Analysis and Development of Antigen-specific T Cells Derived from Peripheral Blood Mononuclear Cells of Healthy Donors.

BACKGROUND/AIM: Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. MATERIALS AND METHODS: CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. RESULTS: As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. CONCLUSION: Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.

Immune repertoire and responses to neoadjuvant TCHP therapy in HER2-positive breast cancer.

Background: Despite the introduction of trastuzumab, pathologic complete response (pCR) is not attained in approximately 30-40% of Human epithelial growth factor receptor-2-positive breast cancer. Tumor-infiltrating lymphocytes (TIL) have been suggested as a predictive marker of treatment response, albeit not always effective. We investigated the relationship between trastuzumab, docetaxel, carboplatin, and pertuzumab (TCHP) treatment and immune repertoire as a treatment response predictor. Design: In all, 35 cases were divided into two experimental groups: 10 and 25 cases in the preliminary and main experiments, respectively. In the preliminary experiment, the biopsy tissues before TCHP treatment and the surgical tissues after TCHP treatment were compared. In the main experiment, the biopsy tissues before TCHP treatment were compared according to the TCHP treatment response. Methods: The T-cell repertoire for TRA, TRB, TRG, and TRD, and B-cell repertoire for immunoglobulin heavy, immunoglobulin kappa, and immunoglobulin lambda were evaluated. Whole transcriptome sequencing was also performed. Results: In the preliminary experiment, the density and richness of the T-cell receptor (TCR) and B-cell receptor (BCR) repertoires decreased after treatment, regardless of TCHP response. In the main experiment, the Shannon's entropy index, density, and length of CDR3 of the TCR and BCR repertoires did not differ significantly in patients who did and did not achieve pCR. The pCR and non-pCR subgroups according to the level of TILs revealed that the non-pCR/lowTIL group had a higher proportion of low-frequency clones than the pCR/lowTIL group in TRA (non-pCR/lowTIL versus pCR/lowTIL, 0.01-0.1%, 63% versus 45.3%; <0.01%, 32.9% versus 51.8%, p < 0.001) and TRB (non-pCR/lowTIL versus pCR/lowTIL, 0.01-0.1%, 26.5% versus 14.7%; <0.01%, 72.0% versus 84.1%, p < 0.001). Conclusions: The role of the diversity, richness, and density of the TCR and BCR repertoires as predictive markers for TCHP response was not identified. Compositions of low-frequency clones could be candidates for predictive factors of TCHP response; however, validation studies and further research are necessary.

Bacterial distribution on the ocular surface of patients with primary Sjögren's syndrome.

Many studies have shown that gut microbial dysbiosis is a major factor in the etiology of autoimmune diseases but none have suggested that the ocular surface (OS) microbiome is associated with Sjögren's syndrome (SS). In this prospective study, we analyzed bacterial distribution on the OS in patients with primary SS. Among the 120 subjects included in this study, 48 patients (group A) had primary SS, whereas 72 subjects (group B) had dry eye symptoms that were unrelated to SS. We evaluated clinical dry eye parameters such as the OS disease index, ocular staining score (OSS), Schirmer's I test, and tear break-up time (TBUT). Conjunctival swabs were used to analyze the microbial communities from the two groups. Bacterial 16S rRNA genes were sequenced using the Illumina MiSeq platform, and the data were analyzed using the QIIME 1.9.1 program. The Shannon index was significantly lower in group A than in group B microbiota (p < 0.05). An analysis of similarity using the Bray-Curtis distance method found no difference in beta-diversity between the two groups (p > 0.05). In group A, Actinobacteria at the phylum level and Corynebacteria at the genus level exhibited low abundance than group B, but the differences were not statistically significant (p > 0.05). SS apparently decreases the diversity of the OS microbial community. These observations may be related to the pathophysiology of SS and should be investigated in future studies.

Effect of CO2 on biogeochemical reactions and microbial community composition in bioreactors with deep groundwater and basalt.

