Avidin-biotin Complex Research Articles

Pattern of Molecular Phenotypes of Breast Carcinomas using Immunohistochemistry in a District Hospital in Nigeria

Background: Cancer of the breast is the most common organ-specific cancer, affecting women worldwide with disproportionately higher mortality seen among women of Africa ancestry. Molecular phenotyping is crucial for tailored treatment of such patients. Aim: The aim of the study is to describe the molecular phenotypes of carcinomas of the breast based on their oestrogen, progesterone, and HER-2/Neu receptors status in our centre. Design: This was a retrospective analysis of 136 histologically diagnosed breast carcinomas, for which oestrogen, progesterone, and HER2 receptors status data were available at the Pathology Unit in a Nigerian District Hospital. Materials and Methods: Relevant biodata and pathology and molecular information of all patients with histologically diagnosed breast cancer (BC) were extracted from patients' forms and reports archived in the department. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue blocks of 136 (54.4%) cases of the BCs utilizing the avidin-biotin complex technique. Results: The ages of the patients ranged from 17 to 74 years, and the mean age was 47.3 ± 11.7 at diagnosis. All affected patients were females except for a male teenager. oestrogen, progesterone, and HER-2/Neu receptor-positive breast carcinomas were 43.4% (59), 39% (53), and 27.2% (37), respectively. Triple-negative breast carcinoma (39%, 53/136) was the most prevalent phenotype, followed by Luminal A (33.8%, 46/136) and Luminal B (14.7%, 20/136), while HER2-enriched (12.5%, 17/136) was the least common molecular subtype in our series. Conclusion: Breast carcinomas are predominantly of the triple-negative molecular phenotype in our setting and commonly affected young women.

Tumor Associated Macrophages and Topoisomerase II Alpha As Prognostic Factors in Advanced Stage Hodgkin's Lymphoma

