Impact of a targeted temperature management quality improvement project on survival and neurologic outcomes in cardiac arrest patients.

BACKGROUND: Targeted temperature management (TTM) is recommended for postresuscitation care of patients with sudden cardiac arrest (SCA) and its implementation remains challenging. This study aimed to evaluate the newly designed Quality Improvement Project (QIP) to improve the quality of TTM and outcomes of patients with SCA. METHODS: Patients who experienced out-of-hospital cardiac arrest (OHCA) and in-hospital cardiac arrest (IHCA) with return of spontaneous circulation (ROSC) and were treated in our hospital between January 2017 and December 2019 were enrolled retrospectively. All included patients received QIP intervention initiated as follows: (1) Protocols and standard operating procedures were created for TTM; (2) shared decision-making was documented; (3) job training instruction was created; and 4) lean medical management was implemented. RESULTS: Among 248 included patients, the postintervention group (n = 104) had shorter duration of ROSC to TTM than the preintervention group (n = 144) (356 vs 540 minutes, p = 0.042); better survival rate (39.4% vs 27.1%, p = 0.04), and neurologic performance (25.0% vs 17.4%, p < 0.001). After propensity score matching (PSM), patients who received TTM (n = 48 ) had better neurologic performance than those without TTM (n = 48) (25.1% vs 18.8%, p < 0.001). OHCA (odds ratio [OR] = 2.705, 95% CI: 1.657-4.416), age >60 (OR = 2.154, 95% CI: 1.428-3.244), female (OR = 1.404, 95% CI: 1.005-1.962), and diabetes mellitus (OR = 1.429, 95% CI: 1.019-2.005) were negative predictors of survival; while TTM (OR = 0.431, 95% CI: 0.266-0.699) and bystander cardiopulmonary resuscitation (CPR) (OR=0.589, 95% CI: 0.35-0.99) were positive predictors. Age >60 (OR= 2.292, 95% CI: 1.58-3.323) and OHCA (OR= 2.928, 95% CI: 1.858-4.616) were negative predictors of favorable neurologic outcomes; while bystander CPR (OR=0.572, 95% CI: 0.355-0.922) and TTM (OR=0.457, 95% CI: 0.296-0.705) were positive predictors. CONCLUSION: A new QIP with defined protocols, documented shared decision-making, and medical management guidelines improves TTM execution, duration from ROSC to TTM , survival, and neurologic outcomes of cardiac arrest patients.

The pathological mechanisms and novel therapeutics for Leber's hereditary optic neuropathy.

Optic neuropathies were estimated to affect 115 in 100,000 population in 2018. Leber's Hereditary Optic Neuropathy (LHON) as one of such optic neuropathy diseases that was first identified in 1871 and can be defined as a hereditary mitochondrial disease. LHON is associated with three mtDNA point mutations which are G11778A, T14484, and G3460A that affect the NADH dehydrogenase subunits of 4, 6, and 1, respectively. However, in most cases, only one point mutation is involved. Generally, in manifestation of the disease, there are no symptoms until the terminal dysfunction in the optic nerve is observed. Due to the mutations, nicotinamide adenine dinucleotide (NADH) dehydrogenase or complex I is absent and thus ATP production is stopped. This further causes the generation of reactive oxygen species and retina ganglion cells apoptosis. Aside from the mutations, there are several environmental factors such as smoking and alcohol consumption that can be pointed out as the risk factors of LHON. Nowadays, gene therapy has been intensively studied for LHON treatment. Disease models using human induced pluripotent stem cells (hiPSCs) have been utilized for LHON research.

Pluripotent Stem Cells in Clinical Cell Transplantation: Focusing on Induced Pluripotent Stem Cell-Derived RPE Cell Therapy in Age-Related Macular Degeneration.

