Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters.

The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines results in serious, potentially life-threatening, adverse effects. By contrast, nucleic acid transfer via electroporation (EP) is a safe and effective method of delivering plasmid-encoded cytokines to tumors. Intratumoral delivery of IL-12 plasmid DNA by electroporation (IT-pIL12-EP) produced objective response rates in Phase 2 clinical trials in metastatic melanoma. However, only 17.9% of patients receiving IT-pIL12-EP show a complete therapeutic response. Here, we sought to improve the antitumor efficacy of our clinical IT-pIL12-EP plasmid electroporation platform. We evaluated multiple plasmid designs for IL-12 expression. IL-12 expression from a plasmid incorporating a picornavirus-derived co-translational P2A site was the most effective in expressing IL-12p70. In addition, modifying the electroporation parameters improved transfection efficiency and expression of plasmid-derived IL-12p70, as well as its downstream effector IFN-γ in vivo. Finally, using a murine melanoma model that is representative of the intended target patient population, we show that combining modified electroporation conditions with the pIL12-P2A plasmid expression enhances the systemic antitumor response. These improvements to the IT-pIL12-EP platform may improve patient clinical response rates and survival when translated to clinical trials.

Modeling a submicrometer electrostatic motor.

Numerical models are developed for a recently proposed submicrometer device that uses the electric field energy of a biased parallel-plate semiconducting capacitor to propel a piston through the open capacitor gap. Through variation of design parameters or applied external bias, actuator forces on the order of hundreds of piconewtons are developed for device size scales ranging from 10(-7) m to 10(-4) m per side. A rotary configuration of the device is also presented.

Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.

In order to investigate the in vivo functions of protein kinase CK2 (CK2), the expression of Myc-tagged versions of the subunits, Myc-CK2alpha and Myc-CK2beta, was carried out in Chinese hamster ovary cells (CHO cells) and in 3T3 L1 fibroblasts. Cell proliferation in these cells was examined. CHO cells that transiently overexpressed the Myc-CK2beta subunit exhibited a severe growth defect, as shown by a much lower value of [(3)H]thymidine incorporation than the vector controls, and a rounded shrunken morphology. In contrast, cells overexpressing Myc-tagged CK2alpha showed a slightly but consistently higher value of [(3)H]thymidine incorporation than the controls. The defect in cell growth and changes in morphology caused by Myc-CK2beta overexpression were partially rescued by coexpression of Myc-tagged CK2alpha. In parallel to the studies in CHO cells, the stable transfection of Myc-CK2alpha and Myc-CK2beta subunits was achieved in 3T3 L1 fibroblast cells. Similarly, the ectopic expression of Myc-CK2beta, but not Myc-CK2alpha, caused a growth defect. By measuring [(3)H]thymidine incorporation, it was found that expression of Myc-CK2beta prolonged the G(1) phase and inhibited up-regulation of cyclin D1 expression during G(1). In addition, a lower mitotic index and lower mitotic cyclin-dependent kinase activities were detected in Myc-CK2beta-expressing cells. Detailed analysis of stable cells that were synchronously released into the cell cycle revealed that the expression of Myc-CK2beta inhibited cells entering into mitosis and prevented the activation of mitotic cyclin-dependent kinases. Taken together, results from both transient and stable expression of CK2 subunits strongly suggest that CK2 may be involved in the control of cell growth and progression of the cell cycle.

Mitogen-activated protein kinase kinase activity is required for the G(2)/M transition of the cell cycle in mammalian fibroblasts.

The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G(0), the level of MAPKK activity was observed to affect the kinetics of progression through both the G(1) and G(2) phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G(0)/G(1) progression, was found to also delay progression of cells through G(2). In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G(2) phase. These data demonstrate a novel role for the MAPK cascade in progression from G(2) into mitosis in NIH 3T3 cells.

The response of crying newborns to sucrose: is it a "sweetness" effect?

