Regulation of transgene expression in genetic immunization

The use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. Essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. Many genetic immunization vectors use promoter elements from pathogenic viruses including SV40 and CMV. Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of IFN-g on transgene expression driven by viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the MHC I promoter, treatment with IFN-g inhibited transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

Control of CD4 gene expression: connecting signals to outcomes in T cell development

The control of CD4 gene expression is essential for proper T lymphocyte development. Signals transmitted from the T-cell antigen receptor (TCR) during the thymic selection processes are believed to be linked to the regulation of CD4 gene expression during specific stages of T cell development. Thus, a study of the factors that control CD4 gene expression may lead to further insight into the molecular mechanisms that drive thymic selection. In this review, we discuss the work conducted to date to identify and characterize the cis-acting transcriptional control elements in the CD4 locus and the DNA-binding factors that mediate their function. From these studies, it is becoming clear that the molecular mechanisms controlling CD4 gene expression are very complex and differ at each stage of development. Thus, the control of CD4 expression is subject to many different influences as the thymocyte develops.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

Effect of the intracerebroventricular administration of GR 113808, a selective 5-HT4 antagonist, on water intake during hyperosmolarity and hypovolemia

We demonstrate here that acute third ventricle injections of GR 113808, a highly selective 5-HT4 antagonist, decrease water intake induced by a previous salt load while potentiating drinking elicited by hypovolemia induced by previous subcutaneous administration of polyethylene glycol in male Wistar rats (200 ± 20 g). At the dose of 160 nmol/rat, third ventricle injections of GR 113808 induced a significant reduction of water intake in salt-loaded animals after 120 min as compared to salt-loaded animals receiving third ventricle injections of saline (salt load + GR = 3.44 ± 0.41 ml, N = 12; salt load + saline = 5.74 ± 0.40 ml, N = 9). At the dose of 80 nmol/rat, GR 113808 significantly enhanced water intake in hypovolemic animals after 120 min as compared to hypovolemic animals receiving third ventricle injections of saline (hypovol + GR = 4.01 ± 0.27 ml, N = 8; hypovol + saline = 2.41 ± 0.23 ml, N = 12). We suggest that central 5-HT4 receptors may exert a positive drive on water intake due to hyperosmolarity and a negative input on drinking provoked by hypovolemia.

Behavioral characteristics of the offspring of adolescent rats

The aim of this study was to test the hypothesis that, during adulthood, the offspring of adolescent rats differ in emotionality, learning and memory from the offspring of adult rats. The behavior of the offspring of adolescent (age, 50-55 days) and adult rats (age, 90-95 days) was tested in the open field, activity cage, and passive and active avoidance apparatus. The latencies during training and testing in the passive avoidance apparatus of the offspring of adolescent parents were shorter than the latencies of control offspring (P<0.001 on both training and testing days). Offspring of adolescent parents showed shorter latency time in acquisition trials during active avoidance testing compared to control offspring (P<0.001). They also showed a higher number of active avoidance responses in the last four blocks of acquisition (P<0.001) and first two blocks of extinction trials (P<0.05 and P<0.001, respectively). The offspring of adolescent parents showed higher latency on the first day of testing in the open field (P<0.01) and a lower latency on the third day of testing (P<0.01). They also showed higher activity during all three days of testing (1st and 2nd day: P<0.01; 3rd day: P<0.05). The spontaneous activity of the offspring of adolescent parents in the activity cage was higher in the last three intervals of testing (P<0.001). In summary, the offspring of adolescent parents were less anxious and tended to be more active. The results of two learning and memory tests were opposite, but could be explained by a higher exploratory drive of the offspring of adolescent parents. This was probably due to chronic malnutrition stress and the disturbed mother-infant relationship in the litters of adolescent mothers.

Thymocyte migration: an affair of multiple cellular interactions?

Cell migration is a crucial event in the general process of thymocyte differentiation. The cellular interactions involved in the control of this migration are beginning to be defined. At least chemokines and extracellular matrix proteins appear to be part of the game. Cells of the thymic microenvironment produce these two groups of molecules, whereas developing thymocytes express the corresponding receptors. Moreover, although chemokines and extracellular matrix can drive thymocyte migration per se, a combined role for these molecules appears to contribute to the resulting migration patterns of thymocytes in their various stages of differentiation. The dynamics of chemokine and extracellular matrix production and degradation is not yet well understood. However, matrix metalloproteinases are likely to play a role in the breakdown of intrathymic extracellular matrix contents. Thus, the physiological migration of thymocytes should be envisioned as a resulting vector of multiple, simultaneous and/or sequential stimuli involving chemokines, adhesive and de-adhesive extracellular matrix proteins, as well as matrix metalloproteinases. Accordingly, it is conceivable that any pathological change in any of these loops may result in the alteration of normal thymocyte migration. This seems to be the case in murine infection by the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease. A better knowledge of the physiological mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells.

