Engineered promiscuous T helper peptides for the induction of immune responses

Following recognition of antigens by T helper (Th) lymphocytes, T cell help is elicited to induce humoral and cellular immune responses. These antigens are presented as short peptides, T helper peptides (THP), bound to MHC class II molecules. Since both endogenous THP (from antigens of interest) or exogenous THP (not encompassed by the sequence of the antigen of interest) are able to elicit T cell help, we decided to engineer promiscuous exogenous THP capable of binding to several HLA-DR molecules, in order to cover an important proportion of the human population. Some of these exogenous THP were able to bind to all seven HLA-DR molecules tested and were immunogenic in vivo in HLA-DR4 transgenic mice. Among them, peptides p37, p62 and p45 elicited Th1 cytokine profiles in vivo, providing help for the induction of potent CTL responses. Finally, in vitro stimulation assays carried out using human cells, showed that these peptides could induce T cell responses using cells obtained from individuals with a broad spectrum of HLA-DR molecules. Thus, engineered exogenous THP may be a valuable tool for the induction of immune responses in a large proportion of human population.     

Gain of multiple copies of the CBFB gene: a new genetic aberration in a case of granulocytic sarcoma

Granulocytic sarcomas (GS) are tumor masses of immature myeloid cells presenting at an extramedullary site, mainly the skin, bone, and lymph node. They are often associated with acute myeloid leukemia (AML) with monoblastic or myelomonocytic differentiation, including either AML M2 with t(8;21)(q22;q22) or AML M4Eo with inv(16)(p13q22). We present a case diagnosed with GS associated with AML M4 that presented a normal karyotype with conventional cytogenetic analysis. Although the myeloblasts did not show the inv(16)(p13q22) (CBFB/MYH11), a gain of multiple copies of the CBFB gene was detected with fluorescence in situ hybridization analysis. To our knowledge, no cases with this rare genetic anomaly have been previously described.     

Reduced activation to implicit affect induction in euthymic bipolar patients: an fMRI study

OBJECTIVE: To examine whether euthymic bipolar patients engage similar or contrasting brain regions as healthy subjects when responding to implicit affect induction. METHODS: The study examined 10 euthymic patients with bipolar I disorder, and 10 age- and gender-matched healthy subjects using event-related functional magnetic resonance imaging (fMRI) while subjects engaged in a modified word-based memory task designed to implicitly evoke negative, positive or no affective change. The activation paradigm involved nominating whether a target word was contained within a previously presented word list using specified response keys. RESULTS: The fMRI task produced significantly greater activation in healthy subjects as compared to patients in response to both negative and positive affect in the anterior and posterior cingulate, medial prefrontal cortex, middle frontal and right parahippocampal gyri. Only negative affect produced significantly greater activation in the postcentral gyrus, inferior parietal lobule, thalamus and putamen and only positive affect achieved the same in the precentral, superior temporal and lingual gyri, precuneus, cuneus, caudate, pons, midbrain and cerebellum. There were no brain regions in which responses were greater in patients as compared to healthy subjects. There were no statistically significant differences between the groups with respect to speed or accuracy. CONCLUSIONS: Diminished prefrontal, cingulate, limbic and subcortical neural activity in euthymic bipolar patients as compared to healthy subjects is suggestive of emotional compromise that is independent of cognitive and executive functioning. This finding is of clinical importance and has implications both for the diagnosis and treatment of bipolar disorder. Future studies should aim to replicate these findings and examine the development of bipolar disorder, investigating in particular the effects of medication.     

Oxidative stress impairs heme detoxification in the midgut of the cattle tick, Rhipicephalus (Boophilus) microplus

