Decontaminating N95 and SN95 masks with ultraviolet germicidal irradiation does not impair mask efficacy and safety.

Inadequate supply of filtering facepiece respirators (FFRs) for healthcare workers during a pandemic such as the novel coronavirus outbreak (SARS-CoV-2) is a serious public health issue. The aim of this study was to synthesize existing data on the effectiveness of ultraviolet germicidal irradiation (UVGI) for N95 FFR decontamination. A systematic review (PROSPERO CRD42020176156) was conducted on UVGI in N95 FFRs using Embase, Medline, Global Health, Google Scholar, WHO feed, and MedRxiv. Two reviewers independently determined eligibility and extracted predefined variables. Original research reporting on function, decontamination, or mask fit following UVGI were included. Thirteen studies were identified, comprising 54 UVGI intervention arms and 58 N95 models. FFRs consistently maintained certification standards following UVGI. Aerosol penetration averaged 1.19% (0.70-2.48%) and 1.14% (0.57-2.63%) for control and UVGI arms, respectively. Airflow resistance for the control arms averaged 9.79 mm H2O (7.97-11.70 mm H2O) vs 9.85 mm H2O (8.33-11.44 mm H2O) for UVGI arms. UVGI protocols employing a cumulative dose >20,000 J/m2 resulted in a 2-log reduction in viral load. A >3-log reduction was observed in seven UVGI arms using >40,000 J/m2. Impact of UVGI on fit was evaluated in two studies (16,200; 32,400 J/m2) and no evidence of compromise was found. Our findings suggest that further work in this area (or translation to a clinical setting) should use a cumulative UV-C dose of 40,000 J/m2 or greater, and confirm appropriate mask fit following decontamination.

Cosmic-ray antinuclei as messengers of new physics: status and outlook for the new decade.

The precise measurement of cosmic-ray antinuclei serves as an important means for identifying the nature of dark matter and other new astrophysical phenomena, and could be used with other cosmic-ray species to understand cosmic-ray production and propagation in the Galaxy. For instance, low-energy antideuterons would provide a "smoking gun" signature of dark matter annihilation or decay, essentially free of astrophysical background. Studies in recent years have emphasized that models for cosmic-ray antideuterons must be considered together with the abundant cosmic antiprotons and any potential observation of antihelium. Therefore, a second dedicated Antideuteron Workshop was organized at UCLA in March 2019, bringing together a community of theorists and experimentalists to review the status of current observations of cosmic-ray antinuclei, the theoretical work towards understanding these signatures, and the potential of upcoming measurements to illuminate ongoing controversies. This review aims to synthesize this recent work and present implications for the upcoming decade of antinuclei observations and searches. This includes discussion of a possible dark matter signature in the AMS-02 antiproton spectrum, the most recent limits from BESS Polar-II on the cosmic antideuteron flux, and reports of candidate antihelium events by AMS-02; recent collider and cosmic-ray measurements relevant for antinuclei production models; the state of cosmic-ray transport models in light of AMS-02 and Voyager data; and the prospects for upcoming experiments, such as GAPS. This provides a roadmap for progress on cosmic antinuclei signatures of dark matter in the coming years.

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-gamma in lung cancer cells by downregulation of DNMT3B.

The prometastatic oncogene synuclein-gamma (SNCG) is not expressed in normal lung tissues, but it is highly expressed in lung tumors. Here, we show that cigarette smoke extract (CSE) has strong inducing effects on SNCG gene expression in A549 lung cancer cells through demethylation of SNCG CpG island. CSE treatment also augments the invasive capacity of A549 cells in an SNCG-dependent manner. To elucidate the mechanisms underlying the demethylating effects of CSE, we examined expression levels of DNA methyltransferases (DNMTs), 1, 3A and 3B in CSE-treated cells. We show that the mRNA expression of DNMT3B is specifically downregulated by CSE with a kinetics concurrent to SNCG reexpression. Utilizing siRNA to knockdown DNMT3B expression, we show that inhibition of DNMT3B directly increases SNCG mRNA expression. We further show that exogenous overexpression of DNMT3B in an SNCG-positive lung cancer cell line H292 suppresses SNCG mRNA and protein expression and induces de novo methylation of SNCG CpG island, whereas overexpression of DNMT1 or DNMT3A has no effects. Taken together, these new findings demonstrate that tobacco exposure induces the abnormal expression of SNCG in lung cancer cells through downregulation of DNMT3B. This work sheds light on the molecular understanding of demethylation of this oncogene during cancer progression.

The effect of DFMO induced uptake of [3H] putrescine on human glioma cells.

