Expression of acidic fibroblast growth factor cDNA confers growth advantage and tumorigenesis to Swiss 3T3 cells.

Acidic fibroblast growth factor (aFGF), a polypeptide with a mol. wt of approximately 16,000, is a potent mitogen for a variety of cells and shares 55% amino acid sequence identity with basic FGF. The recent isolation of three new oncogenes which share 35-45% amino acid sequence similarity with the FGFs suggests that the coding sequences for the FGFs themselves may be oncogenic under certain circumstances. To test this hypothesis, we cotransfected 3T3 NR6 cells with factors expressing the aFGF coding sequence and the bacterial neomycin gene. The aFGF produced by cotransfected cells was found only in the cellular homogenate and not in medium conditioned by the cells. Cells expressing aFGF grew to 10 times the density of control cells at saturation and were multilayered and disorganized, similar to transformed cells. The cotransfected cells do not grow in soft agar, but show enhanced soft agar growth relative to controls in the presence of added aFGF and heparin. The aFGF-producing cells formed small, non-progressive tumors when injected subcutaneously into nude mice. Our data suggest that expression of aFGF in NR6 cells results in enhanced growth, and that several traits characteristic of the transformed phenotype are partially expressed.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Epitope Mapping of Broadly Neutralizing HIV-2 Human Monoclonal Antibodies

Recent studies have shown that natural infection by HIV-2 leads to the elicitation of high titers of broadly neutralizing antibodies (NAbs) against primary HIV-2 strains (T. I. de Silva, et al., J. Virol. 86:930–946, 2012; R. Kong, et al., J. Virol. 86:947–960, 2012; G. Ozkaya Sahin, et al., J. Virol. 86:961–971, 2012). Here, we describe the envelope (Env) binding and neutralization properties of 15 anti-HIV-2 human monoclonal antibodies (MAbs), 14 of which were newly generated from 9 chronically infected subjects. All 15 MAbs bound specifically to HIV-2 gp120 monomers and neutralized heterologous primary virus strains HIV-2_7312A and HIV-2_ST. Ten of 15 MAbs neutralized a third heterologous primary virus strain, HIV-2_UC1. The median 50% inhibitory concentrations (IC₅₀s) for these MAbs were surprisingly low, ranging from 0.007 to 0.028 μg/ml. Competitive Env binding studies revealed three MAb competition groups: CG-I, CG-II, and CG-III. Using peptide scanning, site-directed mutagenesis, chimeric Env constructions, and single-cycle virus neutralization assays, we mapped the epitope of CG-I antibodies to a linear region in variable loop 3 (V3), the epitope of CG-II antibodies to a conformational region centered on the carboxy terminus of V4, and the epitope(s) of CG-III antibodies to conformational regions associated with CD4- and coreceptor-binding sites. HIV-2 Env is thus highly immunogenic in vivo and elicits antibodies having diverse epitope specificities, high potency, and wide breadth. In contrast to the HIV-1 Env trimer, which is generally well shielded from antibody binding and neutralization, HIV-2 is surprisingly vulnerable to broadly reactive NAbs. The availability of 15 human MAbs targeting diverse HIV-2 Env epitopes can facilitate comparative studies of HIV/SIV Env structure, function, antigenicity, and immunogenicity.     

Potent autologous and heterologous neutralizing antibody responses occur in HIV-2 infection across a broad range of infection outcomes

Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC(50)], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC(50)s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.     

Introns and reading frames: correlation between splicing sites and their codon positions

Computer analyses of the entire GenBank database were conducted to examine correlation between splicing sites and codon positions in reading frames. Intron insertion patterns (i.e., splicing site locations with respect to codon positions) have been analyzed for all of the 74 codons of all the eukaryote taxonomic groups: primates, rodents mammals, vertebrates, invertebrates, and plants. We found that reading frames are interrupted by an intron at a codon boundary (as opposed to the middle of a codon) significantly more often than expected. This observation is consistent with the exon shuffling hypothesis, because exons that end at codon boundaries can be concatenated without causing a frame shift and thus are evolutionarily advantageous. On the other hand, when introns interrupt at the middles of codons, they exist in between the first and second bases much more frequently than between the second and third bases, despite the fact that boundaries between the first and second bases of codons are generally far more important than those between the second and third bases. The reason for this is not clear and yet to be explained. We also show that the length of an exon is a multiple of 3 more frequently than expected. Furthermore, the total length of two consecutive exons is also more frequently a multiple of 3. All the observations above are consistent with results recently published by Long, Rosenberg, and Gilbert (1995).      