In the cattle tick Rhipicephalus (Boophilus) microplus digestion of blood is intracellular, accomplished by the so-called digest cells that fill the midgut lumen. Hydrolysis of hemoglobin in the digestive vesicles of these cells results in the release of large amounts of heme, a pro-oxidant compound, whose iron atom, together with H(2)O(2), may participate in the Fenton reaction and lead to the production of hydroxyl radicals. Here, we investigated the role of catalase, an enzyme responsible for H(2)O(2) detoxification. Fully engorged female ticks injected with 3-amino-1,2,4-triazole (AT), a catalase inhibitor, showed increased H(2)O(2) in the gut, together with diminished life span and lower egg-laying rates. Increased mortality observed upon AT injection was reversed by further injection of exogenous catalase, 2 days after AT treatment, confirming that increased death was due to inhibition of this enzyme by AT. In primary cultures of digest cells, intracellular H(2)O(2) is limited to specific organelles, while treatment with AT in vitro resulted in increased H(2)O(2) spreading all over the cell, confirming the role of catalase in regulating H(2)O(2) levels. Ticks fed on a calf that had been injected with AT showed marked inhibition of catalase activity in the gut and diminished life span, oviposition and engorgement. Digest cells of these ticks had an altered morphology, showing heme spread all over the cytosol, instead of being limited to the hemosomes. The amount of aggregated heme found in isolated hemosome was also strongly decreased in AT-treated cattle. All together, our results indicate that catalase performs an important role in the control of redox balance in R. microplus, which dramatically affects hemosome formation and stability. This enzyme may be a target in the development of new methods for tick control.     

Stress-reactivity in psychosis: evidence for an affective pathway to psychosis

This paper will review a series of studies using the Experience Sampling Method that suggest that altered sensitivity to stress is an endophenotype for psychosis. The Experience Sampling Method is a structured diary technique allowing the assessment of emotional reactivity to stressors occurring in normal daily life. Elevated emotional reactivity to stress was found in subjects vulnerable to psychosis, suggesting that affective responses to stressors in the flow of daily life are an indicator of genetic and/or environmental liability to psychosis. Indeed, the small stressors in daily life associated with affective responses also predict more intense moment-to-moment variation of subtle positive psychotic experiences. Increased emotional reactivity was found to be independent from cognitive impairments, and argued to constitute evidence of an affective pathway to psychosis that may underlie a more episodic, reactive, good-outcome type of psychosis. Evidence for this hypothesis was found in data suggesting that the experience of stressful life events and early trauma were associated with increased stress-sensitivity, and that women were more likely to display elevated stress-reactivity. These findings are discussed in the light of recent biological and psychological mechanisms.     

Design and implementation of a water phantom for IMRT, arc therapy, and tomotherapy dose distribution measurements

The aim of this paper is to present a new phantom for arc therapy, intensity-modulated radiation therapy (IMRT), and tomotherapy dose distribution measurement in pretreatment verification. The presented phantom is innovative for its use of water as the tissue equivalent material, together with a technical solution specifically designed to support radiographic or radiochromic film and ionization chambers in any desired position. The phantom comprise a Plexiglas container, whose present shape and dimensions offer the possibility to simulate a human torso or abdomen; the container can be filled with water by opening the upper cover. On the internal side of the cover, a set of carbon pipes can support film in the desired coronal, axial, or sagittal planes. At one of the two ends of the phantom, an ionization chamber can be positioned parallel to the rotation axis of the accelerator gantry in all possible positions within a 20 cm diameter cylinder, for film calibration purposes. Inhomogeneities can be inserted into the phantom using the same carbon pipes and plastic sheets used to support film. An example of vertebra-shaped inserts made of bone equivalent material is reported. Radiochromic film can be dipped in water, while radiographic film must be protected to prevent damage. To accomplish this, radiographic film is laminated using a cold laminating film. In order to assess the effects of both the lamination itself and the effects of water on laminated Kodak EDR2 film, the optical density (OD) of conventional, laminated, and laminated film immersed in water and exposed to a range of doses from 0 to 300 cGy were compared. The OD of the three samples receiving the same radiation dose did not present any significant difference, thus proving that laminated EDR2 film can also be used in water. A prerequisite for any dosimetric comparison between planned and measured data is a proper film to plan registration. The solution proposed here is an extrinsic in-plane registration technique using four reference points marked on each film in predefined positions. The four points and the millimeter scales fixed on the carbon pipes that support the film are designed and manufactured so as to transfer onto the film the same reference system used during the planning procedure, thus allowing a straightforward registration. Tests to assess the accuracy of the proposed registration method demonstrate that the distances between measured and intended marker positions, evaluated for coronal, axial, and sagittal planes, were about 1 mm for both anteroposterior and lateral projections.     