Polyamine synthesis inhibitors, such as a-difluoromethylornithine (DFMO), inhibit tumor cell growth in vitro and in vivo. However, upon cessation of treatment, tumor growth resumes. We hypothesized that incorporation of radioactive polyamines might kill the growth-arrested cells. This hypothesis was previously tested in rat 9L brain tumor cells in which DFMO increased both the uptake and the retention of [3H] putrescine. In these rat cells, DFMO-induced retention of high-specific-activity [3H] putrescine for 20 days resulted in several logs killing. In the present studies all of the 5 different human glioma cell lines tested with DFMO treatment also showed enhanced uptake of exogenous [3H] putrescine, reduced cell counts and enhanced killing of colony forming cells (CSF). Extending the time of DFMO treatment of cells that had taken up high-specific-activity (80 Ci/mmol) [3H] putrescine further increased the killing. A 10-day extension resulted in a 10,000-fold reduction in cumulative cell growth. A 5-day extension resulted in a 2-3 log decrease in numbers of surviving CFC. These data further support the hypothesis and suggest that DFMO-induced cell cycle arrest enhances cellular retention of [3H] putrescine, increasing the effective internal radiation dose enough to cause proliferative death. In a clinical setting, the short (approximately 1 microm) path-length of the tritium beta particle should limit effects to the tumor cells and spare adjacent normal cells. These results support the concept that treatment with the combination of polyamine inhibitors and radioactive polyamines might be a useful adjunct to current therapies for glioblastoma multiforme.

Enhanced uptake of [3H] spermidine by 9L rat brain tumors after direct intratumoral infusion of inhibitors of enzymes of the polyamine biosynthetic pathway.

We have been exploring the feasibility of delivering ionizing radiation to brain tumor cells by using tritium labeled polyamines. Polyamines are taken up preferentially by dividing cells and form noncovalent bonds with DNA. Their uptake can be enhanced by drugs which deplete endogenous polyamines. To test this in vivo, 9L cells were implanted in the striatal region of the brain in male Fisher 344 rats. Osmotic pumps containing trace amounts of [3H] spermidine or [3H] putrescine with either difluoromethylornithine or combinations of 3 inhibitors of enzymes of the polyamine biosynthetic pathway were implanted subcutaneously and were connected to intratumoral cannulas. After 14-16 days the brains were removed and sliced in the coronal plane. The diameters of the tumors were measured and tumor tissue was dissected from each slice, weighed and lysed for scintillation counting. It was found that difluoromethylornithine enhanced the uptake of [3H] putrescine while a combination of inhibitors of enzymes of the polyamine biosynthetic pathway enhanced the uptake of [3H] putrescine and [3H] spermidine producing a localized region of radioactivity in the 9L tumor. It is estimated that if the [3H] polyamines were at higher specific activity (commercially available), instead of the trace dose given here, the [3H] polyamine uptake would be sufficient to kill 9L tumor cells within a 2 to 3 week period.

Characterization of the stage in natural killer cell development in 14.5-day mouse fetal liver using adult bone marrow stroma.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.

Bone marrow as a potential source of hepatic oval cells.

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

Expression of murine CD34 by fetal liver NK cell progenitors.

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma.

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.

Glutathione levels determine apoptosis in macrophages.

Spermine NONOate (SpNO, a nitric oxide donor) induced apoptosis and caspase-3 activity in the macrophage cell line RAW 267.4. RES cells that have been derived from RAW 267.4 cells by repeated exposure to lipopolysaccharide and interferon-gamma (LPS/INF-gamma), followed by outgrowth of viable cells, are resistant to apoptosis and caspase-3 activation upon exposure to SpNO. In this study we have determined that RES cells have lower levels of glutathione (GSH) and a higher oxidative state than RAW cells. Subsequently, RAW and RES cells were depleted of GSH by using l-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of GSH synthesis. GSH depleted cells did not undergo apoptosis nor demonstrate caspase-3 activity when they were exposed to SpNO. These results suggest that the redox status of the cell is one of the key factors mediating the apoptotic pathway in which glutathione plays a critical role in mediating apoptosis via NO* and reactive oxygen species (ROS).

A methodology report of a randomized prospective clinical trial to assess velopharyngeal function for speech following palatal surgery.

