As topical pre-exposure prophylaxis (PrEP) has been shown to cause immune modulation in rectal or cervical tissue, our aim was to examine the impact of oral PrEP on lymphoid and myeloid changes in the foreskin in response to dosing and timing of drug administration.
HIV-negative male individuals ( n  = 144) were recruited in South Africa and Uganda into an open-label randomized controlled trial in a 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 ratio to control arm (with no PrEP) or one of eight arms receiving emtricitabine-tenofovir disoproxil fumarate (F/TDF) or emtricitabine-tenofovir alafenamide (F/TAF) at one of two different doses, 5 or 21 h before undergoing voluntary medical male circumcision (VMMC).
After dorsal-slit circumcision, foreskin tissue sections were embedded into Optimal Cutting Temperature media and analysed, blinded to trial allocation, to determine numbers of CD4 + CCR5 + , CD1a + cells and claudin-1 expression. Cell densities were correlated with tissue-bound drug metabolites and p24 production after ex-vivo foreskin challenge with HIV-1 bal .
There was no significant difference in CD4 + CCR5 + or CD1a + cell numbers in foreskins between treatment arms compared with the control arm. Claudin-1 expression was 34% higher ( P  = 0.003) in foreskin tissue from participants receiving PrEP relative to controls, but was no longer statistically significant after controlling for multiple comparisons. There was neither correlation of CD4 + CCR5 + , CD1a + cell numbers, or claudin-1 expression with tissue-bound drug metabolites, nor with p24 production after ex-vivo viral challenge.
Oral doses and timing of on-demand PrEP and in-situ drug metabolite levels in tissue have no effect on numbers or anatomical location of lymphoid or myeloid HIV target cells in foreskin tissue.

The objective of this study was to evaluate the expression of several cell membrane markers in oral squamous cell carcinomas (OSCC) and to examine their prognostic influence.
We analyzed the immunohistochemical expression of claudin-1 (CLDN-1), claudin-4 (CLDN-4), claudin-5 (CLDN-5), claudin-7 (CLDN-7), occludin (OCLN), and E-cadherin (CDHE) in 60 patients with OSCC treated in a central hospital Center of Oporto. The prognostic significance of these biomarkers in cancer-specific survival and recurrence-free survival were evaluated using multivariate analysis.
Claudin-1 was observed in the membrane of tumor cells in 51 cases (89.5%), CLDN-4 in 36 cases (63.2%), and CLDN-7 in 48 cases (80%). Claudin-5 was detected in the cytoplasm of tumor cells in 46 cases (78%) and OCLN in 40 cases (70.2%). In a multivariate analysis, the combined evaluation of OCLN and CLDN-1 revealed a significant and independent association with cancer-specific survival and recurrence-free survival. We found a low extent score for OCLN and a high intensity score for CLDN-1, presenting the hazard ratios of 15.48 (P = .014) and 9.446 (P = .012), respectively.
The CLDN-1 and OCLN proteins could be involved in tumor progression of OSCC. Their combined deregulated expression showed an adverse effect on survival and therefore they could be regarded as important prognostic biomarkers in OSCC.