Central estrogen-like effect of genistein on growth hormone secretion in the ewe

The present study tested a hypothesis, whether plant-derived genistein influences the secretion of growth hormone (GH) in ewes, acting directly within the central nervous system (CNS). Starting six weeks after ovariectomy, ewes were infused intracerebroventricularly with genistein (n = 5) or 17beta-estradiol (n = 5), both in a total dose of 40 microg/400 microl/4 h, or with a vehicle (control, n = 5). All infusions were performed from 10:00 AM to 2:00 PM and blood samples were collected from 8:00 AM to 8:00 PM at 10-min intervals. Five genistein- and three vehicle-infused ewes were slaughtered the following morning. The plasma GH concentration was assayed by the radioimmunoassay method, and immunoreactivity of GH in the adenohypophysis was determined by immunohistochemistry. In genistein-infused ewes, mean plasma GH concentration was significantly higher during the whole period of infusion than the concomitant concentration in vehicle-infused ewes. However, examining data within group, GH secretion rose gradually, reaching a significant value during the second phase of genistein infusion. In 17beta-estradiol-infused animals, a significant increase in GH concentration was noted during the first two hours of the infusion, in comparison with vehicle-infused and also in comparison with genistein-infused ewes. Although a gradual increase in basic GH secretion continued in all treated groups during the afternoon and evening, mean plasma GH concentrations in genistein- and 17beta-estradiol-infused ewes were still significantly higher than in the vehicle-infused. The percentage of GH-positive cells in the adenohypophysis and the density of immunoreactive material in these cells decreased significantly in genistein-infused ewes, compared to the control, indicating diminished hormone storage. In conclusion, genistein as 17beta-estradiol, is an effective stimulator of GH secretion in ewes and may exert its effect at the level of the CNS.     

Site directed mutagenesis of Drosophila flightin disrupts phosphorylation and impairs flight muscle structure and mechanics

Flightin is a myosin rod binding protein that in Drosophila melanogaster is expressed exclusively in the asynchronous indirect flight muscles (IFM). Hyperphosphorylation of flightin coincides with the completion of myofibril assembly and precedes the emergence of flight competency in young adults. To investigate the role of flightin phosphorylation in vivo we generated three flightin null (fln ⁰ ) Drosophila strains that express a mutant flightin transgene with two (Thr158, Ser 162), three (Ser139, Ser141, Ser145) or all five potential phosphorylation sites mutated to alanines. These amino acid substitutions result in lower than normal levels of flightin accumulation and transgenic strains that are unable to beat their wings. On two dimensional gels of IFM proteins, the transgenic strain with five mutant sites (fln ^5STA ) is devoid of all phosphovariants, the transgenic strain with two mutant sites (fln ^2TSA ) expresses only the two least acidic of the nine phosphovariants, and the transgenic strain with three mutant sites (fln ^3SA ) expresses all nine phosphovariants, as the wild-type strain. These results suggest that phosphorylation of Thr158 and/or Ser162 is necessary for subsequent phosphorylation of other sites. All three transgenic strains show normal, albeit long, IFM sarcomeres in newly eclosed adults. In contrast, sarcomeres in fully mature fln ^5STA and fln ^2TSA adults show extensive breakdown while those in fln ^3SA are not as disordered. The fiber hypercontraction phenotype that characterizes fln ⁰ is fully evident in fln ^5STA and fln ^2TSA but partially rescued in fln ^3SA . Mechanics on skinned fibers from newly eclosed flies show alterations in viscous modulus for fln ^5STA and fln ^2TSA that result in a significant reduction in oscillatory power output. Expression of fln ^5STA and fln ^2TSA , but not fln ^3SA , in a wild-type (fln ⁺ /fln ⁺ ) background resulted in a dominant negative effect manifested as flight impairments and hypercontracted IFM fibers. Our studies indicate that Thr158 and/or Ser162 are (is) indispensable for flightin function and suggest that phosphorylation of one or both residues fulfills an essential role in IFM structural stability and mechanics.