Indirect Immunofluorescence Staining Research Articles

Ex Vivo Analysis of Cell Differentiation, Oxidative Stress, Inflammation, and DNA Damage on Cutaneous Field Cancerization.

Cutaneous field cancerization (CFC) refers to a skin region containing mutated cells' clones, predominantly arising from chronic exposure to ultraviolet radiation (UVR), which exhibits an elevated risk of developing precancerous and neoplastic lesions. Despite extensive research, many molecular aspects of CFC still need to be better understood. In this study, we conducted ex vivo assessment of cell differentiation, oxidative stress, inflammation, and DNA damage in CFC samples. We collected perilesional skin from 41 patients with skin cancer and non-photoexposed skin from 25 healthy control individuals. These biopsies were either paraffin-embedded for indirect immunofluorescence and immunohistochemistry stain or processed for proteins and mRNA extraction from the epidermidis. Our findings indicate a downregulation of p53 expression and an upregulation of Ki67 and p16 in CFC tissues. Additionally, there were alterations in keratinocyte differentiation markers, disrupted cell differentiation, increased expression of iNOS and proinflammatory cytokines IL-6 and IL-8, along with evidence of oxidative DNA damage. Collectively, our results suggest that despite its outwardly normal appearance, CFC tissue shows early signs of DNA damage, an active inflammatory state, oxidative stress, abnormal cell proliferation and differentiation.

Elevated levels of anti-Golgi antibodies: An early sign of seronegative rheumatoid arthritis.

Anti-Golgi antibodies are uncommon antibodies that exhibit specific, polarized cytoplasmic staining on the Hep-2 substrate. The objective of our study was to identify the clinical and laboratory features associated with anti-Golgi antibodies. We examined 4.5 years of data from a Turkish tertiary hospital in this retrospective cohort analysis. The indirect immunofluorescence staining patterns, antinuclear antibody (ANA) titres and clinical data of all patients were obtained from the hospital record system. A total of 146,055 ANAs were detected, of which 224 patients (0.15%) exhibited anti-Golgi antibody staining. In total, 39.4% of diagnosed patients had autoimmune diseases (AIDs). Of the AIDs, 26 (46.4%) were rheumatoid arthritis (RA). This is a very high rate and another remarkable point is that 17 (65.3%) of these patients had seronegative RA. High-titre results (1 ≥ 1/320) were more common in patients with AID. Anti-Ro52 was prevalent in 50% of extractable nuclear antigen (ENA)-positive patients, making it a remarkable finding. The majority of individuals with high-titre anti-Golgi antibodies had AID, particularly RA. The majority of these patients also tested negative for anti-cyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF). Finally, high-titre anti-Golgi antibodies may be an important serologic marker for seronegative RA in the Turkish population.

Abstract 5166: A multiplexed immunofluorescence assay to assess Rb and phospho-Rb as predictive biomarkers for CDK4/6 inhibitors in HR+/HER2- breast cancer

Abstract Introduction: Cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) are used as the first-line treatment of advanced/metastatic or high-risk HR+/HER2- breast cancer (BC) that make up about 60-70% of all cases. However, nearly 50% of patients exhibit drug resistance. Therefore, biomarkers that predict CDK4/6i response are needed to spare patients from toxicities and high costs. Resistance to CDK4/6i [palbociclib, ribociclib, and abemaciclib] is associated with the aberrant or loss of Retinoblastoma (Rb). A decline in phospho-Rb (pRb) levels on therapy is also associated with CDK4/6i response. We aimed to develop a multiplex immunofluorescence (IF) assay to assess Rb and pRb levels that can be used on circulating tumor cells (CTCs) to predict tumor response to CDK4/6i. Here, we report the selection of internal controls for Rb and pRb, assay repeatability and reproducibility, as well as compatibility of the CTC isolation protocol with the assay. Methods: Rb and pRb expression was evaluated by western blotting (WB) and IF in parental (P), estrogen deprivation-resistant (EDR), palbociclib-resistant (PalboR), and EDR/PalboR derivatives of two HR+/HER2- cell lines (MCF7 and T47D), Rb1 KO cells of MCF7 and T47D, as well as triple-negative cell lines, BT549 and MDA-MB-468. IF-stained cells were imaged using Leica STED SP8 confocal microscope and Zeiss Axioscan 7. The concordance between IF and WB results was assessed using a Bland-Altman plot. Selectivity and specificity of the assay as well as repeatability and reproducibility between investigators were also assessed. Blood from healthy subjects was used to test the compatibility of the ScreenCell® filtration system to isolate CTC with the IF assay. Results: Our initial approach utilized indirect IF staining for Rb and pRb using the same primary antibodies as WB. On the Bland-Altman plot, most data points were relatively close to the bias line, suggesting that the difference between the two methods was acceptable. The analytical range for mean Rb and pRb staining intensity was 0.744 - 46.779 and 1.001 - 52.919, respectively. Based on WB and IF data, we nominated T47D PalboR and MDA-MB-468 cells as negative controls for Rb and pRb expression. The selectivity and specificity were 100% and 70%, respectively, for the Rb expression and 99% and 61% for pRb, respectively. The IF assay was repeatable and reproducible between two investigators. The ScreenCell® CTC isolation method was not compatible with the assay, as Rb and pRb expression was either not detected or limited when processed using FC2 or PBTFC buffer, irrespective of the antigen retrieval. Conclusion: We have selected internal cell line controls for Rb and pRb and are in the process of optimizing signal-to-noise ratio and specificity using conjugated antibodies. We are also testing the compatibility of other CTC isolation methods with the IF assay. Citation Format: Mantasha Tabassum, Shivaani Suresh Kanna, Wangjia Cao, Mothaffar Rimawi, George E Miles, Meghana V Trivedi. A multiplexed immunofluorescence assay to assess Rb and phospho-Rb as predictive biomarkers for CDK4/6 inhibitors in HR+/HER2- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5166.

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Human papillomavirus E7 protein induces homologous recombination defects and PARPi sensitivity

PurposeCervical cancer is a common gynecological malignancy, pathologically associated with persistent infection of high-risk types of human papillomavirus (HPV). Previous studies revealed that HPV-positive cervical cancer displays genomic instability; however, the underlying mechanism is not fully understood.MethodsTo investigate if DNA damage responses are aggravated in precancerous lesions of HPV-positive cervical epithelium, cervical tissues were biopsied and cryosectioned, and subjected to immunofluorescent staining. Cloned HA-tagged E6 and E7 genes of HPV16 subtype were transfected into HEK293T or C33A cells, and indirect immunofluorescent staining was applied to reveal the competency of double strand break (DSB) repair. To test the synthetic lethality of E7-indued HRD and PARP inhibitor (PARPi), we expressed E7 in C33A cells in the presence or absence of olaparib, and evaluated cell viability by colony formation.ResultsIn precancerous lesions, endogenous DNA lesions were elevated along with the severity of CIN grade. Expressing high-risk viral factor (E7) in HPV-negative cervical cells did not impair checkpoint activation upon genotoxic insults, but affected the potential of DSB repair, leading to homologous recombination deficiency (HRD). Based on this HPV-induced genomic instability, the viability of E7-expressing cells was reduced upon exposure to PARPi in comparison with control cells.ConclusionIn aggregate, our findings demonstrate that HPV-E7 is a potential driver for genome instability and provides a new angle to understand its role in cancer development. The viral HRD could be employed to target HPV-positive cervical cancer via synthetic lethality.

Effect of dental composite dust on human gingival keratinocytes.

