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Super Typhoon Mangkhut, which traversed the North Equatorial Current (NEC; 8-17 °N) in the western North Pacific in 2018, was the most intense Category-5 tropical cyclone (TC) with the longest duration in history-3.5 days. Here we show that the combination of two factors-high ocean heat content (OHC) and increased stratification - makes the NEC region the most favored area for a rapid intensification (RI) of super typhoons, instead of the Eddy Rich Zone (17-25 °N), which was considered the most relevant for RI occurrence. The high OHC results from a northward deepening thermocline in geostrophic balance with the westward-flowing NEC. The stratification is derived from precipitation associated with the Inter-Tropical Convergence Zone in the summer peak typhoon season. These factors, which are increasingly significant over the past four decades, impede the TC-induced sea surface cooling, thus enhancing RI of TCs and simultaneously maintaining super typhoons over the NEC region., (© 2024. The Author(s).)

Predicting the consequences of a major coastal storm is increasingly difficult as the result of global climate change and growing societal dependence on critical infrastructure (CI). Past storms are no longer a reliable predictor of future weather events, and the traditional approach to vulnerability assessment presents accumulated loss in largely quantitative terms that lack the specificity local emergency managers need to develop effective plans and mitigation strategies. The Rhode Island Coastal Hazards Modeling and Prediction (RI-CHAMP) system is a geographic information system (GIS)-based modeling tool that combines high-resolution storm simulations with geolocated vulnerability data to predict specific consequences based on local concerns about impacts to CI. This case study discusses implementing RI-CHAMP for the State of Rhode Island to predict impacts of wind and inundation on its CI during a hurricane, tropical storm, or nor'easter. This paper addresses the collection and field verification of vulnerability data, along with RI-CHAMP's process for integrating those data with storm models. The project deeply engaged end-users (emergency managers, facility managers, and other stakeholders) in developing RI-CHAMP's ArcGIS Online dashboard to ensure it provides specific, actionable data. The results of real and synthetic storm models are presented along with discussion of how the data in these simulations are being used by state and local emergency managers, facility owners, and others.

In March 2010, a sequence of three major rainfall events in New England (United States) led to a record-breaking flooding event in the Pawtuxet River Watershed with a peak flow discharge of about 500-year return period. After development of hydrological and hydraulic models, a number of factors that played important roles in the impact of this flooding and other extreme events including river structures (reservoirs, historical textile mill dams, and bridges) were investigated. These factors are currently omitted within risk assessments tools such as flood insurance rate maps. Some management strategies that should be considered for future flood risk mitigation were modeled and discussed. Furthermore, to better understand possible future risks in a warmer climate, another extreme flood event was simulated. The synthetic/hypothetical storm (Hurricane Rhody with two landfalls) was created based on the characteristics of the historical hurricanes that severely impacted this region in the past. It was shown that while the first landfall of this hurricane did not lead to significant flood risk, the second landfall could generate more rain and flooding equivalent to a 500-year event. Results and the methodology of this study can be used to better understand and assess future flood risk in similar watersheds., (© 2020 The Authors. Journal of Flood Risk Management published by Chartered Institution of Water and Environmental Management and John Wiley & Sons Ltd.)

Despite significant improvement in computational and observational capabilities, predicting intensity and intensification of major tropical cyclones remains a challenge. In 2017 Hurricane Maria intensified to a Category 5 storm within 24 h, devastating Puerto Rico. In 2019 Hurricane Dorian, predicted to remain tropical storm, unexpectedly intensified into a Category 5 storm and destroyed the Bahamas. The official forecast and computer models were unable to predict rapid intensification of these storms. One possible reason for this is that key physics, including microscale processes at the air-sea interface, are poorly understood and parameterized in existing forecast models. Here we show that surfactants significantly affect the generation of sea spray, which provides some of the fuel for tropical cyclones and their intensification, but also provides some of the drag that limits intensity and intensification. Using a numerical model verified with a laboratory experiment, which predicts spray radii distribution starting from a 100 μm radius, we show that surfactants increase spray generation by 20-34%. We anticipate that bio-surfactants affect heat, energy, and momentum exchange through altered size distribution and concentration of sea spray, with consequences for tropical cyclone intensification or decline, particularly in areas of algal blooms and near coral reefs, as well as in areas affected by oil spills and dispersants.