Human pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), represent valuable cell sources to replace diseased or injured tissues in regenerative medicine. iPSCs exhibit the potential for indefinite self-renewal and differentiation into various cell types and can be reprogrammed from somatic tissue that can be easily obtained, paving the way for cell therapy, regenerative medicine, and personalized medicine. Cell therapies using various iPSC-derived cell types are now evolving rapidly for the treatment of clinical diseases, including Parkinson's disease, hematological diseases, cardiomyopathy, osteoarthritis, and retinal diseases. Since the first interventional clinical trial with autologous iPSC-derived retinal pigment epithelial cells (RPEs) for the treatment of age-related macular degeneration (AMD) was accomplished in Japan, several preclinical trials using iPSC suspensions or monolayers have been launched, or are ongoing or completed. The evolution and generation of human leukocyte antigen (HLA)-universal iPSCs may facilitate the clinical application of iPSC-based therapies. Thus, iPSCs hold great promise in the treatment of multiple retinal diseases. The efficacy and adverse effects of iPSC-based retinal therapies should be carefully assessed in ongoing and further clinical trials.

Autophagy reprogramming stem cell pluripotency and multiple-lineage differentiation.

The cellular process responsible for the degradation of cytosolic proteins and subcellular organelles in lysosomes was termed "autophagy." This process occurs at a basal level in most tissues as part of tissue homeostasis that redounds to the regular turnover of components inside cytoplasm. The breakthrough in the autophagy field is the identification of key players in the autophagy pathway, compounded under the name "autophagy-related genes" (ATG) encoding for autophagy effector proteins. Generally, the function of autophagy can be classified into two divisions: intracellular clearance of defective macromolecules and organelles and generation of degradation products. Therapeutic strategies using stem cell-based approach come as a promising therapy and develop rapidly recently as stem cells have high self-renewability and differentiation capability as known as mesenchymal stem cells (MSCs). They are defined as adherent fibroblast-like population with the abilities to self-renew and multi-lineage differentiate into osteogenic, adipogenic, and chondrogenic lineage cells. To date, they are the most extensively applied adult stem cells in clinical trials. The properties of MSCs, such as immunomodulation, neuroprotection, and tissue repair pertaining to cell differentiation, processes to replace lost, or damaged cells, for aiding cell repair and revival. Autophagy has been viewed as a remarkable mechanism for maintaining homeostasis, ensuring the adequate function and survival of long-lived stem cells. In addition, authophagy also plays a remarkable role in protecting stem cells against cellular stress when the stem cell regenerative capacity is harmed in aging and cellular degeneration. Understanding the under-explored mechanisms of MSC actions and expanding the spectrum of their clinical applications may improve the utility of the MSC-based therapeutic approach in the future.

Pulmonary arterial hypertension in the elderly population.

Pulmonary arterial hypertension (PAH) was a disease predominantly affecting young females about 40 years ago; however, it has been increasingly diagnosed in elderly individuals. Few studies have investigated the features of elderly patients with PAH. This review provides an overview of the characteristics of elderly patients with PAH compared to young patients. The examination of the changing demographics of the population with PAH revealed that the mean age has increased over the years. In addition, the investigation into the diagnostic challenges in elderly patients with PAH revealed the difficulty in differentiating PAH from pulmonary hypertension secondary to diastolic heart failure. Moreover, it was noted that elderly patients underwent combination drug regimens less frequently and exhibited poorer treatment responses than young patients. Finally, it was found that elderly PAH patients experienced poorer survival than young patients. The differences among five survival prediction models and their applicability in predicting the prognosis of PAH patients are discussed.

Single pulley double-strand anchor suture fixation of the coronoid process in terrible triad of the elbow injury.

BACKGROUND: Terrible triad of the elbow injury is difficult to manage, and the role of the coronoid process in instability is very important. We describe a simple, modified suture technique to fix a coronoid process fragment using suture anchor fixation. METHODS: Eight patients (three female and five male) with coronoid process injuries with the fragment involving <50% of the total height (Regan-Morrey type I/II) in terrible triad of elbow injury were included. Patients were treated operatively via a lateral Kocher's approach, and coronoid process fractures were repaired with a single pulley double-strand suture technique. Structures were addressed in a sequential fashion-the coronoid process, radial head, lateral ulnar collateral ligament. RESULTS: All patients were treated with the single pulley double-strand anchor suture technique and the coronoid process fragment was found to be in good contact with the original avulsion site using the method. The final Mayo Elbow Performance Score was excellent (> 90) in six patients and good (between 85 and 89) in two patients after operation 6 months. CONCLUSION: The single pulley double-strand suture tie method using a suture anchor is a less invasive and simpler fixation method for the repair of coronoid process fractures in patients with terrible triad of the elbow injuries and results in good outcomes.

