Diverse effects of clarithromycin and proposal of its clinical application for treating COVID-19 as a repurposing drug.

Outbreak of SARS-CoV-2 has been declared a pandemic, which is a serious threat to human health. The disease was named coronavirus disease 2019 (COVID-19). Until now, several vaccines and a few drugs have been approved for the prevention and treatment for COVID-19. Recently, the effect of some macrolides including clarithromycin (CAM) on COVID-19 has attracted attention. CAM is known to have diverse effects including immunomodulatory and immunosuppressive effects, autophagy inhibition, steroid sparing effect, reversibility of drug resistance, antineoplastic effect, antiviral effect as well as bacteriostatic/bactericidal effect. Many patients with COVID-19 died due to an overwhelming response of their own immune system characterized by the uncontrolled release of circulating inflammatory cytokines (cytokine release syndrome [CRS]). This CRS plays a major role in progressing pneumonia to acute respiratory distress syndrome (ARDS) in COVID-19 patients. It is noteworthy that CAM can suppress inflammatory cytokines responsible for CRS and also has anti-SARS-CoV-2 effect. Considering the rapidly progressive global disease burden of COVID 19, the application of CAM for treating COVID-19 needs to be urgently evaluated. Recently, an open-labeled non-randomized trial using CAM for treating COVID-19 (ACHIEVE) was initiated in Greece in May, 2020. Its results, though preprint, indicated that CAM treatment of patients with moderate COVID-19 was associated with early clinical improvement and containment of viral load. Thus, treatment with CAM as a single agent or combined with other anti-SARS CoV-2 drugs should be tried for treating COVID-19. In this article, we discussed the significance and usefulness of CAM in treating COVID-19.

Mitotic catastrophe and cell death induced by depletion of centrosomal proteins.

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Growth suppression and mitotic defect induced by JNJ-7706621, an inhibitor of cyclin-dependent kinases and aurora kinases.

Aurora kinases and cyclin-dependent kinases, which play critical roles in the cell cycle and are frequently overexpressed in a variety of tumors, have been suggested as attractive targets for cancer therapy. JNJ-7706621, a recently identified dual inhibitor of these kinases, is reported to induce cell cycle arrest, endoreduplication, and apoptosis. In the present study, we further investigated the molecular mechanisms underlying these effects. The inhibitor arrested various cells at G2 phase at low concentration, and at both G1 and G2 phases at high concentration. JNJ-7706621 did not prevent localization of Aurora A to the spindle poles, but did inhibit other centrosomal proteins such as TOG, Nek2, and TACC3 in early mitotic phase. Similarly, the drug did not prevent localization of Aurora B to the kinetochore, but did inhibit other chromosomal passenger proteins such as Survivin and INCENP. In the cells exposed to JNJ-7706621 after nocodazole release, Aurora B, INCENP, and Survivin became relocated to the peripheral region of chromosomes, but Plk1 and Prc1 were localized on microtubules in later mitotic phase. Treatment of nocodazole-synchronized cells with JNJ-7706621 was able to override mitotic arrest by preventing spindle checkpoint signaling, resulting in failure of chromosome alignment and segregation. Injection of the drug significantly inhibited the growth of TC135 Ewing's sarcoma cells transplanted into athymic mice by cell cycle arrest and apoptosis. JNJ-7706621 is a unique inhibitor regulating cell cycle progression at multiple points, suggesting that it could be useful for cell cycle analysis and therapy of various cancers, including Ewing's sarcoma.

Tumor-infiltrating macrophages induce apoptosis in activated CD8(+) T cells by a mechanism requiring cell contact and mediated by both the cell-associated form of TNF and nitric oxide.

We have investigated the ability of different cells present in murine tumors to induce apoptosis of activated CD8(+) T cells in vitro. Tumor cells do not induce apoptosis of T cells; however, macrophages that infiltrate tumors are potent inducers of apoptosis. Tumor macrophages express cell surface-associated TNF, TNF type I (CD120a) and II (CD120b) receptors, and, upon contact with T cells which induces release of IFN-gamma from T cells, secrete nitric oxide. Killing of T cells in vitro is blocked by Abs to IFN-gamma, TNF, CD120a, or CD120b, or N-methyl-L-arginine. In concert with that finding, tumor macrophages isolated from either TNF type I or type II receptor -/- mice are not proapoptotic and do not produce nitric oxide upon contact with activated T cells. Control macrophages do not express TNF receptors or release nitric oxide. Tumor cells or tumor-derived macrophages do not express FasL, and blocking Abs to either Fas or FasL have no effect on macrophage-mediated T cell killing. These results demonstrate that macrophages which infiltrate tumors are highly proapoptotic and may be responsible for elimination of activated antitumor T cells within the tumor bed.