Cold-heat combination is a common method in the treatment of ulcerative colitis, which is represented by classic drug pair, Coptidis Rhizoma and Zingiberis Rhizoma.The present study explored the synergetic effects of berberine and 6-shogaol, the primary components of Coptidis Rhizoma and Zingiberis Rhizoma, respectively, on intestinal inflammation and intestinal flora in mice with ulcerative colitis to reveal the effect and mechanism of cold-heat combination in the treatment of ulcerative colitis.The ulcerative colitis model was induced by dextran sulfate sodium(DSS) in mice.The model mice were administered with berberine(100 mg·kg~(-1)), 6-shogaol(100 mg·kg~(-1)), and berberine(50 mg·kg~(-1)) combined 6-shogaol(50 mg·kg~(-1)) by gavage, once per day.After 20 days of drug administration, mouse serum, colon tissues, and feces were sampled.Hematoxylin-eosin(HE) staining was used to observe histopathological changes in colon tissues.Alcian blue/periodic acid-Schiff(AB/PAS) staining was used to observe the changes in the mucus layer of colon tissues.Enzyme-linked immunosorbent assay(ELISA) was employed to detect the serum content of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6).Immunohistochemical method was adopted to detect the protein expression of macrophage surface markers F4/80, mucin-2, claudin-1, and zonula occludens-1(ZO-1) in colon tissues.High-throughput Meta-amplicon library sequencing was used to detect changes in the intestinal flora of mice.The results indicated that the 6-shogaol group, the berberine group, and the combination group showed significantly relieved intestinal injury, reduced number of F4/80-labeled positive macrophages in colon tissues, increased protein expression of mucin-2, claudin-1, and ZO-1, and decreased serum le-vels of TNF-α, IL-1β, and IL-6.Shannon, Simpson, Chao, and Ace indexes of the intestinal flora of mice in the 6-shogaol group and the combination group significantly increased, and Chao and Ace indexes in the berberine group significantly increased.As revealed by the bioinformatics analysis of intestinal flora sequencing, the relative abundance of Verrucomicrobia at the phylum, class, and order levels decreased significantly in all treatment groups after drug administration, while that of Bacillibacteria gradually increased.In the 6-shogaol group and the combination group, Akkermansia muciniphila completely disappeared, but acid-producing bacillus still existed in large quantities.As concluded, both 6-shogaol and berberine can inhibit intestinal inflammation, reduce the infiltration and activation of macrophages, relieve intestinal damage, reduce intestinal permeability, improve the structure of flora, and promote intestinal microecological balance.The combined application of berberine and 6-shogaol has a significant synergistic effect.

Chemotherapy treatment of cancer in children can influence formation of normal tissues, leading to irreversible changes in their structure and function. Tooth formation is susceptible to several types of chemotherapy that induce irreversible changes in the structure of enamel, dentin and dental root morphology. These changes can make the teeth more prone to fracture or to caries when they have erupted. Recent studies report successful treatment of brain tumors with the alkylating drug temozolomide (TMZ) in combination with veliparib (VLP) in a glioblastoma in vivo mouse model. Whether these drugs also affect tooth formation is unknown.
In this study the effect of TMZ/VLP on incisor formation was investigated in tissue sections of jaws from mice and compared with mice not treated with these drugs.
The following aspects were studied using immunohistochemistry of specific protein markers including: (1) proliferation (by protein expression of proliferation marker Ki67) (2) a protein involved in paracellular ion transport (expression of tight junction (TJ) protein claudin-1) and (3) in transcellular passage of ions across the dental epithelium (expression of Na+, K+ 2Cl- cotransporter/NKCC1).
Chemotherapy with TMZ/VLP strongly reduced immunostaining for claudin-1 in distal parts of maturation ameloblasts. No gross changes were found in the treated mice, either in cell proliferation in the dental epithelium at the cervical loop or in the immunostaining pattern for NKCC1 in (non-ameloblastic) dental epithelium. The salivary glands in the treated mice contained strongly reduced immunostaining for NKCC1 in the basolateral membranes of acinar cells.
Based on the reduction of claudin-1 immunostaining in ameloblasts, TMZ/VLP may potentially influence forming enamel by changes in the structure of TJs structures in maturation ameloblasts, structures that are crucial for the selective passage of ions through the intercellular space between neighboring ameloblasts. The strongly reduced basolateral NKCC1 staining seen in fully-grown salivary glands of TMZ/VLP-treated mice suggests that TMZ/VLF could also influence ion transport in adult saliva by the salivary gland epithelium. This may cause treated children to be more susceptible to caries.

The differential diagnosis between perineurioma (PN) and meningioma (MEN) can be difficult by histology and immunohistochemistry (IHC) because the perineurium and arachnoid have the same embryological origin. However, there are no comparative studies determining conclusive parameters for the differential diagnosis. The aim of this study is to compare IHC of PN and MEN and their ultra-structural characteristics to elucidate which are the useful data that allow differentiate both entities. Thirty-five MEN were analyzed, and 15 PN, (11 skin and soft tissues and four oral cavity). IHC for epithelial membrane antigen (EMA), Claudin-1, GLUT-1, somatostatin-2 receptor (SSTR-2), and progesterone receptor (RP) was performed. Ultrastructural studies were performed on 8 MEN and 15 PN. Only in PN Claudin-1 was positive in 9/11 (90%) cases and GLUT-1 in 7/11 (63%) cases. In MEN, the progesterone receptor was expressed in 21/35 (60%) cases and no case expressed Claudin-1 and GLUT-1; EMA was expressed in all MEN cases and 93% of PN. SSTR-2 was expressed weakly in six cases of MEN (17%), and it was not considered useful for differential diagnosis. On ultrastructure, PN showed thin and parallel processes, some caveolae, and lacked cell junctions. The cellular processes were surrounded by a collagenous stroma in 94% of the cases. MEN were characterized by curved cytoplasmic cell processes showing desmosomes in 75% of cases. Ultrastructural findings aid in the differential diagnosis between PN and MEN, especially if molecular studies are not available.