Integrated hydrological and hydrodynamic modeling study has been conducted to investigate hurricane impact on Woonasquatucket River, Rhode Island, USA. Model simulation was conducted for the case study of 2010 storm event. The hydrological model simulates the runoff from the heavy rainstorm, while the river hydrodynamic model simulates the flood waves affected by the interactions of upstream rainfall runoff and downstream storm surge. Results indicate that the river floods was dominant by rainfall runoff in upper river reaches, but dominant by storm surge in the lower river area near the estuary., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

Tropical storm intensity prediction remains a challenge in tropical meteorology. Some tropical storms undergo dramatic rapid intensification and rapid decline. Hurricane researchers have considered particular ambient environmental conditions including the ocean thermal and salinity structure and internal vortex dynamics (e.g., eyewall replacement cycle, hot towers) as factors creating favorable conditions for rapid intensification. At this point, however, it is not exactly known to what extent the state of the sea surface controls tropical cyclone dynamics. Theoretical considerations, laboratory experiments, and numerical simulations suggest that the air-sea interface under tropical cyclones is subject to the Kelvin-Helmholtz type instability. Ejection of large quantities of spray particles due to this instability can produce a two-phase environment, which can attenuate gravity-capillary waves and alter the air-sea coupling. The unified parameterization of waveform and two-phase drag based on the physics of the air-sea interface shows the increase of the aerodynamic drag coefficient C d with wind speed up to hurricane force ( U 10 ≈ 35 m s -1 ). Remarkably, there is a local C d minimum-"an aerodynamic drag well"-at around U 10 ≈ 60 m s -1 . The negative slope of the C d dependence on wind-speed between approximately 35 and 60 m s -1 favors rapid storm intensification. In contrast, the positive slope of C d wind-speed dependence above 60 m s -1 is favorable for a rapid storm decline of the most powerful storms. In fact, the storms that intensify to Category 5 usually rapidly weaken afterward., (© 2017. The Authors.)

Tropical cyclone track prediction is steadily improving, while storm intensity prediction has seen little progress in the last quarter century. Important physics are not yet well understood and implemented in tropical cyclone forecast models. Missing and unresolved physics, especially at the air-sea interface, are among the factors limiting storm predictions. In a laboratory experiment and coordinated numerical simulation, conducted in this work, the microstructure of the air-water interface under hurricane force wind resembled Kelvin-Helmholtz shear instability between fluids with a large density difference. Supported by these observations, we bring forth the concept that the resulting two-phase environment suppresses short gravity-capillary waves and alters the aerodynamic properties of the sea surface. The unified wave-form and two-phase parameterization model shows the well-known increase of the drag coefficient (Cd) with wind speed, up to ~30 ms(-1). Around 60 ms(-1), the new parameterization predicts a local peak of Ck/Cd, under constant enthalpy exchange coefficient Ck. This peak may explain rapid intensification of some storms to major tropical cyclones and the previously reported local peak of lifetime maximum intensity (bimodal distribution) in the best-track records. The bimodal distribution of maximum lifetime intensity, however, can also be explained by environmental parameters of tropical cyclones alone.

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable "off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe, animal product-free medium containing 2%, 5%, and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery, viability, apoptosis, proliferation rate, expression of a broad panel of MSC markers, and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO, respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO, respectively, were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity, ALP surface expression and Ca⁺⁺ deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution, also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery, respectively, less than 10% of apoptotic cells, and normal proliferation, marker expression, and osteogenic potential. Overall, our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents.