CD8(+) tumor-infiltrating T cells are deficient in perforin-mediated cytolytic activity due to defective microtubule-organizing center mobilization and lytic granule exocytosis.

Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8(+) TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-gamma is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction.

CD8+ tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ.

Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN-gamma mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-gamma. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-gamma, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G(1), do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death.

Nuclear factor-kappaB activates dual inhibition sites in the regulation of tumor necrosis factor-alpha-induced neutrophil apoptosis.

The objective of this study was to elucidate the role of the nuclear factor-kappaB (NF-kappaB) pathway in tumor necrosis factor-alpha (TNF-alpha)-induced neutrophil apoptosis. A single treatment with TNF-alpha produced significant caspase-3 activation in a time- and concentration-dependent manner, while no significant morphological change in neutrophils was observed. After pretreatment of neutrophils with cycloheximide or actinomycin D, TNF-alpha produced morphologically typical apoptosis in a time- and concentration-dependent manner. Similarly, following pretreatment of neutrophils with the specific NF-kappaB inhibitors, pyrrolidine dithiocarbamate or SN50, TNF-alpha also produced neutrophil apoptosis (assessed morphologically). Caspase-3 activation by TNF-alpha was significantly enhanced by pretreatment with both cycloheximide and pyrrolidine dithiocarbamate. TNF-alpha-induced a rapid phosphorylation and degradation of IkappaB-alpha in neutrophils. Furthermore, TNF-alpha increased NF-kappaB DNA binding, which was abolished by pretreatment with pyrrolidine dithiocarbamate. These results indicate that the NF-kappaB pathway is crucial for neutrophil survival against TNF-alpha cell toxicity. Furthermore, it is proposed that NF-kappaB-induced proteins act on dual inhibitory sites, both upstream and downstream of caspase-3, to protect against apoptosis.

Diagnostic utility of CSF soluble CD27 for primary central nervous system lymphoma in immunocompetent patients.

The aim of this study is to determine the diagnostic utility of cerebrospinal fluid (CSF) soluble CD27 (sCD27) as a tumor marker for primary central nervous system lymphoma (PCNSL) in immunocompetent patients. A total of 93 CSF samples were collected from the following four patient groups: the PCNSL group, 13 patients (26 samples) with PCNSL, 12 samples obtained at initial diagnosis, 10 during therapy, four at complete remission; the other brain tumors (OBT) group, 30 patients (30 samples) with other brain tumors; the other neurological diseases (OND) group, 25 (25 samples) with other neurological diseases; the inflammatory neurological diseases (IND) group, 12 patients (12 samples) with inflammatory neurological diseases. sCD27 levels were determined by sandwich enzyme-linked immunosorbent assay. The optimal cut-off value was found to be 15 U ml-1. The CSF sCD27 levels were over 15 U ml-1 in 23 of the 26 PCNSL samples and were significantly higher than those in the OBT and OND groups in which all samples were below 15 U ml-1. Elevated CSF sCD27 levels were also observed in 11 of 12 IND samples. In the two PCNSL patients whose CSF sCD27 levels were studied longitudinally, the sCD27 levels correlated very well with remission and relapse of the disease. CSF sCD27 is useful as a tumor marker for PCNSL in immunocompetent patients, and is also useful to evaluate the effect of various types of treatment. Although there was a large cross-reactivity in the CSF sCD27 levels between PCNSL and IND group, white blood cell count in the CSF is helpful to distinguish these two diseases.

Role of rat adrenal antioxidant defense systems in the aldosterone turn-off phenomenon.

The mechanism(s) of the "aldosterone turn-off phenomenon", hypoaldosteronemia following chronic ACTH administration, remains unclear. Our previous observation that antioxidants prevented turn-off prompted us to evaluate the chronic effect of ACTH on the enzymatic antioxidant system as well as P450aldo activity and expression of CYP11B2 in adrenal zona glomerulosa. Male Wistar rats were administered ACTH-Z for 5 days with or without antioxidants, vitamin E or DMSO. Adrenal capsules were prepared for P450aldo activity measurement and mRNA content determination by competitive RT-PCR, and immunoreactivity of Mn-SOD in whole adrenals was evaluated. ACTH decreased the P450aldo activity and mRNA level of CYP11B2 in adrenal capsules, while co-administration of vitamin E or DMSO partially blocked this inhibition. ACTH increased Mn-SOD mRNA and immunoreactivity but decreased GPx mRNA. These results suggest that prolonged ACTH treatment increases oxidative stress in the zona glomerulosa and an imbalance in the ratio of Mn-SOD to GPx, possibly via corticosterone overproduction in the zona fasciculata, resulting in the downregulation of CYP11B2. Vitamin E and DMSO might thus protect CYP11B2 expression through their antioxidant actions.

Epidural Bilharzioma mansoni compressing the spinal cord: case report.