Background : Classic Hodgkin's lymphoma (cHL) is associated with a high cure rate, but approximately 20% of patients are refractory to initial treatment and 10-30% of patients will relapse after achieving complete remission (CR) , the search of new prognostic factors to identify these high risk patients remains challenging in daily practice, the aim of this study is to evaluate CD68 positive Tumor associated macrophages(TAM) and the cell proliferation marker topoisomerase II alpha as prognostic factor in advanced stage (cHL). Patient and Methods: This study included 82 patients with Advanced stage classic Hodgkin lymphoma presented to Medical Oncology Departments of the National Cancer Institute, Cairo University and Zagazig University hospitals in the period from 2012 to 2017. All patients received ABVD regimen as induction. Immunohistochemical (IHC) analysis was performed for all cases using Tissue microarrays (TMAs) sections and stained with anti‐ CD68 (clones KP1 and PGM1, Dako, Glostrup,Denmark). For Topo II testing, IHC staining was performed on deparaffinized sections using the avidin-biotin complex immunoperoxidase technique with separate antibodies recognizing CD30 and the isoform of Topo II. The percentage of cells expressing cytoplasmic CD30 and nuclear Topo II staining simultaneously was determined for each case. We examined four cutoff points of CD68 expression (<= 20% versus >20%), <5% low (score 1), 5-15% intermediate (score 2) & > 15% high (score 3), <5% low (score 1), 5-25% intermediate (score 2) & > 25% high (score 3) and <= 50% versus > 50%. For Topo II expression, two cut off points were analyzed (<= 30% versus >30%) and (<= 75% versus >75%). Results: The study included 41 males and 41 females with a ratio of 1: 1. The median age was 30.5 years (range: 18-60 years). CRwas achieved in 64.6% (53/82 of patients) and 35.4% of all patients (29/82) were refractory. Relapse was reported in17% of the CR cases (9/53). The significant statistical difference in disease-free survival (DFS) for CD68+was observed at a cut off value of 50%, where 60 patients had CD68 <= 50% and 22 patients had CD68 >50%. The mean DFS for patients with CD68 <= 50% was 49.33 months ± 2.18 months (confidence interval: 45.05 to 53.60 months) vs. 35.255 ± 4.94 months (confidence interval: 25.55 to 44.95 months) in patients with CD68 > 50% (p = 0.015). The low CD68 group included more patients in CR (67.9%, n=36 vs. 32.1%, n=17) , male to female ratio was equal(1:1) in both groups (<= 50% ,n=30:30) and (> 50% ,n=11:11), B symptoms also was equal in both groups (<= 50% , 81% ,n=49) and (> 50% 81% ,n=18), patients with CD68<= 50% had more extra nodal disease (22%, n=13 vs. 18%, n=4) and also more bulky disease (20%, n=12 vs. 4.5% n=1) compared with high (CD68>50%) group. The overall survival (OS) was not significant for all CD68 cutoff points. Higher CD68 is associated with decreased OS: 48 vs. 52 month (CI: 42 to 53 month, p=0.42) at cut off 50%. Topo II expression using the two cut off points was not statistically significant in relation to both DFS and OS , the higher topoII group included more patients in CR (80% ,n=42 topo II >30% vs. 20%, n=10 topo II<=30%) also,(67%, n=53 Topo II >= 75% vs. 33%, n=17 Topo II < 75%). Higher Topo II is associated with prolonged DFS (45 months vs. 36 month) confidence intervals: 40 to 50 months vs 27 to 46. Months) at cut off 30% (p=0.8). At cutoff 75%, the DFS was 46 vs. 38 months (Confidence intervals: 41.73 to 52.10 months vs. 30 to 47.7 months, p= 0.36). Higher Topo II is associated with decreased OS: 50 vs.54 months (CI: 46 to 54 month) at cutoff 75% (p=0.19). CD68 was only statistically significant in correlation to patient age (p = 0.05), and non significant to serum albumin ,hemoglobin level , white blood cells count ,international prognostic score (IPS). Topo II was not statistically significant with the previous patient characteristics. Conclusion : High level of CD68 expression in patients with advanced stage HL (>50%) was significantly associated with inferior DFS but not OS, Topo II does not have significant prognostic value in relation to both DFS or OS of advanced HL cases. DisclosuresNo relevant conflicts of interest to declare.

Heat-enhancing aggregation of gold nanoparticles combined with loop-mediated isothermal amplification (HAG-LAMP) for Plasmodium falciparum detection

Malaria infection represents a major public health and economic issue that leads to morbidity and mortality globally. A highly effective and uncomplicated detection tool is required for malaria control in geographical hotspots of transmission. We developed a simple and more sensitive novel approach for the detection of the 18S rRNA gene of Plasmodium falciparum based on loop-mediated isothermal amplification (LAMP) and visualization using colorimetric, streptavidin-functionalized gold nanoparticles (SA-GNPs). Two loop primers of LAMP were biotinylated to produce biotin-containing products during amplification. After the addition of SA-GNPs, clusters of avidin-biotin complexes were established in the LAMP structure. While the positive reactions remained wine red, the negative reactions became colorless with partial aggregations induced by hydrochloric acid (HCl) under heat enhancement (60 °C). All steps of the assay were completed within 50 min, its detection limit was 1 parasite/μL, and it was highly specific for P. falciparum. This effortless detection system with high sensitivity and specificity could provide an alternative choice for malaria diagnostics in resource-limited regions.

Electroacupuncture improves lung function by suppressing mucin-5AC mediated EGFR-p38MAPK signaling and inflammation reaction in chronic obstructive pulmonary disease rats