Changes in subsurface microbiology following CO2 injection have the potential to impact carbon trapping in CO2 storage reservoirs. However, much remains to be learned about responses of natural microbial consortia to elevated CO2 in basaltic systems. This study asks: how will microbes from deep (700 m) groundwater change along a gradient in CO2 (0-20 psi) in batch reactor systems containing basalt chips and groundwater amended with lactate? Reactors incubated for 87 days at 23 °C. Results for reactors with low CO2 (0 and 3 psi) differed considerably from those with high CO2 (10 and 20 psi). In reactors with low CO2, pH was >6.5 and lactate started to be used within 24 days. By 40 days, lactate was completely consumed and acetate increased to ~4 mM. As lactate was consumed, sulfate decreased from 0.16 to 0 mM after 40 days. In contrast, in reactors with high CO2, pH was <6.5, lactate and sulfate concentrations varied little and acetate was not produced. Biogeochemical modeling and community analyses indicate that differences between reactors with low and high CO2 reflect tolerances of reactor microbes to CO2 exposure. Communities in the low CO2 reactors carried out syntrophic lactate oxidation coupled with methanogenesis and sulfate reduction. Bacteroidota and Firmicutes became dominant phyla after 24 days and groups capable of sulfate reduction and methanogenesis were detected. In reactors with high CO2, however, biogeochemical activity was insignificant, no groups capable of sulfate reducion or methanogenesis were observed, and the community became less diverse during the incubation. These findings show that the response of microbial consortia can vary sharply along a CO2 gradient, creating significant differences in community composition and biogeochemistry, and that the timescale of basalt weathering is likely not rapid enough to prevent significant stress following a rapid increase in CO2 abundance.

Geochemical and microbial characteristics of seepage water and mineral precipitates in a radwaste disposal facility impacted by seawater intrusion and high alkalinity.

The construction of an underground facility can dramatically change the quality, flow direction, and level of groundwater. It may also impact subsurface microbial composition and activity. Groundwater quality was monitored over eight years in two observational wells near an underground disposal facility on the east coast of South Korea. The results showed dramatic increases in dissolved ions such as O2, Na, Ca, Mg, and SO4 during facility construction. Seepage water samples downgradient from the silos and tunnels, and precipitates deposited along the seepage water flow path were collected to determine the impact inside the disposal facility. X-ray analysis (powder X-ray diffraction (pXRD) and X-ray absorption fine structure (XAFS)) were used to characterize the mineral precipitates. Microbial community composition was determined by 16S rRNA gene sequencing. The seepage water composition was of two types: Ca-Cl and Ca-Na-HCO3. The ratio of Cl and δ18O showed that the Ca-Cl type seepage water was influenced by groundwater mixed with seawater ranging from 2.7% to 15.1%. Various sulfate-reducing bacteria were identified in the Ca-Cl type seepage water, exhibiting relatively high sulfate content from seawater intrusion. Samples from the Ca-Na-HCO3 type seepage water had an extremely high pH (>10) and abundance of Hydrogenophaga. The precipitates observed along the flow path of the seepage water included calcite, ferrihydrite, green rust, and siderite, depending on seepage water chemistry and microbial activity. This study suggests that the construction of underground structures creates distinct, localized geochemical conditions (e.g., high alkalinity, high salinity, and oxic conditions), which may impact microbial communities. These biogeochemical changes may have undesirable large-scale impacts such as water pump clogging. An understanding of the process and long-term monitoring are essential to assess the safety of underground facilities.

Waste nutrient solutions from full-scale open hydroponic cultivation: Dynamics of effluent quality and removal of nitrogen and phosphorus using a pilot-scale sequencing batch reactor.

