HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.

Escherichia coli transcript cleavage factors GreA and GreB stimulate promoter escape and gene expression in vivo and in vitro.

The process of RNA chain initiation by RNA polymerases plays a central role in the regulation of transcription. In this complex phase of transcription, short oligomers are synthesized and released from the enzyme-promoter complex in a reaction termed abortive initiation. The polymerase undergoes many cycles of abortive initiation prior to completion of the initiation process, which is signaled by the translocation of the enzyme away from the promoter, release of sigma factor, and formation of an elongation complex in which the RNA is stably bound. We have studied the parameters that affect escape from the promoter by Escherichia coli RNA polymerase for the phage T7 A1 promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25antiDSR. The latter site contains a synthetic initial transcribed region that reduces its ability to synthesize RNA both in vivo and in vitro. Clearance from T5 N25antiDSR can be stimulated up to 10-fold in vitro by addition of the E. coli transcript cleavage factor GreA or GreB, but these factors have little effect on transcription from the normal T7 A1 or T5 N25 promoters. Using an E. coli strain lacking GreA and GreB, we were also able to show stimulation of transcription by the Gre factors from the T5 N25antiDSR promotor in vivo. The stimulation of RNA chain initiation by Gre factors, together with their known biochemical properties in the transcription elongation reaction, suggests some specific models for steps in the transcription initiation reaction.

TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

Directed seed dispersal by bellbirds in a tropical cloud forest

A fundamental goal of plant population ecology is to understand the consequences for plant fitness of seed dispersal by animals. Theories of seed dispersal and tropical forest regeneration suggest that the advantages of seed dispersal for most plants are escape from seed predation near the parent tree and colonization of vacant sites, the locations of which are unpredictable in space and time. Some plants may gain in fitness as a fortuitous consequence of disperser behavior if certain species of dispersers nonrandomly place seeds in sites predictably favorable for seedling establishment. Such patterns of directed dispersal by vertebrates long have been suggested but never demonstrated for tropical forest trees. Here we report the pattern of seed distribution and 1-year seedling survival generated by five species of birds for a neotropical, shade-tolerant tree. Four of the species dispersed seeds to sites near the parent trees with microhabitat characteristics similar to those at random locations, whereas the fifth species, a bellbird, predictably dispersed seeds under song perches in canopy gaps. The pattern of seedling recruitment was bimodal, with a peak near parent trees and a second peak, corresponding to bellbird song perches, far (>40 m) from parent trees. Seedling survival was higher for seeds dispersed by bellbirds than by the other species, because of a reduction in seedling mortality by fungal pathogens in gaps. Thus, bellbirds play a significant role in seed dispersal by providing directed dispersal to favorable sites and therefore may influence plant recruitment patterns and species diversity in Neotropical forests.

β-Chemokines and neutralizing antibody titers correlate with sterilizing immunity generated in HIV-1 vaccinated macaques

One of the obstacles to AIDS vaccine development is the variability of HIV-1 within individuals and within infected populations, enabling viral escape from highly specific vaccine induced immune responses. An understanding of the different immune mechanisms capable of inhibiting HIV infection may be of benefit in the eventual design of vaccines effective against HIV-1 variants. To study this we first compared the immune responses induced in Rhesus monkeys by using two different immunization strategies based on the same vaccine strain of HIV-1. We then utilized a chimeric simian/HIV that expressed the envelope of a dual tropic HIV-1 escape variant isolated from a later time point from the same patient from which the vaccine strain was isolated. Upon challenge, one vaccine group was completely protected from infection, whereas all of the other vaccinees and controls became infected. Protected macaques developed highest titers of heterologous neutralizing antibodies, and consistently elevated HIV-1-specific T helper responses. Furthermore, only protected animals had markedly increased concentrations of RANTES, macrophage inflammatory proteins 1α and 1β produced by circulating CD8+ T cells. These results suggest that vaccine strategies that induce multiple effector mechanisms in concert with β-chemokines may be desired in the generation of protective immune responses by HIV-1 vaccines.

Sodium channel selectivity filter regulates antiarrhythmic drug binding

Local anesthetic antiarrhythmic drugs block Na+ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel’s selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na+ channel (μ1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na+ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.

Increased liposome extravasation in selected tissues: effect of substance P.

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.

Macrophage killing is an essential virulence mechanism of Salmonella typhimurium.

Phagocytic cells are a critical line of defense against infection. The ability of a pathogen to survive and even replicate within phagocytic cells is a potent method of evading the defense mechanisms of the host. A number of pathogens survive within macrophages after phagocytosis and this contributes to their virulence. Salmonella is one of these pathogens. Here we report that 6-14 hr after Salmonella enters the macrophage and replicates, it resides in large vacuoles and causes the destruction of these cells. Furthermore, we identified four independently isolated MudJ-lacZ insertion mutants that no longer cause the formation of these vacuoles or kill the macrophages. All four insertions were located in the ompR/envZ regulon. These findings suggest that killing and escape from macrophages may be as important steps in Salmonella pathogenesis as are survival and replication in these host cells.

Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG.

Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light.

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ± 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.