Objective: To investigate the damage and mechanism of artemisia annua pollen on tight junction of human nasal mucosa epithelial cells (HNEpC). Methods: HNEpC were cultured in vitro. Different concentrations of artemisia annua pollen (0, 20, 40, 80, 100, 160, 200 μg/ml) were used to intervene the cells for 24 h, and the cell proliferation activity was detected by the CCK-8 method. The expression and phosphorylation of p38MAPK signaling pathway were detected by Western Blot before and after the intervention of SB203580, a p38MAPK inhibitor in HNEpC. Immunofluorescence chemical staining, Western Blot and quantitative real-time PCR (qPCR) were used to observe the expression and distribution of tight junctions Occludin and Claudin-1. SPSS 21.1 software was used for statistical analysis. Results: CCK-8 results showed that, compared with the control group, the proliferation activity of HNEpC increased after 6 h intervention with different concentrations of artemisia annua pollen (all P<0.05). After 12 h of intervention, the proliferation activity of HNEpC in the 20, 40, 80, 100 and 160 μg/ml groups was not significantly changed (all P>0.05), while that in the 200 μg/ml group was decreased (P<0.05). After the intervention for 24 h, the proliferation activity of cells in the 20 and 40 μg/ml groups was not significantly changed (all P>0.05), while that in the 80, 100, 160 and 200 μg/ml groups was decreased (all P<0.05). Immunofluorescence staining showed that the Occludin and Claudin-1 proteins in the normal control group were localized on the cell membrane and expressed more and formed a ring structure around the cell membrane. However, under the intervention of high concentration artemisia annua pollen, its expression level decreased, appeared broken, fuzzy, and nonuniform distribution. Western Blot and qPCR results showed that after 24 h of intervention, the expression levels of HNEpC Claudin-1 protein and its mRNA in the pollen groups (40, 80, 100, 160, 200 μg/ml) of artemisia annua decreased compared with those of those of the control group (mRNA expression levels were 0.567±0.214, 0.443±0.109, 0.462±0.160, 0.497±0.134, 0.388±0.076 compared with 1.001±0.067, respectively, all P<0.05). However, the mRNA of Occludin protein and its mRNA only decreased in the 200 μg/ml treatment group (mRNA expression level was 0.631±0.109 compared with 1.016±0.026, P<0.05), while all the other treatment groups increased (mRNA expression levels were 1.258±0.134, 1.827±0.103, 2.429±0.077, 1.707±0.085, 1.477±0.066 compared with 1.016±0.026, respectively, all P<0.05). Western Blot showed that p-p38MAPK expression increased after intervention with 100, 160, 200 μg/ml artemisia annua pollen for 24 h. SB203580 could inhibit the decreasing expression of Occludin caused by artemisinin pollen (mRNA expression was 1.255±0.179 compared with 0.631±0.109, P<0.05), but had no effect on Claudin-1 protein expression. Conclusion: Pollen from artemisia annua may activate p38MAPK signaling pathway and destroy the close connection of HNEpC.
目的： 探讨黄花蒿花粉对鼻黏膜上皮细胞（HNEpC）紧密连接的损伤作用及机制。 方法： 体外培养HNEpC，采用不同质量浓度黄花蒿花粉（0、20、40、80、100、160、200 μg/ml）分别干预细胞24 h，CCK-8法检测细胞增殖活性；p38丝裂原素活化蛋白激酶（p38MAPK）抑制剂SB203580干预HNEpC前后，Western Blot法检测p38MAPK信号通路表达及其磷酸化水平；免疫荧光化学染色法、Western Blot法和实时荧光定量聚合酶链反应（qPCR）观察细胞紧密连接Occludin、Claudin-1的表达和分布情况。采用SPSS 21.0软件分析数据。 结果： CCK-8结果显示，与对照组相比，不同质量浓度黄花蒿花粉干预6 h后，HNEpC增殖活性均增高（P值均<0.05）；干预12 h后，20、40、80、100、160 μg/ml组的HNEpC增殖活性未见明显改变（P值均>0.05），200 μg/ml组降低（P<0.05）；干预24 h后，20、40 μg/ml组的细胞增殖活性未见明显改变（P值均>0.05），80、100、160、200 μg/ml组降低（P值均<0.05）。免疫荧光染色可见正常对照组Occludin和Claudin-1蛋白定位于细胞膜上，表达多，围绕细胞膜形成环状结构；而在高浓度黄花蒿花粉干预下，其表达水平下降，出现断裂、模糊，分布不均匀。Western Blot和qPCR实验结果表明，干预24 h后，黄花蒿花粉（40、80、100、160、200 μg/ml）干预组HNEpC Claudin-1蛋白及其mRNA表达水平较对照组降低（mRNA表达量分别为0.567±0.214、0.443±0.109、0.462±0.160、0.497±0.134、0.388±0.076比1.001±0.067，P值均<0.05），而Occludin蛋白及其mRNA仅在200 μg/ml黄花蒿花粉处理组降低（mRNA表达量为0.631±0.109比1.016±0.026，P<0.05），其他处理组均增高（mRNA表达量分别为1.258±0.134、1.827±0.1034、2.429±0.077、1.707±0.085、1.477±0.066比1.016±0.026，P值均<0.05）。Western Blot结果表明，100、160、200 μg/ml黄花蒿花粉干预24 h后，磷酸化p38MAPK（p-p38MAPK）表达增加。SB203580能够抑制黄花蒿花粉引起的紧密连接蛋白Occludin表达下降（mRNA表达量为1.255±0.179比0.631±0.109，P<0.05），而对Claudin-1蛋白表达无影响。 结论： 黄花蒿花粉可能通过激活p38MAPK信号通路，破坏HNEpC的紧密连接。.

