IL-6 mediates olfactory dysfunction in a mouse model of allergic rhinitis.

BACKGROUND: Immune-inflammatory response is a key element in the occurrence and development of olfactory dysfunction (OD) in patients with allergic rhinitis (AR). As one of the core factors in immune-inflammatory responses, interleukin (IL)-6 is closely related to the pathogenesis of allergic diseases. It may also play an important role in OD induced by diseases, such as Sjögren's syndrome and coronavirus disease 2019. However, there is no study has reported its role in OD in AR. Thus, this study aimed to investigate the role of IL-6 in AR-related OD, in an attempt to discover a new target for the prevention and treatment of OD in patients with AR. METHODS: Differential expression analysis was performed using the public datasets GSE52804 and GSE140454 for AR, and differentially expressed genes (DEGs) were obtained by obtaining the intersection points between these two datasets. IL-6, a common differential factor, was obtained by intersecting the DEGs with the General Olfactory Sensitivity Database (GOSdb) again. A model of AR mice with OD was developed by sensitizing with ovalbumin (OVA) to verify the reliability of IL-6 as a key factor of OD in AR and explore the potential mechanisms. Furthermore, a supernatant and microglia co-culture model of nasal mucosa epithelial cells stimulated by the allergen house dust mite extract Derp1 was established to identify the cellular and molecular mechanisms of IL-6-mediated OD in AR. RESULTS: The level of IL-6 in the nasal mucosa and olfactory bulb of AR mice with OD significantly increased and showed a positive correlation with the expression of olfactory bulb microglia marker Iba-1 and the severity of OD. In-vitro experiments showed that the level of IL-6 significantly increased in the supernatant after the nasal mucosa epithelial cells were stimulated by Derp1, along with significantly decreased barrier function of the nasal mucosa. The expression levels of neuroinflammatory markers IL-1ß and INOS increased after a conditioned culture of microglia with the supernatant including IL-6. Then knockdown (KD) of IL-6R by small interfering RNA (siRNA), the expression of IL-1ß and INOS significantly diminished. CONCLUSION: IL-6 plays a key role in the occurrence and development of OD in AR, which may be related to its effect on olfactory bulb microglia-mediated neuroinflammation.

P2X7 receptor of microglia in olfactory bulb mediates the pathogenesis of olfactory dysfunction in a mouse model of allergic rhinitis.

The pathogenesis of allergic rhinitis (AR)-related olfactory dysfunction (OD) remains unknown. Inhibiting microglial response in olfactory bulb (OB) can ameliorate AR-related OD, but no precise targets have been available. In this study, we established a mouse model of ovalbumin (OVA)-induced AR and combined with the application of P2X7 receptor (P2X7R)-specific antagonists and cell culture in conditioned medium to investigate the role and mechanism of OB microglial P2X7R in AR-related OD. Serum IgE and IL-5 levels determined via ELISA and federated the number of nose-scratching to affirm the success of OVA-induced AR mouse model. Buried food pellet test was used to evaluate the olfactory function of mice. The changes of IBA1, GFAP, P2X7R, IL-1ß, IL-1Ra, and CASPASE 1 were detected by quantitative polymerase chain reaction and western blotting. The levels of adenosine triphosphate (ATP) were determined by the commercialized kit. The morphological changes of microglia were assessed using immunofluorescence staining and Sholl analysis. Findings showed that AR-related OD was associated with OB microglia-mediated imbalance between IL-1ß and IL-1Ra. Treatment with BBG improved the olfactory function in AR mice with restoring the balance between IL-1ß and IL-1Ra. In vitro, the conditioned medium obtained after HNEpC treatment with Der p1 could activate HMC3 to arise inflammatory reaction basing on "ATP-P2X7R-Caspase 1" axis, while inhibition of its P2X7R suppressed the reaction. In brief, microglial P2X7R in OB is a direct effector molecule in AR-related OD and inhibition of it may be a new strategy for the treatment of AR-related OD.

