Correlation between Pneumonia Severity and Pulmonary Complications in Middle East Respiratory Syndrome

This nationwide, prospective cohort study evaluated pulmonary function and radiological sequelae according to infection severity in 73 survivors from the 2015 Middle East respiratory syndrome (MERS) outbreak in Korea. Patients with severe pneumonia in MERS-coronavirus infection had more impaired pulmonary function than those with no or mild pneumonia at the 1-year follow-up, which was compatible with the radiological sequelae. Severe pneumonia significantly impairs pulmonary function and makes long radiological sequelae in MERS.

In vitro Expansion of Natural Regulatory T Lymphocytes Useful for Cell Therapy in Allotransplantation

Immunotherapy with regulatory T lymphocytes is considered to be an attractive new therapeutic modality to prevent allograft rejection. The success of this new therapy is critically dependent on the preparation of highly effective and enough number of regulatory T cells. Here, we tried to establish a proper strategy for the ex vivo expansion of regulatory T cells and evaluated their characteristics. CD4+CD25h+CD62L+ T cells were isolated from the recipient mice and weekly stimulated with various stimuli in the presence of IL-2. The most efficient protocol for the expansion of regulatory T cells maintaining Foxp3 expression and regulatory activity was the three cycles stimulation with donor bone marrow-derived dendritic cells (BM-DCs) which yielded around 400 fold expansion of regulatory T cells. The in vitro-expanded regulatory T cells expressing lymph node homing receptors on their cell surface, were composed of polyclonal population, and did not acquire the ability to produce effector cytokines. Importantly, these expanded regulatory T cells induced a modest prolongation of skin allograft survival when combined with transient T cell depletion in recipient mice. These data indicate that our protocol could be used to obtain an effective population of natural regulatory T cells available for the regulatory T cell therapy to prevent allograft rejection.

Experimental Micro Encapsulation of Pancreatic Islets with Air-driven Droplet Generator and Alginate / 대한이식학회지

PURPOSE: Transplantation of microencapsulated islets is proposed as an ideal therapy for the treatment of type 1 diabetes mellitus without immunosuppression. This is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. The aim of this study is to establish an ideal condition of microencapsulation by using an air-driven droplet generator and alginate in vitro. METHODS: Islets were prepared from Sprague Dawley rat and semi SPF-micro pig. Alginate concentrations were changed from 1.5% to 3.0%, and inflow rate of alginate was varied from 10 mL/hr to 40 mL/hr. CO2 flow rate was regulated from 2.0 L/min to 4.0 L/min. Viability was checked by dithizone and FDA/PI staining. Secretory function was tested with glucose challenge and insulin stimulation index was investigated. RESULTS: The optimal conditions for islet encapsulation were revealed with alginate inflow rate of 10 mL/hr, CO2 flow rate of 2.0 L/min in concentration of 2% alginate. In concentration of 2.5% alginate, alginate inflow rate of 20 mL/hr, CO2 flow rate 3.0 L/min was ideal, and alginate inflow rate of 40 mL/hr, CO2 flow rate of 4.0 L/min showed good conditions of microcapsules in concentration of 3% alginate. Viability of encapsulated islets was higher than 90% in both rat and porcine. In terms of insulin secretion, encapsulated islets secreted insulin in response to glucose in static culture medium. However there was no normal response to low and high glucose challenge with stimulation index of less than 2.0. CONCLUSION: Microencapsulation of islets in rat and pig was successful with air-driven droplet generator and alginate in vitro. Further studies about biocompatibility and glucose control in vivo should be followed to be a useful tool for treatment of diabetes mellitus patients in clinical setting.

Standardization of Isolation Procedure and Analysis of Variables on Successful Isolation of Islet from the Human Pancreas / 대한내분비학회지

BACKGROUND: Identifying the donor and isolation-related factors during the islet isolation would be greatly helpful to improve the result of human islet isolation for successful clinical islet transplantation. METHODS: Sixty-nine pancreata from cadaveric donors were isolated with standard protocol and analyzed to identify the donor factors and isolation variables for successful isolation. Islet isolations recovered > or = 100,000 Islet Equivalent (IEQ, n=53) were compared to islet mass less than 100,000 IEQ (n=16). RESULTS: The mean islet recovery was 216.0 x 10(3) +/- 173.7 x 10(3) (IEQ) before purification and 130.6 x 10(3) +/- 140.2 x 10(3) (IEQ) after purification. Mean purity was 54 +/- 31%. Mean age of donor was 31.2 +/- 13.2 year and mean cold ischemic time was 6.9 +/- 6.2 hour. Quality of isolated islets was acceptable in terms of bacterial culture, viability and secretory function in vitro and in vivo. In univariate analysis on successful isolation, status of pancreas was the only significant factor and sex, duration of collagenase expansion and digestion time were marginal factors. Stepwise multivariate logistic regression analysis showed donor sex, status of pancreas and digestion time were significant factors for the successful islet isolation. CONCLUSION: This study confirms some donor factors and variables in isolation process can influence the ability to obtain the successful isolation of human islet. Enough experiences and pertinent review of donor and isolation factors can make islet isolation successful, supporting the clinical islet transplantation without spending of cost.

Alpha-Melanocyte Stimulating Hormone Protects Pancreatic Islet Dysfunction by Peripheral Blood Mononuclear Cells in vitro / 대한이식학회지

PURPOSE: The alpha-melanocyte-stimulating hormone (alpha-MSH) has been shown to interact with various cells of the immune and inflammatory system and down-regulate either the production or the action of the pro-inflammatory cytokines. In this study, we investigated the potential of alpha-MSH on preventing pancreatic islet cell from death and dysfunction by inflammatory cytokines released from peripheral blood mononuclear cells (PBMCs) in rat. METHODS: Rat pancreatic islets were co-cultured with PBMCs, stimulated by phorbol myrstic acid and ionomycin. alpha-MSH was treated to PBMCs for 2 hours before co-culture. Viability and apoptosis of islets were observed by MTT and FACS. Inflammatory cytokines and nitric oxide (NO) were measured. Insulin release from islet co-cultured with mononuclear cells was checked for the islet function. RESULTS: In comparison to control group, viability of islets with alpha-MSH treated mononuclear cells was increased and apoptosis was reduced significantly. Inflammatory cytokines such as TNF-alpha and IL-1beta were reduced in alpha-MSH-treated group. NO production in alpha-MSH-treated group was decreased. Insulin secretory function of islet was recovered in condition of alpha-MSH treatment. CONCLUSION: This study demonstrates that alpha-MSH protects cell death and preserves the secretory function of pancreatic islet cells from the pro-inflammatory reaction of mononuclear cells, and may have the potential to improve the graft survival in clinical islet transplantation.

Experimental Study on Cryopreservation of Pancreas Islet Cell in Rat / 대한이식학회지

PURPOSE: Cryopreservation of pancreas islet cells can facilitate the clinical islet transplantation by giving a means of storage of islets and immunomodulation on pancreatic islet preparations. METHODS: Pancreatic islets were isolated by standard technique using collagenase in rat. Cryopreservation was performed by using DMSO as a cryoprotectant after one day or 48 hr culture. Recovery rate, viability and insulin release in assay in vitro and vivo were checked under the various conditions, such as concentration of DMSO (1.5 M or 2.0 M), culture condition (1 day or 2 day), and taurine treatment. RESULTS: Percentage of recovery of cryopreserved islet was 56.8+/-10% after thawing. Viability was decreased from 97.2+/-1.1% before cryopreservation to 82.7+/-9.9% after thawing. Glucose stimulation index was reduced from 1.7+/-0.2 before cryopreservation to 1.2+/-0.8 after thawing. Nucleation method by metal rod showed better viability than control (no nucleation) or chamber nucleation method. Mean viability and glucose stimulation index was 81% and 1.5 in one day culture, and 84.2% and 1.2 in 2 day culture before cryopreservtion. Islet treated with taurine showed better insulin release and intracellular insulin content compared with non treated islets before and after cryopreservation. When 4000 IEQ (Islet Equivalent) of islets treated with taurine and non treated cryopreserved islets were transplanted into syngenic streptozotocin induced diabetic rat, all showed normoglycemia over 60 days. CONCLUSION: Cryopreservation of islets could give a tool of storage with preservation of islet secretion function. However pertinent effort to improve the recovery is needed in order to be used in the clinical islet transplantation.

