured Substrates.

The aim of the present work was to evaluate the responses of rat muscle-derived stem cells (rMDSCs) to growth on silica nanostructured substrates (SN) with nanoscale topographic surfaces. SN of different sizes (SN-60, SN-150, SN-300, SN-500, and SN-700) were prepared using silica nanoparticles with sizes of 60-700 nm. The prepared SN showed roughness at the nanoscale level. The total number of adherent cells on SN increased with increasing nanoscale level and incubation time. The rMDSCs attached to SN-500 and SN-700 were extensively flattened, whereas those grown on SN-60, SN-150, and SN-300 were more rounded. The rank order of the cell length and height of attached rMDSCs at 5 d on different surfaces was SN-60 ≈ SN-150 >> SN-300 > SN-500 > SN-700 > glass. Compared with rMDSCs grown on SN-60, SN-150, or SN-300, those attached to SN-500 and SN-700 exhibited a distinct morphology with filopodial extensions and stronger expression of focal adhesion, integrin, and actin. An evaluation of the gene expression of adhered rMDSCs showed that rMDSCs grown on SN-300 exhibited a higher environmental stress response than those grown on glass or SN-700. Collectively, our data provide fundamental insight into the cellular response and gene expression of rMDSCs grown on nanostructured substrates.

Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches.

Previously, we detected that 14-3-3 protein epsilon (YWHAE) was involved in the pathogenesis of atopic dermatitis (AD) and tyrosinase-mediated pigmentation. In this study, we aimed to identify critical factors associated with YWHAE in human keratinocytes using high-throughput screening (HTS) approaches to reveal its functions in skin. We overexpressed YWHAE in human HaCaT keratinocytes and then conducted serial HTS studies, including RNA sequencing integrated with antibody arrays and the implementation of bioinformatics algorithms. Cumulatively, these approaches identified several novel genes in keratinocytes associated with the function of YWHAE including KRT9, KRT1, KRT6C, BST2, CIB2, APH1B, ACTC1, IFI27, TUBA1A, CAPN6, UTY, MX2, and MAPK15, based on RNA sequencing data, and MAPK1, MMP2, TYK2, NOS3, and CASP3, based on antibody array data. In particular, CD37 is a unique gene that was detected and validated in all the methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of YWHAE in skin keratinocytes. The approach used here could contribute to the clinical understanding of YWHAE-associated applications in the treatment of AD disease. AbbreviationsDAVIDthe database for annotation, visualization and integrated discoveryHTSHigh-throughput screeningKEGGKyoto Encyclopedia of Genes and GenomesPPIprotein-protein interactionsCommunicated by Ramaswamy H. Sarma.

An OMICS-based study of the role of C3dg in keratinocytes: RNA sequencing, antibody-chip array, and bioinformatics approaches.

Previously, we have identified the C3dg protein as an important player in the pathogenesis of atopic dermatitis (AD). In this study, we aimed to identify critical factors associated with C3dg in human keratinocytes based on high-throughput screening (HTS) approaches. We overexpressed C3dg in HaCaT human keratinocytes and conducted serial HTS studies, including RNA sequencing analysis integrated with antibody-chip arrays and implementation of bioinformatics algorithms (PPI mappings). Cumulatively, these approaches identified several novel C3dg-associated genes and proteins that are thought to be significantly involved in skin diseases including AD. These novel genes and proteins included LPA, PROZ, BLK, CLDN11, and FGF22, which are believed to play important roles in C3dg-associated skin functions in keratinocytes, as well as genes related to the two important pathways of systemic lupus erythematosus and Staphylococcus aureus infection. In particular, FGF22 is a unique gene that was detected and validated in all methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of C3dg in keratinocytes. The approach used here contributes to clinical understanding of C3dg-associated applications and may also be applicable to treatment of AD.

Evaluation of small intestine submucosa and poly(caprolactone-co-lactide) conduits for peripheral nerve regeneration.

The present study employed nerve guidance conduits (NGCs) only, which were made of small intestine submucosa (SIS) and poly(caprolactone-co-lactide) (PCLA) to promote nerve regeneration in a peripheral nerve injury (PNI) model with nerve defects of 15 mm. The SIS- and PCLA-NGCs were easily prepared by rolling of a SIS sheet and a bioplotter using PCLA, respectively. The prepared SIS- and PCLA-NGCs fulfilled the general requirement for use as artificial peripheral NGCs such as easy fabrication, reproducibility for mass production, suturability, sterilizability, wettability, and proper mechanical properties to resist collapsing when applied to in vivo implantation. The SIS- and PCLA-NGCs appeared to be well integrated into the host sciatic nerve without causing dislocations and serious inflammation. All NGCs stably maintained their NGC shape for 8 weeks without collapsing, which matched well with the nerve regeneration rate. Staining of the NGCs in the longitudinal direction showed that the regenerated nerves grew successfully from the SIS- and PCLA-NGCs through the sciatic nerve-injured gap and connected from the proximal to distal direction along the NGC axis. SIS-NGCs exhibited a higher nerve regeneration rate than PCLA-NGCs. Collectively, our results indicate that SIS- and PCLA-NGCs induced nerve regeneration in a PNI model, a finding that has significant implications in the future with regard to the feasibility of clinical nerve regeneration with SIS- and PCLA-NGCs prepared through an easy fabrication method using promising biomaterials.