Hydroponic cultivation is revolutionizing agricultural crop production techniques all over the world owing to its minimal environmental footprint, enhanced pest control, and high crop yield. However, waste nutrient solutions (WNS) generated from hydroponic systems contain high concentrations of N and P; moreover, they are discharged into surface and subsurface environments, leading to eutrophication and subsequent ecosystem degradation. In this study, the nutrient concentrations in WNS from 10 hydroponic indoor tomato, capsicum, and strawberry farms (greenhouses) were monitored for up to six months. The concentrations of N and P in WNS discharged from these farms were 48.0-494.0 mg L-1 and 12.7-96.9 mg L-1, respectively, which exceeded the Korean water quality guidelines (40.0 mg L-1 N and 4.0 mg L-1 P) for effluents. These concentrations were varied and dependent on the supplied nutrient concentrations, crop types, and growth stages. In general, the concentrations of N and P were in the following order: tomato > capsicum > strawberry. High N as NO3- and P as PO43- but low organic C in WNS warrant subsequent treatment before discharge. Therefore, this study tested a pilot-scale sequencing batch reactor (SBR) system as a potential technology for WNS treatment. The SBR system had BOD, COD, nitrate, and phosphate removal efficiency of 100, 100, 89.5, and 99.8%, respectively. In addition, the SBR system removed other cations such as Ca2+, dissolved Fe, K+, Mg2+, and Na+ and the removal efficiencies of those ions were 48, 67, 18, 14 and 15%, respectively. Lower methanol addition (0.63 mg L-1) and extended aeration (~30 min) improved SBR performance efficiency of C, N, and P removal. Thus, SBR showed significant promise as a treatment alternative to WNS pollutants originating from hydroponic systems.

Inhibitory Effects of Pterocarpus santalinus Extract against IgE/Antigen-Sensitized Mast Cells and Atopic Dermatitis-Like Skin Lesions.

Pterocarpus santalinus has been traditionally used as a medicinal plant owing to its anti-inflammatory, anthelmintic, tonic, antihyperglycemic, and diaphoretic properties. We hypothesized that P. santalinus might have therapeutic potential in alleviating allergy and atopic dermatitis. Thus, we investigated the inhibitory effects of P. santalinus extract against allergic responses and 2,4-dinitrochlorobenzene-induced atopic dermatitis-like dorsal skin lesions using immunoglobulin E-sensitized rat basophilic leukemia-2H3 mast cells and NC/Nga mice. Degranulation and enzyme-linked immunosorbent assays were conducted to measure degranulation, proinflammatory cytokine levels, and prostaglandin E2 concentrations in immunoglobulin E/antigen-sensitized RBL-2H3 mast cells. The therapeutic efficacy of P. santalinus extract in 2,4-dinitrochlorobenzene-induced atopic dermatitis was evaluated through morphological, physiological, and immunological analysis. P. santalinus extract inhibited ß-hexosaminidase and histamine release and reduced tumor necrosis factor-α, interleukin-4, and prostaglandin E2 secretion. Furthermore, P. santalinus extract suppressed atopic dermatitis-like skin lesions by regulating the serum levels of immunoglobulin E and immunoglobulin G2a, and messenger ribonucleic acid expression of T helper cell 1- and T helper cell 2-related mediators in the skin lesions. Histopathological analyses showed a decrease in epidermal thickness and intradermal inflammatory cell infiltration. These results suggested that P. santalinus extract might have beneficial effects in treating allergic and atopic dermatitis-like skin disorders.

Distribution and Diversity of Ocular Microbial Communities in Diabetic Patients Compared with Healthy Subjects.

PURPOSE: The aim of this study was to identify differences in the major (core vs. variable) microbial genera of human subjects with and without diabetes. METHODS: Bacterial 16S rRNA genes obtained from conjunctival swabs of 19 healthy subjects and 30 diabetic patients were sequenced using the Illumina MiSeq platform, and the sequencing data were analyzed using QIIME 1.9.1. To elucidate the microbial diversity in the ocular surface (OS), test programs from various bioinformatics domains were used. RESULTS: Diversity index and rarefaction analysis showed that the microbial community of the diabetic patients was more diverse than that of the healthy subjects. Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria and Bacteroidetes were the dominant taxa present in the OS, and there was a significant difference in the relative abundance of the bacterial phyla between the diabetic patients and control subjects. Proteobacteria were more abundant in the diabetic group, whereas Firmicutes was more abundant in the control group. Analysis of bacterial taxa at the genus level showed that the core microbiome of diabetic patients comprised Acinetobacter, Burkholderia, Sphingomonas, and Ralstonia, whereas that of the controls comprised Bradyrhizobiaceae, Staphylococcus, Corynebacterium, Pseudomonas, Novosphingobium, Neisseriaceae, and Acinetobacter. CONCLUSIONS: There was a significant difference in the microbial community composition between diabetic patients and healthy subjects. A high abundance of Acinetobacter in the OS of diabetic patients may arise from the unique characteristics of the OS compared with those of other organ surfaces.

