The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background

In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen.

Materials and methods

BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 × LD₅₀) of tetanus toxin.

Results

The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p<0.05).

Conclusion

Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively.     

EPA对草鱼前体脂肪细胞增殖分化的影响

体外培养草鱼前体脂肪细胞,并用不同浓度(0-100 μmol/L)二十碳五烯酸(Eicosapentaenoic acid,EPA)进行处理,噻唑兰比色法(Methyl thiazolte trazoliu,MTT)和油红O染色提取法检测EPA对细胞增殖及分化的影响,Real-time qPCR检测过氧化物酶增殖激活受体家族(Peroxidase proliferation activated receptor,PPARs)、脂蛋白酯酶(Lipoprotein lipase,LPL)、过氧化物酶体增殖激活受体γ辅助活化因子1α (Peroxisome proliferatoractivated receptor gamma coactivator-1α,PGC-1α)等基因的表达情况.结果显示,不同浓度EPA在2d内均显著促进草鱼前体脂肪细胞增殖(P＜0.05);不同浓度EPA处理ld后均显著抑制草鱼前体脂肪细胞分化(P＜0.05),且100μmol/L EPA处理细胞2d可显著促进LPL和PGC-1α基因的表达(P＜0.05).研究表明,EPA可促进草鱼前体脂肪细胞增殖,抑制其分化,该抑制作用与其调控PGC-1α、LPL等脂代谢基因的表达有关.     

Conjunctival amelanotic melanoma--a case report

Conjunctival melanoma is a relatively rare malignancy. It is presented as pigmented nodule in any area of conjunctiva, amelanotic tumors are pink with smooth appearance. The authors describe an amelanotic melanoma of the conjunctiva in an 82-year-old female patient. Cytological, histopathological and immunohistochemical studies revealed an invasive amelanotic melanoma exhibiting S-100 and MART-1 positivity. The patient undervent surgical and chemotherapy treatment and three years after the initial treatment is in the terminal stage of metastatic disease. Absence of pigmentation delayed early clinical detection and treatment. Awareness of this nonpigmented melanoma is crucial for early recognition and appropriate management.     

Protease inhibitors as possible pitfalls in proteomic analyses of complex biological samples

Sample preparation, especially protein and peptide fractionation prior to identification by mass spectrometry (MS), is typically applied to reduce sample complexity. The second key element in this process is proteolytic digestion, which is performed most often with trypsin. Optimization of this step is an important factor in order to achieve both speed and better performance of proteomic analysis, and tryptic digestion prior to the MS analysis has been a topic of many studies. To date, only a few studies have paid attention to the negative interaction between the proteolytic enzyme and sample components, and sample losses caused by these interactions. In this study, we demonstrated impaired activity after "in solution" tryptic digestion of plasma proteins caused by a potent trypsin inhibitor family, inter-alpha inhibitor proteins. Sample boiling followed by gel electrophoretic separation and "in-gel" digestion drastically improved both the number of identified proteins and the sequence coverage in subsequent LC-ESI-MS/MS. The present investigations show that a thorough validation is necessary when "in solution" digestion followed by LC-MS analysis of complex biological samples is performed. The parallel use of two or more different mass spectrometers can also yield additional information and contribute to further method validation.     

The phenanthridine biguanides efficiently differentiate between dGdC, dAdT and rArU sequences by two independent, sensitive spectroscopic methods

At submicromolar concentrations two novel phenanthridine biguanides exhibit distinctly different spectroscopic signals for dGdC and dAdT sequences, respectively, by opposite fluorimetric changes (quenching for dGdC and increase for dAdT) and especially the bis-biguanide derivative gives an opposite ICD response (negative ICD for dGC and strong positive for dAdT). This specific signalling was explained by the ability of compounds to switch the binding mode from intercalation into dGdC to minor groove binding into dAdT sequences. Both compounds bind to rArU by intercalation, yielding different fluorimetric and CD response in comparison to any of aforementioned ds-DNA. Moreover, both compounds revealed significantly higher affinity toward ds-polynucleotides in comparison to previously studied alkylamine- and urea-analogues. Furthermore, DNA/RNA binding properties of novel compounds could be controlled by pH, due to the protonation of heterocyclic nitrogen. Low in vitro cytotoxicity of both compounds against human cell lines makes them interesting spectrophotometric probes.     

FAB, ESI and MALDI Mass Spectrometric methods in the study of metallo-drugs and their biomolecular interactions

This review surveys the soft ionisation mass spectrometric methods that are most commonly used for the investigation of the mechanism of interaction between metallo-drugs and biomolecules. In the first part of the review, an overview of the applications of transition metal complexes in the therapy of various diseases (arthritis, cancer, diabetes) is given, whereas the second part focuses on the obtained results dealing with various aspects of the interaction between metal complexes and different types of biomolecules. Possibilities and limitations of each mass spectrometric method-namely, fast atom bombardment (FAB), electrospray ionisation (ESI) and matrix-assisted laser desorption and ionisation (MALDI)--are discussed in the third part of this review along with the examples of their application for the analysis of metal complexes, as well as of the products of their interaction with biomolecules.     

PGC-1α determines light damage susceptibility of the murine retina

The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases.