Upregulation of the expression of Toll-like receptor 9 in basophils in patients with allergic rhinitis: An enhanced expression by allergens.

It was reported that the expression of Toll-like receptor (TLR) 9 may be related to Th2-type allergic inflammation including allergic rhinitis (AR). However, little is known about the expression of TLR9 in the basophils in AR. In the present study, the expression of TLR9 was examined by flow cytometry analysis, and the expression of TLR9 mRNA in KU812 was determined by quantitative real-time PCR. The results showed that the percentage of TLR9+ CCR3+ cells in blood granulocytes increased by 46% in patients with AR, but not in peripheral blood mononuclear cells (PBMCs). Allergens namely Dermatophagoide allergen extract (DAE) and Platanus pollen allergen extract (PPAE) upregulated the expression of TLR9 in CCR3+ granulocytes by 76% and 84%, respectively. DAE and PPAE also enhanced the proportions of TLR9+ CD123+ HLA-DR- cells and TLR9+ CCR3+ CD123+ HLA-DR- cells in granulocytes and PBMCs of patients with AR. In order to investigate the actions of allergens on basophils, KU812 cells were used. It was observed that all KU812 cells expressed TLR9, and the expression intensity of TLR9 in a single KU812 cell was elevated by CpG. IL-37, IL-31, IL-33, Artemisia sieversiana wild allergen extract (ASWAE), DAE, OVA and Der p 1 induced an increase in the expression of TLR9 mRNA and IL-6 production in KU812 cells. It was shown that the percentage of TLR9-expressing basophils increased in the blood of ovalbumin (OVA)-sensitized mice. In conclusion, an increased expression of TLR9 and the production of IL-6 in basophils implicate that the contribution of basophils to AR is likely via TLR9.

[Up-regulated expression of NK1R in eosinophil-enriched blood cells from patients with chronic spontaneous urticaria].

Objective To investigate the expressions of substance P (SP) and neurokinin-1 receptor (NK1R) in eosinophil-enriched blood cells from patients with chronic spontaneous urticaria (CSU). Methods Peripheral venous blood samples were collected from patients with CSU and healthy controls (HCs), and then stimulated with crude extracts of Artemisia pollen, dust mite, and Platanus pollen (all at concentrations of 0.1 and 1.0 µg/mL). The expressions of SP and NK1R in eosinophil-enriched blood cells were detected by flow cytometry. Results Compared with HCs, eosinophil proportion in peripheral blood of CSU patients increased 1.2-fold. Percentage of NK1R+ eosinophils in the patients with CSU was elevated up to 66% compared with HCs when cultured in the medium only. However, the level of SP decreased by 40% in the CSU patients. In eosinophil-enriched blood cells from the CSU patients, the crude extract of dust mite at 0.1 µg/mL induced approximately 1.11-fold increase of NK1R expression. Conclusion Expression of NK1R increases in the eosinophils of CSU patients. Blockers of NK1R might be used for CSU treatment.

Analysis of anti-asthmatic drug patents published in China between 2004 and 2013.

INTRODUCTION: We previously reported that 789 anti-allergic patents were granted in China between 1988 and 2008, but the number of patents seems to have grown much faster in China in recent years. Therefore, it is necessary to analyse the patents for anti-asthmatic products between 2004 and 2013 to give pharmaceutical companies and individuals a better understanding of potential candidates for anti-asthmatic drug development from patents published in China. AREAS COVERED: The current report analyses the scientific progress that supports anti-asthmatic drug patent applications and reviews the published patent literature in China from 2004 to 2013. EXPERT OPINION: The rapid increase in the number of anti-asthmatic patents in China indicates that more specific discoveries have been made and that more people are aware of the importance of intellectual property protection in China. Holding patents may guarantee protection for an innovative new product.

Analysis of antibiotic patents issued in China from 1992 to 2011.