The aim was to investigate the effect of particles released during grinding of dental composites on human gingival keratinocytes (HGK). Specimens from Filtek™ Supreme XTE and ceram.x® universal were prepared and ground to dust. The dust was filtered (≤ 5µm) and the particle size distribution was examined using NANO-flex®-180° dynamic light scattering (DLS). Suspensions at five concentrations (3, 10, 30, 100 and 300µg/mL) were prepared using keratinocyte growth medium (KGM). These suspensions, as well as a positive (CuO) and a negative control (KGM) were added to HGK. The cells treated with Filtek™ Supreme XTE suspensions were analyzed by real-time monitoring using RTCA iCELLigence™. In addition, light and scanning electron microscopic images of the exposed cells were taken. Indirect immunofluorescence staining was performed to detect the extracellular matrix protein fibronectin. In distilled water, DLS showed similar particles' range (171.9nm- 2.7µm) for both composites. In saliva, larger particles were detected (Filtek™ Supreme XTE: 243nm-6,5µm; ceram.x® universal: 204nm- 4,6µm). iCELLigence™ revealed similar results of cell growth parameters for HGK incubated with composite dust (≤ 5µm) at different concentrations. The microscopic images indicated unaltered cell structures and formation of large agglomerates with high particle concentration (> 100µg/mL). Exposure to composite dust resulted in upregulation of fibronectin expression. Grinding of dental composite materials generates dust particles of different sizes. The particle size distribution seems to be more influenced by the suspending medium than the material itself. While cell growth of HGK seem not to be affected by the particles, an upregulation of fibronectin in the intercellular space concomitant by increasing particle concentration may indicate an increase of cell migration/mobility.

Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina.

Investigation of nuclear lamina architecture relies on superresolved microscopy. However, epitope accessibility, labeling density, and detection precision of individual molecules pose challenges within the molecularly crowded nucleus. We developed iterative indirect immunofluorescence (IT-IF) staining approach combined with expansion microscopy (ExM) and structured illumination microscopy to improve superresolution microscopy of subnuclear nanostructures like lamins. We prove that ExM is applicable in analyzing highly compacted nuclear multiprotein complexes such as viral capsids and provide technical improvements to ExM method including three-dimensional-printed gel casting equipment. We show that in comparison with conventional immunostaining, IT-IF results in a higher signal-to-background ratio and a mean fluorescence intensity by improving the labeling density. Moreover, we present a signal-processing pipeline for noise estimation, denoising, and deblurring to aid in quantitative image analyses and provide this platform for the microscopy imaging community. Finally, we show the potential of signal-resolved IT-IF in quantitative superresolution ExM imaging of nuclear lamina and reveal nanoscopic details of the lamin network organization-a prerequisite for studying intranuclear structural coregulation of cell function and fate.

HBx-induced PLA2R overexpression mediates podocyte pyroptosis through the ROS-NLRP3 signaling pathway

Introduction Hepatitis B virus-associated glomerulonephritis (HBV-GN) is one of the main types of secondary glomerular diseases, and podocyte injury is an important pathogenic mechanism of HBV-GN, participating in the occurrence and development of HBV-GN. However, the specific mechanism of podocyte injury remains to be studied. Methods Human renal podocytes cultured in vitro were divided into six groups. The podocyte morphology was observed under a transmission electron microscope, and the expression of M-type phospholipase A2 receptor (M-PLA2R) on the podocyte membrane was observed by indirect immunofluorescence staining under a fluorescence microscope. The pyroptosis rate and reactive oxygen species (ROS) of podocytes were assessed by FLICA/PI double staining and flow cytometry. Western blot (WB) and quantitative real-time PCR (qPCR) were used to determine the expression of PLA2R, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β, and IL-18. Results Hepatitis B virus X (HBx) transfected into human renal podocytes in vitro induced the overexpression of PLA2R. Moreover, the overexpressed PLA2R combined with secretory phospholipase A2 group IB (sPLA2-IB) aggravated podocyte injury and increased the pyroptosis rate. In addition, the expression of ROS, the NLRP3 inflammasome and downstream inflammatory factors was increased. In contrast, after inhibiting the expression of PLA2R and ROS, podocyte damage was alleviated, and the pyroptosis rate and the expression of genes related to the ROS-NLRP3 signaling pathway were decreased. Conclusion HBx-induced PLA2R overexpression on the podocyte membrane can significantly upregulate the ROS-NLRP3 signaling pathway, thereby mediating podocyte pyroptosis.

