Drug development strategies for asthma: in search of a new paradigm

The spiraling costs of asthma treatment seem set to continue rising, given the equivocal performance of the latest generation of specific anti-inflammatory drugs in trials in adult asthmatics. We argue that the continuation of this trend is inevitable unless there is a substantial realignment of entrenched drug development policy in the pharmaceutical industry and a parallel shift in licensing policy by regulatory authorities to encourage the development of drugs capable of halting the progression from acute to chronic asthma when the disease first manifests in childhood. The theoretical framework for such an approach, including proof-of-principle data from studies in children with early-stage disease and a range of candidate drugs, already exists. What is needed is informed debate on the risks versus potential benefits of this approach.     

Advanced 1H Solid‐State NMR Spectroscopy on Hydrogels, 1

Summary: The nature of the pH dependent collapse of poly(methacrylic acid) (PMAA) hydrogels is investigated using recent ¹H solid‐state NMR methods. In aqueous solution, PMAA changes from an expanded conformation at high pHs to a compact contracted form at low pHs, where hydrogen bonds play a central role. In solid‐state ¹H NMR spectra, recorded under fast magic angle spinning (MAS), dried PMAA samples previously collapsed at low pHs show characteristic signals in the spectral region of the carboxylic acid protons. With the aid of 2D ¹H‐¹H double‐quantum (DQ) MAS NMR spectra, three signals can be distinguished at 8, 10.5 and 12.5 ppm, which are attributed to free carboxylic groups and two different types of hydrogen bonded forms, respectively. The 12.5 ppm signal arises from the hydrogen bond with the shortest H? H distance, corresponding to the form that is most stable with respect to increasing temperature and pH. The weaker hydrogen‐bonded form (with a signal at 10.5 ppm) requires a slightly lower pH, while the free acid signal (at 8 ppm) emerges under the most acidic medium. Moreover, the stabilities of the hydrogen‐bonded carboxylic acid dimers can be inferred from the proton‐proton distances within the dimers, i.e. (275 ± 5) pm and (295 ± 15) pm for the protons at 12.5 and 10.5 ppm, respectively, which are determined by means of DQ MAS sideband patterns. Both the stability of the hydrogen bonds and the acidity of the protons may be related to the stereochemistry and the conformation of the PMAA chains.

     

Inflammatory responses and cell adhesion to self-assembled monolayers of alkanethiolates on gold

The acute inflammatory response and the adhesion of cells to self-assembled monolayers (SAMs) of well-defined surface chemistry was studied in vivo using a rodent air-pouch model of inflammation. SAMs with three different terminal functional groups (OH, COOH and CH3) were implanted in subcutaneous air pouches induced in BALB/c mice. After 24 h, inflammatory cells were recovered from the air pouches and the implants were removed and prepared for observation by scanning electron microscopy (SEM). The implants coated with OH and CH3, were found to cause the highest recruitment of inflammatory cells into the subcutaneous pouches. Polymorphonuclear neutrophils (PMNs) leukocytes predominated over mononuclear cells in inflammatory exudates of SAMs-coated implants, the opposite being found in uncoated implants (controls). CH3-coated implants induced the highest number of inflammatory cells and also the largest percentage of PMNs seen in the subcutaneous pouches. Control and OH-covered implants presented the higher densities of attached inflammatory cells detected by SEM. In contrast, the CH3-coated implants showed a very low density of cells adherent to the implant surface. We conclude that the chemical nature and the degree of hydrophobicity of the surface of implants modulate both the local acute inflammatory reaction and the adhesion of leukocytes.     

Why are human newborns so fat? Relationship between fatness and brain size at birth [retracted article]

The plumpness of the human newborn has long been recognized as a trait in need of explanation among researchers. Using a linear regression analysis, we find that head circumference is significantly and positively associated with BMI at birth, after gestational age and birthlength were controlled for, in a sample of 1,069 healthy liveborn routinely delivered at the University Hospital of Coimbra (partial correlation r = 0.409, P < 0.0001). This significant association is consistent with the idea that newborn fatness is related to the higher need of lipids in newborn humans as an energetic and plastic substrate during its accelerated brain growth period. As birthweight and birth head size are associated with head size and cognitive abilities in childhood and adult life, it could be postulated that these cognitive abilities could have acted as selective pressure responsible for the newborn fatness increase in our lineage. Am. J. Hum. Biol. 16:24–30, 2004. © 2003 Wiley‐Liss, Inc.     

