V3 peptide binding pattern and HIV-1 transmission route in Rio de Janeiro

To characterize antibody binding to a panel of V3 loop peptides representing diverse HIV-1 neutralization epitopes, 149 HIV-1 infected individuals from Rio de Janeiro (RJ) were investigated. Results were analyzed with respect to risk factors for infection and other epidemiological and clinical data. Peptide reactivity was not associated with sex, clinical status, CD4 counts, antigenemia or ß2-microglobulin serum level. A segregation of peptide reactivity according to route of infection was encountered. This finding suggests that more then one viral strain may be circulating in RJ, in subjects with different risk factors for HIV-1 infection. An investigation of prevalent HIV-1 genotypes, serotypes and immunotypes may be of importance for the design and selection of potential vaccines to be used in Brazil as well as for the selection of populations to be included in future vaccine efficacy trials.

A molecular framework for light and gibberellin control of cell elongation.

Cell elongation during seedling development is antagonistically regulated by light and gibberellins (GAs). Light induces photomorphogenesis, leading to inhibition of hypocotyl growth, whereas GAs promote etiolated growth, characterized by increased hypocotyl elongation. The mechanism underlying this antagonistic interaction remains unclear. Here we report on the central role of the Arabidopsis thaliana nuclear transcription factor PIF4 (encoded by PHYTOCHROME INTERACTING FACTOR 4) in the positive control of genes mediating cell elongation and show that this factor is negatively regulated by the light photoreceptor phyB (ref. 4) and by DELLA proteins that have a key repressor function in GA signalling. Our results demonstrate that PIF4 is destabilized by phyB in the light and that DELLAs block PIF4 transcriptional activity by binding the DNA-recognition domain of this factor. We show that GAs abrogate such repression by promoting DELLA destabilization, and therefore cause a concomitant accumulation of free PIF4 in the nucleus. Consistent with this model, intermediate hypocotyl lengths were observed in transgenic plants over-accumulating both DELLAs and PIF4. Destabilization of this factor by phyB, together with its inactivation by DELLAs, constitutes a protein interaction framework that explains how plants integrate both light and GA signals to optimize growth and development in response to changing environments.

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

Combination of transient lymphodepletion with busulfan and fludarabine and peptide vaccination in a phase I clinical trial for patients with advanced melanoma.

Taking advantage of homeostatic mechanisms to boost tumor-specific cellular immunity is raising increasing interest in the development of therapeutic strategies in the treatment of melanoma. Here, we have explored the potential of combining homeostatic proliferation, after transient immunosuppression, and antigenic stimulation of Melan-A/Mart-1 specific CD8 T-cells. In an effort to develop protocols that could be readily applicable to the clinic, we have designed a phase I clinical trial, involving lymphodepleting chemotherapy with Busulfan and Fludarabine, reinfusion of Melan-A specific CD8 T-cell containing peripheral blood mononuclear cells (exempt of growth factors), and Melan-A peptide vaccination. Six patients with advanced melanoma were enrolled in this outpatient regimen that demonstrated good feasibility combined with low toxicity. Consistent depletion of lymphocytes with persistent increased CD4/CD8 ratios was induced, although the proportion of circulating CD4 regulatory T-cells remained mostly unchanged. The study of the immune reconstitution period showed a steady recovery of whole T-cell numbers overtime. However, expansion of Melan-A specific CD8 T-cells, as measured in peripheral blood, was mostly inconsistent, accompanied with marginal phenotypic changes, despite vaccination with Melan-A/Mart-1 peptide. On the clinical level, 1 patient presented a partial but objective antitumor response following the beginning of the protocol, even though a direct effect of Busulfan/Fludarabine cannot be completely ruled out. Overall, these data provide further ground for the development of immunotherapeutic approaches to be both effective against melanoma and applicable in clinic.

Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro.

There is a clinical need to enhance functional recovery of injured peripheral nerves. Local administration of neurotrophic factors (NTFs) after surgical repair has been proposed for this purpose. Little is known, however, on the optimal local dose and dosing frequency of NTFs in a peripheral nerve defect. For increasing our knowledge on biologically relevant local NTFs concentrations and for making available an in vitro assay for assessing the bioactivity of NTFs in connection with implantable localized delivery systems, we developed in this study a bioassay for NTFs, which is based on dorsal root ganglion (DRG) explants from E9 (9 days old) chicken embryos. Axonal elongation and extent of axonal branching was analyzed microscopically after addition of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), each alone and in combination. GDNF significantly promoted axonal elongation, but only little axonal branching, whereas NGF induced extensive axonal branching with modest axonal elongation. The combination of GDNF and NGF exerted a synergistic effect on the axonal elongation, axonal branching and growth kinetics. GDNF and NGF also enhanced the expression of their respective functional receptors Ret and TrkA on the DRG neurons. This information should be relevant for the development of implants containing NTFs and on drug therapy of damaged peripheral nerves.

Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels.

BACKGROUND The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR). RESULTS Our aim was to analyze and compare angiogenic factor expression levels in both subcutaneous (SC) and omentum (OM) adipose tissues from morbidly obese patients (n = 26) with low (OB/L-IR) (healthy obese) and high (OB/H-IR) degrees of IR, and lean controls (n = 17). Another objective was to examine angiogenic factor correlations with obesity and IR.Here we found that VEGF-A was the isoform with higher expression in both OM and SC adipose tissues, and was up-regulated 3-fold, together with MMP9 in OB/L-IR as compared to leans. This up-regulation decreased by 23% in OB/-H-IR compared to OB/L-IR. On the contrary, VEGF-B, VEGF-C and VEGF-D, together with MMP15 was down-regulated in both OB/H-IR and OB/L-IR compared to lean patients. Moreover, MMP9 correlated positively and VEGF-C, VEGF-D and MMP15 correlated negatively with HOMA-IR, in both SC and OM. CONCLUSION We hereby propose that the alteration in MMP15, VEGF-B, VEGF-C and VEGF-D gene expression may be caused by one of the relevant adipose tissue processes related to the development of IR, and the up-regulation of VEGF-A in adipose tissue could have a relationship with the prevention of this pathology.

Unconjugated TAT carrier peptide protects against excitotoxicity.

We report in this article for the first time the neuroprotective effects of unconjugated TAT carrier peptide against a mild excitotoxic stimulus both in vitro and in vivo. In view of the widespread use of TAT peptides to deliver neuroprotectants into cells, it is important to know the effects of the carrier itself. Unconjugated TAT carrier protects dissociated cortical neurons against NMDA but not against kainate, suggesting that TAT peptides may interfere with NMDA signaling. Furthermore, a retro-inverso form of the carrier peptide caused a reduction in lesion volume (by about 50%) in a rat neonatal cerebral ischemia model. Thus, even though TAT is designed merely as a carrier, its own pharmacological activity will need to be considered in the analysis of TAT-linked neuroprotectant peptides.

C3-symmetric peptide scaffolds are functional mimetics of trimeric CD40L.

Interaction between CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ligand CD40L, a 39-kDa glycoprotein, is essential for the development of humoral and cellular immune responses. Selective blockade or activation of this pathway provides the ground for the development of new treatments against immunologically based diseases and malignancies. Like other members of the TNF superfamily, CD40L monomers self-assemble around a threefold symmetry axis to form noncovalent homotrimers that can each bind three receptor molecules. Here, we report on the structure-based design of small synthetic molecules with C3 symmetry that can mimic CD40L homotrimers. These molecules interact with CD40, compete with the binding of CD40L to CD40, and reproduce, to a certain extent, the functional properties of the much larger homotrimeric soluble CD40L. Architectures based on rigid C3-symmetric cores may thus represent a general approach to mimicking homotrimers of the TNF superfamily.

Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers.

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4(+) T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b(+) cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4(+) T cells, include central and transitional memory polyfunctional populations, and do not include CD4(+)CD25(+)CD127(-) regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.

Kinetic study of neuropeptide Y (NPY) proteolysis in blood and identification of NPY3-35: a new peptide generated by plasma kallikrein.

There is little information on how neuropeptide Y (NPY) proteolysis by peptidases occurs in serum, in part because reliable techniques are lacking to distinguish different NPY immunoreactive forms and also because the factors affecting the expression of these enzymes have been poorly studied. In the present study, LC-MS/MS was used to identify and quantify NPY fragments resulting from peptidolytic cleavage of NPY(1-36) upon incubation with human serum. Kinetic studies indicated that NPY(1-36) is rapidly cleaved in serum into 3 main fragments with the following order of efficacy: NPY(3-36) >> NPY(3-35) > NPY(2-36). Trace amounts of additional NPY forms were identified by accurate mass spectrometry. Specific inhibitors of dipeptidyl peptidase IV, kallikrein, and aminopeptidase P prevented the production of NPY(3-36), NPY(3-35), and NPY(2-36), respectively. Plasma kallikrein at physiological concentrations converted NPY(3-36) into NPY(3-35). Receptor binding assays revealed that NPY(3-35) is unable to bind to NPY Y1, Y2, and Y5 receptors; thus NPY(3-35) may represent the major metabolic clearance product of the Y2/Y5 agonist, NPY(3-36).

Effects of intranasal administration of synthetic (4-28)human atrial natriuretic peptide to normal volunteers.