Tissue-engineered bone grafts seeded with mesenchymal stem cells (MSCs) have been sought as a replacement for bone grafts currently used for bone repair. For production of osteogenic constructs, MSCs are isolated from bone marrow (BM) or other tissues, expanded in culture, then trypsinized, and seeded on a scaffold. Predifferentiation of seeded cells is often desired. We describe here bone progenitor cells (BPCs) obtained by direct osteogenic differentiation of unprocessed BM bypassing isolation of MSCs. Human BM aspirates were incubated for 2 weeks with a commonly used osteogenic medium (OM), except no fetal calf serum, serum substitutes, or growth factors were added, because responding stem and/or progenitor cells were present in the BM milieu. The adherent cells remaining after the culture medium and residual BM were washed out, expressed high levels of bone-specific alkaline phosphatase (ALP) on their surface, demonstrated high ALP activity, were capable of mineralization of the intercellular space, and expressed genes associated with osteogenesis. These parameters in BPCs were similar and even at higher levels compared to MSCs subjected to osteogenic differentiation for 2 weeks. The yield of BPCs per 1 mL BM was 0.71±0.39×10(6). In comparison, the yield of MSCs produced by adhesion of mononuclear cells derived from the same amount of BM and cultured in a commercial growth medium for 2 weeks was 0.3±0.17×10(6). When a scaffold was added to the BM-OM mixture, and the mixture was cultured in a simple rotational bioreactor; the resulting BPCs were obtained already seeded on the scaffold. BPCs seeded on scaffolds were capable of proliferation for at least 6 weeks, keeping high levels of ALP activity, expressing osteogenic genes, and mineralizing the scaffolds. Autologous rat BPCs seeded on various scaffolds were transplanted into critical-size calvarial defects. Six weeks after transplantation of polylactic acid/polyglycolic acid scaffolds, 76.1%±18.3% of the defects were filled with a new bone, compared to 37.9%±28.4% in the contralateral defects transplanted with the scaffolds without cells.

Expansion of plastic-adherent bone marrow-derived mesenchymal stem cells (MSCs) results in gradual loss of osteogenic potential after passage 5-6. One explanation is contamination of MSC cultures with mature cells including fibroblasts. Identification and elimination of fibroblasts from MSC cultures could improve MSC yield and differentiation potential and also prevent tumor formation after MSC transplantation. However, no specific markers currently exist that can reliably discriminate between MSCs and fibroblasts. Flow cytometry analysis demonstrated that markers currently used to define MSCs, such as CD105, CD166, CD90, CD44, CD29, CD73, and CD9, are also expressed on human skin or lung fibroblasts. However, the level of expression of CD166 was significantly higher and that of CD9 was significantly lower in MSCs than in fibroblasts. CD146 was expressed only in MSCs. Using small focused microarrays, new markers differentially expressed in MSCs and fibroblasts were identified. Real-time polymerase chain reaction confirmed that expression of CD106, integrin alpha 11, and insulin-like growth factor-2 in MSCs was at least 10-fold higher than in fibroblasts; whereas expression of matrix metalloproteinase 1 and matrix metalloproteinase 3 was almost 100-fold lower. Flow cytometry and immunostaining demonstrated that CD106 protein expression on cell surface could be upregulated in MSCs but not in fibroblasts by the treatment with tumor necrosis factor-alpha. Comparison of surface expression of commonly used and newly identified MSC markers in MSCs cultures of passage 2 and passage 6 demonstrated that CD106 (with and without tumor necrosis factor-alpha treatment), integrin alpha 11, and CD146 were downregulated in MSCs of passage 6, and CD9 was upregulated; whereas all other markers did not change. Newly identified markers that have robust differences of expression in MSCs and fibroblasts on gene and protein level could be used for quality control of MSC cultures after expansion, cryopreservation, gene transfection, and other manipulations.

After 24-hour middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats, brain ceramide level increased from baseline reached 595% (ischemic core) and 460% (perifocal/penumbral areas); brain glucosylceramide synthase (GCS) activities in these areas simultaneously decreased by 70% and 50%, respectively. Ten-minute MCAO preconditioning significantly attenuated 24-hour MCAO-induced ceramide accumulation by 40% to 60% in ischemic core and perifocal areas, and GCS activities improved by 60% to 70% in both areas. Thus, potentially toxic levels of brain ceramide induced by MCAO were attenuated to intermediate levels in preconditioned animals; brain GCS activity was relatively preserved. In ischemic tolerance, GCS appears to modulate otherwise high levels of brain ceramide.

We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.

Human embryonic stem (huES) cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation of huES cells and factors regulating cell development. We have used high-quality microarrays containing 16 659 seventy-base pair oligonucleotides to examine gene expression in 6 of the 11 available huES cell lines. Expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All 6 cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient > 0.85).A common subset of 92 genes was identified that included Nanog, GTCM-1, connexin 43 (GJA1), oct-4, and TDGF1 (cripto). Gene expression was confirmed by a variety of techniques including comparison with databases, reverse transcriptase-polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry. Comparison with published "stemness" genes revealed a limited overlap, suggesting little similarity with other stem cell populations. Several novel ES cell-specific expressed sequence tags were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES cells and provide a unique set of markers to profile and better understand the biology of huES cells.