Transcriptomic analysis of LMH cells in response to the overexpression of a protein of Eimeria tenella encoded by the locus ETH_00028350.

A protein of Eimeria tenella (encoded by the locus ETH_00028350) homologous to Toxoplasma gondii dense granule protein 9, designated as EtHGRA9 hereafter, was reported to be expressed in all life cycle stages of E. tenella. However, no data are currently available regarding its functional properties. In the present study, a recombinant vector harboring a 741 bp gene segment encoding the mature form of EtHGRA9 was constructed and transfected into leghorn male hepatoma (LMH) cells. Then, transcriptomic analysis of the transfected LMH cells was carried out by using a high-throughput RNA-seq technology. The LMH cells overexpressing EtHGRA9 was validated by means of Western blotting as well as indirect immunofluorescence staining. The results demonstrated that the expression of 547 genes (275 upregulated genes and 272 downregulated genes) was altered by EtHGRA9. The quantitative real-time polymerase chain reaction (qRT-PCR) validation of the ten genes with differential expression between the two groups was consistent with the transcriptome analysis. According to pathway enrichment analysis for the obtained differentially expressed genes, seven pathways were significantly affected by EtHGRA9, such as cytokine-cytokine receptor interaction, MAPK signaling pathway, and protein processing in endoplasmic reticulum. Our data reveal several possible roles of EtHGRA9 in immune or inflammatory responses, which paves the way for a better understanding of the molecular interplay between E. tenella and its host.

Colocalization of senescent biomarkers in deep, superficial, and ovarian endometriotic lesions: a pilot study

Endometriosis is a prevalent gynecological condition with deleterious effects on women’s quality of life in terms of physical, emotional, and social compromise. It is an inflammatory disease characterized by the presence of endometrial-like tissue outside the uterus, and its presentation varies from superficial peritoneal lesions to deep infiltrative endometriosis and ovarian endometrioma. In our previous study, endometriotic lesions were implicated in cellular senescence as their inflammatory pattern could potentially compromise surrounding tissue integrity, thereby inducing a senescent state in cells. P16Ink4a and lamin b1 are biomarkers used to assess cellular senescence. Indirect immunofluorescence staining is a broad technique used to assess cellular structure and behavior driven by protein–protein interactions that provide valuable information about cell functioning. The etiopathogeny of endometriosis is not completely understood and diagnostic approaches still rely on invasive methods; therefore, it is important to use validated methods to increase our understanding of the disease and the development of novel diagnostic tools. However, indirect immunofluorescence protocols are often tissue specific and, if neglected, can lead to misinterpretation of results. Moreover, no valid endometriotic tissue-specific colocalization immunofluorescence protocols have been established. Thus, we have validated a well-funded and suitable protocol to allow precise evaluation of the three presentations of endometriosis lesions using indirect immunofluorescence aiming to support further investigations in endometriosis lesions.