Cleft lip and palate occurs in approximately 1 in every 750 live human births, making it one of the most common congenital malformations. Surgical closure of the palatal cleft does not always result in a velopharyngeal port capable of supporting normal speech. The University of Florida (UF), in collaboration with the University of São Paulo (USP), is engaging in a 5-year prospective, randomized controlled study to compare velopharyngeal function for speech outcomes between patients undergoing palatoplasty for complete unilateral cleft lip and palate performed using the von Langenbeck procedure with intravelar velarplasty and those receiving the Furlow double-reversing Z-plasty palatoplasty. The von Langenbeck procedure was selected as the time-tested standard against which the Furlow procedure could be judged. The Furlow procedure, a relatively new operation, has been reported to yield substantially higher rates of velopharyngeal competency for speech than have most other reported series and theoretically should result in less disturbance to midfacial growth. A total of 608 patients will be entered into one of two age categories. Inclusion of two age groups will allow a comparison of results between patients having surgery before 1 year of age (9-12 months) and patients undergoing surgery at approximately 1.5 years of age (15-18 months). Speech data will be collected and will be available for definitive analysis throughout the last 3 years of the study. Collection of preliminary growth data will require more than 5 years; growth analysis is anticipated to continue until all patients have reached maturity. The Hospital for Research and Rehabilitation of Patients with Cleft Lip and Palate at the University of São Paulo (USP-HPRLLP) in Bauru, Brazil, is uniquely situated for conducting this study. The well-equipped and modern facilities are staffed by well-trained specialists representing all disciplines in cleft-palate management. In addition, an already existing social services network throughout Brazil will ensure excellent follow-up of study cases. The clinical caseload at this institution currently exceeds 22,000, and more than 1200 new cases are added annually. This project represents a unique opportunity to obtain prospective data from a large number of subjects while controlling the variables that have traditionally plagued cleft-palate studies. This study is designed to determine which of the two proposed surgical procedures is superior in constructing a velum capable of affecting velopharyngeal competency for the development of normal speech.

Suppression of natural killer cell differentiation by activated T lymphocytes in long-term cultures of mouse bone marrow.

The goal of the present work was to study the regulatory role of T lymphocytes on natural killer (NK) cell generation in NK long-term bone marrow cultures (LTBMCs), an established mouse long-term bone marrow (BM) culture system used for the study of NK cell differentiation from precursors. Activation of the few T cells present in NK-LTBMCs by addition of anti-CD3 monoclonal antibody (mAb) together with interleukin (IL)-2 inhibited the generation of NK cells. Coculture with NK-LTBMCs of a pure population of preactivated BM T cells completely inhibited NK cell development even when the T cells were separated from the NK-LTBMCs by transwells. Depletion of IL-2 by activated T cells was not the mechanism of the negative regulation because anti-CD3 mAb added to the cultures inhibited the generation of NK cells even in the presence of 10-fold higher concentrations of exogenous IL-2 than that used in controls. Medium from cultures in which suppression had occurred was also suppressive, suggesting that one or more soluble factors released in the medium was responsible. That this effect was exerted on NK cell development from precursors was indicated by the finding that T cell-conditioned medium stimulated proliferation of mature NK cells. In our experimental conditions, monoclonal antibodies to IL-10, IL-13, transforming growth factor-beta, and tumor necrosis factor receptor failed to reverse the inhibitory effect.

Lack of natural killer cell precursors in fetal liver of Ikaros knockout mutant mice.

The role of Ikaros in early stages of natural killer (NK) cell differentiation was investigated using an in vitro system that promotes proliferation and differentiation of NK cell precursors into mature NK1.1+ cells. Day 14.5 fetal liver cells from mice, either homozygous for Ikaros Null or dominant negative (DN) mutations, had severe 55- to 79-fold reductions in functional NK cell precursors. Although there was no statistically significant difference between values for +/+ and +/- Null mice, the mean precursor frequency for DN mutant (+/-) mice was significantly above that for DN -/- mice and below that for DN +/+ mice. The NK activity values for cells generated from the NK cell precursors followed the same respective relationships found for NK cell precursor frequencies. These data suggest that the deficiency of mature NK cells in Ikaros mutant mice is related to lack of functional precursors.

Stage II breast cancer: differences between four coping patterns in side effects during adjuvant chemotherapy.

Fifty-six women with stage II breast cancer receiving adjuvant chemotherapy were recruited for a study evaluating and comparing coping patterns for differences in physical and psychological side effects during treatment with adjuvant chemotherapy. Cluster analyses were used to split women into confrontive, avoidant-confrontive, avoidant-resigned, and resigned coping clusters. Side-effect measurements were taken on the day of adjuvant chemotherapy infusion and 3 and 7 days later. Repeated measures ANCOVAs indicated that coping clusters predicted significant variance in physical, psychological, and total side effects when variance in covariates was held constant. Confrontive subjects reported significantly fewer psychological and physical symptoms than avoidant-confrontive and avoidant-resigned copers. Confrontive copers also reported fewer side effects than resigned copers, but this difference was not significant when differences in covariate distributions were controlled. Particularly robust differences were noted when confrontive copers were compared with avoidant-confrontive copers. Results suggest that a critical component in optimal coping may be a willingness to discuss and think about illness.