Autophagy-related Proteins in Genome Stability: Autophagy-Dependent and Independent Actions.

It is emerging that autophagy-related proteins regulate the adaptive response to DNA damage in maintaining genome stability at multiple pathways. Here, we discuss recent insights into how autophagy-related proteins participate in DNA damage repair processes, influence chromosomal instability, and regulate the cell cycle through autophagy-dependent and independent actions. There is increasing awareness of the importance of these pathways mediated by autophagy-related proteins to DNA damage response (DDR), and disturbances in these regulatory connections may be linked to genomic instability participated in various human diseases, such as cancer and aging.

Ablation of Liver X receptor ß in mice leads to overactive macrophages and death of spiral ganglion neurons.

Age-related hearing loss is the most common type of hearing impairment, and is typically characterized by the loss of spiral ganglion neurons (SGNs). The two Liver X receptors (LXRs) are oxysterol-activated nuclear receptors which in adults, regulate genes involved in cholesterol homeostasis and modulation of macrophage activity. LXRß plays a key role in maintenance of health of dopaminergic neurons in the substantia nigra, large motor neurons in the spinal cord, and retinal ganglion cells in adult mice. We now report that LXRß is expressed in the SGNs of the cochlea and that loss of LXRß leads to age-related cochlea degeneration. We found that in the cochlea of LXRß-/- mice, there is loss of SGNs, activation of macrophages, demyelination in the spiral ganglion, decrease in glutamine synthetase (GS) expression and increase in glutamate accumulation in the cochlea. Part of the cause of damage to the SGNs might be glutamate toxicity which is known to be very toxic to these cells. Our study provides a so far unreported role of LXRß in maintenance of SGNs whose loss is a very common cause of hearing impairment.

Silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in liver cell lines.

Alcoholic liver disease has become one of the main causes of liver injury, and its prevention and cure are important medical tasks. Silibinin, a natural flavonoid glycoside, is a conventional hepatic protectant. This study elucidates the modulation of ferroptosis in silibinin's protective effects on ethanol- or acetaldehyde-induced liver cell damage by using human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells. Our results show that ferroptosis is induced in the cells treated with ethanol or acetaldehyde, as evidenced by the increased ROS stress and iron level. Silibinin resolves the oxidative stress and reduces iron level. Ferroptosis induced by ethanol- or acetaldehyde involving nuclear receptor co-activator 4 (NCOA4)-dependent autophagic degradation of ferritin, a protein for storing iron is rescued by silibinin. PINK1 and Parkin-mediated mitophagy is arrested in ethanol- or acetaldehyde-treated cells but reversed by silibinin. Ferritin degradation and ROS level are further increased when PINK1 or Parkin is silenced in the cells treated with ethanol or acetaldehyde. Collectively, our study reveals that silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in two liver cell lines, HepG2 and HL7702 cells, providing new therapeutic strategies for alcoholic liver injury.

Protective effects of silibinin against ethanol- or acetaldehyde-caused damage in liver cell lines involve the repression of mitochondrial fission.

Silibinin is a natural polyphenolic flavonoid, isolated from the seeds of the milk thistle of Silybum marianum (L.) Gaertn. Silibinin has been widely used clinically as a traditional medicine for liver diseases. This study investigated the protective role of silibinin in ethanol- or acetaldehyde-induced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells, focusing on elucidation of the underlying mechanism in vitro. The toxicity of ethanol or acetaldehyde was evaluated by MTT assay. Apoptosis-related proteins, mitochondrial fission-associated proteins and mitochondrial fusion-associated proteins were analyzed by western blotting and immunofluorescence microscopy. Present experimental results demonstrated that silibinin improved cell viability, reduced the enzyme activities of AST/ALT and ALDH/ADH, inhibited apoptosis and recovered mitochondrial function in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. Silibinin reduced the expression of mitochondrial fission-associated proteins, dynamin-related protein 1 (DRP1), but increased mitochondrial fusion-associated proteins, optic atrophy 1 (OPA1) and mitofusin 1 (MFN1). Accordingly, inhibition of DRP1 activity with its pharmacological inhibitor or siDRP1 efficiently attenuated ethanol- or acetaldehyde-induced apoptosis, whereas activation of DRP1 by using staurosporine (STS) further increased apoptosis in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. The results show that silibinin protects cells against ethanol- or acetaldehyde-induced mitochondrial fission that results in apoptosis.