Pandemic aspect of dexamethasone: Molecular mechanisms and clinical application.

The rapid spread of coronavirus disease (COVID-19) in many countries has caused inconvenience in conducting daily life activities, and even deaths. Dexamethasone is a corticosteroid applied in clinical medicine since 1957, especially in immune therapy fields. Herein, we present the characteristics of Dexamethasone, from molecular mechanisms such as genomic and nongenomic pathways by cellular signal regulations, to clinical applications in various phases of the disease. During COVID-19 pandemic, Dexamethasone given to patients who required oxygen or ventilation therapy showed improved life efficacy.

Enhancing induced pluripotent stem cell toward differentiation into functional cardiomyocytes.

BACKGROUND: Heart diseases, especially myocardial ischemia, remain one of the leading causes of mortality worldwide and usually result in irreparable cardiomyocyte damage and severe heart failure. Recent advances in induced pluripotent stem cell (iPSC) technologies for applied regenerative medicine and stem cell research, especially for iPSC-derived cardiomyocytes have increased the hope for heart repair. However, the driver molecules of myocardial differentiation and the functional reconstruction capacity of iPSC-derived cardiomyocytes are still questionable. METHODS: Herein, we established a rapid differentiated platform that is involved in cardiomyogenic differentiation and maturation from iPSCs in vitro. Functional analysis is performed in miR-181a-transfected iPSC-derived cardiomyocyte (iPSC-cardio/miR-181a) under a time-lapse microscope. In addition, we calculated the beating area and frequency of iPSC-cardio/miR-181a cells in the presence of HCN4 shRNA or miR-181a SPONGE. RESULTS: miR-181a enhanced the beating area and maintained the beating frequency of iPSC-derived cardiomyocytes by enhancing HCN4 expression. CONCLUSION: miR-181a would play a key role on maintaining proper beating function in iPSC-derived cardiomyocytes.

Non-coding RNA and lung cancer progression.

Lung cancer (LC) is a major killer disease globally. This situation is further supported by yearly increase in new LC cases and its poor 5-year survival which is less than 15%. Although a large percentage of LC cases have been attributed to smoking, a considerable amount of nonsmokers also develops this disease, thereby suggesting a genetic and/or epigenetic undertone to LC development. Several growth-related genes such as epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) as well as tumor suppressor genes such as p53 have been implicated in LC pathogenesis and progression. Likewise, the genome only contains approximately 1% of coding regions. Hence, noncoding portion of the genome such as noncoding RNAs (ncRNAs) has been studied and discovered to play a cogent role in LC pathogenesis. More precisely, microRNAs (miRNAs) and long ncRNAs (lncRNAs) have been studied for decades. Posttranscriptional gene modulation function of miRNAs is well established and characterized. Likewise, the antagonizing interaction between lncRNAs and miRNAs had also been proven to further control gene expression during healthy and disease conditions like LC. More recently, renewed attention toward circular RNAs [circular RNAs (circRNAs)] study showed that circRNAs can also sponge miRNAs to modulate gene expressions too. Hence, miRNAs, lncRNAs, and circRNAs seem to function within a circuit to optimally determine which gene is needed to be upregulated or downregulated in biological system. Therefore, this review will discuss important ncRNAs, namely miRNA, lncRNA, and circRNA in LC progression. Paracrine effect of exosomal ncRNA will be also reviewed. In addition, the prospect of these ncRNAs in enhancing better LC treatment will be highlighted as well.

Generation of induced pluripotent stem cells from a patient with Best Dystrophy carrying 11q12.3 (BEST1 (VMD2)) mutation.

Best disease (BD), also termed Best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and central visual loss. In this report, we generated an induced pluripotent stem cell (iPSC) line, TVGH-iPSC-012-04, from the peripheral blood mononuclear cells of a female patient with BD by using the Sendai virus delivery system. The resulting iPSCs retained the disease-causing DNA mutation, expressed pluripotent markers and could differentiate into three germ layers. We believe that BD patient-specific iPSCs provide a powerful in vitro model for evaluating the pathological phenotypes of the disease.

The different modes of binding of the dust mite allergens, Der f 7 and Der p 7, on a monoclonal antibody WH9 contribute to the differential reactivity.

