An association between a positive direct antiglobulin test and HLA-DR12 in COVID-19.

SARS-CoV-2 infection has been reported to be associated with a positive direct antiglobulin test (DAT). In this study, an analysis of 40 consecutive coronavirus disease 2019 (COVID-19) cases from December 2020 to September 2021 in Japan revealed that patients of 70 years and over were predisposed to a positive DAT. DAT positivity was related to a decrease in the hemoglobin level. Anemia in DAT-positive COVID-19 patients was attributed to hemolysis, which was corroborated by high reticulocyte counts and an increase in the red blood cell distribution width. Human leukocyte antigen (HLA)-DRB1*12:01 and DRB1*12:02 were exclusively found in DAT-positive COVID-19 patients. In silico assays for the Spike protein of SARS-CoV-2 predicted several common core peptides that met the criteria for a B cell epitope and strong binding to both HLA-DRB1*12:01 and DRB1*12:02. Among these peptides, the amino acids sequence TSNFR, which is found within the S1 subunit of SARS-CoV-2 Spike protein, is shared by human blood group antigen Rhesus (Rh) CE polypeptides. In vitro analysis showed that the expression of HLA-DR in CD4+ T cells and CD8+ T cells from a DAT-positive patient was increased after pulsation with TSNFR-sequence-containing peptides. In summary, positive DAT is related to enhanced anemia and to HLA-DR12 in the Japanese population. A peptide sequence within SARS-CoV-2 Spike protein may act as an epitope for IgG binding to RBCs in DAT-positive COVID-19 patients.

Synthesis of fluoro-functionalized diaryl-λ3-iodonium salts and their cytotoxicity against human lymphoma U937 cells.

Conscious of the potential bioactivity of fluorine, an investigation was conducted using various fluorine-containing diaryliodonium salts in order to study and compare their biological activity against human lymphoma U937 cells. Most of the compounds tested are well-known reagents for fluoro-functionalized arylation reactions in synthetic organic chemistry, but their biological properties are not fully understood. Herein, after initially investigating 18 fluoro-functionalized reagents, we discovered that the ortho-fluoro-functionalized diaryliodonium salt reagents showed remarkable cytotoxicity in vitro. These results led us to synthesize more compounds, previously unknown sterically demanding diaryliodonium salts having a pentafluorosulfanyl (SF5) functional group at the ortho-position, that is, unsymmetrical ortho-SF5 phenylaryl-λ3-iodonium salts. Newly synthesized mesityl(2-(pentafluoro-λ6-sulfanyl)phenyl)iodonium exhibited the greatest potency in vitro against U937 cells. Evaluation of the cytotoxicity of selected phenylaryl-λ3-iodonium salts against AGLCL (a normal human B cell line) was also examined.

Agaritine derived from Agaricus blazei Murrill induces apoptosis via mitochondrial membrane depolarization in hematological tumor cell lines.

Objectives: Agaritine (AGT) is a hydrazine-containing compound derived from the mushroom Agaricus blazei Murill. We previously reported the antitumor effect of AGT on hematological tumor cell lines and suggested that AGT induces apoptosis in U937 cells via caspase activation. However, the antitumor mechanism of AGT has not been fully understood. Methods: Four hematological tumor cell lines (K562, HL60, THP-1, H929) were used in this study. The cells were incubated in the presence of 50 µM AGT for 24 h and analyzed for cell viability, annexin V positivity, caspase-3/7 activity, mitochondrial membrane depolarization, cell cycle, DNA fragmentation, and the expression of mitochondrial membrane-associated proteins (Bax and cytochrome c). Results: In HL60, K562, and H929 cells, AGT reduced cell viability and increased annexin V- and dead cell-positive rates; however, it did not affect THP-1 cells. In K562 and HL60 cells, caspase-3/7 activity, mitochondrial membrane depolarization, and expression of mitochondrial membrane proteins, Bax and cytochrome c, were all increased by AGT. Cell cycle analysis showed that only K562 exhibited an increase in the proportion of cells in G2/M phase after the addition of AGT. DNA fragmentation was also observed after the addition of AGT. Conclusions: These results indicate that AGT induces apoptosis in K562 and HL60 cells, like U937 reported previously, but showed no effect on THP-1 cells. It was suggested that AGT-induced apoptosis involves the expression of Bax and cytochrome c via mitochondrial membrane depolarization.

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937.

BACKGROUND: Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine. METHODS: Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells. RESULTS: Nuclear damage, DNA fragmentation, was observed by Wright-Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10µg/mL of agaritine for 48h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment. CONCLUSIONS: These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria. GENERAL SIGNIFICANCE: This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.

Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans, Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and ß share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3ß were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and ß-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells.