The deacetylation of Foxk2 by Sirt1 reduces chemosensitivity to cisplatin.

In multiple types of cancer, decreased tumour cell apoptosis during chemotherapy is indicative of decreased chemosensitivity. Forkhead box K2 (FOXK2), which is essential for cell fate, regulates cancer cell apoptosis through several post-translational modifications. However, FOXK2 acetylation has not been extensively studied. Here, we evaluated the effects of sirtiun 1 (SIRT1) on FOXK2 deacetylation. Our findings demonstrated that SIRT1 inhibition increased FOXK2-induced chemosensitivity to cisplatin and that K223 in FOXK2 was acetylated. Furthermore, FOXK2 K223 deacetylation reduced chemosensitivity to cisplatin in vitro and in vivo. Mechanistically, FOXK2 was acetylated by the acetyltransferase cAMP response element binding protein and deacetylated by SIRT1. Furthermore, cisplatin attenuated the interaction between FOXK2 and SIRT1. Cisplatin or SIRT1 inhibition enhanced FOXK2 acetylation, thereby reducing the nuclear distribution of FOXK2. Additionally, FOXK2 K223 acetylation significantly affected the expression of cell cycle-related and apoptosis-related genes in cisplatin-stimulated cancer cells, and FOXK2 K223 hyperacetylation promoted mitotic catastrophe, which enhanced chemosensitivity to cisplatin. Overall, our results provided insights into the mechanisms of SIRT1-mediated FOXK2 deacetylation, which was involved in chemosensitivity to cisplatin.

Terpenoids from stigma maydis (Zea mays L.) alleviate hydrogen peroxide-induced SH-SY5Y cell injury by activating Nrf2.

Five terpenoids (1-5), including three new ent-kaurane diterpenoids (1-3), one new ent-rosane type diterpenoid (4) and one known triterpenoid (5), were isolated from stigma maydis (Zea mays L.). The structures of the compounds were elucidated by comprehensive spectroscopic analyses. The relative configurations of stigmanes A-D (1-4) were determined by NOESY experiments. In addition, the relative configuration of stigmane D (4) was further established by NMR calculations. The absolute configurations of these compounds were identified by a comparison of experimental and calculated specific rotations. The neuroprotective effects of these compounds against H2O2-induced injury in human neuroblastoma SH-SY5Y cells were evaluated, and the results showed that among the compounds, 2 exhibited the most significant neuroprotection. Further study demonstrated that 2 could activate nuclear factor E2-related factor (Nrf2), downregulate apoptosis and reactive oxygen species (ROS) generation, and increase antioxidant enzyme activities in SH-SY5Y cells. However, the neuroprotective effect was reversed when Nrf2 was silenced. In conclusion, this study suggested that terpenoids from stigma maydis exerted neuroprotective effects through Nrf2 activation.

[Clinical Characteristics and Risk Factors of ESKAPE Pathogens Bloodstream Infection in Cancer Patients].

OBJECTIVE: To identify the risk factors of ESKAPE pathogens infection and related death in cancer patients, and to supply evidence for clinical precaution and diagnosis. METHODS: A retrospective study of clinical and experimental data of cancer patients with bloodstream infection were carried out in Sichuan Cancer Hospital from 2013 to 2018. The clinical feature, predisposing factors and risk factors of death in ESKAPE group and non-ESKAPE group were analyzed by univariate analysis and multivariate logistic regression. RESULTS: A total of 753 patients were enrolled in the study. Totally 795 pathogenic bacteria strains were isolated from blood culture and there were 278 ESKAPE strains, which took up 34.97% of isolated strains. Univariate analysis and multivariate logistic regression analysis showed that gender of male, multiple pathogens, history of exposure to enzyme inhibitors and agranulocytosis were independent risk factors of ESKAPE pathogens bloodstream infection. Peritoneal infection and combined fungal infection were independent risk factors of ESKAPE bloodstream infection related death. CONCLUSION: The bloodstream infection of ESKAPE pathogens is a problem worthy of clinical attention for cancer patients with neutrophil deficiency, previous antibiotic exposure, and fungal infection and peritoneal infection.