Human ES (hES) cell lines have only recently been generated, and differences between human and mouse ES cells have been identified. In this manuscript we describe the properties of two human ES cell lines, BG01 and BG02. By immunocytochemistry and reverse transcription polymerase chain reaction, undifferentiated cells expressed markers that are characteristic of ES cells, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4. Both cell lines were readily maintained in an undifferentiated state and could differentiate into cells of all three germ layers, as determined by expression of beta-tubulin III neuron-specific molecule (ectoderm), cardiac troponin I (cardiomyocytes, mesoderm), and alpha-fetoprotein (endoderm). A large-scale microarray (16,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG01 and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE01, GE09, and pooled samples derived from GE01, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming growth factor-beta superfamily were employed to examine differentially expressed genes. Several growth factors, receptors, and components of signaling pathways that regulate embryonic development, in particular the nodal signaling pathway, were detected in both BG01 and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG01 and BG02 human ES cell lines.

Studies in animal models have suggested a role for stem cells in repair and regeneration of the nervous system. Human equivalents of stem and precursor cells have been isolated and their efficacy is being evaluated in rodent and primate models. Difficulties exist in translating results of these preclinical models to therapy in humans. Evolutionary differences among rodents, primates, and humans; fundamental differences in the anatomy and physiology; differences in immune responses in xenotransplant models; the paucity of good transplant models of chronic disease; and allelic variability in the cells themselves make any study evaluating the efficacy of cells in transplant models difficult to interpret. As no better alternatives to testing in animals exist, we suggest that at this early stage a considered step-by-step approach to testing and comparison of different transplant strategies in isolation will prepare us better for clinical trials than simple evaluation of functional outcomes in various models of disease. We emphasize that we do not recommend delaying or abandoning clinical trials; rather, we suggest that one anticipate failures and design experiments and data collection such that we learn from these failures to ensure future success in as rapid a time frame as possible.

Hematopoietic stem cells, unlike neural stem cells, can be readily identified and isolated from developing and adult cell populations using positive and negative selection criteria. Isolating stem cells and progenitor cells from neural tissue has been more difficult because of difficulties in separating cells in solid tissue, the limited numbers of stem cells that persist in the adult, and the paucity of rigorously characterized markers. Nevertheless, strategies that have worked successfully in hematopoietic stem cell isolation can be adapted to isolate multiple classes of stem and progenitor cells from neural tissue. Neural stem cells also share cellular and molecular properties with other stem cell populations that may serve as surrogate identifiers of multipotentiality. Such potential markers are described. Unlike hematopoietic stem cells, tracking neural cells after transplantation is both necessary and more difficult. It will therefore be necessary to develop invasive and non-invasive strategies to follow transplanted cells and develop useful quantifiable readouts. Some potential strategies are described and current results are discussed.

Fetal neural stem cells (NSCs) have received great attention not only for their roles in normal development but also for their potential use in the treatment of neurodegenerative disorders. To develop a robust method of assessing the state of stem cells, we have designed, tested, and validated a rodent NSC array. This array consists of 260 genes that include cell type-specific markers for embryonic stem (ES) cells and neural progenitor cells as well as growth factors, cell cycle-related genes, and extracellular matrix molecules known to regulate NSC biology. The 500-bp polymerase chain reaction products amplified and validated by using gene-specific primers were arrayed along with positive controls. Blanks were included for quality control, and some genes were arrayed in duplicate. No cross-hybridization was detected. The quality of the arrays and their sensitivity were also examined by using probes prepared by conventional reverse transcriptase or by using amplified probes prepared by linear polymerase replication (LPR). Both methods showed good reproducibility, and probes prepared by LPR labeling appeared to detect expression of a larger proportion of expressed genes. Expression detected by either method could be verified by RT-PCR with high reproducibility. Using these stem cell chips, we have profiled liver, ES, and neural cells. The cell types could be readily distinguished from each other. Nine markers specific to mouse ES cells and 17 markers found in neural cells were verified as robust markers of the stem cell state. Thus, this focused neural stem array provides a convenient and useful tool for detection and assessment of NSCs and progenitor cells and can reliably distinguish them from other cell populations.