Tracheal cellular immune response in chickens inoculated with Mycoplasma synoviae vaccine, MS-H or its parent strain 86079/7NS

Mycoplasma synoviae causes respiratory tract disease in chickens characterised by mild to moderate lymphoplasmacytic infiltration of the tracheal mucosa. MS-H (Vaxsafe1 MS, Bioproperties Pty Ltd.) is an effective live attenuated vaccine for M. synoviae, but the immunological basis for its mechanism of protection has not been investigated, and the phenotypes of lymphocytes and associated cytokines involved in the local adaptive immune response have not been described previously. In this study, specific-pathogen-free chickens were inoculated intra-ocularly at 3 weeks of age with either M. synoviae vaccine strain MS-H or vaccine parent strain 86079/7NS (7NS), or remained uninoculated. At 2-, 7- and 21 days post-inoculation (dpi), tracheal mucosal pathology, infiltrating lymphocytes subsets and transcription levels of mRNA encoding 8 cytokines were assessed using light microscopy, indirect immunofluorescent staining and RT-qPCR, respectively. After inoculation, tracheal mucosal thickness, tracheal mucosal lesions, and numbers of infiltrating CD4+CD25- cells, B-cells, and macrophages were greater in MS-H- and 7NS-inoculated chickens compared with non-inoculated. Inoculation with 7NS induced up-regulation of IFN-γ, while vaccination with MS-H induced up-regulation of IL-17A, when compared with non-inoculated birds. Both inoculated groups had a moderate infiltrate of CD4+CD25+ T cells in the tracheal mucosa. These findings reveal that the tracheal local cellular response after MS-H inoculation is dominated by a Th-17 response, while that of 7NS-inoculated chickens is dominated by a Th-1 type response.

Occurrence of autoantibodies against skin proteins in patients with hereditary epidermolysis bullosa predisposes to development of autoimmune blistering disease.

Skin blistering disorders are associated with inherited defects in proteins involved in the dermal-epidermal adhesion or autoantibodies targeting those proteins. Although blistering in hereditary epidermolysis bullosa (EB) is pathogenetically linked to genetic deficiency of distinct proteins of the epidermis or the dermal-epidermal junction, circulating autoantibodies against these proteins have also been identified in EB patients. So far, autoantibodies have been considered bystanders in EB and active pathogenicity of them in EB has not been disclosed. In sera of a cohort of 258 EB patients, we found by ELISA in 22% of the patients autoantibodies against the bullous pemphigoid antigen BP180. The titers correlated negatively with collagen VII skin expression and positively with disease severity. Among those patients, we identified six (2.33%) with clinical features of an autoimmune bullous disorder (AIBD) and positive indirect immunofluorescence (IIF) staining. In literature, we found four more cases of EB patients developing disease-aggravating AIBD. Co-existence of these two rare skin disorders suggests that EB patients have a predisposition for the development of AIBD. Our work highlights that EB patients with increased itch or blister formation should be evaluated for additional AIBD and repeated screening for changes in autoantibody titers and skin-binding specificities is advised.

Improved in vitro endothelialization on nanostructured titania with tannin/glycosaminoglycan-based polyelectrolyte multilayers

Blood compatibility of cardiovascular implants is still a major concern. Rapid endothelialization of these implant surfaces has emerged as a promising strategy to enhance hemocompatibility and prevent complications such as thrombus formation and restenosis. The successful endothelialization of implant surfaces mostly depends on the migration of endothelial cells (ECs), the differentiation of stem cells, and the inhibition of smooth muscle cell (SMC) proliferation. Our previous study demonstrated that nanostructured titania surfaces modified with polyelectrolyte multilayers based on tanfloc (a cationic tannin derivative) and glycosaminoglycans (heparin and hyaluronic acid) have improved antithrombogenic properties. In this work, we used in vitro cell culture of ECs and SMCs to investigate the outcomes of these surface modifications on endothelialization. The cells were seeded on the surfaces, and their viability, adhesion, and proliferation were evaluated after 1, 3, and 5 days. Indirect immunofluorescent staining was used to determine the cellular expression of ECs through the presence of specific marker proteins after 7 and 10 days, and EC migration on the NT surfaces was also investigated. The surfaces modified with tanfloc and heparin showed enhanced EC adhesion, proliferation, and migration. However, SMC proliferation is not promoted by the surfaces. Therefore, these surfaces may promote endothelialization without stimulating SMC proliferation, which could improve the hemocompatibility without enhancing the risk of SMC proliferation leading to restenosis. The surface modification here proposed is a promising candidate to be used in cardiovascular applications due to enhanced antithrombogenic and endothelialization properties.