BACKGROUND: Mushrooms of the genus Agaricus are a common folk remedy against carcinoma. The active ingredients, polysaccharides and protein-polysaccharide complexes containing beta-glucan, have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation. METHODS: The diffusible fraction of a hot-water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumor activity against leukemic cells in vitro. The structure of the anti-tumor substance was determined by NMR and MS analyses. RESULTS: We purified a tumorcidal substance from the diffusible fraction of ABM and identified it as agaritine, beta-N-(gamma-l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267 Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562 with IC(50) values of 2.7, 9.4, 13.0, and 16.0 microg/mL, respectively, but showed no significant effect on normal lymphatic cells at concentrations up to 40 microg/mL. Although agaritine has been suspected of having genotoxic or carcinogenic properties, agaritine did not activate the umu gene of Salmonella, which reacts to carcinogens. GENERAL SIGNIFICANCE: The results indicate that agaritine from ABM has direct anti-tumor activity against leukemic tumor cells in vitro. This is in contrast to the carcinogenic activity previously ascribed to this compound. Our results also show that this activity is distinct from that of beta-glucan, which indirectly suppresses proliferation of tumor cells.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a concentration-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Reliability of serological tests for COVID-19: comparison of three immunochromatography test kits for SARS-CoV-2 antibodies.

BACKGROUND: Several immunochromatographic serological test kits have been developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, but their relative performance and potential clinical utility is unclear. METHODS: Three commercially available serological test kits were evaluated using 99 serum samples collected from 29 patients diagnosed with coronavirus disease 2019 (COVID-19) and 100 serum samples collected from 100 healthy volunteers in 2017 as negative controls. RESULTS: The specificity of the IgM and IgG antibodies showed comparable results among the three immunochromatographic serological test kits. The specificity for IgM antibody was 98.0%, 98.0%, and 97.0%, and the specificity for IgG antibody was identical among the three kits (99.0%). The IgM antibody-positive rates of the three test kits for samples taken at the early stage of the disease (0-4 days after onset) were consistent with all three kits (18.2%); however, the IgM antibody-positive rates thereafter showed considerable differences among the kits, making it difficult to interpret the kinetics of IgM response against SARS-CoV-2. The IgG antibody-positive rates for samples taken after 13 days of onset were 100.0%, 97.6%, and 97.6%, respectively. CONCLUSION: There were large differences among the results of the three test kits. Only few cases showed positive results for IgM, suggesting that at least 2 of these kits used in this study were unsuitable for diagnosis of COVID-19. The IgG antibody was positive in almost all samples after 13 days of onset, suggesting that it may be useful for determining infections in the recent past.

Biological evaluation of both enantiomers of fluoro-thalidomide using human myeloma cell line H929 and others.

Over the last few years, thalidomide has become one of the most important anti-tumour drugs for the treatment of relapsed-refractory multiple myeloma. However, besides its undesirable teratogenic side effect, its configurational instability critically limits any further therapeutic improvements of this drug. In 1999, we developed fluoro-thalidomide which is a bioisostere of thalidomide, but, in sharp contrast to the latter, it is configurationally stable and readily available in both enantiomeric forms. The biological activity of fluoro-thalidomide however, still remains virtually unstudied, with the exception that fluoro-thalidomide is not teratogenic. Herein, we report the first biological evaluation of fluoro-thalidomide in racemic and in both (R)- and (S)-enantiomerically pure forms against (in vitro) H929 cells of multiple myeloma (MM) using an annexin V assay. We demonstrate that all fluoro-thalidomides inhibited the growth of H929 MM cells without any in-vivo activation. Furthermore, we report that the enantiomeric forms of fluoro-thalidomide display different anti-tumour activities, with the (S)-enantiomer being noticeably more potent. The angiogenesis of fluoro-thalidomides is also investigated and compared to thalidomide. The data obtained in this study paves the way towards novel pharmaceutical research on fluoro-thalidomides.

Synthesis of chiral (tetrazolyl)methyl-containing acrylates via silicon-induced organocatalytic kinetic resolution of Morita-Baylis-Hillman fluorides.

Reported herein is a new approach for the asymmetric installation of a (tetrazolyl)methyl group via Si/F activation using organocatalytic kinetic resolution of racemic MBH-fluorides.

A synergistic increase of apoptosis utilizing Fas antigen expression induced by low doses of anticancer drug.

Anticancer drugs have been known to enhance both Fas receptor and Fas ligand expression on tumor cells. Recently, low doses of cytosine arabinoside (ara-C) were reported to enhance Fas antigen expression in the human myeloid leukemia cell line HL60. Here, we showed that low doses of ara-C (LD-ara-C) and etoposide (LD-VP-16) but not vincristine (LD-VCR) induce Fas expression in the human monocytic leukemia cell line U937. We determined the concentrations of ara-C, VP-16 and VCR as 10, 100 and 1 ng/ml, respectively. The ratios for Fas antigen expression induced in non-treated U937 by 24 h incubations with ara-C, VP-16 or VCR were 1.90, 1.36 and 1.00, respectively. Utilizing the Fas antigen expression induced by low doses of anticancer drugs, we examined whether anti-Fas IgM monoclonal antibody (CH-11) combined with LD-ara-C, LD-VP-16 or LD-VCR enhances apoptosis. When CH-11 and LD-anticancer drug were added simultaneously, the ratios of annexin V positive cells were 67.8 +/- 2.4% with ara-C, 70.0 +/- 1.6% with VP-16 and 54.2 +/- 1.3% with VCR. Thus, the ratios of annexin V positive cells significantly increased when CH-11 was simultaneously added to the cells with ara-C (p < 0.0001) and VP-16 (p < 0.0001), but not with VCP (p = 0.5559), compared with the sums of annexin V positive ratios of CH-11 and LD-anticancer drug added separately. We examined whether a broad-range caspase inhibitor (C.I.) can inhibit the Fas expression enhanced by LD-anticancer drugs. However, the Fas expression enhanced by LD-ara-C or LD-VP-16 was not inhibited by a broad-range caspase inhibitor. We demonstrated that apoptosis induced by LD-ara-C or LD-VP-16 is synergistically increased by the addition of CH-11 in U937.