Nutrient removal from hydroponic wastewater by a microbial consortium and a culture of Paracercomonas saepenatans.

The potential of microbial processes for removal of major nutrients (e.g., N, P) and inorganic cations (e.g., Ca2+, Mg2+, and Fe2+) from hydroponic systems was investigated. Microbial consortium- and axenic culture-based experiments were conducted in a waste nutrient solution (WNS). A microbial consortium grown in the WNS and selected microalgae species of Paracercomonas saepenatans were inoculated in two different synthetic media (Bold's Basal Medium (BBM) and synthetic WNS) in batch systems, and the microbial growth characteristics and the rate and extent of nutrient removal were determined for each system. No toxicity or growth inhibition was observed during microbial growth in either media. Both the waste-nutrient-grown microbial consortium and Paracercomonas saepenatans can be grown effectively in BBM and WNS, and both remove most ions from both media (e.g.,>99% removal of NO3- and 41-100% removal of PO43-) within 16days. Significant nutrient removal was observed during the growth phase of the microbial communities (4-10days period), indicating major nutrient utilization for microbial growth as well as chemical mineral precipitation. Furthermore, MINEQL+4.6 modeling showed higher PO43- removal in WNS during microbial growth (compared to BBM) due to precipitation of phosphate minerals (e.g., hydroxyapatite, vivianite). The dominant microbial species in both systems were also identified. DNA sequencing showed that Vorticella (58%) and Scenedesmus (33%) in WNS and Scenedesmus (89%) in BBM were the predominant species. This study demonstrates the potential application of microbial consortium (predominantly algae and protozoan)-based treatment techniques for hydroponic systems.

Application of an in-situ soil sampler for assessing subsurface biogeochemical dynamics in a diesel-contaminated coastal site during soil flushing operations.

Subsurface biogeochemistry and contaminant dynamics during the remediation of diesel-contamination by in-situ soil flushing were investigated at a site located in a coastal region. An in-situ sampler containing diesel-contaminated soils separated into two size fractions (<0.063- and <2-mm) was utilized in two monitoring wells: DH1 (located close to the injection and extraction wells for in-situ soil flushing) and DH2 (located beyond sheet piles placed to block the transport of leaked diesel). Total petroleum hydrocarbon (TPH) concentrations and biogeochemical properties were monitored both in soil and groundwater for six months. A shift occurred in the groundwater type from Ca-HCO3 to Na-Cl due to seawater intrusion during intense pumping, while the concentrations of Ni, Cu, Co, V, Cr, and Se increased substantially following surfactant (TWEEN 80) injection. The in-situ sampler with fine particles was more sensitive to variations in conditions during the remedial soil flushing process. In both wells, soil TPH concentrations in the <0.063-mm fraction were much higher than those in the <2-mm fraction. Increases in soil TPH in DH1 were consistent with the expected outcomes following well pumping and surfactant injection used to enhance TPH extraction. However, the number of diesel-degrading microorganisms decreased after surfactant injection. 16S-rRNA gene-based analysis also showed that the community composition and diversity depended on both particle size and diesel contamination. The multidisciplinary approach to the contaminated site assessments showed that soil flushing with surfactant enhanced diesel extraction, but negatively impacted in-situ diesel biodegradation as well as groundwater quality. The results also suggest that the in-situ sampler can be an effective monitoring tool for subsurface biogeochemistry as well as contaminant dynamics.

Geochemical Influence on Microbial Communities at CO2-Leakage Analog Sites.