BACKGROUND: Der f 7 and Der p 7 are important house dust mite allergens. An IgE-binding inhibition monoclonal antibody WH9 reacts ten folds stronger against Der p 7 than to Der f 7. The purpose of this study is to identify the antigenic determinant(s) and the structural basis of Der f 7 recognize by WH9. METHODS: WH9-reactive determinant(s) on Der f 7 was identified by immunoblot and immunoblot inhibition. The 3-D binary complex structures of WH9 and the group 7 allergens were simulated with homology modeling and docking methods. RESULTS: WH9 reacted with the Der f 7 f9 fragment. Among the five site-directed Der f 7 mutants, WH9 showed reduced immunoblot reactivity against Der f 7 S156A, D159A and P160A mutants. Only the wild-type protein and the Der f 7 I157A and L158A mutants can inhibit significantly the WH9-binding against Der f 7. The structural model of the Der f 7-WH9 complex suggests residues S156 and D159 of Der f 7 can bind to WH9 via potential hydrogen bonds. CONCLUSION: The structure models of Der f 7-WH9 and Der p 7-WH9 complexes revealed that the differential modes of binding of Der p 7 and Der f 7 allergens on WH9 contribute to the differential reactivity of WH9 against the Der f 7 and the Der p 7 mite allergens.

Dectin-1-Mediated Pathway Contributes to Fusarium proliferatum-Induced CXCL-8 Release from Human Respiratory Epithelial Cells.

Fusarium species are causative agents of human respiratory disorders and are distributed widely in our environment. Little is known of their interaction with human respiratory epithelial cells, which may contribute to allergic airway responses. In this study, we report on the release of C-X-C motif chemokine ligand 8 (CXCL-8) from human bronchial epithelial BEAS-2B cells upon stimulation with Fusarium proliferatum extracts. F. proliferatum-induced cytokine release from BEAS-2B cells was determined by cytokine array and CXCL-8 enzyme-linked immunosorbent assay (ELISA) kits. Blocking antibodies and signaling pathway inhibitors were employed to delineate cell surface receptors and signaling pathways participating in CXCL-8 release. F. proliferatum extracts induced the release of CXCL-8 in a time-dependent manner. The dectin-1 receptor ligands, curdlan and laminarin, reduced CXCL-8 release. Cells pre-treated with anti-Dectin-1 antibodies (2 µg/mL) decreased CXCL-8 release by 24%. Furthermore, F. proliferatum-stimulated CXCL-8 release was reduced by 32%, 53%-81%, 40% and 26% after BEAS-2B cells were pretreated with activation inhibitors of spleen tyrosine kinase (Syk)-piceatannol-, mitogen-activated protein kinases (MAPKs)-PD98059, U0126, SB202190, SP600125-, phosphatidylinositol-3-kinase (PI3K)-LY294002-and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-BAY117082-, respectively. These results suggest that Dectin-1-mediated activation of the Syk, MAPKs, PI3K and NF-κB signaling pathways contributes to F. proliferatum-stimulated CXCL-8 release from BEAS-2B cells and provides an important basis for developing novel therapeutic strategies in clinical allergy.

Vacuolar Serine Protease Is a Major Allergen of Fusarium proliferatum and an IgE-Cross Reactive Pan-Fungal Allergen.

PURPOSE: Fusarium species are among prevalent airborne fungi and causative agents of human respiratory atopic disorders. We previously identified a 36.5-kDa F. proliferatum component recognized by IgE antibodies in 9 (53%) of the 17 F. proliferatum-sensitized atopic serum samples. The purpose of this study is to characterize the 36.5-kDa allergen of F. proliferatum. METHODS: Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning/expression and immunoblot inhibition studies. RESULTS: Based on the finding that the 36.5-kDa IgE-binding component reacted with the mouse monoclonal antibody FUM20 against fungal vacuolar serine protease allergens, the cDNA of F. proliferatum vacuolar serine protease (Fus p 9.0101) was subsequently cloned. Nine serum samples from respiratory atopic patients with IgE binding to the vacuolar serine protease allergen of Penicillium chrysogenum (Pen ch 18) also showed IgE-immunoblot reactivity to rFus p 9.0101. The purified rFus p 9.0101 can inhibit IgE and FUM20 binding to the 36.5-kDa component of F. proliferatum. Thus, a novel and important Fus p 9.0101 was identified. The rPen ch 18 can inhibit IgE binding to Fus p 9.0101. It indicates that IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 also exists. Furthermore, neither rFus p 9.0101 K88A nor rPen ch 18 K89A mutants inhibited IgE binding to rFus p 9.0101. Lys88 was considered a critical core amino acid in IgE binding to r Fus p 9.0101 and a residue responsible for IgE cross-reactivity between Fus p 9.0101 and Pen ch 18 allergens. CONCLUSIONS: Results obtained from this study indicate that vacuolar serine protease may be a major allergen of F. proliferatum and an important IgE cross-reactive pan-fungal allergen, and provide important bases for clinical diagnosis of fungal allergy.

