Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation

Cytokine-producing cells in gut-associated lymphoid tissues of rhesus macaques with chronic enterocolitis were studied. The confocal microscopy technique that we developed enables simultaneous in situ visualization of multiple extra- and/or intracellular antigens at a resolution higher than that allowed by light or epifluorescence microscopy. The presence of interleukin-6 (IL-6)-, tumor necrosis factor alpha (TNF-alpha)-, and IL-1-alpha-producing cells was focally intense in the colon lamina propria of the affected animals. The IL-1-alpha-producing cells were T lymphocytes (CD3+), while the TNF-alpha-producing cells were both macrophages (CD68+/HAM56+/LN5+) and T lymphocytes (CD3+). The IL-6-producing cells within the colon consisted of T lymphocytes and macrophages. The amount of IL-6-producing cells seen in macaques with enterocolitis was significantly higher (P<0.001) than that seen in the healthy control animal, while TNF-alpha- and IL-1-alpha-producing cells were seen only in macaques with enterocolitis. Most of the T lymphocytes that produced cytokines were detected in the lamina propria, while the macrophages were most prominent in highly inflamed regions of the lamina propria. Taken together, our findings indicate that there might be immunological similarity between chronic enterocolitis of rhesus macaques and humans, suggesting the potential use of the nonhuman primate model for the validation of novel therapies.     

Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have previously shown that treatment of ovarian cancer cells, NOM1, with fibronectin (FN) stimulated matrix metalloproteinase (MMP)-9 secretion and thereby activated the invasiveness of cells via the FAK/Ras signaling pathway. By use of chemical inhibitors, we investigated the downstream effectors critical for FN-dependent secretion of MMP-9. Treatment of cells with MEK1 inhibitors, U0126 and PD98059, dramatically suppressed the secretion of MMP-9 activated by FN. Similarly, PI-3 kinase inhibitors, Wortmannin and LY294002, strongly suppressed the FN-dependent secretion of MMP-9 together with the inhibition of Akt activation. In contrast, a specific PKC inhibitor (GF109203X) showed no inhibitory effect on the FN-dependent MMP-9 secretion. Moreover, we found that both the MEK1 inhibitor and the PI3-K inhibitor, but not the PKC inhibitor, strongly suppressed the invasiveness of NOM1 cells. Taken together, our results suggest that activation of dual signaling pathways, MEK1-MAPK and PI3K-Akt, is required for the FN-dependent activation of MMP-9 secretion. Our results suggest the importance of these signaling molecules as a chemotherapeutic target for cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Clinical and Epidemiological Relevance of Quantitating Hepatitis E Virus-Specific Immunoglobulin M

Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.     

PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted,but Functional in Patients with COVID-19

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Novel second-generation rexinoid induces growth arrest and reduces cancer cell stemness in human neuroblastoma patient-derived xenografts

IntroductionThe poor therapeutic efficacy seen with current treatments for neuroblastoma may be attributed to stem cell-like cancer cells (SCLCCs), a subpopulation of cancer cells associated with poor prognosis and disease recurrence. Retinoic acid (RA) is a differentiating agent used as maintenance therapy for high-risk neuroblastoma but nearly half of children treated with RA relapse. We hypothesized that 6-Methyl-UAB30 (6-Me), a second-generation rexinoid recently developed with a favorable toxicity profile compared to RA, would reduce cancer cell stemness in human neuroblastoma patient-derived xenografts (PDXs).MethodsCells from three neuroblastoma PDXs were treated with 6-Me and proliferation, viability, motility, and cell-cycle progression were assessed. CD133 expression, sphere formation, and mRNA abundance of stemness and differentiation markers were evaluated using flow cytometry, in vitro extreme limiting dilution analysis, and real-time PCR, respectively.ResultsTreatment with 6-Me decreased proliferation, viability, and motility, and induced cell-cycle arrest and differentiation in all three neuroblastoma PDXs. In addition, 6-Me treatment led to decreased CD133 expression, decreased sphere-forming ability, and decreased mRNA abundance of Oct4, Nanog, and Sox2, indicating decreased cancer cell stemness.Conclusions6-Me decreased oncogenicity and reduced cancer cell stemness of neuroblastoma PDXs, warranting further exploration of 6-Me as potential novel therapy for neuroblastoma.     

Ultrastructural study of the effects of 6-hydroxydopamine on the sinuatrial and atrioventricular nodes of the heart of the monkey (Macaca fascicularis)

The present study examined the ultrastructural changes in the sinuatrial and atrioventricular nodes of the heart of the monkey (Macaca fascicularis) after 6-hydroxydopamine (6-OHDA) at survival times of 1, 3, 5, 7 and 14 days. Changes ranged from dissolution of the cytoplasm to amorphous appearance and darkening of the nodal cells. Initially, the ultrastructural changes were quite similar in both sinuatrial (SA) and atrioventricular (AV) nodal cells but in the later stage, especially at fourteen days, affected SA nodal cells showed empty-looking appearance while affected AV nodal cells displayed a darkened appearance. The cardiac neurons also showed ultrastructural changes such as diffuse accumulation of glycogen particles, distended cisternae of the rough endoplasmic reticulum and increased lipofuscin granules. Some of the vacuolated axonal profiles containing large dense-cored vesicles were in close association with the somata of the cardiac neurons. There were also changes in the non-neuronal cells such as darkening and vacuolation of the cells capping the neurons. Macrophages and Schwann cells were activated to engulf the degenerating nodal cells and axonal profiles. The ultrastructural changes in the nodal cells and the cardiac neurons reflect a disturbance in the cell metabolism presumably brought about by the impairment of the sympathetic nervous system.     

The pancreatic ductal epithelium serves as a potential pool of progenitor cells

Abstract:  With the increasing success of islet transplantation, β-cell replacement therapy has had renewed interest. To make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin-producing cells must become readily available. The most promising sources are stem cells, whether embryonic or adult stem cells. Clearly identifiable adult pancreatic stem cells have yet to be characterized. Although considerable evidence suggests their possibility, recent lineage-tracing experiments challenge their existence. Even in light of these lineage-tracing experiments, we suggest that evidence for neogenesis or new islet formation after birth remains strong. Our work has suggested that the pancreatic duct epithelium itself serves as a pool for progenitors for both islet and acinar tissues after birth and into adulthood and, thus, that the duct epithelium can be considered 'facultative stem cells'. We will develop our case for this hypothesis in this perspective.     

Screening for Inhibition of Vibrio cholerae VipA-VipB Interaction Identifies Small-Molecule Compounds Active against Type VI Secretion

The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an α-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A₁ activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.     

Genotoxic and oxidative responses in coelomocytes of Eisenia fetida and Hediste diversicolor exposed to lipid‐coated CdSe/ZnS quantum dots and CdCl2

The emerging of Quantum Dots utilization in industrial or medicinal fields involved a potentially increase of these nanoparticles in environment. In this work, the genotoxic (comet assay) and oxidative effects (SOD activity, TBARS) of functionalized‐QDs and cadmium chloride were investigated on Hediste diversicolor and Eisenia fetida coelomocytes. Results demonstrated that functionalized‐QDs (QDNs) and cadmium chloride induced DNA damages through different mechanisms that depended on the nano‐ or ionic nature of Cd. The minimal genotoxic concentrations for H. diversicolor (<0.001ng/g for QDNs and CdCl₂) were lower than for E. fetida (between 0.01 and 0.1 ng/g for QDNs, and between 0.001 and 0.01 ng/g for CdCl₂). These results showed that H. diversicolor was more sensitive than E. fetida. The two contaminants had a low impact on the oxidative stress markers. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 918–926, 2015.