To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36) on the expression of epidermal growth factor receptor (EGFR), tumor necrosis factor α(TNF-α) transfer growth factor α(TGF-α), interleukin-8(IL-8), p38 mitogen-activated protein kinases (p38MAPK), mucin-5AC (MUC5AC) and other related factors in chronic obstructive pulmonary disease (COPD) rats, so as to reveal its underlying mechanisms in improving COPD. A total of thirty male SD rats were randomly divided into normal control, model and EA groups, with 10 rats in each group. The COPD model was replicated using a combined method of tracheal infusion of lipopolysaccharide (LPS) and forced smoke-inhaling. EA (1-3 mA, 4 Hz/20 Hz) was applied to bilateral ST36 for 30 min, once daily for two consecutive weeks. The lung ventilation activities including the forced vital capacity (FVC) and forced expiratory volume (FEV) at 0.1 and 0.3 s (FEV0.1, FEV0.3) were detected. Histopathological changes of the middle lobe and bronchus of the right lung were observed after H.E. staining. The contents of TGF-α, TNF-α and IL-8 in the serum, bronchoalveolar lavage fluid (BALF) and superior lobe of the right lung were assayed by using ELISA, and the expression levels of EGFR, p38MAPK and MUC5AC proteins (inferior lobe of the left lung) and mRNAs (inferior lobe of the right lung) detected using Western blot, immunohistochemistry (strept avidin-biotin complex, SABC method) and real-time quantitative PCR, respectively. Compared with the normal group, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased (P<0.01), while the contents of TNF-α, TGF-α and IL-8 in the serum, BALF and lung tissues, expression levels of EGFR, p38MAPK and MUC5AC mRNAs and proteins, and the immunoactivity of EGFR, p38MAPK and MUC5AC in the lung tissues were significantly increased in the model group (P<0.01). After EA intervention, the decreased levels of the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC, and the increased levels of the abovementioned genes and proteins were all reversed in the EA group (P<0.01, P<0.05). After modeling, the bronchial walls were thickened, with enlarged alveolar cavities, fractured alveolar walls, obvious inflammatory cell infiltration, and rich mucus secretion in the lumen, which was relatively milder in the EA group. EA of ST36 can improve the ventilation function in COPD rats, which may be associated with its function in down-regulating the levels of TNF-α, TGF-α, IL-8, EGFR, p38MAPK and MUC5AC mRNAs and proteins in the lung tissues, inhibiting EGFR-p38MAPK signaling mediated expression of MUC5AC.

Evaluation of MMP-9 and iNOS expressions in sheep with encephalitic listeriosis

This study aimed to correlate Matrix Metalloproteinase-9 and iNOS expressions with the severity of histopathological findings in tissue samples taken from sheep with encephalitic listeriosis. Thus, the role of these molecules in the pathogenesis of the disease can be elucidated. After systemic necropsy, tissue samples of adult sheeps with meningoencephalitis were investigated by the culture, histopathological and immunohistochemical methods in the presence of Listeria spp. isolation from tissues was performed in accordance with the USDA-FSIS method with some modifications. Tissue samples were fixed in a 10% buffered formaldehyde solution. Following routine procedures, tissue sections at 5 μm were stained with Hematoxylin and Eosin, investigated under light microscope and photographed. Immunohistochemical staining was performed on the tissues using the avidin-biotin immune peroxidase complex method. Listeria spp. were obtained in 20 (83.3%) of 24 tissue samples with the presence of bright grey-black centred smooth colonies on Listeria Selective Agar and identified as Listeria monocytogenes through the phenotypically supportive tests. Liquefaction necrosis, purulent meningoencephalitis, perivascular cuffing, microabscesses and glial nodules were the most important histopathological findings. MMP-9 immunpositive reactions were observed in the cytoplasm of microglial cells and neurons in areas where inflammatory and necrotic areas are concentrated in medulla oblongata and pons. In perivascular cuffing areas, immune reactions in endothelial cells were detected. We detected iNOS positive reactions in the medulla oblangata and pons, especially in inflammatory cells in the microabscesses. Consequently, a positive correlation (p &amp;lt; 0.05) was found between MMP-9 expression and the severity of histopathological findings in sheep with encephalitic listeriosis. expression. In addition, we found that iNOS expression increased in parallel with the increase in MMP-9

Co-Inhibitors of Second Signal of Lymphocyte Response in Human Renal Transplants: PD-L2, GITR, and ILT-2/3/5 Positive Cells from Aspiration Biopsies Associate with Acute Rejection-Freedom

