Proteomic analysis of adult T-cell leukemia/lymphoma: A biomarker identification strategy based on preparation and in-solution digestion methods of total proteins.

Adult T-cell leukemia/lymphoma (ATL), caused by human T-cell leukemia virus type-1 (HTLV-1) infection, is a malignant hematologic cancer that remains difficult to cure. We herein established a biomarker identification strategy based on the total cell proteomics of cultured ATL cells to search for novel ATL biomarkers. Four protocols with a combination of selected conditions based on lysis buffers and addition agents for total cell proteomics were used for a differential analysis between the ATL cell group (consisting of 11 cell lines), HTLV-1-infected cell group (consisting of 6 cell lines), and HTLV-1-negative cell group (consisting of 6 cell lines). In the analysis, we identified 24 and 27 proteins that were significantly increased (ratio ≥2.0, p < 0.05) and decreased (ratio ≤ 0.5, p < 0.05), respectively, in the ATL group. Previously reported CCL3 and CD30/TNFRSF8 were confirmed to be among significantly increased proteins. Furthermore, correlation analysis between identified proteins and Tax suggested that RASSF2 and GORASP2 were candidates of novel Tax-regulated factors. The biomarker identification strategy established herein is expected to contribute to the identification of biomarkers for ATL and other diseases.

Whole-genome CRISPR screening identifies molecular mechanisms of PD-L1 expression in adult T-cell leukemia/lymphoma.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.

Rapid increase in salivary IgA and broad recognition of spike protein following SARS-CoV-2 vaccination.

Saliva is a key component of mucosal immunity, which protects the oral cavity from viral infections. However, salivary immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in terms of immunoglobulin dynamics and recognition, have not been investigated sufficiently. In this study, saliva samples were collected from individuals that received SARS-CoV-2 vaccine, and immunoglobulin G (IgG), IgM, and IgA against whole spike protein and S1 protein were measured. IgA against whole spike protein increased significantly following vaccination, while IgA against S1 protein did not. Of note, the IgA response was evident two weeks after the first vaccine dose and continued to rise thereafter. On the contrary, IgG antibodies against S1 increased significantly at four weeks after vaccination. These results reveal the dynamics and recognition antigens of immunoglobulins in saliva, indicating the function of IgA in the mucosal immune system. These findings may pave the way for further studies on mucosal immune response induced by vaccination.

Performance evaluation of Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test for different diagnostic situations.

IMPORTANCE: The World Health Organization estimated that 5-10 million people are infected with human T-cell leukemia virus type 1 (HTLV-1). This number is likely to be underestimated because reliable endemic data are available for only approximately 1.5 billion people worldwide. The point-of-care test is a powerful tool for the easy and quick detection of infections without the requirement for expensive instruments and laboratory equipment. Espline HTLV-I/II, a newly developed rapid immunochromatographic antibody test that was evaluated in this study, might significantly advance our understanding of the global epidemiology of HTLV-1 infection.

The evaluation of a rapid microfluidic immunofluorescence antigen test in detecting the infectiousness of COVID-19 patients.

BACKGROUND: A test-based strategy against coronavirus disease 2019 (COVID-19) is one of the measures to assess the need for isolation and prevention of infection. However, testing with high sensitivity methods, such as quantitative RT-PCR, leads to unnecessary isolation, whereas the lateral flow antigen test shows low sensitivity and false negative results. The purpose of this study was to evaluate the performance of the LumiraDx SARS-CoV-2 Ag test (Lumira Ag), a rapid microfluidic immunofluorescence method, in assessing infectivity. METHODS: This study was performed from March 2022 to July 2022. A pair of nasopharyngeal swab samples were obtained from each patient with mild COVID-19. One swab was used for Lumira Ag testing, and the other for quantitative RT-PCR testing and virus culture. RESULTS: A total of 84 patients were included in the study. Among them, PCR, Lumira Ag test, and virus culture indicated positivity for 82, 66, and 24 patients, respectively. When comparing the Lumira Ag test to virus culture, its sensitivity was 100.0% (24/24), specificity, 30.0% (18/60); positive predictive value, 36.3% (24/66); and negative predictive value (NPV), 100.0% (18/18). The positive sample for virus culture was observed until the ninth day from the onset of symptoms, while the Lumira Ag test was observed until day 11. CONCLUSIONS: The Lumira Ag test showed high sensitivity and NPV (100% each) compared to virus culture. A test-based strategy using the Lumira Ag test can effectively exclude COVID-19 infectiousness.