The transaldolase, a novel allergen of Fusarium proliferatum, demonstrates IgE cross-reactivity with its human analogue.

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.

Epitope mapping and in silico characterization of interactions between Der p 7 allergen and MoAb WH9.

Der p 7 is an important house dust mite allergen. However, antigenic determinants of Der p 7 are largely unknown. The purpose of this study is to analyze the determinants of Der p 7 and determine the structural basis of interactions between Der p 7 and WH9, an IgE-binding inhibition mouse monoclonal antibody (MoAb). IgE and WH9-reactive determinant(s) was identified by immunoblot using allergen mutants. A 3-D binary complex structure of Der p 7 and WH9 was simulated with homology modeling and docking methods. Our results obtained showed that among the five Der p 7 mutants (S156A, I157A, L158A, D159A, P160A), serum no. 1045 with IgE-binding against Der p 7 exhibited a reduced IgE immunoblot reactivity against Der p 7 L158A and D159A mutants. WH9 showed reduced immunoblot reactivity against S156A, L158A, D159A and P160A and the observation was confirmed by immunoblot inhibition. The WH9-binding determinant on Der p 7 containing S156, L158, D159 and P160 assumes a loop-like structure. The structural model of the Der p 7-WH9 complex suggests residues S156, I157, L158, D159 and P160 of Der p 7 contribute to WH9 binding via potential hydrogen bonds, electrostatic and hydrophobic interactions. In conclusion, MoAb WH9 interacts with critical residues L158 and D159 of Der p 7 and inhibits IgE-binding to Der p 7. Results obtained advance our understanding on molecular and structural bases of the antigenicity of Der p 7, its interactions with MoAb WH9 and facilitate the design of safer immunotherapy of human atopic disorders.

Homology modeling and monoclonal antibody binding of the Der f 7 dust mite allergen.

The group 7 allergens are important allergenic specificities for mite-sensitive patients and may need to be incorporated into new diagnostic and therapeutic strategies. However, little is known about their biological and structural features. Position-specific iterative BLAST showed that they had strong ancestral homology to two related families of lipid-binding proteins, namely, the bactericidal permeability-increasing (BPI) proteins and the odorant-binding protein. A three-dimensional model of Der f 7 made with the Phyre and SWISS-MODEL homology-modeling servers showed a close match with the human BPI coordinates used for its construction. The binding of the monoclonal antibody HD12 known to block IgE binding could be blocked by the linear sequence (46GILDF50) with a critical role for L48 or F50. These hydrophobic residues were located on a surface loop of the model. The properties of Der f 7 that can be deduced from the model provide avenues for further characterizing these allergens, their IgE binding structures and biological properties that can enhance allergenicity.

Pen ch 13 major fungal allergen decreases CD44 expression in human bronchial epithelial cells.

BACKGROUND: Pen ch 13 is an alkaline serine protease major allergen from Penicilliumchrysogenum. CD44 adhesion molecules play important roles in resolving lung inflammation and repairing epithelial damages during bronchial asthma. The purpose of this study was to investigate the effects of Pen ch 13 on CD44 of human bronchial epithelial cells. METHODS: Cells of the SV40-transformed immortalized bronchial epithelial cell line 16HBE14o- and primary cultures of human bronchial epithelial cells were exposed to purified Pen ch 13. CD44 expression on Pen ch 13-treated cells was analyzed by immunoblot analysis and flow cytometry. The release of soluble CD44 (sCD44) into culture supernatants was determined using human sCD44std ELISA kits. RESULTS: Pen ch 13 (0.01-1.0 µg/ml) dose-dependently down-regulates CD44 expression in 16HBE14o- cells. In addition, the decrease in CD44 expression can be abolished by pre-treating Pen ch 13 with a serine protease inhibitor, phenylmethyl-sulfonyl fluoride. Results from flow-cytometric analysis showed that the population mean fluorescence intensity for CD44 was significantly lower (p < 0.05) in Pen ch 13 (1.0 µg/ml)-treated 16HBE14o- cells (18 ± 4) than that of non-treated control cells (41 ± 7). Furthermore, Pen ch 13 induced increased shedding of sCD44 into the culture media compared with the shedding of non-treated 16HBE14o- and primary bronchial epithelial cells. CONCLUSIONS: Pen ch 13 allergen down-regulated CD44 protein expression in airway epithelial cells. It may contribute to atopic asthma by influencing the resolution of lung inflammation and prolonging the repair response of damaged bronchial epithelial cells.