Bis(pentafluorosulfanyl)phenyl azide as an expeditious tool for click chemistry toward antitumor pharmaceuticals.

The inclusion of fluorine in pharmaceutical agents is a well- established means of improving their druglike properties. Different substituents have been used to introduce fluorine, including trifluoromethyl and trifluoromethylthio groups; however, the pentafluorosulfanyl remains relatively underutilized although it is considered to be a "super" trifluoromethyl group. Here, a series of pentafluorosulfanyl-containing 1,4-disubstituted-1,2,3-triazoles were synthesized by click reaction from alkynes and 3,5-bis(pentafluorosulfanyl)phenyl azide in excellent yields. Their biological activities were evaluated against human leukemic monocyte lymphoma U937 cells. In particular, 1-(3,5-bis(pentafluorosulfanyl)phenyl)-4-(4-fluorophenyl)-1H-1,2,3-triazole exhibited potent efficacy in cell viability assays at a concentration of 60 µM and was shown to activate caspase-3 activity, indicating induction of apoptosis. An analogous fluorenol-substituted triazole also exhibited promising cytotoxic effects against U937 cells, with an IC50 value of 6.29 µM. Given these preliminary results, these pentafluorosulfanyl-containing triazoles represent useful building blocks for the further development of novel antitumor agents.

Heat shock protein 60: specific binding of lipopolysaccharide.

Human heat shock protein 60 (HSP60) has been shown to bind to the surface of innate immune cells and to elicit a proinflammatory response. In this study we demonstrate that the macrophage stimulatory property of recombinant human HSP60 is tightly linked to the HSP60 molecule and is lost after protease treatment. However, inhibition of macrophage stimulation was reached by the LPS-binding peptide magainin II amide. Indeed, HSP60 specifically bound [(3)H]LPS. [(3)H]LPS binding to HSP60 was saturable and competable by the unlabeled ligand. To identify the epitope region of the HSP60 molecule responsible for specific LPS binding, we analyzed the effect of several anti-HSP60 mAbs on HSP60-induced production of inflammatory mediators from macrophages. We identified only one mAb, clone 4B9/89, which blocked the macrophage stimulatory activity of the chaperone. The epitope specificity of this mAb points to the region aa 335-366 of HSP60. Clone 4B9/89 also strongly inhibited [(3)H]LPS binding to HSP60. A more detailed analysis was performed by screening with selected overlapping 20-mer peptides of the HSP60 sequence, covering the region aa 331-380. Only one peptide blocked LPS binding to HSP60, thereby restricting the potential LPS-binding region to aa 351-370 of HSP60. Finally, analysis of selected 15-mer peptides and a 13-mer peptide of the HSP60 sequence revealed that most of the LPS-binding region was accounted for by aa 354-365 of HSP60, with the motif LKGK being critical for binding. Our studies identified a defined region of HSP60 involved in LPS binding, thereby implicating a physiological role of human HSP60 as LPS-binding protein.

Heat shock protein 60 elicits abnormal response in macrophages of diabetes-prone non-obese diabetic mice.

Heat shock protein 60 (hsp60) is a target antigen in autoimmune diabetes and injections of human hsp60 for tolerance induction were found to protect non-obese diabetic (NOD) mice, an animal model of human type 1 diabetes, from disease development. We tested whether innate immune cells of NOD mice exhibit an abnormal response to extracellular hsp60. Bone marrow derived macrophages (BMM) were grown from NOD, C57BL/6J, non-obese non-diabetic (NON) mice, and NOD-related congenic variants differing in the Idd-3, Idd-10/18, or major histocompatibility complex (MHC) region. Hsp60-stimulated BMM of NOD mice were found to produce high levels of interleukin (IL)-12(p70). The addition of IL-10 downregulated, whereas cyclooxygenase inhibitors elevated, IL-12(p70) production of activated BMM. BMM of NON, NON-NOD-H-2(g7) as well as of NOD-NON-H-2(nbl) mice produced significantly less IL-12(p70) than BMM of NOD mice, indicating that an interaction between the MHC haplotype and non-MHC genes of the NOD mouse is required for hyperresponsiveness to hsp60.