Comparative Study on Biologic and Immunologic Characteristics of the Pancreas Islet Cell between 24degrees C and 37degrees C Culture in the Rat / 대한이식학회지

PURPOSE: Islet cell transplantation, as an alternative approach to endocrine cell replacement to treat the diabetes mellitus, has received significant attention because it holds several advantages over whole gland transplantation. However cell damage from islet isolation and immunologic rejection after transplantation prevent from successful clinical application for diabetic patients. Culture of cells at low temperature has known to stabilize the cell viability, and to decrease the immunologic antigenicity. Aim of this study is to investigate the effect of culture at 24oC on cell viability, cellular function, immunogenicity and cytokine profiles in rat pancreas islet. METHODS: Pancreas islets were isolated from Lewis rat and cultured at 24oC or 37oC during 14 days. Islet recovery after culture period was counted as islet equivalent number, and islet viability was examined with fluorescent vital staining (FDA/PI). Islet function was measured with glucose stimulation test. Annexin V expression and MHC class I and II expression were measured with flow cytometric assay for apoptosis and immunogenicity respectively. Lymphocyte cell proliferation through mixed lymphocyte islet culture was examined with WST-1 proliferation assay. Cytokine profiles were analyzed with quantitative real time RT-PCR. All these parameters were measured on 1, 3, 5, 7, 14 culture days after islet isolation. RESULTS: Islet recovery was higher in islet cultured at 24oC than in islet cultured at 37oC without change of viability. Insulin secretion after glucose stimulation was more effective in 24oC culture condition. Decrease of apoptotic cell death was demonstrated in 24oC cultured islet. MHC class I and II expression on islets and lymphocyte proliferation when cocultured with islets were less prominent in 24oC cultured islet. TNF-alpha and IL-4 cytokine expression was higher in islet cultured at 24oC than in islet cultured at 37oC. IL-1beta and IL-10 cytokine expression were similar in both culture condition. CONCLUSION: This study demonstrated that cell recovery and function are increased in islet cultured at 24oC than in islet cultured at 37oC while antigenicity and proinflammatory cytokine expression are decreased. Low temperature culture can be a good approach to prevent the loss of islet mass, and to reduce the immunologic rejection of transplanted islet for successful clinical islet transplantation.

Requirements for the Differentiation of CD4+ Cytotoxic T cells Specific to Mycobacterial Antigens

The cytolytic activity of CD4' T cells, both human and murine, has been clearly demonstrated in the immune response to mycobacterial infection and suggested to play a significant role in the protection and immunopathology. However, Uttle is known about the differentiation of CD4' CTL. In order to address this issue, we examined the influences of some factors on the generation of CD4' CTL specific to mycobacterial antigens. After 7 days' stimulation of PBMCs from healthy tuberculin reactors with mycobacterial antigens, the cytolytic activity of purised CD4' T cells toward autologous macrophages infected with mycobacteria was measured by Cr release assay. First, we found that both of live M. tubeiculosis and soluble antigens (ST-CF) induced the cytolytic activity of CD4' T cells, although the inducibility of the former was slightly greater than the latter. Second, the cytolytic activity was maximally induced at the relatively low antigen concentration (0.2:1 bacteria:monocyte ratio or 0.5 mg/ml of ST-CF). Finally, in the presence of increasing amounts of neutralizing anti-IL-12 or anti-IFN-r MoAb, the cytolytic activity of CD4+ T cells was decreased in a dose-dependent manner. These results suggest that low dose of antigen, its particulate type give mycobacteria), IL-12, and IFN-r give some positive signals for the generation of CD4+ CTL.