Decreased production of MCP-1 and MMP-2 by keloid-derived fibroblasts.

Keloid tissue results from aberrant wound healing. The underlying mechanism responsible for keloid formation remains unclear. The aim of this study was to analyse the production of tissue inflammatory mediators by normal and keloid-derived human dermal fibroblasts cultured in vitro. Production of monocyte chemoattractant protein 1 (MCP-1), matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2), with and without the addition of interleukin-1beta, was determined by enzyme-linked immunosorbant assay. Our results showed that IL-1beta (10 ng/ml) stimulated a statistically significant increase in MCP-1 and MMP-2 production and decreased production of TIMP-2 by both normal and keloid-derived fibroblasts (Student's t-test, p<0.05), but to differing extents. This may contribute to factors leading to keloid formation.

Lys89, Lys90, and Phe91 are critical core amino acid residues of the Pen ch 18 major fungal allergen recognized by human IgE antibodies.

A vacuolar serine protease (Pen ch 18) has been identified as a major allergen of Penicillium chrysogenum. The molecular features of antigenic determinant(s) on Pen ch 18 recognized by human IgE antibodies, however, have remained unclear. Here, we show that a dominant IgE epitope on the N-terminally processed Pen ch 18 allergen was narrowed down to residues 83-91. In addition, Lys89, Lys90, and possibly Phe91 were identified as the core residues. Substitution of Lys89, Lys90, or Phe91 with alanine can significantly reduce IgE-binding to Pen ch 18. Immunoblot inhibition confirmed that Lys89 and Phe91 played a significant role in IgE-binding against Pen ch 18. Molecular modeling suggests they are located on a loop-like structure at or near the surface of the major fungal allergen.

Vacuolar serine protease is a major allergen of Cladosporium cladosporioides.

BACKGROUND: Cladosporium is an important allergenic fungus worldwide. We report here a major allergen of C. cladosporioides. METHODS: Major C. cladosporioides allergens were characterized by immunoblotting, N-terminal amino acid sequencing, protein purification and cDNA cloning. RESULTS: Seventy-four sera (38%) from 197 bronchial asthmatic patients demonstrated IgE binding against C. cladosporioides extracts. Among these 74 sera, 41 (55%) and 38 (51%) showed IgE binding against a 36- and a 20-kDa protein of C. cladosporioides, respectively. Both IgE-reacting components reacted with FUM20, a monoclonal antibody against fungal serine proteases. N-terminal amino acid sequencing results suggest that they are vacuolar serine proteases, and the 20-kDa component is possibly a degraded product of the 36-kDa allergen. A corresponding 5'-truncated 1,425-bp cDNA fragment was isolated. The mature protein after N-terminal processing starts with an N-terminal serine that is the ninth residue encoded by the 5'-truncated cDNA. The protein sequence deduced shares 69-72% sequence identity with Penicillium vacuolar serine proteases and was designated as Cla c 9. The purified 36-kDa Cla c 9 allergen showed proteolytic activity with peptide Z-Ala-Ala-Leu-pNA as substrate. IgE cross-reactivity was detected between the purified Cla c 9 and serine protease allergens from Aspergillusfumigatus and Penicillium chrysogenum. CONCLUSION: We identified a vacuolar serine protease as a major allergen of C. cladosporioides (Cla c 9) and a major pan-allergen of prevalent airborne fungi. IgE cross-reactivity among these highly conserved serine protease pan-fungal allergens was also detectable.