The clinical characteristics and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among very elderly people in Japan.

AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial infection-causing pathogen. The clonal shift from staphylococcal cassette chromosome mec (SCCmec) type II MRSA to SCCmec type IV MRSA has occurred rapidly in acute-care hospitals. However, the epidemiology and clinical impacts of MRSA in geriatric hospitals are poorly documented. We performed a molecular epidemiological analysis of the clinical isolates and retrospectively investigated the clinical characteristics of SCCmec type IV MRSA in elderly individuals. METHODS: MRSA isolates were grouped according to the SCCmec type and virulence genes (tst, sea, seb, sec, and lukS/F-PV), and multi-locus sequence typing (MLST) was performed. RESULTS: Of the 145 MRSA isolates obtained from patients with a median age of 85 years, 100 (69.0%) were obtained from sputum samples, 22 (15.2%) from skin and soft tissues, and seven (4.8%) from blood samples. The most prevalent clone was SCCmec type IV/clonal complex (CC)1/sea+ (59.3%), followed by SCCmec type I/sequence type (ST) 8 (17.3%). Of the 17 (11.7%) strains to which an anti-MRSA drug was administered by a physician, only three were SCCmec type IV/CC1/sea+ (17.6%) and five were SCCmec type I/ST8 (29.4%). SCCmec type IV/CC1/sea+ MRSA was more frequently isolated in long-term care wards than were SCCmec type I/ST8 strains (odds ratio: 2.85, 95% confidence interval: 1.08-7.54) and was less frequently treated as the cause of MRSA infections (odds ratio: 0.15, 95% confidence interval: 0.03-0.73). CONCLUSIONS: SCCmec type IV/CC1/sea+ MRSA was the predominant clone and could be easily transmissible and be capable of colonization. Geriatr Gerontol Int 2023; 23: 744-749.

Therapeutic efficacy of the resorcylic acid lactone LL-Z1640-2 for adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) remains incurable. The NF-κB and interferon regulatory factor 4 (IRF4) signalling pathways are among the critical survival pathways for the progression of ATL. TGF-ß-activated kinase 1 (TAK1), an IκB kinase-activating kinase, triggers the activation of NF-κB. The resorcylic acid lactone LL-Z1640-2 is a potent irreversible inhibitor of TAK1/extracellular signal-regulated kinase 2 (ERK2). We herein examined the therapeutic efficacy of LL-Z1640-2 against ATL. LL-Z1640-2 effectively suppressed the in vivo growth of ATL cells. It induced in vitro apoptosis and inhibited the nuclear translocation of p65/RelA in ATL cells. The knockdown of IRF4 strongly induced ATL cell death while downregulating MYC. LL-Z1640-2 as well as the NF-κB inhibitor BAY11-7082 decreased the expression of IRF4 and MYC at the protein and mRNA levels, indicating the suppression of the NF-κB-IRF4-MYC axis. The treatment with LL-Z1640-2 also mitigated the phosphorylation of p38 MAPK along with the expression of CC chemokine receptor 4. Furthermore, the inhibition of STAT3/5 potentiated the cytotoxic activity of LL-Z1640-2 against IL-2-responsive ATL cells in the presence of IL-2. Therefore, LL-Z1640-2 appears to be an effective treatment for ATL. Further studies are needed to develop more potent compounds that retain the active motifs of LL-Z1640-2.

Clinical and microbiological characteristics of bloodstream infection caused by Klebsiella pneumoniae harboring rmpA in Japanese adults.