Neuroprotective terpenoids from the leaves of Viburnum odoratissimum.

As a part of our ongoing search for neuroprotective compounds from natural products, two new iridoid glycosides, vibsansuspenside A-B (1-2), along with five known terpenoids (3-7), were isolated from the dry leaves of Viburnum odoratissimum. Their chemical structures were well determined by means of NMR spectroscopic data as well as HRESIMS analysis. All compounds were detected for their neuroprotective effects against H2O2-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y). Among them, compound 3 displayed the most potent neuroprotective ability, and further investigation by Annexin V/PI and Western blot analysis demonstrated that compound 3 could protect SH-SY5Y cells from oxidative damage through inhibiting cell apoptosis.[Formula: see text].

Targeting ERß in Macrophage Reduces Crown-like Structures in Adipose Tissue by Inhibiting Osteopontin and HIF-1α.

Proinflammatory processes in adipose tissue contribute to development of breast cancer and insulin resistance. Crown-like structures (CLS) are histologic hallmarks of the proinflammatory process in adipose tissue. CLS are microscopic foci of dying adipocytes surrounded by macrophages mostly derived from monocytes in blood. Estrogen receptor ß (ERß) is expressed in microglia, macrophages within the central nervous system (CNS), where it evokes an anti-inflammatory response. The present study investigates the function of ERß in macrophages within CLS. We report that even though monocytes in the blood have no detectable levels of ERß, macrophages in CLS do express ERß. In ERß-/- mice, there was a significant increase in the number of CLS in both subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). CLS in these mice were dominated by pro-inflammatory macrophages (M1 macrophages) with higher expression of osteopontin (OPN) and an increase in number of proliferating macrophages. In mice made obese by Western diet, treatment with an ERß selective agonist (LY3201) reduced the number of CLS in both SAT and VAT with downregulation of OPN, activated hypoxia-inducible factor-1α (HIF-1α), proliferation and upregulation prolyl hydroxylase 2 (PHD2), the enzyme which prevents activation of HIF1α, in macrophages. We conclude that ERß expression is induced in macrophages in CLS within adipose tissue where it plays a pivotal role in suppression of CLS. Thus ERß agonists may be used to alleviate CLS-related breast cancer and insulin resistance in adipose tissue.

Eclalbasaponin I causes mitophagy to repress oxidative stress-induced apoptosis via activation of p38 and ERK in SH-SY5Y cells.

Oxidative stress accompanying excessive accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction leads to the occurrence of neurodegenerative diseases. Our previous study showed that Eclalbasaponin I (EcI), a triterpene saponin isolated from Aralia elata (Miq.) Seem. (A. elata), repressed oxidative stress in human neuroblastoma SH-SY5Y cells. However, the detailed mechanism remains unclear. In this study, pretreatment with EcI in SH-SY5Y cells significantly activated the p38-mitogenactivated protein kinase (p38), the extracellular regulated protein kinase (ERK), whereas it did not affect the c-jun NH2 terminal kinases (JNK). In accordance with the initial findings, EcI-induced neuroprotective effect was attenuated by SB203580 (SB, a p38 inhibitor) or FR180204 (FR, an ERK inhibitor), being further confirmed by specific small interfering RNA (siRNA). Inhibition of either p38 or ERK up-regulated the apoptosis induction in EcI- and H2O2-cotreated cells. Furthermore, p38 or ERK suppression enhanced intracellular and mitochondrial ROS generation, decreased the activities of endogenous antioxidant defences as well as the mitochondrial membrane potential (MMP), resulting in dysfunction of mitochondria. In addition, EcI-induced autophagy and mitophagy were obviously down-regulated when p38 or ERK activation was blocked. Cumulatively, these findings supported that EcI-caused mitophagy contributed to the neuroprotective effect through p38 or ERK activation. Mitophagy induction might be an effective therapeutic intervention in neurodegenerative diseases.