Following organ transplantation, the outcome of the encounter between an APC and a T lymphocyte is strongly dependent on the presence of costimulatory and co-inhibitory molecules, the former associated with allograft rejection and the latter with allograft acceptance. We evaluated the expression of PD-L2, GITR, ILT-2/3/5, and ILT-4 on graft-infiltrating cells procured by Fnab from human KTx under different immunosuppressive regimens. Methods: Fnab biopsies were performed on days 7 or 14 - 30 in stable KTx and on the day of acute rejection diagnosis. Cytopreparations were studied by the enzymatic avidin biotin complex staining. Results: Acute rejection group showed a significant down-regulated expression of PD-L2, GITR, and ILT-2/3/5 as compared to stable group, while for ILT-4 we did not find significant difference. Anti-IL2αR and rapamicyn treatment trend to down-regulate ILT-4 expression, although meaningless. A significantly positive correlation was observed between PD-L2 and GITR expression in Fnab. The PPV for acute rejection diagnosis for both PD-L2 and GITR was clearly above 0.8. Conclusions: Our findings point to an early entrance of cells expressing PD-L2, GITR and ILT-2/3/5 inside human KTx who are going to remain rejection-free. Both PD-L2 and GITR shared a high ability to rule-in and rule-out acute rejection.

Understanding the avidin-biotin binding based on polarized protein-specific charge.

In this study, charge updating schemes based on the local polarized protein-specific charge (LPPC) were introduced to vary the atomic charges of the biotin molecule and the residues in close contact during the simulation of the avidin-biotin complexes. The need of the charge variation of the ligand in response to changes in its surroundings was thoroughly studied. The results show that the calculated binding energy difference between biotin (BTN1) and 2'-iminobiotin (BTN2) and avidin is in excellent agreement with the experimental value, thus verifying the feasibility of updating the atomic charges of ligands during the simulation.

CD16+ Cells and Costimulatory Molecules of Lymphocyte Activation Present inside Human Kidney Grafts and in Blood Circulation

Background: We studied the expression of important costimulatory molecules of lymphocyte activation and the presence of CD16+ cells on aspiration biopsies of kidney transplants, measured three soluble factors and when indicated tested their robustness in diagnosing acute rejection. Methods: Fine-needle aspiration biopsies were performed either on days seven or 14 - 30 post-transplantation among stable kidney transplants and on the day of acute rejection diagnosis, while a sample of peripheral blood was collected simultaneously. The cyto preparations were studied by the enzymatic avidin biotin complex staining. The immunocytochemistry was directed to CD16, CD28, CD152, ICOS, CD40, CD154, CD26 and CD27. We performed the analysis in the peripheral blood by ELISA for soluble(s) CD16, CD26, and CD154. Results: The group of acute rejection cases showed a significant up-regulated expression of CD16, CD26, ICOS and CD40 as compared to the group of stable cases. Both sCD16 and sCD154 were significantly higher in the blood samples of the group with acute rejection. Thymoglobulin down-regulated CD154 and sCD16. CD16, CD26 and ICOS exhibited very high sensitivity and specificity for acute rejection diagnosis. Conclusions: The presence of CD16+ cells inside the graft emerged as a distinct player in acute rejection, confirming other previous reports whereas we first document that in human kidney transplants, ICOS and CD26 are significantly up-regulated and both reached positive predictive values for acute rejection ≥ 80%. The other costimulatory molecules, with the exception of CD40, though widely known, did not show robust association with immune events.

Histological study of gonadal tissues of adult Artemia salina (Linnaeus 1758) and immunohistochemistry by Caspase 3 and HSP70 to detect specific apoptosis markers on gonadal tissues after exposure to TBTCl.