Metabolome and metaboproteome remodeling in nuclear reprogramming

Nuclear reprogramming resets differentiated tissue to generate induced pluripotent stem (iPS) cells. While genomic attributes underlying reacquisition of the embryonic-like state have been delineated, less is known regarding the metabolic dynamics underscoring induction of pluripotency. Metabolomic profiling of fibroblasts vs. iPS cells demonstrated nuclear reprogramming-associated induction of glycolysis, realized through augmented utilization of glucose and accumulation of lactate. Real-time assessment unmasked downregulated mitochondrial reserve capacity and ATP turnover correlating with pluripotent induction. Reduction in oxygen consumption and acceleration of extracellular acidification rates represent high-throughput markers of the transition from oxidative to glycolytic metabolism, characterizing stemness acquisition. The bioenergetic transition was supported by proteome remodeling, whereby 441 proteins were altered between fibroblasts and derived iPS cells. Systems analysis revealed overrepresented canonical pathways and interactome-associated biological processes predicting differential metabolic behavior in response to reprogramming stimuli, including upregulation of glycolysis, purine, arginine, proline, ribonucleoside and ribonucleotide metabolism, and biopolymer and macromolecular catabolism, with concomitant downregulation of oxidative phosphorylation, phosphate metabolism regulation, and precursor biosynthesis processes, prioritizing the impact of energy metabolism within the hierarchy of nuclear reprogramming. Thus, metabolome and metaboproteome remodeling is integral for induction of pluripotency, expanding on the genetic and epigenetic requirements for cell fate manipulation.     

Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (～50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.     

Natural killer cell function is well preserved in asymptomatic human immunodeficiency virus type 2 (HIV-2) infection but similar to that of HIV-1 infection when CD4 T-cell counts fall

Natural killer (NK) cells are potent effectors of natural immunity and their activity prevents human immunodeficiency virus type 1 (HIV-1) viral entry and viral replication. We sought to determine whether NK immune responses are associated with different clinical course of HIV-1 and HIV-2 infections. A cross-sectional analysis of NK cell responses was undertaken in 30 HIV-1 and 30 HIV-2 subjects in each of three categories of CD4(+)-T-cell counts (>500, 200 to 500, and <200 cells/microl) and in 50 HIV-uninfected control subjects. Lytic activity and gamma interferon (IFN-gamma) secretion were measured by chromium release and enzyme-linked immunospot assays, respectively. Flow cytometry was used to assess intracellular cytokines and chemokines. Levels of NK cytotoxicity were significantly higher in HIV-2 than in HIV-1 infections in subjects with high CD4(+)-T-cell counts and were similar to that of the healthy controls. In these HIV-2 subjects, cytolytic activity was positively correlated to NK cell count and inversely related to plasma viremia. Levels of intracellular MIP-1beta, RANTES, tumor necrosis factor alpha, and IFN-gamma produced by NK CD56(bright) cells were significantly higher in HIV-2- than HIV-1-infected subjects with high CD4(+)-T-cell counts but fell to similar levels as CD4 counts dropped. The data suggest efficient cytolytic and chemokine-suppressive activity of NK cells early in HIV-2 infection, which is associated with high CD4(+) T-cell counts. Enhancement of these functions may be important in immune-based therapy to control HIV disease.     