We investigated the clinical features of bloodstream infections (BSIs) caused by Klebsiella pneumoniae harboring rmpA and molecular characteristics of the bacteria. We retrospectively investigated adult patients with K. pneumoniae BSI from January 2010 to March 2021 at Nagasaki University Hospital. A matched case-control study in a 1:3 ratio was conducted to clarify the clinical and bacterial characteristics of BSI caused by rmpA-positive K. pneumoniae compared with those caused by rmpA-negative isolates. Antimicrobial susceptibility testing and multilocus sequence typing (MLST) were performed for rmpA-positive isolates. The rmpA was detected in 36 (13.4%) of the 268 isolates. Of these 36 isolates, 31 (86.1%) harbored iucA and 35 (97.2%) each possessed peg-344 and iroB; capsular types were identified as K1 in 9 (25.0%) and K2 in 10 isolates (27.8%). Contrarily, of the 108 rmpA-negative isolates, which were matched for case-control studies, 5 isolates (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%) and 3 isolates (2.8%) had K1 and K2 capsular types, respectively. Among the rmpA-positive isolates, ST23/K1 (eight isolates) was the most frequent, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates). In a multivariate analysis using clinical factors, liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and use of anticancer drugs negatively correlated with rmpA-positive isolates in patients with K. pneumoniae BSI. Considering the correlation between rmpA-positive isolates and clinical features, rmpA can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.

Serological response to a third dose of SARS-CoV-2 vaccine according to previous infection history.

The IgG antibody titer against SARS-CoV-2 receptor binding protein (RBD) after mRNA vaccine were compared between those with and without previous infection (PI) for up to 48 weeks. Though sustained higher IgG-RBD were observed in the PI group after two doses of vaccines, both groups benefited from the booster shots of the third vaccine. This data supports the necessity of the booster shots to those with PI.

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.

RAISING is a high-performance method for identifying random transgene integration sites.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

BioFire FilmArray Pneumonia Panel enhances detection of pathogens and antimicrobial resistance in lower respiratory tract specimens.

BACKGROUND: This study investigated the diagnostic utility of the BioFire FilmArray Pneumonia Panel (PN panel), an automated and multiplexed nucleic acid detection system that rapidly detects 26 pathogens (18 bacteria and eight viruses) and seven antimicrobial resistance markers in a single assay. METHODS: We analyzed the targets in lower respiratory tract specimens using the PN panel and compared the detection results with those of bacterial culture methods and antimicrobial susceptibility testing. RESULTS: Of the 57 samples analyzed, the PN panel detected 97 targets (84 bacteria, four viruses, and nine antimicrobial resistance markers). Detection of bacteria and antimicrobial resistance was three times greater than that of the bacterial culture (25 bacteria and two resistant isolates) against the targets available in the panel. The overall positive and negative percent agreements between the PN panel and culture methods for bacterial detection were 100.0% and 92.9%, respectively. Multiple pathogens were detected by the PN panel in 24 samples (42.1%), ranging from two pathogens in 11 samples (19.3%) to six pathogens in one sample (1.8%). The PN panel semiquantitatively detected higher copies (≥ 106 copies/mL) of bacterial targets if the bacteria were positive by the culture method. In contrast, the semiquantitative values obtained by the panel varied (104 to 107 ≤ copies/mL) among bacteria that were negative by the culture method. CONCLUSIONS: The PN panel enhanced the detection of pathogens and antimicrobial resistance markers in lower respiratory tract specimens.

Acute type adult T-cell leukemia cells proliferate in the lymph nodes rather than in peripheral blood.

A massive increase in the number of mature CD4+ T-cells in peripheral blood (PB) is a defining characteristic of acute type of adult T-cell leukemia (ATL). To date, the site of proliferation of ATL cells in the body has been unclear. In an attempt to address this question, we examined the expression of the proliferation marker, Ki-67, in freshly isolated ATL cells from PB and lymph nodes (LNs) of patients with various types of ATL. Our findings reveal that LN-ATL cells display higher expression of the Ki-67 antigen than PB-ATL cells in acute type patients. The gene expression of T-cell quiescence regulators such as Krüppel-like factor 2/6 and forkhead box protein 1 was substantially high in acute type PB-ATL cells. The expression of human telomerase reverse transcriptase, which is involved in T-cell expansion, was significantly low in PB-ATL cells from acute type patients, similar to that in normal resting T-cells. These findings suggest that ATL cells proliferate in the LNs rather than in PB.

Corrigendum: The Contribution of Serum Complement Component 3 Levels to 90-Day Mortality in Living Donor Liver Transplantation.

[This corrects the article DOI: 10.3389/fimmu.2021.652677.].

TAS-116 (pimitespib), a heat shock protein 90 inhibitor, shows efficacy in preclinical models of adult T-cell leukemia.