P450 Side-Chain Cleavage Enzyme (P450-SCC) Is an Ovarian Autoantigen in a Mouse Model for Autoimmune Oophoritis.

Steroid-producing cells contain key cytochrome P450 enzymes, such as side-chain cleavage (P450-SCC) and 17α-hydroxylase (17α-OH). They are required for steroid hormone synthesis and considered antigens associated with Addison's disease and autoimmune primary ovarian insufficiency (POI). We studied an animal model for human autoimmune POI in mice with autoimmune oophoritis induced by neonatal thymectomy performed at day 3 (TX3). We previously identified an oocyte-specific protein as a major antigen inciting autoimmune oophoritis in mice. In this study, we characterized ovarian steroid-producing cell antigens. Using indirect immunofluorescence staining, we tested immune reactions in mouse ovarian and adrenal tissue sections with sera from TX3 female mice. More than half of the TX3 mice (8 of 15) produced antibodies reacting with both ovarian and adrenal steroid-producing cells, including some that reacted to oocytes as well. We produced recombinant proteins for the three key steroidogenic enzymes 17α-OH, P450-SSC, and 3β-hydroxysteroid dehydrogenase (3β-HSD) and tested their immune reactions with individual mouse sera. By immunoblotting, all mouse sera that reacted with the steroid-producing cells (n = 8) were shown to react with the P450-SCC, but not with the 17α-OH or 3β-HSD recombinant proteins. The sham-operated mouse sera and TX3 mouse sera negative for steroid-producing cells did not react with the P450-SCC recombinant protein. Our findings indicate that the P450-SCC is a specific and unique major antigen within the ovarian steroid-producing cells. Given their similarity of predicted antigenicity, we assume that P450-SCC acts in human autoimmune POI as it does in mouse autoimmune oophoritis.

Frequency of anti-neutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Patients with SLE exhibit multiple serum autoantibodies, including anti-neutrophil cytoplasmic antibodies (ANCAs). There are two main techniques to detect ANCAs: indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA). In this study, an attempt was made to determine the frequency and clinical associations of ANCAs in patients with SLE. MethodsA cross-sectional study was conducted in a tertiary care hospital in Colombia that included 74 patients with SLE. The presence of ANCAs was assessed using IIF with ethanol-fixed slides, and ELISA was used to detect antibody specificities for myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA. ResultsOf the 74 patients with SLE evaluated, 60 (81.1%) of them were ANCA-positive by IIF. By contrast, only one patient showed specificity for PR3-ANCA by ELISA. The relevance of ANCA positivity by IIF and clinical and serological features was significant for renal involvement (p=.0174), and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (p=.0308). ConclusionANCAs are common in the serum of patients with SLE, as detected by ethanol-fixed slides with IIF staining. However, detection of specificity to PR3 and/or MPO is rare, thus highlighting the importance of detecting these autoantibodies by different techniques.

Microvalve Bioprinting of MSC-Chondrocyte Co-Cultures.

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof-of-concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures to investigate whether the use of MSCs in ACI procedures would stimulate enhanced ECM production by chondrocytes. Microvalve-based bioprinting uses small-scale solenoid valves (microvalves) to deposit cells suspended in media in a consistent and repeatable manner. In this case, MSCs and chondrocytes have been sequentially printed into an insert-based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate ECM production. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This study, therefore, provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

PD-1 gene polymorphisms and thyroid expression of PD-1 ligands differ between Graves’ and Hashimoto’s diseases