The effects of intranasal administration of increasing doses of synthetic human natriuretic peptide (4-28 hANP) were studied in six healthy volunteers. The peptide was administered as a nasal spray at doses of 50, 100, 200, and 500 micrograms in ascending order at 48-h intervals. Vehicle was administered by the same route randomly between any two of the doses. Intranasal hANP administration had no effect on either blood pressure, heart rate (HR), or hematocrit. Diuresis did not change consistently, whereas natriuresis tended to rise with vehicle as well as with hANP administration. This was attributed to the infusion of isotonic saline during the experiment. There was no significant increase in plasma ANP levels after intranasal administration of any of the different doses. Thus, no evidence that the atrial natriuretic peptide tested (4-28 hANP) can cross the nasal mucosal barrier was found.

Glucagon-like peptide-I and the control of insulin secretion in the normal state and in NIDDM.

Potentiation of glucose-induced insulin secretion by intestinal factors has been described for many years. Today, two major peptides with potent insulinotropic action have been recognized: gastric inhibitory peptide and truncated forms of glucagon-like peptide I, GLP-I(7-37) or the related GLP-I(7-36)amide. These hormones have specific beta-cell receptors that are coupled to production of cAMP and activation of cAMP-dependent protein kinase. Elevation in intracellular cAMP levels is required to mediate the glucoincretin effect of these hormones: the potentiation of insulin secretion in the presence of stimulatory concentrations of glucose. In addition, circulating glucoincretins maintain basal levels of cAMP, which are necessary to keep beta-cells in a glucose-competent state. Interactions between glucoincretin signaling and glucose-induced insulin secretion may result from the phosphorylation of key elements of the glucose signaling pathway by cAMP-dependent protein kinase. These include the ATP-dependent K+ channel, the Ca++ channel, or elements of the secretory machinery itself. In NIDDM, the glucoincretin effect is reduced. However, basal or stimulated gastric inhibitory peptide and glucagon-like peptide I levels are normal or even elevated, suggesting that signals induced by these hormones on the beta-cells are probably altered. At pharmacological doses, infusion of glucagon-like peptide I but not gastric inhibitory peptide, can ameliorate postprandial insulin secretory response in NIDDM patients. Agonists of the glucagon-like peptide I receptor have been proposed as new therapeutic agents in NIDDM.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

Chaperones and proteases: cellular fold-controlling factors of proteins in neurodegenerative diseases and aging.

The formation of toxic protein aggregates is a common denominator to many neurodegenerative diseases and aging. Accumulation of toxic, possibly infectious protein aggregates induces a cascade of events, such as excessive inflammation, the production of reactive oxygen species, apoptosis and neuronal loss. A network of highly conserved molecular chaperones and of chaperone-related proteases controls the fold-quality of proteins in the cell. Most molecular chaperones can passively prevent protein aggregation by binding misfolding intermediates. Some molecular chaperones and chaperone-related proteases, such as the proteasome, can also hydrolyse ATP to forcefully convert stable harmful protein aggregates into harmless natively refoldable, or protease-degradable, polypeptides. Molecular chaperones and chaperone-related proteases thus control the delicate balance between natively folded functional proteins and aggregation-prone misfolded proteins, which may form during the lifetime and lead to cell death. Abundant data now point at the molecular chaperones and the proteases as major clearance mechanisms to remove toxic protein aggregates from cells, delaying the onset and the outcome of protein-misfolding diseases. Therapeutic approaches include treatments and drugs that can specifically induce and sustain a strong chaperone and protease activity in cells and tissues prone to toxic protein aggregations.

Pharmacological Therapy in the Heart as an Alternative to Cellular Therapy: A Place for the Brain Natriuretic Peptide?

The discovery that stem cells isolated from different organs have the ability to differentiate into mature beating cardiomyocytes has fostered considerable interest in developing cellular regenerative therapies to treat cardiac diseases associated with the loss of viable myocardium. Clinical studies evaluating the potential of stem cells (from heart, blood, bone marrow, skeletal muscle, and fat) to regenerate the myocardium and improve its functional status indicated that although the method appeared generally safe, its overall efficacy has remained modest. Several issues raised by these studies were notably related to the nature and number of injected cells, as well as the route and timing of their administration, to cite only a few. Besides the direct administration of cardiac precursor cells, a distinct approach to cardiac regeneration could be based upon the stimulation of the heart's natural ability to regenerate, using pharmacological approaches. Indeed, differentiation and/or proliferation of cardiac precursor cells is controlled by various endogenous mediators, such as growth factors and cytokines, which could thus be used as pharmacological agents to promote regeneration. To illustrate such approach, we present recent results showing that the exogenous administration of the natriuretic peptide BNP triggers "endogenous" cardiac regeneration, following experimental myocardial infarction.