Assessment of the cardiovascular effects of electroconvulsive therapy in individuals older than 50 years

To evaluate the impact of electroconvulsive therapy on arterial blood pressure, heart rate, heart rate variability, and the occurrence of ischemia or arrhythmias, 38 (18 men) depressive patients free from systemic diseases, 50 to 83 years old (mean: 64.7 ± 8.6) underwent electroconvulsive therapy. All patients were studied with simultaneous 24-h ambulatory blood pressure and Holter monitoring, starting 18 h before and continuing for 3 h after electroconvulsive therapy. Blood pressure, heart rate, heart rate variability, arrhythmias, and ischemic episodes were recorded. Before each session of electroconvulsive therapy, blood pressure and heart rate were in the normal range; supraventricular ectopic beats occurred in all patients and ventricular ectopic beats in 27/38; 2 patients had non-sustained ventricular tachycardia. After shock, systolic, mean and diastolic blood pressure increased 29, 25, and 24% (P < 0.001), respectively, and returned to baseline values within 1 h. Maximum, mean and minimum heart rate increased 56, 52, and 49% (P < 0.001), respectively, followed by a significant decrease within 5 min; heart rate gradually increased again thereafter and remained elevated for 1 h. Analysis of heart rate variability showed increased sympathetic activity during shock with a decrease in both sympathetic and parasympathetic drive afterwards. No serious adverse effects occurred; electroconvulsive therapy did not trigger any malignant arrhythmias or ischemia. In middle-aged and elderly people free from systemic diseases, electroconvulsive therapy caused transitory increases in blood pressure and heart rate and a decrease in heart rate variability but these changes were not associated with serious adverse clinical events.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Sympathetic activation in rats with L-NAME-induced hypertension

We evaluated the hemodynamic pattern and the contribution of the sympathetic nervous system in conscious and anesthetized (1.4 g/kg urethane, iv) Wistar rats with L-NAME-induced hypertension (20 mg/kg daily). The basal hemodynamic profile was similar for hypertensive animals, conscious (N = 12) or anesthetized (N = 12) treated with L-NAME for 2 or 7 days: increase of total peripheral resistance associated with a decrease of cardiac output (CO) compared to normotensive animals, conscious (N = 14) or anesthetized (N = 14). Sympathetic blockade with hexamethonium essentially caused a decrease in total peripheral resistance in hypertensive animals (conscious, 2 days: from (means ± SEM) 2.47 ± 0.08 to 2.14 ± 0.07; conscious, 7 days: from 2.85 ± 0.13 to 2.07 ± 0.33; anesthetized, 2 days: from 3.00 ± 0.09 to 1.83 ± 0.25 and anesthetized, 7 days: from 3.56 ± 0.11 to 1.53 ± 0.10 mmHg mL-1 min-1) with no change in CO in either group. However, in the normotensive group a fall in CO (conscious: from 125 ± 4.5 to 96 ± 4; anesthetized: from 118 ± 1.5 to 104 ± 5.5 mL/min) was observed. The responses after hexamethonium were more prominent in the hypertensive anesthetized group. However, no difference was observed between conscious and anesthetized normotensive rats in response to sympathetic blockade. The present study shows that the vasoconstriction in response to L-NAME was mediated by the sympathetic drive. The sympathetic tone plays an important role in the initiation and maintenance of hypertension.

Can the fractal dimension be applied for the early diagnosis of non-proliferative diabetic retinopathy?

The fractal dimension has been employed as a useful parameter in the diagnosis of retinal disease. Avakian et al. (Curr Eye Res 2002; 24: 274-280), comparing the vascular pattern of normal patients with mild to moderate non-proliferative diabetic retinopathy (NPDR), found a significant difference between them only in the macular region. This significant difference in the box-counting fractal dimension of the macular region between normal and mild NPDR patients has been proposed as a method of precocious diagnosis of NPDR. The aim of the present study was to determine if fractal dimensions can really be used as a parameter for the early diagnosis of NPDR. Box-counting and information fractal dimensions were used to parameterize the vascular pattern of the human retina. The two methods were applied to the whole retina and to nine anatomical regions of the retina in 5 individuals with mild NPDR and in 28 diabetic but opthalmically normal individuals (controls), with age between 31 and 86 years. All images of retina were obtained from the Digital Retinal Images for Vessel Extraction (DRIVE) database. The results showed that the fractal dimension parameter was not sensitive enough to be of use for an early diagnosis of NPDR.

Central chemoreceptors and neural mechanisms of cardiorespiratory control

The arterial partial pressure (P CO2) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO2 from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO2 exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO2 causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO2-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.