The programmed cell death-1 (PD-1)/PD ligand pathway plays a key role in the maintenance of peripheral tolerance by enhancing the suppressive activity of regulatory T (Treg) cells. The promoter activity of the A allele of PD1 rs36084323 G/A polymorphism is lower than that of the G allele. We examined the association of PD1 gene polymorphisms, PD-1 expression on Treg cells, and thyroid PD-1/PD-1 ligand (PD-L1) expression with the pathogenesis of autoimmune thyroid disease (AITD). We classified patients and genotyped PD-1 polymorphisms by using the PCR-RFLP method in a total of 176 Graves’ disease (GD) patients, 150 Hashimoto’s disease (HD) patients with different disease severities and 99 healthy controls. PD-1 expression on Treg cells was analysed by flow cytometry. Indirect immunofluorescence staining was performed in thyroid tissue to detect PD-1, PD-L1, and PD-L2. The frequencies of the A allele and the AA + AG genotypes of the PD1 rs36084323 polymorphism were lower in HD patients than in GD patients, and the frequencies of the AA genotype of the PD1 rs36084323 and of the TT genotype of the PD1 rs2227982 were lower in mild HD patients than in severe HD patients. In patients with severe HD, the titres of TgAb at the onset were higher in patients with the PD1 rs36084323 AA genotype than in patients with the GG genotype. Peripheral PD1+ Treg cells tended to decrease in individuals with the PD1 rs36084323 AA genotype than with the G carrier genotype. Peripheral PD-1+ Treg cells were increased in HD, especially in mild HD. PD-1, PD-L1, and PD-L2 were expressed in thyroid-infiltrating mononuclear cells (TIMCs), and PD-L1 and PD-L2 were expressed in thyroid epithelial cells (TECs) in AITD patients but not in normal controls. Expression of PD-L1 in TIMCs and expression of PD-L2 in TECs were predominant in HD and GD patients, respectively. In conclusion, the functional PD1 rs36084323 polymorphism and the thyroid PD-1/ PD-L1s expression which may enhance the suppressive activity of Treg cells differ between GD and HD, and the PD1 rs36084323 and rs2227982 polymorphisms and PD1+ Treg cells are related to the severity of HD.

Properties of Uncommon Indirect Immunofluorescence Staining Patterns Determined during Antinuclear Antibody Detection on HEp-2 Cells.

In this study, we aimed to assess the prevalence of uncommon staining patterns found during testing for the presence of antinuclear antibodies (ANA) and to determine their association with certain antibodies and clinical diagnoses. Presence of ANA and the staining pattern was determined in 10955 samples using indirect immunofluorescence (IIF) on HEp-2 cells. ANA-positive samples were assessed for presence of 14 specific antibody types using a microbead based system. Demographic data (age, sex) and clinical diagnoses were collected from the referral documentation. Particular staining patterns were then compared with a representative comparison group comprised of samples with common staining patterns using these criteria. There were 22 patterns present in less than 3% of samples each and these were jointly present in 42.43% of ANA-positive samples. Specific antibodies were found in proportions similar to the comparison group (46.06%) and varied significantly between patterns. Likewise, there were significant differences in antibody distribution in particular patterns. Some patterns were associated with presence of rheumatic diseases or inflammatory arthropathies, while in others there was a concurrent diagnosis of liver disease, or a neoplastic process. Many of the uncommon IIF patterns have distinctive characteristics that warrant further investigation in order to determine their role in diagnosing various diseases, not limited only to the illnesses of the rheumatic spectrum. IIF on HEp-2 cells remains an irreplaceable method because of the diversity of ANA, only a number of which can be detected using other standardised methods.

Mucosal immune responses in the trachea after chronic infection with Mycoplasma gallisepticum in unvaccinated and vaccinated mature chickens.