INTRODUCTION: The wide use of antibiotics both within and beyond the medical territory plays a significant role in the development of resistant bacteria. Therefore, there is a pressing need for the development of effective antibiotics worldwide. AREAS COVERED: The current analysis report covers the scientific progress in supporting antibiotic patent application and the granted patent literature in China for the last 20 years. EXPERT OPINION: Among the 2780 patents granted in China last 20 years, ß-lactam antibiotics, macrolides, quinolones, aminoglycosides, sulfonamides, glycopeptides and tetracyclines constitute 44.3, 17.4, 13.5, 4.1, 3.8, 3.0 and 2.1% of total patents, respectively. Scientists have faced challenges in developing new antibiotics against increasingly growing types of drug-resistant bacteria, which may causes serious global public health disaster in future. Poor financial investment in antibiotic research has exacerbated the situation. Therefore, new antibiotic patents will be applied continuously. The combination individual ß-lactam antibiotics with a ß-lactamase inhibitor have been demonstrated clinically beneficial and may suggest a new way for the development of more effective antibiotics in future. Local patent applications of China are stably increasing largely through a dependence on the imitation of Western drugs. The current study may help to better understand the patent situation in China and to invent the more efficient antibiotic therapies.

An update on anti-allergic patents granted in China: 2009 - 2011.

INTRODUCTION: The prevalence of allergic diseases has increased dramatically in recent decades. Therefore, there is a pressing need for the development of effective anti-allergic services worldwide. AREAS COVERED: In previous studies, the authors had analyzed a total of 789 anti-allergic patents granted in China from 1988 to 2008. Herein, they report a further 151 anti-allergic patents issued in China during 2009 - 2011. The current analysis covers the scientific progress in supporting anti-allergic patent applications and granted patent literature, in China, for the last 3 years. EXPERT OPINION: The 151 anti-allergic patents granted from 2009 to 2011 mainly focus on seven types of products. They are: traditional Chinese medicines (TCM), plant extracts, biological products, synthetic compounds, pharmaceutical preparations, medical apparatus and new treatment modalities. Although the overall number of anti-allergic patent applications made between 2009 and 2011 in China is less than that of the USA and Europe, patents on TCM have increased. This suggests that there are demands for modernization of TCMs. Recently, studies of interesting new immunomodulators have also been conducted, and some of these are likely to represent clinically useful advances. In the last 3 years, several patents on these novel potential drugs have also been granted in China. The large number of anti-allergic patents issued in China, in recent times, suggests that the Chinese market is relatively competitive one that will help pharmaceutical companies make proper decisions for their research and development strategies.

Analysis of patents on anti-allergic therapies issued in China from 1988 to 2008.

BACKGROUND: The prevalence of allergic diseases has increased dramatically in recent decades. Therefore, there is a pressing need for the development of effective anti-allergic services worldwide. However, little is known what anti-allergic products have been patented in China and what the potential drug candidates for patents are in China. OBJECTIVE: To analyze the patents of anti-allergic products for the last 20 years and help pharmaceutical companies and individuals to understand the potential candidates for anti-allergic patents in China. METHODS: Data were obtained from the People's Republic of China Country Intellectual Property Rights Bureau website and United States Patent and Trademark Office website. RESULTS: A total of 789 anti-allergic patents have been granted in China during the past 20 years, which all focused on synthetic compounds, traditional Chinese medicines (TCM), combinations of synthetic compounds and TCM, biological products and medical apparatus. It appears that more and more effective therapeutic components of TCM rather than whole herbs have been patented in recent years and alteration of natural molecules to produce more therapeutically effective molecules has emerged as a novel trend for the modernization of TCM. The patents on synthetic anti-allergic compounds in China mainly focus on well-known targets, such as histamine receptor and leukotrienes, which consist of 93% of patents for validated targets. CONCLUSION: The number of anti-allergic patents applied in China is far lower than that in the US. Therefore, there are great opportunities for obtaining anti-allergic patents, particularly patents on active ingredients from TCM in China.

Analysis of trends and opportunities of anti-allergy patents in China from 1998 to 2008.

The prevalence of allergic diseases has increased dramatically in recent decades. Holding patents is one of the means to protect good anti-allergy products. However, little is known of anti-allergy patent situation in China. The paper summarized and analyzed anti-allergy patents issued in China from January 1988 to September 2008. A total of 789 anti-allergy patents have been granted in China during the 20 years. China, European countries, USA, Japan and other countries possesses 44%, 21%, 19%, 12% and 4% of all of these anti-allergy patents respectively. Interestingly, 88% anti-allergy patents issued to Chinese are held by civilians, whereas vast majority of the patents issued to foreigners were held by pharmaceutical companies. All anti-allergy patents are focused on synthetic compounds, Traditional Chinese Medicines (TCM),combinations of synthetic compounds and TCM (CST), biological products and medical apparatus. The anti-allergy patents in China mainly focus on well-known targets, such as histamine receptor and leukotrienes, which consist of 93% of patents for validated targets. Approximately 93% targeting diseases are bronchial asthma, allergic rhinitis and atopic dermatitis. Our analyzing results indicate that there are great opportunities for application of patents on development of novel anti-allergic compounds and modernization of TCM in China.