A case of an epidural Bilharzioma mansoni (epidural granuloma due to Schistosoma mansoni) compressing the spinal cord at T11-T12 is presented. The patient, a 20-year old African man, started complaining of recurrent back pain since 1993 and became paraparetic in 1996. The myelography showed a complete block at T12 and the CT scan showed a mass at T11-T12 compressing the spinal cord. Through a bilateral laminectomy of T 10, T11 and T12, the bilharzioma was completely removed. The histopathology and the laboratory tests confirmed the diagnosis of granuloma due to Schistosoma mansoni. The patient recovered completely and was seen last time more than one year after surgery. Not a similar case has been found in the literature and the authors presume that this is the first case ever successfully treated by surgery and chemotherapy and reported in the world literature.

Evidence for the synthesis of corticosteroid-binding globulin in human placenta.

We demonstrated the expression of corticosteroid-binding globulin (CBG) in human placenta using reverse transcription-polymerase chain reaction-Southern blot analysis and immunohistochemical and immunoblotting studies. In the RT-PCR-Southern blot analysis, only one predicted PCR product was detected without nonspecific products in all samples of human placenta and 3A (tPA-30-1) human placental cells. In Western blot analysis, polyclonal anti-CBG antibodies recognized a protein of approximately 55 kD in the protein extracts prepared from 3A (tPA-30-1) cells. Additionally, CBG mRNA expression was demonstrated by in situ hybridization in the syncytiotrophoblasts. Immunohistochemical studies performed on the placenta demonstrated the presence of specific immunoreactivity in the syncytiotrophoblast layer surrounding the chorionic villi. These findings suggest that CBG is synthesized in human placenta during pregnancy in addition to its synthesis in the liver.

Increased levels of CSF soluble CD27 in patients with primary central nervous system lymphoma.

The aim of this study is to determine the diagnostic value of cerebrospinal fluid (CSF) soluble CD27 (sCD27) as a tumor marker for primary central nervous system lymphoma (PCNSL). We examined sCD27 levels in CSF obtained from various types of brain tumor patients. Forty-two patients were studied (including 12 PCNSL patients) who had not received any therapy for their tumors. In all PCNSL cases, CSF sCD27 levels were more than 15 U/ml (median 84.5 U/ml, range 17-484 U/ml) and in other brain tumor cases, CSF sCD27 levels were all less than 15 U/ml. Our data suggest that CSF sCD27 levels are useful to distinguish PCNSL from other brain tumors.

Induction of M-CSF receptor and its mRNA, and activation of tyrosine kinase in peripheral monocytes by oestradiol-17 beta and progesterone.

In monocytes at the secretory (oestrogen-progesterone dominant) phase of the menstrual cycle, expression of c-fms and macrophage colony-stimulating factor (M-CSF) receptor and activity of tyrosine kinase (TK) were increased by oestradiol with or without progesterone. In vivo, oestrogen may induce expression of c-fms and M-CSF receptor as well as the activation of TK in monocytes under the milieu of the secretory phase. Alternatively, cells of monocyte lineage during the secretory phase might, via various factors, obtain the potency to induce the expression and the function of M-CSF receptors, this potency being effected by oestrogen. Macrophages in peritoneal fluid in pelvic endometriosis (oestrogen predominant) might be activated during the secretory phase of the menstrual cycle, causing infertility.

Growth characteristics of murine B16 melanoma multicellular spheroids: a model for invasion and effects of doxorubicin treatments.

Multicellular spheroids, derived from murine B16 melanoma cells, showed unique growth characteristics: when they reached about 500 microns in diameter, their morphology changed rapidly and they became amoeba-like irregular-shaped aggregates. This morphological characteristic closely resembled that of invasive cancer, and may serve as a model for local invasion. To test the possibility that the changes mentioned above can be inhibited by a drug, spheroids were treated with 0.8 microgram/ml of doxorubicin for one hour and their morphology was observed temporally. Although this concentration of the drug decreased the survival of the melanoma cells in monolayer to about 10(-3), the growth was not delayed nor were the "invasion"-like changes inhibited in the spheroids. We believe this system of multicellular spheroids is a useful model to study the mechanisms of tumour invasion, although doxorubicin could not inhibit "invasion"-like changes in this system.

Formation and growth of multicellular spheroids in media containing low concentrations of agarose.

Human melanoma HMV-1 cells formed efficiently multicellular spheroids in media containing low concentrations (0.001-0.01%) of agarose (LCAM). In addition, the spheroids were prevented from deformation without apparent delay of the growth rate. On the other hand, spheroid formation was inhibited markedly by LCAM in the case of murine melanoma B16 cells. Also, LCAM could not prevent the B16 spheroids from deformation. These results indicate that LCAM acts differently depending on the cell types used for spheroid formation.