Microorganisms influence the chemical and physical properties of subsurface environments and thus represent an important control on the fate and environmental impact of CO2 that leaks into aquifers from deep storage reservoirs. How leakage will influence microbial populations over long time scales is largely unknown. This study uses natural analog sites to investigate the long-term impact of CO2 leakage from underground storage sites on subsurface biogeochemistry. We considered two sites with elevated CO2 levels (sample groups I and II) and one control site with low CO2 content (group III). Samples from sites with elevated CO2 had pH ranging from 6.2 to 4.5 and samples from the low-CO2 control group had pH ranging from 7.3 to 6.2. Solute concentrations were relatively low for samples from the control group and group I but high for samples from group II, reflecting varying degrees of water-rock interaction. Microbial communities were analyzed through clone library and MiSeq sequencing. Each 16S rRNA analysis identified various bacteria, methane-producing archaea, and ammonia-oxidizing archaea. Both bacterial and archaeal diversities were low in groundwater with high CO2 content and community compositions between the groups were also clearly different. In group II samples, sequences classified in groups capable of methanogenesis, metal reduction, and nitrate reduction had higher relative abundance in samples with relative high methane, iron, and manganese concentrations and low nitrate levels. Sequences close to Comamonadaceae were abundant in group I, while the taxa related to methanogens, Nitrospirae, and Anaerolineaceae were predominant in group II. Our findings provide insight into subsurface biogeochemical reactions that influence the carbon budget of the system including carbon fixation, carbon trapping, and CO2 conversion to methane. The results also suggest that monitoring groundwater microbial community can be a potential tool for tracking CO2 leakage from geologic storage sites.

Impacts of leachates from livestock carcass burial and manure heap sites on groundwater geochemistry and microbial community structure.

We investigated the impacts of leachates from a swine carcass burial site and a cow manure heap on the geochemical and microbiological properties of agricultural water samples, including leachate, groundwater from monitoring wells and background wells, and stream water. The leachate from the livestock burial site showed extremely high electrical conductivity, turbidity, and major ion concentrations, but low redox potential and dissolved oxygen levels. The groundwater in the monitoring wells adjacent to both sites showed severe contamination from the leachate, as indicated by the increases in EC, turbidity, Cl-, and SO42-. Bacteria from the phylum Firmicutes and Bacteriodetes and Archaea from the phylum Euryarchaeota were the major phyla in both the leachates and manure heap. However, the class- or genus-level components of these phyla differed markedly between the leachate and manure heap samples. The relative abundance of Firmicutes decreased from 35% to 0.3~13.9% in the monitoring wells and background wells at both sites. The Firmicutes in these wells was unlikely to have originated from the transportation of leachate to the surrounding environment because Firmicutes genera differed drastically between the leachate and monitoring wells. Meanwhile, sulfate-reducing bacteria (SRB) from the livestock carcass burial site were detected in the monitoring wells close to the leachate. This was likely because the release of carcass decomposition products, such as organic acids, to adjacent areas improved the suitability of the local environments for SRB, which were not abundant in the leachate. This study highlights the need to better understand microbial community dynamics along groundwater flow paths to evaluate bacterial transport in subsurface environments and provides new insights into the effective management of groundwater quality at both farm and regional scales.

Geochemical characteristics and microbial community composition in toxic metal-rich sediments contaminated with Au-Ag mine tailings.

The effects of extreme geochemical conditions on microbial community composition were investigated for two distinct sets of sediment samples collected near weathered mine tailings. One set (SCH) showed extraordinary geochemical characteristics: As (6.7-11.5%), Pb (1.5-2.1%), Zn (0.1-0.2%), and pH (3.1-3.5). The other set (SCL) had As (0.3-1.2%), Pb (0.02-0.22%), and Zn (0.01-0.02%) at pH 2.5-3.1. The bacterial communities in SCL were clearly different from those in SCH, suggesting that extreme geochemical conditions affected microbial community distribution even on a small spatial scale. The clones identified in SCL were closely related to acidophilic bacteria in the taxa Acidobacterium (18%), Acidomicrobineae (14%), and Leptospirillum (10%). Most clones in SCH were closely related to Methylobacterium (79%) and Ralstonia (19%), both well-known metal-resistant bacteria. Although total As was extremely high, over 95% was in the form of scorodite (FeAsO4·2H2O). Acid-extractable As was only ∼118 and ∼14 mg kg(-1) in SCH and SCL, respectively, below the level known to be toxic to bacteria. Meanwhile, acid-extractable Pb and Zn in SCH were above toxic concentrations. Because As was present in an oxidized, stable form, release of Pb and/or Zn (or a combination of toxic metals in the sediment) from the sediment likely accounts for the differences in microbial community structure. The results also suggest that care should be taken when investigating mine tailings, because large differences in chemical/biological properties can occur over small spatial scales.