Tracheitis associated with the chronic respiratory disease in chickens caused by Mycoplasma gallisepticum is marked by infiltration of leukocytes into the mucosa. Although cytokines/chemokines are known to play a key role in the recruitment, differentiation, and proliferation of leukocytes, those that are produced and secreted into the trachea during the chronic stages of infection with M. gallisepticum have not been described previously. In this study, the levels of transcription in the trachea of genes encoding a panel of 13 cytokines/chemokines were quantified after experimental infection with the M. gallisepticum wild-type strain Ap3AS in unvaccinated chickens and chickens vaccinated 40-, 48- or 57-weeks previously with the novel attenuated strain ts-304. These transcriptional levels in unvaccinated/infected and vaccinated/infected chickens were compared with those of unvaccinated/uninfected and vaccinated/uninfected chickens. Pathological changes and subsets of leukocytes infiltrating the tracheal mucosa were concurrently assessed by histopathological examination and indirect immunofluorescent staining. After infection, unvaccinated birds had a significant increase in tracheal mucosal thickness and in transcription of genes for cytokines/chemokines, including those for IFN-γ, IL-17, RANTES (CCLi4), and CXCL-14, and significant downregulation of IL-2 gene transcription. B cells, CD3+ or CD4+ cells and macrophages (KUL01+ ) accumulated in the mucosa but CD8+ cells were not detected. In vaccinated birds, the levels of transcription of the genes for IL-6, IL-2, RANTES and CXCL-14 were significantly lower after infection than in the unvaccinated/infected and/or unvaccinated/uninfected birds, while the transcription of the IFN-γ gene was significantly upregulated, and there were aggregations of B cells in the tracheal mucosa. These observations indicated that M. gallisepticum may have suppressed Th2 responses by upregulating secretion of IFN-γ and IL-17 by CD4+ cells and induced immune dysregulation characterized by depletion of CD8+ cells and downregulation of IL-2 in the tracheas of unvaccinated birds. The ts-304 vaccine appeared to induce long-term protection against this immune dysregulation. TAKE AWAY: The ts-304 vaccine-induced long-term protection against immune dysregulation caused by M. gallisepticum Detection of B cells and plasma cells in the tracheal mucosa suggested that long-term protection is mediated by mucosal B cell memory Infection of unvaccinated birds with M. gallisepticum resulted in CD8+ cell depletion and downregulation of IL-2 in the tracheal mucosa, suggestive of immune dysregulation Infection of unvaccinated birds with M. gallisepticum resulted in upregulation of IFN-γ and infiltration of CD4+ cells and antigen presenting cells (B and KUL01+ cells) into the tracheal mucosa, suggesting enhanced antigen processing and presentation during chronic infection Th2 responses to infection with M. gallisepticum may be dampened by CD4+ cells through upregulation of IFN-γ and IL-17 during chronic infection.

GNAI2 Promotes Proliferation and Decreases Apoptosis in Rabbit Melanocytes.

GNAI2 (G protein subunit alpha i2) is a signaling modulator or transducer, involved in several transmembrane signaling systems, that plays a vital role in the melanogenesis signaling pathway. However, whether GNAI2 regulates cell proliferation and apoptosis in rabbit melanocytes is not known. We found that GNAI2 was differentially expressed in rabbits with different coat colors using qRT-PCR and Wes assays. Furthermore, it was observed that the rabbits with black skin had the highest GNAI2 levels, and those with white skin had the lowest expression. The coding sequence of GNAI2 was successfully cloned and inserted into pcDNA3.1 and pcDNA3.1-Myc vectors. It was observed that the GNAI2 protein was mainly localized in the cytoplasm using the indirect immunofluorescence staining assay. Overexpression of GNAI2 significantly increased melanin content, promoted melanocyte proliferation, and inhibited melanocyte apoptosis. On the contrary, the knockdown of GNAI2 using siRNA had the opposite effect. In addition, GNAI2 significantly increased the mRNA expression levels of the melanin-related genes TYR, GPNMB, PMEL, and DCT in rabbit melanocytes. The results suggested that GNAI2 regulated melanocyte development by promoting melanocyte proliferation and inhibiting apoptosis.