Preconditioning with sublethal ischemia results in natural tolerance to ischemic stress, where multiple mediators of ischemic damage are simultaneously counteracted. Tumor necrosis factor alpha (TNF-alpha) has been implicated in development of ischemic tolerance. Using cellular models of ischemic tolerance, we have demonstrated that an effector of TNF-alpha-induced preconditioning is ceramide, a sphingolipid messenger in TNF-alpha signaling. TNF-alpha/ceramide-induced preconditioning protected cultured neurons against ischemic death and cultured astrocytes against proinflammatory effects of TNF-alpha. TNF-alpha activates a transcription factor NF-kappaB that binds promoters of multiple genes, thus ensuring pleiotropic effects of TNF-alpha. We describe here a mechanism that allows selective suppression of TNF-alpha/NF-kappaB-induced harmful genes in preconditioned cells while preserving cytoprotective responses. We demonstrate that in astrocytes activation of an adhesion molecule ICAM-1 by TNF-alpha is regulated through association of the phosphorylated p65 subunit of NF-kappaB with an adapter protein, p300, and that in preconditioned cells p65 remains unphosphorylated and ICAM-1 transcription is inhibited. However, TNF-alpha-activated transcription of a protective enzyme, MnSOD, does not depend on p300 and does not become inhibited in preconditioned cells. This new understanding of TNF-alpha-induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases.

Hypoxia is known to induce extravasation of lymphocytes and leukocytes during ischemic injury and increase the metastatic potential of malignant lymphoid cells. We have recently identified a new adhesion molecule, hypoxia-activated ligand-1/13 (HAL-1/13), that mediates the hypoxia-induced increases in lymphocyte and neutrophil adhesion to endothelium and hypoxia-mediated invasion of endothelial cell monolayers by tumor cells. In this report, we used expression cloning to identify this molecule as the lupus antigen and DNA-dependent protein kinase-associated nuclear protein, Ku80. The HAL-1/13-Ku80 antigen is present on the surface of leukemic and solid tumor cell lines, including T and B lymphomas, myeloid leukemias, neuroblastoma, rhabdomyosarcoma, and breast carcinoma cells. Transfection and ectopic expression of HAL-1/13-Ku80 on (murine) NIH/3T3 fibroblasts confers the ability of these normally nonadhesive cells to bind to a variety of human lymphoid cell lines. This adhesion can be specifically blocked by HAL-1/13 or Ku80-neutralizing antibodies. Loss of expression variants of these transfectants simultaneously lost their adhesive properties toward human lymphoid cells. Hypoxic exposure of tumor cell lines resulted in upregulation of HAL-1/13-Ku80 expression at the cell surface, mediated by redistribution of the antigen from the nucleus. These studies indicate that the HAL-1/13-Ku80 molecule may mediate, in part, the hypoxia-induced adhesion of lymphocytes, leukocytes, and tumor cells.

Previous work in primary cell culture has shown that TNF-alpha and ceramide are involved in the signaling that induces tolerance to brain ischemia (Ginis et al., 1999; Liu et al., 2000). To validate the in vitro studies, the authors administered cell permeable analogs of ceramides intracisternally or intravenously to examine their effect on neuroprotection after focal cerebral ischemia. Permanent middle cerebral artery occlusion (MCAO) was performed in spontaneously hypertensive rats. Infarct volumes were assessed at 24 hours after surgery. D-erythro-N-acetylsphingosine (C2-ceramide) or its vehicle was infused intracisternally for 1 hour before MCAO. In a second set of studies, D-erythro-N-octanoylsphingosine (C8-ceramide) or its vehicle was injected intravenously 48 or 24 hours before MCAO to mimic preconditioning (PC) and was also injected 5 minutes after MCAO. C2-ceramide infusion significantly reduced infarct volumes by approximately 14% (P < 0.05). C8-ceramide injection reduced infarct volumes approximately 17% compared with controls. This effect was constant and significant compared with controls over the time periods examined (P < 0.01). This work supports findings in primary brain cell cultures that implicate ceramide as a downstream signal that is proximate to development of tolerance to brain ischemia. Because the degree of protection represents approximately 50% of the maximal infarct reduction observed in this model, there are probably additional signaling pathways that subserve tolerance.