Adult T-cell leukemia/lymphoma (ATL) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by human T-cell leukemia virus type 1 infection, for which there is an urgent need for more effective therapeutic options. The molecular chaperone heat shock protein 90 (HSP90) plays a crucial role in nuclear factor-κB (NF-κB)-mediated antiapoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti-ATL effects of a novel oral HSP90 inhibitor, TAS-116 (pimitespib), and the mechanisms involved in ex vivo and in vivo preclinical models. TAS-116 achieved IC50 values of less than 0.5 µmol/L in 10 ATL-related cell lines and less than 1 µmol/L in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Given orally, TAS-116 also showed significant inhibitory effects against tumor cell growth in ATL cell-xenografted mice. Furthermore, gene expression profiling of TAS-116-treated Tax-positive or -negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF-κB pathway in Tax-positive cells and cell-cycle arrest in Tax-negative cells and primary ATL cells. TAS-116 suppressed the activator protein-1 and tumor necrosis factor pathways in all examined cells. These findings strongly indicate the efficacy of TAS-116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti-ATL therapeutic agent.

Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.

Rapid detection and surveillance of cfiA-positive Bacteroides fragilis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

OBJECTIVES: To perform surveillance of cfiA-positive Bacteroides fragilis using new subtyping software module, MALDI Biotyper Subtyping Module (MBT Subtyping Module), on MALDI-TOF MS system, and to evaluate the detection ability of the module. METHODS: cfiA-positive strains were presumed using the module against B. fragilis isolated between 2006 and 2019. The cfiA gene was confirmed using PCR. In cfiA-positive B. fragilis, the insertion sequence (IS) elements were examined and the MBT STAR-BL assay was performed to examine meropenem hydrolysis activity. RESULTS: Of the 396 B. fragilis strains included, the MBT Subtyping Module detected 33 presumptive cfiA-positive strains (8.3%), of which 32 harbored the cfiA gene. The sensitivity and specificity of the MBT Subtyping Module for detecting cfiA-positive B. fragilis were 100.0% and 99.7%, respectively. Of the 32 strains harboring the cfiA gene, seven strains possessed IS elements, which were thought to induce high cfiA expression. Meropenem hydrolysis was detected in all seven strains that were positive for both cfiA and IS elements, and they exhibited resistance to meropenem and imipenem. The overall non-susceptibility rates to meropenem and imipenem were 84.8% and 36.4%, respectively, in the 33 presumptive cfiA-positive strains. CONCLUSION: The MBT Subtyping Module can detect cfiA-positive B. fragilis rapidly and accurately, supporting its use for surveillance of cfiA-positive B. fragilis in clinical settings.

Performance of anti-SARS-CoV-2 antibody testing in asymptomatic or mild COVID-19 patients: A retrospective study in outbreak on a cruise ship.

OBJECTIVES: A few studies on antibody testing have focused on asymptomatic or mild coronavirus disease 2019 (COVID-19) patients with low initial anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses. Anti-SARS-CoV-2 antibody-testing performance was evaluated using blood samples from asymptomatic or mild COVID-19 patients. METHODS: Blood samples were collected from 143 COVID-19 patients during an outbreak on a cruise ship 3 weeks after diagnosis. Simultaneously, a follow-up SARS-CoV-2 genetic test was performed. Samples stored before the COVID-19 pandemic were also used to evaluate the lateral flow immunochromatographic assay (LFA) and electrochemiluminescence immunoassay (ECLIA). Titers of anti-SARS-CoV-2 IgM and IgG antibodies against the nucleocapsid and spike proteins were measured using the enzyme-linked immunosorbent assay to confirm which antibodies were influenced on LFA- and ECLIA- false-negative result in crew-member samples. RESULTS: Sensitivity, specificity, positive-predictive, and negative-predictive values of LFA-detected IgM antibodies were 0.231, 1.000, 1.000, and 0.613, respectively; those of LFA-detected IgG antibodies were 0.483, 0.989, 0.972, and 0.601, respectively; and those of ECLIA-detected total antibodies were 0.783, 1.000, 1.000, and 0.848, respectively. All antibody titers measured using ELISA were significantly lower in blood samples with negative results than in those with positive results in both LFA and ECLIA. In the patients with negative results from the follow-up genetic testing, IgM-, IgG-, and total-antibody positivity rates were 22.9%, 47.6%, and 72.4%, respectively. CONCLUSIONS: These findings suggest that anti-SARS-CoV-2 antibody testing has lower performance in asymptomatic or mild COVID-19 patients than required in the guidelines.