Intraoral sucrose (and other sweet carbohydrates) induce rapid and sustained calming in crying newborns and transiently increase mouthing and hand-mouth contact ("sucrose effects"). To investigate whether these effects are due to the sweetness of sucrose, 60 crying newborns were randomized to receive 250 microL of 24% sucrose solution, 0.12% of aspartame solution of equivalent sweetness (to adults), or 24% polycose, a soluble carbohydrate that is only very slightly sweet (to adults), as well as water in a mixed parallel crossover design. Relative to water, sucrose persistently reduced crying, and transiently increased mouthing and hand-mouth contact, as previously demonstrated. Aspartame also reduced crying, and transiently increased mouthing and hand-mouth contact, virtually mimicking the time course and the magnitude of the effects obtained in response to sucrose. By contrast, polycose solution had no specific effects on crying, mouthing, or hand-mouth contact. The results imply that the responses of crying newborns to intraoral sucrose are neither specific to sucrose nor to the general class of carbohydrates, and that these effects are more appropriately understood as "sweetness" effects.

Differential calming responses to sucrose taste in crying infants with and without colic.

BACKGROUND: Colic is a behavioral syndrome of early infancy of unknown etiology whose core symptom is increased crying. Both clinical anecdotal descriptions and controlled observations converge in suggesting that a defining characteristic of the crying behavior is the longer duration of the crying bouts, especially during the second month of life when colic is at its peak. This implies that, once infants with colic begin crying, they do not calm as well as infants without colic. To investigate this difference objectively, we used response to sucrose taste as a probe of colic-normal differences in regulation of crying for three reasons. First, sucrose taste has been shown to be a potent regulator of crying in human newborns. Second, convergent evidence from studies in both nonhuman and human infants suggests that sucrose calming reflects the function of central distress regulatory systems that are opioid-dependent. Third, effectiveness of sucrose calming diminishes in normal infants by 4 to 6 weeks of age, consistent with the developmental increase in crying duration common to infants with and without colic. Consequently we predicted that, if the regulation of crying by sucrose taste is relevant to the crying of infants with colic, calming responses to sucrose taste should be less effective in 6-week-old infants with colic compared with those without. OBJECTIVES: To investigate the clinical observation that infants with and without colic differ in their ability to regulate their crying behavior, our primary objective was to determine if there were differential crying responses to intraoral sucrose tastes (relative to water) in crying infants with and without colic. Based on previous studies of calming responses to sucrose taste, it was predicted that sucrose-specific calming before a feeding would be less effective in infants with colic than in those without. A secondary and more exploratory aim was to assess calming responses to sucrose (relative to water) on spontaneous crying after a feed in both groups. METHODS: Nineteen infants meeting modified Wessel's criteria for colic were compared with 19 age- and sex-matched normal infants without colic in a within-subject controlled observation of calming and mouthing responses to both intraoral sucrose and water tastes. Both before and after two feedings on the same day, each infant was observed until she or he cried continuously for 15 consecutive seconds, at which time three 250-microL tastes of 48% sucrose solution or sterile water were administered 30 seconds apart, and infant responses videotaped. Outcome measures derived from second-by-second coding of the videorecordings were percent time crying per minute for 4 minutes and percent time mouthing per minute for 2 minutes after stimulus administration. RESULTS: As predicted, the crying of infants with colic was less affected than the crying of infants without colic after sucrose but not water tastes before feedings. After feedings, the crying of infants with colic was less affected than the crying of infants without colic for both sucrose and water tastes, and sucrose was more effective than water in both groups of infants. These calming differences could not be attributed to differences in mouthing responses because the calming effects persisted after mouthing ceased, and there were no differences in mouthing responses between groups before or after feedings. CONCLUSIONS: As in newborns, a significant calming effect of sucrose taste that persisted beyond the cessation of mouthing could be elicited in crying 6-week-old infants, but it required a stronger taste stimulus to do so. As predicted, infants with colic were less effectively calmed by sucrose taste than infants without. These differential effects could not be accounted for by differences in crying when the stimulus was applied or by differences in mouthing behavior. Before a feed, these differences in calming were specific to sucrose taste. After a feed, infants with c

[Cognitive therapy for chronic and severe mental disorders.]. / Thérapie cognitive des troubles mentaux graves et persistants.