Timosaponin AIII, a steroidal saponin, exhibits anti-tumor effect on taxol-resistant cells in vitro and in vivo.

Timosaponin AIII (TAIII), a steroidal saponin isolated from the rhizome of Anemarrhena asphodeloides, exerted cytotoxic effect in many cancer cell lines. However, the effect of TAIII on resistant tumor cancer cells was unclear. In this study, MTT assay showed that TAIII exhibited significant cytotoxicity against A549/Taxol and A2780/Taxol cells in vitro. Annexin V-FITC/PI staining revealed that TAIII induced apoptosis in A549/T and A2780/T cells. Furthermore, Western blot analysis demonstrated that TAIII inhibited the expressions of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR) as well as Ras, Raf, mitogen-activated protein kinase (MEPK), extracellular regulated protein kinases (ERK) in two taxol-resistant cancer cell lines. Besides, in vivo studies demonstrated that TAIII inhibited tumor growth in a nude mouse xenograft model. Additionally, TAIII (2.5 and 5 mg/kg) also down-regulated the protein expressions of PI3K/AKT/mTOR and Ras/Raf/MEK/ERK pathways in vivo. Taken together, these findings demonstrated that TAIII exhibited significant anti-tumor effect on taxol-resistant cells.

Discovery of cycloneolignan enantiomers from Isatis indigotica Fortune with neuroprotective effects against MPP+-induced SH-SY5Y cell injury.

A pair of new cycloneolignan enantiomers (1a and 1b) were isolated from the leaves of Isatis indigotica Fortune. Their structures were elucidated by extensive spectroscopic data analysis, including 1D and 2D NMR, HRESIMS, MS/MS analysis, together with theoretical electronic circular dichroism (ECD) calculations. Compounds 1a and 1b were then evaluated for their neuroprotective effects against MPP+-induced SH-SY5Y cell injury. As a result, compounds 1a (77.64%) and 1b (78.62%) exhibited moderate neuroprotective activity at the concentration of 12.5 µM compared with that of MPP+ treated group (62.00% at 1 mM) by MTT assay. Furthermore, Annexin V-FITC/PI analysis showed that apoptosis ratios of 1a and 1b were reduced to 10.99% and 9.31%, respectively.

Quassinoids from Picrasma quassioides and Their Neuroprotective Effects.

Quassinoids are a class of highly oxygenated degraded triterpenoids exclusively discovered from plants of the Simaroubaceae family. In this study, eight new (1-8) and 15 known quassinoids (9-23) were isolated from an extract of the stems of Picrasma quassioides. The structures were elucidated by spectroscopic analysis and electronic circular dichroism spectra combined with quantum chemical calculations. Compounds 4 and 5 represent the first examples of 18-nor-quassinoids from P. quassioides. All isolates were screened for their neuroprotective activities toward H2O2-induced cell damage in SH-SY5Y cells. Further study revealed that the potential protective activities of these compounds appeared to occur via the suppression of cell apoptosis and downregulation of caspase-3 activation.

Inhibition of Heme Oxygenase-1 enhances hyperthermia-induced autophagy and antiviral effect.