OBJECTIVE: Dietary factors and immune dysfunction may induce symptoms in patients with functional dyspepsia (FD). The aim of the study was to evaluate whether gluten consumption impacts symptom onset in patients with FD and to evaluate for possible histologic alterations in the duodenum of patients with FD.
METHODS: We prospectively enrolled 101 patients newly diagnosed with FD and 31 asymptomatic controls. Specific FD symptoms and gluten consumption patterns were evaluated by self-reported questionnaires. Tight junction protein (claudin-1) expression and presence of intraepithelial lymphocyte (IEL) infiltration in the bulb (D1) and second portion (D2) of the duodenum were assessed by immunohistochemistry.
RESULTS: Wheat bun consumption had higher frequency (P = 0.047) and increased average consumption (P = 0.01) scores in patients with FD compared with the control group. Of the 101 patients with FD, early satiety (P = 0.03) was associated with increased wheat bun consumption frequency score. On histologic evaluation, claudin-1 expression was decreased in D1 (0.003 ± 0.001 vs 0.012 ± 0.002, P = 0.003) and D2 (0.002 ± 0.0004 vs 0.012 ± 0.001, P < 0.001), while duodenal IEL counts were increased in D1 (15.5 ± 7.8 vs 3.1 ± 2.5, P < 0.001) and D2 (20.6 ± 7.7 vs 5.8 ± 3.4, P < 0.001) among patients with FD compared with the control group. Finally, Helicobacter pylori infection was associated with increased IELs in D1 (20.6 ± 7.0 vs 14.2 ± 7.4, P = 0.001) among patients with FD.
CONCLUSIONS: Among patients with FD, gluten-rich food may lead to symptom onset, specifically early satiety. Intestinal epithelial barrier dysfunction characterized by decreased claudin-1 expression and mucosal immune activation demonstrated by IEL infiltration may contribute to the pathogenesis of FD.