Apoptotic rate in peripheral T-cell lymphomas. A study using a tissue microarray with validation on full tissue sections

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas with a wide spectrum of clinicopathologic features, and apoptosis mechanisms may have a role in lymphomagenesis. We assessed apoptotic rate (AR) in 112 PTCLs using a tissue microarray developed in our laboratory and a modified terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. The mean AR was 1.47% +/- 1.38% for the entire group of PTCLs (range, 0.06%-5.15%), and AR varied significantly among different tumor types. In mycosis fungoides, the mean AR was 0.74%; angioimmunoblastic T-cell lymphoma, 1.02%; PTCL, not otherwise specified, 1.38%; cutaneous anaplastic large cell lymphoma (ALCL), 1.41%; anaplastic lymphoma kinase protein (ALK)-negative ALCL, 1.43%; extranodal natural killer/T-cell lymphoma of nasal type, 2.04%; ALK-positive ALCL, 2.95%; and enteropathy-type T-cell lymphoma, 3.06%. Mean AR was higher in PTCL with large cell vs small/medium cell morphologic features (1.66% +/- 1.1% vs 0.99% +/- 1.0%). In a subset of 33 PTCLs, the tissue microarray results comparedfavorably with those obtained in full tissue sections. We conclude that the highest ARs in PTCLs are found in enteropathy-type T-cell lymphoma and ALK-positive ALCL, and that AR can be assessed reliably by using a tissue microarray.     

NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia

Changes in high-energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P-nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re-establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen-depleted medium (oxygen concentration below 30 microM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 microM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set-up enabled the acquisition of 31P-spectra with high signal-to-noise ratio within 10-20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C-NMR using [1-13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy.     

Squash Procedure for Protein Immunolocalization in Meiotic Cells

Several techniques have been developed for protein immunolocalization in meiotic cells. However, most of them include treatments that lead to cell disruption and are only suitable for prophase-I cells. We describe a novel squash procedure of cell preparation for protein immunolabelling of different meiotic stages. This procedure is an alternative to both cryosectioning and whole spreading procedures. We present results obtained in mouse spermatocytes with three different antibodies: the MPM-2 mAb against mitotic phosphoepitopes, an anticentromere serum and a polyclonal serum against the SCP3 protein of the axial elements and lateral elements of the synaptonemal complex. The procedure was tested for single and double immunolabelling. With this technique a large number of cells at different meiotic stages can be analysed. Cell stages are easily identified and cell and chromosome structures are preserved. Thus, it allows the study of chromosome behaviour and the relations hips between the different structural elements of the cell throughout meiotic divisions. Our procedure is also suitable for three-dimensional (3D) analyses and proved to be reliable in a wide range of systems including insects and mammals. In addition, the procedure may be interesting to obtain a rapid immunological diagnosis.     

The paraventricular nucleus of hypothalamus mediates the pressor response to noradrenergic stimulation of the medial prefrontal cortex in unanesthetized rats

The medial prefrontal cortex (MPFC) is a structure that is also involved in cardiovascular modulation. The injection of norepinephrine (NE) into the prelimbic (PL) area of the MPFC of unanesthetized rats evokes a pressor response which is mediated by acute vasopressin release. Vasopressin is synthesized by magnocellular cells of the paraventricular (PVN) and supraoptic nucleus (SON) of the hypothalamus. In the present study, we endeavored to determine which vasopressin-synthesizing hypothalamic nucleus is involved in the pressor pathway activated after NE injection into the PL area of the MPFC. We report here that lidocaine microinjection into the SON did not change the pressor response evoked by NE injection into the PL. However, the response to NE was blocked by prior injection of lidocaine or CoCl₂ into the PVN, indicating that this area is responsible for the mediation of this pressor response. A neuroanatomic experiment in which the neuronal tracer biotinylated dextran amine (BDA) was microinjected into the MPFC showed a lack of axons or neuronal cell bodies in the PVN, indicating that there are no direct connections between the PL area of the MPFC and the PVN. The results suggest that the PVN is involved in the mediation of the pressor response to NE in the PL area and that this pathway must relay in other brain structures before reaching the PVN.