Hyperthermia has been clinically utilized as an adjuvant therapy in the treatment of cervical carcinoma. However, thermotolerance induced by heme oxygenase-1 (HO-1), a stress-inducible cytoprotective protein, limits the efficacy of hyperthermic therapy, for which the exact mechanism remains unknown. In the present study, we found that heat treatment induced HO-1 expression and decreased copy number of HPV16 in cervical cancer cells and tissues from cervical cancer and precursor lesions. Knockdown of HO-1 stimulated autophagy accompanied by downregulation of X-linked inhibitor of apoptosis protein. Furthermore, silencing of HO-1 led to cell intolerance to hyperthermia, as manifested by inhibition of cell viability and induction of autophagic apoptosis. Moreover, HO-1 modulated hyperthermia-induced, autophagy-dependent antiviral effect. Thus, the findings indicate that blockade of HO-1 enhances hyperthermia-induced autophagy, an event resulting in apoptosis of cervical cancer cells through an antiviral mechanism. These observations imply the potential clinical utility of hyperthermia in combination with HO-1 inhibition in the treatment of cervical cancer.

Seven new neuroprotective sesquineolignans isolated from the seeds of Crataegus pinnatifida.

The investigation of the ethanol extract of the seeds of Crataegus pinnatifida led to the isolation of seven new 8-O-4' type sesquineolignans crasesquineolignan A-G (1-7), along with a reported analogue, leptolepisol B (8). The chemical structures of these compounds were elucidated based on complex analysis of their MS, 1D and 2D NMR data. All the isolated compounds were tested for their neuroprotective effects against the damage of human neuroblastoma SH-SY5Y cells induced by H2O2, and most of them showed significant neuroprotective activity. Among them, compound 4 (77.58%) showed the best protective effect, even better than the positive control (69.26%) at 25 µM.

Timosaponin AIII: A novel potential anti-tumor compound from Anemarrhena asphodeloides.

Timosaponin AIII, a major steroidal saponin found in Anemarrhena asphodeloides Bge., which has been widely used as anti-pyretic, anti-diabetic, anti-inflammatory, anti-platelet aggregator and anti-depressant agents in traditional Chinese medicine. Recent pharmacological study showed that timosaponin AIII had potent cytotoxicity, which was potential to be developed as an anticancer agent, however the molecular mechanism underlying the anticancer activity has not been fully elucidated. This review aims to give a systematic summary of the study of timosaponin AIII to reveal its anti-tumor activities by investigating invasion and migration, apoptosis, autophagy and reversing multi-drug resistance. Furthermore, we also make an overview of the mechanisms identified till now. These meaningful findings may provide novel insights on exploiting timosaponin AIII as a new anti-tumor agent.

Neolignans from Red Raspberry ( Rubus idaeus L.) Exhibit Enantioselective Neuroprotective Effects against H2O2-Induced Oxidative Injury in SH-SY5Y Cells.

Red raspberry has been well-known for its nutritional purpose. Although this fruit has been reported for its potent antioxidant activity and health-promoting properties, systematic studies responsible for the bioactive constituents were still insufficient. In the current study, three pairs of dihydrobenzofuran-type enantiomeric neolignans (1a/1b-3a/3b), including two new compounds (1b and 2a), were isolated from the fruit of Rubus idaeus. The structures of these enantiomers were determined through spectroscopic methods and quantum mechanical calculations. Biologically, enantiomers 2a and 2b exhibited significant enantioselective protective effects against H2O2-induced neurotoxicity at 50 µM (2a, 86.72 ± 1.17%; 2b, 69.70 ± 1.59%). The underlying mechanism study demonstrated that enantiomer 2a is able to attenuate H2O2-induced apoptosis, reactive oxygen species (ROS) generation, and mitochondrial dysfunction in SH-SY5Y cells. Overall, these findings provide a valuable foundation for the understanding of neuroprotective activities of red raspberry and further investigation on its potential application values.

Stereoisomeric guaiacylglycerol-ß-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells.

Two 8-O-4'-type neolignan epimers erythro-guaiacylglycerol-ß-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-ß-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC50 = 45.56 µM) and 2b (IC50 = 39.02 µM) had more cytotoxic effect than its enantiomers 1a (IC50 = 82.66 µM) and 2a (IC50 = 67.97 µM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 µM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-ß-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.