Erythrocyte Hemolysis and Hemoglobin Oxidation Promote Ferric Chloride-induced Vascular Injury

The release of redox-active iron and heme into the blood-stream is toxic to the vasculature, contributing to the development of vascular diseases. How iron induces endothelial injury remains ill defined. To investigate this, we developed a novel ex vivo perfusion chamber that enables direct analysis of the effects of FeCl₃ on the vasculature. We demonstrate that FeCl₃ treatment of isolated mouse aorta, perfused with whole blood, was associated with endothelial denudation, collagen exposure, and occlusive thrombus formation. Strikingly exposing vessels to FeCl₃ alone, in the absence of perfused blood, was associated with only minor vascular injury. Whole blood fractionation studies revealed that FeCl₃-induced vascular injury was red blood cell (erythrocyte)-dependent, requiring erythrocyte hemolysis and hemoglobin oxidation for endothelial denudation. Overall these studies define a unique mechanism of Fe³⁺-induced vascular injury that has implications for the understanding of FeCl₃-dependent models of thrombosis and vascular dysfunction associated with severe intravascular hemolysis.Iron and heme-containing moieties are indispensable for the normal transport of oxygen in the blood; however, once released into the bloodstream these molecules are highly toxic to the vasculature because of their pro-oxidative effects on the endothelium (1-3). Humans have therefore evolved sophisticated iron transport and sequestration systems as well as heme-metabolizing enzymes to rapidly clear iron and heme from the circulation (4, 5). There is growing evidence that defects in these natural protective mechanisms lead to endothelial dysfunction and vascular disease, and as a consequence, methods that reduce the pro-oxidative effects of iron and heme may have therapeutic benefit (2).Clinical syndromes associated with marked intravascular hemolysis and circulating free hemoglobin, such as sickle cell disease, paroxysmal nocturnal hemoglobinuria, thalassemias, and hereditary spherocytosis, lead to endothelial dysfunction, thrombosis, and vascular disease (5-10). Similarly administration of purified recombinant hemoglobin to humans promotes vascular injury and arterial thrombosis, precipitating acute myocardial infarction (11-13). Some of these vascular effects are related to nitric oxide scavenging by excess plasma hemoglobin, whereas others are linked to cytotoxic, proinflammatory, and pro-oxidant effects of iron-containing hemoglobin and heme (14-19). Interestingly elevated levels of body iron stores are associated with an increased risk of myocardial infarction, and carriers of the hemochromatosis gene have an increased risk of myocardial infarction and cardiovascular death (20, 21). Whether the pro-oxidative effects of iron per se are proatherogenic remains controversial; however, in the context of erythrocyte-dependent release of hemoglobin and heme, redox-active iron is likely to play an important role in promoting vascular dysfunction.The well defined pro-oxidative properties of redox-active iron have been exploited experimentally with topical application of ferric chloride (FeCl₃) widely used to induce vascular injury and thrombosis in experimental animal models (22). High concentrations of FeCl₃ induce profound injury to the vasculature, leading to endothelial denudation, and collagen and tissue factor exposure, leading to the rapid formation of vaso-occlusive thrombi. Histologically FeCl₃-induced thrombi are rich in platelets, fibrin, and red blood cells (23-26). However, the mechanism(s) by which FeCl₃ induces vascular injury has not been clearly defined. FeCl₃ can have direct pro-oxidative effects on endothelial cells as a result of the Fenton reaction, leading to hydroxyl radical generation and lipid peroxidation (1, 3). It can also mediate vascular injury indirectly through oxidative modification of LDL³ (3, 14). A recent study has demonstrated transfer of ferric ions through the vasculature, penetrating the internal elastic membrane and emerging through the endothelium via an endocytic/exocytic pathway, leading to the development of ferric oxide aggregates in the vascular lumen (27). Although the direct cytotoxic effects of redox-active iron on endothelial cells have been well established in vitro, the importance of this mechanism to the severe vascular injury and thrombus formation induced by topical FeCl₃ in vivo remains unclear.To gain insight into this, we developed a novel ex vivo perfusion chamber that enables direct analysis of the effects of FeCl₃ on the vasculature. Our studies demonstrated that FeCl₃ alone induces relatively mild injury to endothelial cells with severe vascular injury only observed in the presence of flowing blood. Whole blood fractionation studies revealed that FeCl₃-mediated vascular injury is dependent on erythrocyte hemolysis and hemoglobin oxidation, defining a unique mechanism of iron-induced vascular injury.