In vivo biofunctionality comparison of different topographic PLLA scaffolds.

In this work, the in vivo biodegradation of, biocompatibility of, and host response to various topographic scaffolds were investigated. Randomly oriented fibrous poly(L-lactide) (PLLA) nanofibers were fabricated using the electrospinning technique. A PLLA scaffold was obtained by salt leaching. Both the electrospun PLLA nanofibers and the salt-leaching PLLA scaffolds formed three-dimensional pore structures. Cytotoxicity studies, in which rat muscle-derived stem cells (rMDSCs) were grown on electrospun PLLA nanofibers or the salt-leaching PLLA scaffolds, revealed that the rMDSCs cell count on the PLLA nanofibers was slightly higher than that on the salt-leaching PLLA scaffolds. An in vivo study was carried out by implanting the scaffolds subcutaneously into rats to test the biodegradation, biocompatibility, and host response at regular intervals over 0-4 weeks. The degradation of the PLLA nanofibers 1, 2, and 4 weeks after initial implantation was more extensive than that observed for the salt-leaching PLLA scaffolds. PLLA nanofibers seeded the growth of larger fibrous tissue masses due to in vivo cellular infiltration into the randomly oriented fibrillar structures of the PLLA nanofibers. In addition, the inflammatory cell accumulation in PLLA nanofibers was lower than that in the salt-leaching PLLA scaffolds. These results indicate that the electrospun PLLA nanofibers may serve as a good scaffold to elicit fibrous cellular infiltration, to minimize host response, and to enhance tissue-scaffold integration.

Tissue engineered regeneration of completely transected spinal cord using human mesenchymal stem cells.

The present study employed a combinatorial strategy using poly(D,L-lactide-co-glycolide) (PLGA) scaffolds seeded with human mesenchymal stem cells (hMSCs) to promote cell survival, differentiation, and neurological function in a completely transected spinal cord injury (SCI) model. The SCI model was prepared by complete removal of a 2-mm length of spinal cord in the eighth-to-ninth spinal vertebra, a procedure that resulted in bilateral hindlimb paralysis. PLGA scaffolds 2 mm in length without hMSCs (control) or with different numbers of hMSCs (1 × 10(5), 2 × 10(4), and 4 × 10(3)) were fitted into the completely transected spinal cord. Rats implanted with hMSCs received Basso-Beattie-Bresnahan scores for hindlimb locomotion of about 5, compared with ~2 for animals in the control group. The amplitude of motor-evoked potentials (MEPs) averaged 200-300 µV in all hMSC-implanted SCR model rats. In contrast, the amplitude of MEPs in control group animals averaged 135 µV at 4 weeks and then declined to 100 µV at 8 weeks. These results demonstrate functional recovery in a completely transected SCI model under conditions that exclude self-recovery. hMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating in vivo survival of implanted hMSCs. Immunohistochemical staining revealed differentiation of implanted hMSCs into nerve cells, and immunostained images showed clear evidence for axonal regeneration only in hMSC-seeded PLGA scaffolds. Collectively, our results indicate that hMSC-seeded PLGA scaffolds induced nerve regeneration in a completely transected SCI model, a finding that should have significant implications for the feasibility of therapeutic and clinical hMSC-delivery using three-dimensional scaffolds, especially in the context of complete spinal cord transection.

Regeneration of completely transected spinal cord using scaffold of poly(D,L-lactide-co-glycolide)/small intestinal submucosa seeded with rat bone marrow stem cells.