This article presents an overview of cognitive therapy applied to the treatment of three psychotic symptoms: delusions, hallucinations and negative symptoms often resistant to medication. It describes the essential therapeutic conditions for the success of such a therapy including therapeutic alliance. Clinical vignettes illustrate how sessions work. Finally, the article concludes with results on the efficiency of this intervention.

Computer-assisted psychotherapy.

The rationale for using computers in psychotherapy includes the possibility that therapeutic software could improve the efficiency of treatment and provide access for greater numbers of patients. Computers have not been able to reliably duplicate the type of dialogue typically used in clinician-administered therapy. However, computers have significant strengths that can be used to advantage in designing treatment programs. Software developed for computer-assisted therapy generally has been well accepted by patients. Outcome studies have usually demonstrated treatment effectiveness for this form of therapy. Future development of computer tools may be influenced by changes in health care financing and rapid growth of new technologies. An integrated care delivery model incorporating the unique attributes of both clinicians and computers should be adopted for computer-assisted therapy.

Differential response to intraoral sucrose, quinine and corn oil in crying human newborns.

Intraoral sucrose induces rapid and sustained calm in crying newborns and transiently increases mouthing and hand-mouth contact. To determine whether these effects are specific to sucrose and to explore which properties of orogustatory stimuli might contribute to this effect, 60 crying newborns were randomized to receive 250 ul of 24% sucrose solution, 0.25% quinine hydrochloride solution, or corn oil as well as water in a mixed parallel crossover design. Relative to water, sucrose persistently reduced crying, and transiently increased mouthing and hand-mouth contact as previously demonstrated. While quinine produces a "disgust" face in calm infants, in crying infants it transiently decreased crying and increased mouthing, but did not affect hand-mouth contact. Corn oil had no specific effect on crying, mouthing or hand-mouth contact. The results imply that crying newborns respond differentially to orogustatory stimuli, that taste "salience" rather than positive hedonic valence may account for initial crying reduction and increased mouthing, and that these behavioral effects are not induced by at least one lipid nutrient.

A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation.

SHP-1 and SHP-2 are intracellular protein tyrosine phosphatases containing two adjacent src homology 2 domains that target these phosphatases to cell surface receptor signaling complexes and play a role in receptor signal transduction. In this report the PC12 cell system was used to investigate the potential roles of SHP-1 and SHP-2 in the induction of neuronal differentiation by nerve growth factor (NGF). By using neurite outgrowth as a marker for differentiation, the effects of transfected constructs of SHP-1 and SHP-2 were assessed. Overexpression of a catalytically inactive SHP-2, but not a catalytically inactive SHP-1, blocked NGF-stimulated neurite outgrowth. The mitogen-activated protein kinase (MAPK) signaling cascade is important for the morphological differentiation in PC12 cells, and both SHP-1 and SHP-2 have been implicated to act upstream of MAPK in other receptor signaling systems. A positive role for SHP-2 but not SHP-1 in the activation of MAPK by NGF was demonstrated by introduction of the SHP-2 phosphatase mutants along with hemagglutinin-tagged MAPK. Coexpression studies with the SHP-2 mutant along with mutant forms of MAPK kinase suggested that SHP-2 functions upstream of MAPK kinase and MAPK in NGF-induced neurite outgrowth.

Ornithine decarboxylase overexpression leads to increased epithelial tumor invasiveness.

Ornithine decarboxylase (ODC) overexpression cooperates with genetic lesions such as an activated c-rasHa to enhance epithelial tumorigenesis. To assess the invasiveness of ODC-overexpressing cells, two noninvasive epidermal cell lines, nontumorigenic BK-1 cells, and the papilloma-derived cell line SP-1 were infected with a replication-defective retrovirus that overexpresses ODC, inoculated into deepithelialized rat tracheas, and transplanted into athymic nude mice. After 5 weeks, ODC-overexpressing BK-1 cells remained localized on the luminal surface of the tracheal xenotransplants, whereas the ODC-overexpressing SP-1 cells were extremely invasive, with the whole tracheal wall penetrated. This invasiveness of ODC-overexpressing SP-1 cells was accompanied by elevated proteinase expression, including increased urokinase plasminogen activator activity in ODC-overexpressing cells and elevated stromelysin-1 mRNA expression in the stromal cells of invaded tracheal transplants.