Distribution of mec Regulator Genes in Methicillin - Resistant Staphylococci

In order to understand the role of mec regulator genes in the evolution of methicillin-resistant S. aureus (MRSA), the distribution of the mec regulator genes among the 66 clinical isolates of MRSA was analysed. And also the correlation between gene mutation and degree of phenotypic expression of resistance was studied. Fifty strains carried whole mec regulator region, while the mecI gene and nearly half of the 3'-end of the mecR#l gene were deleted in fifteen strains. The mecRl MS gene was detected among all of the mecA carried strains, but the mecRl PB gene was carried by 77% of the MRSA strains. At least a portion of the 5'-end region of the mecRl gene was carried by all MRSA strains tested. Forty-seven strains were finally confirmed to have mecI gene and each mecI gene of above strains was sequenced for identification of the relationship between repressor function of mecI gene on mecA transcription and MIC level of methicillin. Point mutations were detected in 11 strains of 47 strains. In 8 strains, there was one nucleotide substitution (C to T at position 202) that produced a new termination codon at position 201. In 3 strains, one nucleotide substitution from G to T at position 43 caused an amino acid substitution from Val to Phe. The MIC of methicillin of strains carrying mutated mecI genes ranged 256 ug/ ml to 1024 ug/ml. Transcription level of amplified cDNA corresponding to mecA was determined by the method of RT-PCR of extracted RNA. Total RNA was extracted from two strains with mutated mecI gene and a strain with intact mecI gene. Deletional loss or the mutational inactivation of the mecI gene did not affect the level of mecA transcription. Role of mecI gene as a strong repressor function on mecA gene seemed to be skeptical.

Eukaryotic Kxpression of the Major Antigenic Determinants Evoking Neutralizing Antibodies in Human Cytomegalovirus ( HCMV ) Isolated in Korea

Human cytomegalovirus (HCMV) isolated from Korean patients is different in the antigenic and genomic structure of gB from the laboratory-adapted strain. To dissect the reactivity to HCMV glycoprotein B (gB) domains, each domain gene of gB of HCMV SNUCH1, Korean isolate, was amplified from the extracted DNA of the virus-infected fibroblasts with the specific primers by polymerase chain reaction (PCR). Amplified DNA was cloned into pcDNA3. Immunofluorescent staining and western blot analysis revealed that the expressed gB in mammalian cells was immunoreactive and equivalent to the naturally expressed gB in virus-infected fibroblasts. The antigenic component reactive with monoclonal antibodies, MCMVA 57, 88, and 98 appeared at the D3 domain of gB molecule, and that with MCMVA 66 and 135 at the D2b domain. Antibody titer was measured with HCMV-infected fibroblasts and the domains of gB expressed in mammalian cells. There was no correlation between the antibody titer to the whole HCMV and neutralizing antibody titer, and between the antibody titer to whole HCMV and whole gB. It was more reasonable to use whole gB than whole HCMV in the comparison with the neutralizing antibody titer. D3 was representative domain in gB molecule in the anti-gB reactivity. Conclusively it is highly recommendable to use the representing isolates in Korea and its domains for the detection of antibody or the analysis of antigen in the aspect of immunological properties and molecular structures.

Isolation and Identification of the Antigen Recognized by Human Cytomegalovirus Specific Monoclonal Antibody SCMVM 34

Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM 34, recognizes early antigen confined to the nucleus of HCMV-infected cells. This study was performed to identify the antigen reactive to SCMVM 34 with purification and amino acid sequencing. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 M NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE. The molecular weight of the reactive proteins was 52 kD, 40 kD and 34 kD. The modified or blocked amino termini of 52 kD and 40 kD showed resistance to Edman degradation. The internal peptide fragments were isolated by tryptic digeytion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from HPLC profile revealed that the antigens recognized by SCMVM 34 was ppUIA4.

Gene cloning of the human cytomegalovirus (HCMV) antigen reactive with the serum from a HCMV-infected patient

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.

The role of CDI on the antigen recognition of human CD3+CD4-CD8 T lymphocyte clone specific to M.tyberculosis

No abstract available.

Production and characterization of human T-cell clones reactive to mycobacterium tuberculosis

No abstract available.

Production and characterization of human T cell hybridomas

No abstract available.