Multiplex immunoassays confer several advantages over widely adopted singleplex immunoassays including increased efficiency at a reduced expense, greater output per sample volume ratios and higher throughput predicating more resolute, detailed diagnostics and facilitating personalised medicine. Nonetheless, to date, relatively few protein multiplex immunoassays have been validated for in vitro diagnostics in clinical/point‐of‐care settings. This review article will outline the challenges, which must be ameliorated prior to the widespread integration of multiplex immunoassays in clinical settings: (i) biomarker validation; (ii) standardisation of immunoassay design and quality control (calibration and quantification); (iii) availability, stability, specificity and cross‐reactivity of reagents; (iv) assay automation and the use of validated algorithms for transformation of raw data into diagnostic results. A compendium of multiplex immunoassays applicable to in vitro diagnostics and a summary of the diagnostic products currently available commercially are included, along with an analysis of the relative states of development for each format (namely planar slide based, suspension and planar/microtitre plate based) with respect to the aforementioned issues.

Background/aims: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. Methods: Systematic serial 5–7 μm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the “stem cell” marker ABCG2 transporter protein. Results: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. Conclusions: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

BACKGROUND/AIMS The antimicrobial activity of the tear film exceeds the activity of its known constituents. The authors postulate that this excess activity is the result of antimicrobial peptides called defensins, and they aimed to look for defensins in the human eye. METHODS Evidence of defensin production was sought by reverse transcriptase polymerase chain reaction (RT-PCR). Intron spanning primers were designed for β defensins 1 and 2, and α defensins 5 and 6. RT-PCR was performed on cornea, conjunctiva, and lacrimal gland samples, and reaction products were size fractionated and sequenced to confirm their identity. A monoclonal antibody was utilised for the detection of α defensins 1, 2, and 3 in tissue sections and in immunoblots of tears. RESULTS RT-PCR revealed β defensin 1 message in samples of conjunctiva, cornea, and lacrimal gland. β Defensin 2 message was detected in the conjunctiva and cornea but was absent from the lacrimal gland. α Defensin 5 and 6 message was absent in these tissues but α defensins 1, 2, and 3 were detected in normal tears, lacrimal gland, and inflamed conjunctiva by immunochemistry. CONCLUSION The data suggest the human eye innately produces a spectrum of antimicrobial defensin peptides. Defensins hold therapeutic potential in ocular infections as they have a broad spectrum of antimicrobial activity (bacteria fungi and viruses ) and accelerate epithelial healing.

Interleukin‐12 (IL‐12, p70) a heterodimeric cytokine of p40 and p35 subunits, important for Th1‐type immune responses, has been attributed a prominent role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Recently, the related heterodimeric cytokine, IL‐23, composed of the same p40 subunit as IL‐12 and a unique p19 subunit, was shown to be involved in Th1 responses and EAE. We investigated whether astrocytes and microglia, CNS cells with antigen‐presenting cell (APC) function can present antigen to myelin basic protein (MBP)‐reactive T cells, and whether this presentation is blocked with antibodies against IL‐12/IL‐23p40. Interferon (IFN)‐γ‐treated APC induced proliferation of MBP‐reactive T cells. Anti‐IL‐12/IL‐23p40 antibodies blocked this proliferation. These results support and extend our previous observation that astrocytes and microglia produce IL‐12/IL‐23p40. Moreover, we show that stimulated astrocytes and microglia produce biologically active IL‐12p70. Because IL‐12 and IL‐23 share p40, we wanted to determine whether astrocytes also express IL‐12p35 and IL‐23p19, as microglia were already shown to express them. Astrocytes expressed IL‐12p35 mRNA constitutively, and IL‐23 p19 after stimulation. Thus, astrocytes, under inflammatory conditions, express all subunits of IL‐12/IL‐23. Their ability to present antigen to encephalitogenic T cells can be blocked by neutralizing anti‐IL‐12/IL‐23p40 antibodies.

Purpose. Amniotic membrane (AM) transplantation is an accepted procedure in ocular surgery. However, little is known of the interdonor and intradonor variability within the membrane. In addition, the effects of the methods of processing, storage, and preoperative preparation on the membrane are not fully elucidated. The purpose of this study was to use TGF-β as an example to investigate interdonor and intradonor variability and to determine the effect of “handling ” on TGF-β1 within fresh, processed and stored, and transplantation-ready AM (TRAM). methods. Seventeen human AMs, both fresh and handled, were analyzed for TGF-β1 by real-time polymerase chain reaction, immunohistochemistry, SDS-PAGE, and Western blotting. results. TGF-β1 was the highest normalized expressed isoform of TGF-β in all samples, but it varied between membranes of different donors and at different sites within the same membrane. The highest concentration was noted in the spongy layer. Removal of the spongy layer successfully removed the bulk of TGF-β1 from TRAM. Latency-associated protein (LAP) and a latent TGF-β–binding protein (LTBP) were also detected. conclusion. TGF-β1 is present in various regulatory forms in the AM. A degree of intermembrane and intramembrane variation is modified by handling. Unless a standardized protocol is adopted that delivers a membrane with consistent constituents, clinical outcomes may vary and comparisons may be invalid.

Aims: To investigate the regulation of cannabinoid receptors CB₁ and CB₂ on immune cells by pro‐inflammatory cytokines and its potential relevance to the inflammatory neurological disease, multiple sclerosis (MS).CB₁ and CB₂ signalling may be anti‐inflammatory and neuroprotective in neuroinflammatory diseases. Cannabinoids can suppress inflammatory cytokines but the effects of these cytokines on CB₁ and CB₂ expression and function are unknown.Methods: Immune cells from peripheral blood were obtained from healthy volunteers and patients with MS. Expression of CB₁ and CB₂ mRNA in whole blood cells, peripheral blood mononuclear cells (PBMC) and T cells was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR). Expression of CB₁ and CB₂ protein was determined by flow cytometry. CB₁ and CB₂ signalling in PBMC was determined by Western blotting for Erk1/2.Results: Pro‐inflammatory cytokines IL‐1β, IL‐6 and TNF‐α (the latter likely NF‐κB dependently) can upregulate CB₁ and CB₂ on human whole blood and peripheral blood mononuclear cells (PBMC). We also demonstrate upregulation of CB₁ and CB₂ and increased IL‐1β, IL‐6 and TNF‐α mRNA in blood of patients with MS compared with controls.Conclusion: The levels of CB₁ and CB₂ can be upregulated by inflammatory cytokines, which can explain their increase in inflammatory conditions including MS.