Difluoromethylornithine enhanced uptake of tritiated putrescine in 9L rat brain tumors.

Difluoromethylornithine (DFMO) depletes endogenous putrescine and enhances the uptake of and retention of [3H] putrescine in vitro. To determine if DFMO also enhances uptake of [3H] putrescine in vivo, DFMO and trace doses of [3H] putrescine, dissolved in artificial CSF, were infused into growing (6-9 day) 9L brain tumors by means of osmotic pumps. When 7-day osmotic pumps were loaded with 1 microCi [3H] putrescine, with or without 10 or 100 mM DFMO, pumped at 1 microl/h, the mean uptake after 3 days was 168 +/- 62 cpm/mg tumor (17 rats) without DFMO, 300 +/- 197 cpm/mg tumor (11 rats) with 10 mM DFMO and 1088 +/- 421 cpm/mg tumor (11 rats) with 100 mM DFMO (p < or = 0.05 vs. control). Significantly less radioactivity was detected in the contralateral brain and in nonbrain tissues (0.5 +/- 0.1 to 14 +/- 5 cpm/mg). To measure the extent of [3H] putrescine distribution in the tumor, the same dose of drugs was delivered for a longer period of time, using 14-day pumps to allow tumors to become large enough to be divided into 1.4 mm thick transections. The mean radioactivity in the sections from eight control rats receiving [3H] putrescine without DFMO were not significantly different between the sections (174 +/- 61 cpm/mg tumor for sections containing the cannulas, 273 +/- 61 and 259 +/- 91 cpm/mg for adjacent sections). In the six rats given 100 mM DFMO there was a significant increase in mean radioactivity in the cannula containing section (2251 +/- 919 cpm/mg tumor). Mean counts from adjacent sections in these rats were 97 +/- 44 and 33 +/- 13 cpm/mg. Values for contralateral corpus striatum and nonbrain tissues ranged from 0.7 +/- 0.3 to 4.3 +/- 1.5 cpm/mg tissue. When DFMO was delivered directly to the tumors while [3H] putrescine was infused intraperitoneally, the uptake in the tumor slices was low (5-10 cpm/mg in different slices). These results demonstrate that infusion of DFMO directly into growing 9L brain tumors can selectively enhance the uptake of exogenous [3H] putrescine by rapidly dividing cells which are within a 1.4 mm diameter area at the cannula tip. Although these studies used [3H] putrescine at trace doses, it is estimated that infusion of higher doses of [3H] putrescine plus DFMO will selectively kill tumor cells.

Constitutive systemic expression of IL-1Ra or soluble TNF receptor by genetically modified hematopoietic cells suppresses LPS induction of IL-6 and IL-10.

We have been developing both local and systemic gene therapy approaches to treat inflammatory and autoimmune diseases. To determine if systemic, constitutive expression of biologically active anti-inflammatory agents is therapeutic and/or has associated toxicity, mouse hematopoietic stem cells were infected with retroviral vectors carrying the genes for human IL-1 receptor antagonist (IL-1Ra), human soluble TNF receptor p75 (sTNFR), or the beta-galactosidase (lacZ) gene, and transplanted into lethally irradiated recipients. The serum levels of human IL-1Ra and human sTNFR in the long-term reconstituted mice, 2-7 months after transplantation, were 596 and 158 ng/ml respectively. The long-term expression of human IL-1Ra had minimal effects on the PBMC profile whereas human sTNFR expression increased the percentage of B220 and Mac.1 stained cells and decreased slightly the specific T cell subsets. The ability of these proteins to protect the transplanted mice from endotoxin treatment was determined by measuring serum interleukin-6 (IL-6) and interleukin-10 (IL-10) responses after LPS injection at 1.5, 3, 4.5 and 24 h after treatment. The IL-1Ra group showed diminished IL-10 levels and less mortality after injection of LPS. These results demonstrate that constitutive, systemic expression of IL-1Ra and sTNFR is able to confer partial protective effects following treatment with endotoxin. These results further demonstrate that gene transfer methods which result in systemic, long-term expression of immunodulatory proteins could be applied to the treatment of inflammatory diseases.

Effects of recombinant cytokines on colony formation by irradiated human cord blood CD34+ hematopoietic progenitor cells.