Background:Several types of research have been recently carried out on the biological effects of TBTs, including investigations of genitals in invertebrates in response to exposure to TBTs in marine water.Aim:The objective of this research was to investigate the acute effects of tributyltin chloride (TBTCl) on gonads in the adult stage of Artemia salina by use normal histology and immunohistochemistry (IHC) (Caspase 3 and HSP70) to see specific apoptosis markers.Methods:After exposure of A. salina to different concentrations of TBTCl (25, 50, 100, 200, and 300 ng.l−1), 50 adult A. salina (25 male and 25 female) were selected randomly from each concentration to histologically study the gonads. The gonad tissue was sectioned (5 μm) and some slides were stained with hematoxylin and eosin and others were stained with IHC avidin–biotin complex, and were examined under a light microscope.Results:The results showed significant differences (p < 0.05) in histological lesions between different concentrations of TBTCl. The histological lesions in the testis and ovary section were undifferentiated cells, degenerating yolk globules, and follicle cells enveloping the oocyte which was then compared with control tissue, and these effects were found to be increased in females more than in males with the highest concentration of TBTCl. Immunohistochemistry (IHC) showed that positive immunostaining was observed in the testis and ovary as brownish deposits to Caspase 3 and HSP70 antibody after exposure to TBTCl, while the testis and ovary section in control tissue had no immunoreactivity to Caspase 3 and HSP70 antibody; these effects were profoundly increased with the highest concentration of TBTCl in females more than in males. Finally, the histological lesions and IHC (Caspase 3 and HSP70) revealed that the apoptosis and immune system stress of A. salina gonad tissue damage in females were more sensitive to TBTCl toxicity as compared to white males.Conclusion:In general, the present study aimed to observe the effects TBTCl on A. salina gonads by using histological sections and IHC (Caspase 3 and HSP70), which were evaluated for the first time and have been proven to possess an important function in apoptosis marker and immune system stress in Artemia. Finally, the specific mechanisms through which TBTCl affects A. salina Caspase 3 and HSP70 expression need further investigation.

Highly sensitive and rapid detection of porcine circovirus 2 by avidin-biotin complex based lateral flow assay coupled to isothermal amplification.

In this study, a new platform for the detection of porcine circovirus 2 was developed by avidin-biotin complex based lateral flow assay (LAMP-LFA). Improved detection sensitivity was attained by using loop mediated isothermal amplification (LAMP) with a low limit of detection (LOD), so the platform can be used to detect even early or asymptomatic stages of infection. LFA, which requires no specialized equipment, facilitates the use of point-of-care (POC) tests. Therefore, by applying LFA, the result can be confirmed accurately with the naked eye. Moreover, this platform has a unique structure using a single-tag detection system. The avidin-biotin interaction is the strongest interaction between proteins and has a higher Kd value than antigen-antibody interactions. Thus, the results are stable and can be clearly confirmed. The high sensitivity of LAMP-LFA enables all steps to be completed in 30 min. As a result, it could be applied to different targets, such as other pathogens. Future POC diagnostic studies are expected to be of great practical benefit.

Innate Immune Response in Human Kidney Transplantation: IRF3 and IRF7 Together with Interferon-Alpha Are Significantly Up-Regulated in Acute Rejection

Background: Ischemia-reperfusion injury of organ transplantation activates several mediators which may link the innate to the adaptive immune response. Down the cascade of TLRs, we selected to study the expression of Interferon Regulatory Factors (IRF)-3 and -7 inside human Kidney Transplanted (KTx) organs and the synthesis of IFNα, the main growth factor induced by them, in KTx aspiration biopsy cultures. Simultaneously, we tested their robustness in diagnosing Acute Rejection (AR). Methods: Fine-needle aspiration biopsies (F-nab) were performed either on day 7 or 14 post-KTx among stable patients or on the day of AR diagnosis. On Fnab cytopreparations, we studied IRF3 and IRF7 by the enzymatic avidin-biotin complex staining, and in a different group of cases we quantified IFNα by ELISA in 48 hours Fnab culture supernatants. Results: AR group showed a significantly up-regulated expression for IRF3 and IRF7, reaching Positive Predictive Values (PPV) of 0.824 and 0.8, respectively, as well as Negative Predictive Values (NPV) above 0.9 for both; IFNα presented a PPV = 1.0 and a NPV = 0.9. A variation in the results was noticed according to different immunosuppressive therapies. Conclusions: Our findings suggest that IRF3 and IRF7, and IFNα which they promote, may be very important players in the early days post-KTx, linking the innate with an adaptive response and triggering acute rejection. These differences were very clear-cut, lending consistency to our speculation. It would be important to scrutinize for other potential effects derived from these IRFs up-regulation which could be of clinical relevance.