Preconditioning brain with tumor necrosis factor alpha (TNF-alpha) can induce tolerance to experimental hypoxia and stroke and ceramide is a downstream messenger in the TNF-alpha signaling pathway. A hypoxic-ischemic (HI) insult in the immature rat injures brain primarily through apoptosis. Apoptosis is regulated by Bcl-2 family proteins. The authors explored whether ceramide protects against HI in the immature rat, and whether Bcl-2 family protein expression is involved. Hypoxia-ischemia was produced in seven-day-old rats by ligating the right carotid artery, followed by 2 hours of 8% oxygen exposure. Thirty minutes after HI, C2-ceramide (150 microg/kg) was injected intraventricularly. Infarct volume was measured 5 days later. C2-ceramide reduced HI-induced brain damage by 45% to 65% compared with HI/dimethyl sulfoxide (DMSO) (vehicle control) or HI only groups. In separate experiments, brains of sham-operated control and HI only animals and animals subjected to HI plus C2-ceramide or DMSO infusion were sampled 6 hours, 24 hours, and 5 days after treatments and analyzed for Bcl-2, Bcl-xl, and Bax expression (Western blotting), and apoptosis (TUNEL assay). Augmented Bcl-2 and Bcl-xl levels in the C2-ceramide treated group were associated with a significant decrease in TUNEL-positive cells. The results support a protective role for ceramide in neonatal HI.

Background: Tumor necrosis factor alpha (TNFalpha) plays a key role in pathogenesis of brain injury. However, TNFalpha exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFalpha cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFalpha in causing cell death., Materials and Methods: Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFalpha alone or together with different doses of H2O2, and apoptotic cell death and DNA fragmentation were measured by means of 3'-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H2O2 on TNFalpha-induced activation of nuclear factor kappa B (NF-kappaB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-kappaB (IkappaB) and anti-p65 subunit of NF-kappaB antibodies. Nuclear translocation of NF-kappaB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies., Results: TNFalpha alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to 100 ng/ml. Co-treatment with 5-10 microM of H2O2 caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1-3 microM) of H2O2 initiated early DNA fragmentation. H2O2 inhibited TNFalpha-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IkappaB in cytoplasm. Immunostaining confirmed that H2O2 inhibited p65 transport to the nucleus., Conclusions: Reactive oxygen species could act synergistically with TNFalpha in causing cytotoxicity via inhibition of a cytoprotective branch of TNFalpha signaling pathways, which starts with NF-kappaB activation.

Brief "preconditioning" ischemia induces ischemic tolerance (IT) and protects the animal brain from subsequent otherwise lethal ischemia. Identification of the signaling steps most proximal to the development of the IT will allow induction of the resistance to ischemia shortly after the onset of stroke. Animal studies demonstrate a key role of tumor necrosis factor-alpha (TNF-alpha) in induction of IT. The sphingolipid ceramide is known as a second messenger in many of the multiple effects of TNF-alpha. We hypothesized that ceramide could mediate IT. We demonstrate that preconditioning of rat cortical neurons with mild hypoxia protects them from hypoxia and O(2)-glucose deprivation injury 24 h later (50% protection). TNF-alpha pretreatment could be substituted for hypoxic preconditioning (HP). HP was attenuated by TNF-alpha-neutralizing antibody. HP and TNF-alpha pretreatment cause a two- to threefold increase of intracellular ceramide levels, which coincides with the state of tolerance. Fumonisin B(1), an inhibitor of ceramide synthase, attenuated ceramide upregulation and HP. C-2 ceramide added to the cultures right before the hypoxic insult mimicked the effect of HP. Ceramide did not induce apoptosis. These results suggest that HP is mediated via ceramide synthesis triggered by TNF-alpha.

Ischemic stroke is a leading cause of death and disability in developed countries. Yet, in spite of substantial research and development efforts, no specific therapy for stroke is available. Several mechanism for neuroprotection have been explored including ion channels, excitatory amino acids and oxygen radicals yet none has culminated in an effective therapeutic effect. The review article on "inflammation and stroke" summarizes key data in support for the possibility that inflammatory cells and mediators are important contributing and confounding factors in ischemic brain injury. In particular, the role of cytokines, endothelial cells and leukocyte adhesion molecules, nitric oxide and cyclooxygenase (COX-2) products are discussed. Furthermore, the potential role for certain cytokines in modulation of brain vulnerability to ischemia is also reviewed. The data suggest that novel therapeutic strategies may evolve from detailed research on some specific inflammatory factors that act in spatial and temporal relationships with traditionally recognized neurotoxic factors. The dual nature of some mediators in reformatting of brain cells for resistance or sensitivity to injury demonstrate the delicate balance needed in interventions based on anti-inflammatory strategies.