Reduction of CD4 positive T cells and improvement of pathological changes of collagen-induced arthritis by FTY720.

FTY720 belongs to a new class of immunosuppressants. Little is known about its influence on T cell subtypes and pathological changes in arthritis. Here we illustrated the effect of FTY720 on peripheral T cell subsets and joint damage of collagen-induced arthritis rats. Rats were administered FTY720 or prednisone daily from day 0 to day 28. Body weight, hind paw swelling and arthritis index were measured. Bone destruction was determined by micro-computed tomography and histopathology, and T cell subsets were analyzed by flow cytometry and immunohistochemistry. The results showed that FTY720 inhibited the development of arthritis. Radiological analysis revealed that FTY720 treated collagen-induced arthritic rats had much less joint damage in comparison to untreated collagen-induced arthritic rats. Histological study showed that collagen-induced arthritic rats suffered from inflammatory cell infiltration and synovial hyperplasia in their joints, and FTY720 treatment clearly reduced these pathological changes. Immunohistochemical analysis showed that FTY720 treatment significantly decreased the number of CD4(+) T cells in the synovium of collagen-induced arthritic rats. Collagen-induced arthritic rats appeared to have more CD4(+), but not CD8(+) T cells in their peripheral blood than normal control rats. Following FTY720 treatment, peripheral blood CD3(+) and CD4(+) T cells in collagen-induced arthritic rats were significantly decreased. In conclusion, FTY720 is an effective compound in the treatment of collagen-induced arthritic rats and in reducing CD4(+) T cells in collagen-induced arthritic rats.

Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L.

AIM: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LvCao in Chinese) and short ragweed (Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. METHODS: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3'cDNA ends were identified by using the 3'-RACE method. The gene function conferred by the full-length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET-44 RosettaBlue cells were subsequently characterized by N-terminal end sequencing, IgE binding, and cross-reactivity. RESULTS: Three full-length cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%-90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in non-fusion forms and were confirmed by using the N-terminal end sequence identity. Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia, but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross-reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full-length cDNA demonstrated that alternative nucleotide degeneracy occurred. CONCLUSION: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little-studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.

Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration.

AIM: To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production. METHODS: Gene fragments corresponding to the gene specific region and the cDNA ends of pollen allergens of short ragweed (Rg, Ambrosia artemisiifolia L.) were obtained by pan-degenerate primer-based PCR and rapid amplification of the cDNA ends (RACE), and the products were mixed to serve as the bridging PCR (BPCR) template. The full-length gene was then obtained. Partially overlapping primer-based PCR (POP-PCR) method was developed to overcome the other problem, i.e., the non-specific amplification of the ORF with routine long primers for expression insert decoration. Northern blot was conducted to confirm pollen sources of the gene. The full-length coding region was evaluated for its gene function by homologue search in GenBank database and Western blotting of the recombinant protein Amb a 8(D106) expressed in Escherichia coli pET-44 system. RESULTS: The full-length cDNA sequence of Amb a 8(D106) was obtained by using the above procedure and deduced to encode a 131 amino acid polypeptide. Multiple sequence alignment exhibited the gene D106 sharing a homology as high as 54-89% and 79-89% to profilin from pollen and food sources, respectively. The expression vector of the allergen gene D106 was successfully constructed by employing the combined method of BPCR and POP-PCR. Recombinant allergen rAmb a 8(D106) was then successfully generated. The allergenicity was hallmarked by immunoblotting with the allergic serum samples and its RNA source was confirmed by Northern blot. CONCLUSION: The combined procedure of POP-PCR and BPCR is a powerful method for full-length allergen gene retrieval and expression insert decoration, which would be useful for recombinant allergen production and subsequent diagnosis and immunotherapy of pollen and food allergy.

[Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens(Lour.) Merr by degenerate primer].

AIM: To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants. METHODS: Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences. RESULTS: Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%-85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR. CONCLUSION: Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.