The role of recombinant hematopoietic growth factors in radiation repair has become a subject of increasing interest in both clinical and basic radiobiology. Combinations of cytokines such as hepatocyte growth factor, interleukin (IL)-3, IL-11, kit ligand, GM-CSF and erythropoietin were used to study the in vitro radiation dose response of human cord blood CD34+ hematopoietic progenitor cells using clonogenic survival assays. CD34+ cells were isolated by immunomagnetic selection and irradiated at 8 cGy/min. Irradiated cells were plated in methylcellulose with or without added cytokines, and hematopoietic colonies including CFU-GM, BFU-E and CFU-GEMM were scored on day 14. The radiation response characteristics of BFU-E and CFU-GEMM were similar for all culture conditions tested. The D0 values for BFU-E ranged between 1.29 and 2.40 Gy and n between 1.0 and 1.4. The D0 values for CFU-GEMM ranged from 86 cGy to 2.02 Gy and n between 1.0 and 1.5. The D0 for CFU-GM grown without added factors was 1.03 Gy. With single cytokine stimulation (IL-3, IL-11 or varying concentrations of HGF), D0 values ranged from 1.11 to 1.44 Gy. With the combination of IL-3, GM-CSF, kit ligand and HGF, D0 values were not significantly altered and ranged between 1.61 and 2.60 Gy. In contrast, the combination of IL-11 and HGF produced an increase in the shoulder of the radiation survival curve (n = 3.35). No increase in the shoulder was detected for any of the other conditions tested (n = 1.0-1.7). Thus the combination of HGF and IL-11 increased the radiation survival of hematopoietic progenitor cells forming CFU-GM. Understanding the mechanism by which combinations of early-acting growth factors support postirradiation recovery of primitive clonogenic hematopoietic cells may be relevant to the design of clinical protocols for improving hematopoietic recovery after total-body irradiation.

lacZ transduction of 9L rat tumor cells without cloning.

Several laboratories have introduced the lacZ gene into 9L cells to improve the usefulness of this already popular rat brain tumor model. However, these laboratories were not concerned about possible changes in the phenotypic characteristics of the 9L cell line which can be induced by the selection of lacZ-expressing clones. Here, we describe a method for introducing the lacZ gene into 9L cells without selective cloning. The 9L parent cells (passaged the same number of times) and 9L/lacZ cells were compared in a number of tests and found to have the same phenotype. Specifically, they had the same sensitivity to radiation from external gamma or internal beta radiation, the same growth rates with or without frequent media changes and the same patterns of growth in rat brain. We demonstrated that the 9L/lacZ cells could be sorted from dissociated tumors by flow cytometry and the percentage of nonmalignant versus malignant cells determined. These percentages were variable from rat to rat. The colony-forming efficiency could be determined on the basis of whole tumor or, by using the percent of lacZ-positive cells, on the basis of malignant cells in a tumor. These novel approaches should render the 9L tumor model even more useful.

Peroxynitrite protects RAW 264.7 macrophage from Lipopolysaccharide/Interferon-gamma-induced cell death.

Peroxynitrite, formed by the interaction of superoxide with nitric oxide, has previously been implicated mostly as a cytotoxic agent. In contrast, its physiological and, possibly, beneficial effects are largely unknown. We have previously shown [Journal of Biological Chemistry, 1997, 272, 7253] that RAW 264.7 macrophages can be selected to be resistant toward lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-induced cytotoxicity. Resistant cells produced comparable amount of nitric oxide, but showed increased formation of superoxide, which might lead to increased production of peroxynitrite. We utilized this well characterized cell model to seek evidence that peroxynitrite might cause protection of RAW cells from cytokine toxicity. Exogenous peroxynitrite (30-50 microM), applied to RAW cells before cytokine stimulation, dramatically reduced LPS/IFN-gamma toxicity. Measurement of cell viability after overnight incubation with a mixture of LPS (10 microg/ml) and IFN-gamma (100 U/ml), showed that pretreatment with 40 microM peroxynitrite completely reverted LPS/IFN-gamma cytotoxicity. Differently, pretreatment of RAW cells with peroxynitrite (10-60 microM) did not prevent cytotoxicity induced by the nitric oxide-donors S-Nitroso-L-glutathione (0.2-1 mM), or spermine NONOate (0.2-2 mM), and by Actimomycin D (0.5-1 microg/ml), suggesting that the protective effect is specific for the LPS/IFN-gamma pathway. These results were confirmed through extensive controlled studies aimed to optimize cell exposure to peroxynitrite, and showed that peroxynitrite protects macrophages from cytokine-induced cytotoxicity.

Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.