Distribution of Antigens Defined by OKB Monoclonal Antibodies on Benign and Malignant Lymphoid Cells and on Nonlymphoid Tissues

AbstractMonoclonal antibodies OKB1, OKB2, OKB4 and OKB7 have been previously shown to detect distinctive antigens displayed on B, but not on T, lymphocytes. Benign and malignant lymphoid cells were investigated for their reactivity with these antibodies in cell suspension by indirect immunofluorescence and in cryostat tissue sections by the avidin-biotin complex immunoperoxidase technique. Fetal liver pre-B cells and pre-B and common type acute lymphoblastic leukemia cells isolated from 15 patients were OKB1− OKB2 OKB4+ OKB7−. All mature lymphoid tissue B cells and the neoplastic cell surface immunoglobulin-positive (SIg+) B cells isolated from each of 47 B cell neoplasms were OKB2+. OKB1 and OKB7 were expressed by interfollicular, follicular center, and many, but not all, mantle zone B cells. OKB4 was expressed by follicular center cells, but not by mantle zone or interfollicular B cells. The neoplastic SIg+ B cells isolated from 45 of 47 B cell malignancies were OKB1+ OKB4+, and those isolated from 45 of 46 B cell malignancies were OKB7+. The neoplastic B cells of one mantle zone lymphoma were OKB1−, of one small lymphocytic cell lymphoma were OKB7−, of one large cell lymphoma were OKB4−, and of one small lymphocytic cell lymphoma with a monoclonal gammopathy were OKB1− OKB4−. Normal and myeloma plasma cells were OKB−. The malignant T cells isolated from 12 T cell neoplasms were OKB2− OKB4−, but were OKB1+ and/ or OKB7+ in 3 cases. Thus, the OKB antibodies appear to detect distinctive antigens that may be expressed at different stages of B cell differentiation. In addition, OKB4 reacted with selected renal and respiratory epithelium, and OKB2 reacted with a wide range of epithelial tissues. The OKB antibodies should prove useful in the investigation of B cell differentiation and may aid in the identification and characterization of lymphoproliferative malignancies with significant therapeutic and prognostic differences not identifiable by conventional histopathologic and immunologic methods.

Diagnostic Utility of BRCA1-Associated Protein 1 (BAP1) in Distinguishing Reactive and Neoplastic Mesothelial Proliferation

Background: Malignant mesothelioma (MM) is an aggressive tumor which arises from the lining of serous cavities. Distinguishing between begnin and malignant mesothelial proliferation on effusions is a challenge. This study aims to evaluate the diagnostic utility of BAP1 in distinguishing MM from reactive mesothelial proliferation.Material and methods: This is a retrospective study on 61 cases; pleural biopsies (n=36) and pleural cell blocks (n=25). Included were synchronous cytology/ biopsy pair samples. All cases were stained with BAP-1antibody using avidin-biotin complex. BAP1 immunohistochemistry was evaluated using cut off value; negative staining indicate malignancy. Statistical analysis was done using SPSS (version 20), P value (P value <0.05 was considered statistically significant). ROC curve to predict cut off value.Results: In MM cases 61.1% showed BAP1 negative nuclear expression. In reactive mesothelial proliferation cases; 20% showed BAP1 negative expression. In atypical mesothelial proliferation cases, 80% showed BAP1 negative nuclear expression. synchronous cytology/ biopsy pairs (13 cases) showed BAP1 matching results. There was a highly statistically significant correlation between BAP1 expression and the study groups (P-value <0.001). There was statistically significant correlation between BAP-1 expression and histological types of mesothelioma (P-value <0.05) and stage (P-value <0.05). In cell blocks; sensitivity was 80% and specificity was 80% for atypical mesothelial proliferation. In tissue biopsy; sensitivity was 61.1% and Specificity was 80% for mesothelioma.Conclusion: In effusions, negative BAP1 strongly support the diagnosis of malignant mesothelioma, so BAP1 may be included in immunohistochemical panels for malignant mesothelioma cytodiagnosis.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy

Mesenchymal stem cells (MSCs) have a tumor-homing ability—they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats using the Thymidine Analog BrdU.

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The immunodetection of thymidine analogs (such as 5-Bromo-2'-deoxyuridine (BrdU)) is a standard technique used for visualizing these newly generated cells. Therefore, BrdU is usually injected in small animals intraperitoneally, so the thymidine analog gets incorporated into dividing cells during DNA synthesis. Detection is performed by immunohistochemical analysis of brain slices. Every research group that has been using this technique can appreciate that it requires special attention to minute details to achieve a successful stain. For instance, an important step is DNA denaturation with HCl, which allows it to reach the cell nucleus to stain it. However, the existing scientific reports describe very few of such steps in detail. Therefore, standardizing the technique is challenging for new laboratories as it can take several months to yield positive and successful outcomes. The purpose of this work is to describe and elaborate the steps to obtain positive and successful outcomes of the immunostaining technique in detail when working with the thymidine analog BrdU. The protocol includes the reagent preparation and setup, administration of thymidine analog in a rodent, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, use of avidin-biotin complex, immunofluorescence, counterstaining, microscopy imaging, and cell analysis.

Detection of feline coronavirus RNA, spike gene mutations, and feline coronavirus antigen in macrophages in aqueous humor of cats in the diagnosis of feline infectious peritonitis.

Uveitis is common in cats, and is often a feature of feline infectious peritonitis (FIP). We evaluated 3 tools for detection of feline coronavirus (FCoV) in aqueous humor: 1) a 7b gene reverse-transcription real-time PCR (7b-RT-rtPCR) assay to detect FCoV RNA, 2) a spike gene mutation RT-rtPCR (S-RT-rtPCR) assay to detect 2 point mutations in the spike gene of FCoV in cats positive by 7b-RT-rtPCR, and 3) immunocytochemistry (ICC) for detection of FCoV antigen in aqueous humor macrophages. We studied 58 cats, including 31 cats with FIP and 27 control cats. FIP was excluded by postmortem examination and negative immunohistochemistry (IHC). Aqueous humor samples obtained postmortem were assessed using 7b-RT-rtPCR in all cats, and positive samples were evaluated with S-RT-rtPCR. ICC evaluation of aqueous humor samples from 36 of the 58 cats was done using an avidin-biotin complex method and monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, and negative and positive predictive values were calculated including 95% CIs. 7b-RT-rtPCR had a specificity of 100.0% (95% CI: 87.2-100.0) and sensitivity of 35.5% (95% CI: 19.2-54.6). Specificity of S-RT-rtPCR could not be determined because there were no FCoV 7b-RT-rtPCR-positive samples in the control group. Sensitivity of S-RT-rtPCR was 12.9% (95% CI 3.6-29.8). Sensitivity and specificity of ICC were 62.5% (95% CI: 40.6-81.2) and 80.0% (95% CI: 44.4-97.5), respectively. The combination of 7b-RT-rtPCR and IHC could be useful in diagnosing FIP; S-RT-rtPCR did not add value; and ICC of aqueous humor samples cannot be recommended for the diagnosis of FIP.