Tight junction and epithelial barrier disruption is a common trait of many gastrointestinal pathologies, including chemotherapy-induced gut toxicity. Currently, there are no validated in vitro models suitable for the study of chemotherapy-induced mucosal damage that allow paralleled functional and structural analyses of tight junction integrity. We therefore aimed to determine if a transparent, polyester membrane insert supports a polarized T84 monolayer with the phenotypically normal tight junctions. T84 cells (passage 5-15) were seeded into either 0.6 cm(2), 0.4 µm pore mixed-cellulose transwell hanging inserts or 1.12 cm(2), 0.4 µm pore polyester transwell inserts at varying densities. Transepithelial electrical resistance was measured daily to assess barrier formation. Immunofluoresence for key tight junction proteins (occludin, zonular occludens-1, claudin-1) and transmission electron microscopy were performed to assess tight junction integrity, organelle distribution, and polarity. Reverse transcription-polymerase chain reaction was performed to determine expression of toll-like receptor 4 (TLR4). Liquid chromatography was also conducted to assess SN38 degradation in this model. Polyester membrane inserts support a polarized T84 phenotype with functional tight junctions in vitro. Transmission electron microscopy indicated polarity, with apico-laterally located tight junctions. Immunofluorescence showed membranous staining for all tight junction proteins. No internalization was evident. T84 cells expressed TLR4, although this was significantly lower than levels seen in HT29 cells (P = .0377). SN38 underwent more rapid degradation in the presence of cells (-76.04 ± 1.86%) compared to blank membrane (-48.39 ± 4.01%), indicating metabolic processes. Polyester membrane inserts provide a novel platform for paralleled functional and structural analysis of tight junction integrity in T84 monolayers. T84 cells exhibit the unique ability to metabolize SN38 as well as expressing TLR4, making this an excellent platform to study clinically relevant therapeutic interventions for SN38-induced mucosal damage by targeting TLR4.

Mal de Meleda (MdM, MIM: 248300) is a rare autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis with onset in early infancy. The gene responsible for MdM, ARS, encodes for Secreted Lys6/Plaur domain-containing protein 1 which is essential for epidermal homeostasis. Tight junctions have been proposed to have two mutually exclusive functions: a fence function which prevents the mixing of membrane proteins between the apical and basolateral membranes; and a gate function which controls the paracellular passage of ions and solutes between cells. In this study we report immunohistochemical investigations of tight junction proteins claudin-1 and occludin in MdM Tunisian families. Nine skin biopsies from patients with MdM were analyzed. The control group was formed by skin biopsies belonging to healthy individuals. Immunohistochemical study was performed on fixed sections from biopsies of four microns with the following polyclonal antibodies: anti-claudin-1 and anti-occludin. In control skin, claudin-1 exhibited membrane expression throughout the epidermis with increasing and upward intensity, whereas occludin was detected in the cell membrane of keratinocytes of the stratum granulosum. In MdM skin, claudin-1 was expressed throughout the thickness of the spinous layers with membrane staining, and occludin had cytoplasmic staining in the granular layer. The immunohistochemical expression of TJ proteins in MdM patients harbors premature expression of occludin and decreased expression of claudin-1, highlighting further evidence for disorders in epidermal homeostasis.

BACKGROUND: Perineurioma is an infrequent and benign cutaneous neoplasm characterized by proliferation of perineurial cells. It is classified into two main types: intraneural and the extraneural or soft tissue perineurioma, in which the sclerosing variant is included. Sclerosing perineurioma is more frequently found on acral skin. Clinically, they are well-circumscribed,skin colored, nodular tumors.
OBJECTIVE: Describe and communicate clinicopathologic findings from a case series of sclerosing acral perineurioma.
METHODS: This is a clinical, morphological and immunohistologic case study of eight patients with the diagnosis of sclerosing perineurioma.
RESULTS: It included five men and five women, with ages ranging between nine and 66 years. All of them had lesion on acral skin. At microscopy study, the lesions showed a proliferation of epithelioid and spindle-shaped perineurial cells, arranged in small aggregates and short fascicles between thickened collagen bundles. Immunohistochemistry studies revealed that the proliferating cells expressed EMA, Claudin-1 and Glut-1, and were negative for S-100 protein.
CONCLUSIONS: It is important to report these infrequent skin tumors, so they can be taken into account in the differential diagnoses of acral lesions.