Using a complete spinal cord transection model, the present study employed a combinatorial strategy comprising rat bone marrow stem cells (rBMSCs) and polymer scaffolds to regenerate neurological function after spinal cord injury (SCI) of different lengths. SCI models with completely transected lesions were prepared by surgical removal of 1 mm (SC1) or 3 mm (SC3) lengths of spinal cord in the eighth-to-ninth spinal vertebrae, a procedure that resulted in bilateral hindlimb paralysis. A cylindrical poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold 1 or 3 mm in length with or without rBMSCs was fitted into the completely transected lesion. Rats in SC1 and SC3 groups implanted with rBMSC-containing scaffolds received Basso-Beattie-Bresnahan scores for hindlimb locomotion of 15 and 8, respectively, compared with ∼3 for control rats in SC1-C and SC3-C groups implanted with scaffolds lacking rBMSCs. The amplitude of motor-evoked potentials recorded in the hindlimb area of the sensorimotor cortex after stimulation of the injured spinal cord averaged ∼100 µV in SC1-C and 10-50 µV in SC3-C groups at 4 weeks, and then declined to nearly zero at 8 weeks. In contrast, the amplitude of motor-evoked potentials increased from ∼300 to 350 µV between 4 and 8 weeks in SC1 rats and from ∼200 to ∼250 µV in SC3 rats. These results demonstrate functional recovery in rBMSC-transplanted rats, especially those with smaller defects. Immunohistochemically stained sections of the injury site showed clear evidence for axonal regeneration only in rBMSC-transplanted SC1 and SC3 models. In addition, rBMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating cell survival in SCI. Collectively, our results indicate that therapeutic rBMSCs in a poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold induced nerve regeneration in a complete spinal cord transection model and showed that functional recovery further depended on defect length.

In vivo efficacy of an intratumorally injected in situ-forming doxorubicin/poly(ethylene glycol)-b-polycaprolactone diblock copolymer.

The effectiveness of systemically administered anticancer treatments is limited by difficulties in achieving therapeutic doses within tumors, a problem that is complicated by dose-limiting side effects to normal tissue. This work examined injectable in situ-forming gels as a localized drug-delivery system. An MPEG-PCL (MP) solution containing doxorubicin (Dox) existed in an emulsion-sol state at room temperature and rapidly gelled in vitro and in vivo at body temperature. The release of Dox from Dox-loaded MP gels was sustained in vitro over 20 days after an initial burst, indicating that the MP gel acted as a drug depot. Dox-loaded MP gels exhibited remarkable in vitro anti-proliferative activities against B16F10 cancer cells. In vivo experiments employing B16F10 cancer cell xenograft-bearing mice showed that a single intratumoral injection of Dox-loaded MP gel inhibited the growth of tumors as effectively as repeated injections of free Dox, and more effectively than a single dose of free Dox, or saline or gel alone. Consistent with the observed suppression of tumor growth, intratumorally injected free Dox or Dox released from Dox-loaded MP gels caused apoptosis of tumor cells. The tumor biodistribution of free Dox after 1 day was ∼90%, which dropped to ∼15% after 4 days. The biodistribution of Dox following a single injection of Dox-loaded MP gel was also ∼90% on day 1, but remained at ∼13%, even after 15 days. Only a small amount of Dox was found in other organ tissues following intratumoral injection, implying fewer off-target side effects.

Upregulation of interleukin-1ß production by 1,25-dihydroxyvitamin D(3) in activated human macrophages.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) plays important roles in the immune system. In contrast to its well known function in the adaptive immune system, much less is known about the immunoregulatory effects of 1,25(OH)(2)D(3) in the innate immune system, especially on activated human macrophages. Here we found that 1,25(OH)(2)D(3) strongly stimulated the production of interleukin-1ß (IL-1ß) in PMA-differentiated U937 cells and human monocyte-derived macrophages treated with lipopolysaccharide (LPS) or PMA. In this study, Erk1/2 appeared to mediate 1,25(OH)(2)D(3)-induced expression of IL-1ß. Parallel to the increased production of IL-1ß, 1,25(OH)(2)D(3) increased the expression and phosphorylation of the CCAAT enhancer-binding protein ß (C/EBPß), which is one of the key transcriptional regulatory factors for IL-1ß transcription. These results suggest that 1,25(OH)(2)D(3) may function as a proinflammatory molecule in inflammatory macrophages.

In vivo biocompatibility study of electrospun chitosan microfiber for tissue engineering.

In this work, we examined the biocompatibility of electrospun chitosan microfibers as a scaffold. The chitosan microfibers showed a three-dimensional pore structure by SEM. The chitosan microfibers supported attachment and viability of rat muscle-derived stem cells (rMDSCs). Subcutaneous implantation of the chitosan microfibers demonstrated that implantation of rMDSCs containing chitosan microfibers induced lower host tissue responses with decreased macrophage accumulation than did the chitosan microfibers alone, probably due to the immunosuppression of the transplanted rMDSCs. Our results collectively show that chitosan microfibers could serve as a biocompatible in vivo scaffold for rMDSCs in rats.