Cognitive therapy with inpatients.

Psychotherapeutic interventions often play a major role in the treatment of patients who are hospitalized for depression. Much of the "therapeutic milieu" of the inpatient unit includes patient participation in group psychotherapy and in one-on-one psychotherapy with staff members. These interventions are designed not only to be primary treatments for depression, but are also used to enhance patients' compliance with pharmacotherapy. Cognitive therapy (CT) has been adapted for use with inpatients and has been used as an organizing theory for the hospital milieu in several inpatient units. Research on inpatient CT suggests that it is a beneficial treatment that enhances continuity of care after discharge from the hospital. This paper describes the general principles of inpatient CT, and discusses the various types of inpatient cognitive therapy units (CTUs) that have been developed. The benefits of such programs are described, and research regarding the effectiveness of inpatient CT is discussed.

Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.

"Sucrose analgesia" and diphtheria-tetanus-pertussis immunizations at 2 and 4 months.

In human newborns, small amounts of sucrose reduce crying with procedural pain by about 50%. To determine whether "sucrose analgesia" could be extended to painful procedures beyond the newborn period, 57 infants were randomly assigned to receive three 250-microliters doses of 50% sucrose solution (g/100 mL) or water before their diphtheria-tetanus-pertussis immunizations at 2 and 4 months of age. Crying during and after injection was measured separately to determine whether sucrose modified crying during the noxious stimulus, recovery from the stimulus, or both. Sucrose was effective in reducing crying only from 83 to 69%, and the reduction was limited to the postinjection period. We conclude that, although sucrose continues to have some effect beyond the newborn period, the effect is limited to recovery from the noxious stimulus, is clinically modest, and is probably smaller than in the newborn period.

Protein-DNA interactions in soluble telosomes from Saccharomyces cerevisiae.

Telomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210). The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays. Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes. The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes. Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes. These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt. In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210; Conrad, M. N., Wright, J. H., Wolf, A. J. and Zakian, V. A. (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

A switch from stromal to tumor cell expression of stromelysin-1 mRNA associated with the conversion of squamous to spindle carcinomas during mouse skin tumor progression.

We previously reported that the expression of stromelysin-1 (ST-1), a matrix-degrading metalloproteinase, correlates with tumor progression in the mouse skin model of carcinogenesis. Using in situ hybridization techniques, we confirmed in this study the expression of ST-1 mRNA in mouse skin keratinocytes treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and also observed dramatic expression of ST-1 message in underlying fibroblastic cells. Benign tumors formed by an initiation/promotion protocol expressed low levels of ST-1 mRNA, which was localized exclusively to stromal tissue surrounding the tumor cells. Squamous cell carcinomas, produced either by chemical carcinogenesis or by injection of cultured cells derived from chemically initiated squamous cell tumors, expressed high levels of ST-1 mRNA, which was also localized to adjacent stromal tissues. In contrast, aggressive, highly metastatic spindle cell tumors expressed ST-1 mRNA in the tumor cells as well as in normal, adjacent stroma. These results suggest that the change from ST-1 expression in surrounding stromal cells to its expression in the tumor cells themselves is associated with the conversion of squamous to spindle carcinomas and may play a causal role in the ability of these cells to invade and metastasize.

The structure of attentional control: contingent attentional capture by apparent motion, abrupt onset, and color.

Five spatial cuing experiments tested 2 hypotheses regarding attentional capture: (a) Attentional capture is contingent on endogenous attentional control settings, and (b) attentional control settings are limited to the distinction between dynamic and static discontinuities (C. L. Folk, R. W. Remington, & J. C. Johnston, 1992). In Experiments 1 and 2, apparent-motion precues produced significant costs in performance for targets signaled by motion but not for targets signaled by color or abrupt onset. Experiment 3 established that this pattern is not due to differences in the difficulty of target discrimination. Experiments 4 and 5 revealed asymmetric capture effects between abrupt onset and apparent motion related to stimulus salience. The results support the hypotheses of Folk et al. (1992) and suggest that stimulus salience may also play a role in attentional capture.