Myointimoma of the penis

Myointimoma is an uncommon, benign soft-tissue tumor derived from the intimal cells of blood vessels. Since little is known about this rare tumor entity, our aim is to describe an additional case and to perform the first literature review on this topic. A 49-year-old Caucasian man presented with a 12-month history of a palpable, firm, solitary lesion involving the glans penis. On physical examination, there was a 1 cm palpable, endophytic well-circumscribed nodule located to the left side of glans penis, close to the coronal sulcus, with disease-free external urethral orifice. The patient was submitted to complete excisional biopsy. A skin rhombus measuring 1.1 × 0.8 × 0.3 cm was removed and the biopsy sample, fixed in 10% formaldehyde, sent to Pathology. At the 18-month follow-up visit, the patient was clinically disease free. Histopathology revealed a multinodular intravascular proliferation of the corpus spongiosum. This myointimal proliferation comprised bland predominantly spindle cells in an abundant fibromyxoid stroma. Immunostains for smooth muscle actin (1A4), cytokeratins (AE1/AE3, CAM5.2), and CD34 were carried out using the avidin-biotin complex (ABC) immunoperoxidase method. Lesional cells displayed positivity for smooth muscle actin and negativity for cytokeratins and CD34. Myointimoma is confirmed to be a penile benign lesion that may be adequately treated with excisional biopsy. Even after incomplete or marginal removal, the penile lesion has been shown to remain stable overtime or regress. Differential diagnosis is essential to exclude similar histologic entities that could be more aggressive or have possible systemic implications.

Decreased expression of cytokeratin 15 and tropoelastin in men with androgenetic alopecia and its relationship with increased expression of p15/p16

IntroductionAndrogenetic alopecia (AGA) or male pattern baldness is a polygenic disease with a high prevalence in the Caucasian population, with psychosocial implications. However, the molecular mechanisms involved are unknown. The aim of this study was to assess the possible changes in protein expression of cytokeratin 15 (CK15), tropoelastin (TE) and cellular senescence markers (p15/p16) in AGA patients.Material and methodsAn observational, analytical and prospective cohort study was performed in 57 men with AGA (42.31 ±3.01 years). Two tissue biopsies were taken: the control area (C) and the alopecia area (A). Cell viability was assessed using scalp explants from the patients, maintaining explants long term (90 days). Protein expression analysis was performed by immunohistochemistry by detecting antigen-antibody reactions (avidin-biotin complex).Resultshe results showed a significantly higher percentage of dead cells in area A (17.00 ±1.09% C vs. 28.50 ±1.41% A, *p &lt; 0.05). The area affected by alopecia showed significantly lower CK15 and TE protein expression in hair follicles, sebaceous glands and epidermis (*p &lt; 0.05). Expression of the senescence marker p15/p16 was significantly higher in hair follicles, hair bulbs and the epidermis (*p &lt; 0.05).ConclusionsThe results suggest that patients with AGA suffer tissue damage that affects different components of hair follicle stem cells as well as the extracellular matrix itself.

Expression of PCNA, MMP-9, p53, Bax and Bcl-2 in canine transmissible venereal tumors

In this study, we aimed to evaluate the proliferative, metastatic and apoptotic capacities of TVT cases, which are in various phases of development, by using immunohistochemical markers. The material of this study consisted of twelve female and six male dogs diagnosed with TVT brought to our department between 2007 and 2020 years. Diff-quick staining was applied to the smear taken from tumoral masses for cytological examinations. Tumoral tissues from dogs were fixed in a 10% neutral buffered formaldehyde solution. After routine tissue procedures Hematoxylin &amp;amp; Eosin stain was applied to the sections. Tissue sections were investigated under a light microscope and photographed. Immunohistochemical staining was performed on the tissues using the avidin-biotin immune peroxidase complex method. As a result of macroscopic, cytological and histopathological examinations, TVT positive cases were divided into three according to their developmental stages. While the expression of PCNA, MMP-9, mutant p53 and Bcl-2 increased significantly in progressive cases compared to regressive and stable cases, Bax expression increased significantly in regressive cases compared to progressive and stable cases. In conclusion, we thought that the mentioned markers are very useful for understanding the prognosis of TVT, the tumor aggressiveness and the survival of the malignant cells.

Exhausted phenotype of follicular CD8 T cells in CVID

Exhausted phenotype of follicular CD8 T cells in CVID