Genetic Approaches to Hypothalamic-Pituitary-Adrenal Axis Regulation

The normal function of the hypothalamic-pituitary-adrenal (HPA) axis, and resultant glucocorticoid (GC) secretion, is essential for human health. Disruption of GC regulation is associated with pathologic, psychological, and physiological disease states such as depression, post-traumatic stress disorder, hypertension, diabetes, and osteopenia, among others. As such, understanding the mechanisms by which HPA output is tightly regulated in its responses to environmental stressors and circadian cues has been an active area of investigation for decades. Over the last 20 years, however, advances in gene targeting and genome modification in rodent models have allowed the detailed dissection of roles for key molecular mediators and brain regions responsible for this control in vivo to emerge. Here, we summarize work done to elucidate the function of critical neuropeptide systems, GC-signaling targets, and inflammation-associated pathways in HPA axis regulation and behavior, and highlight areas for future investigation.     

In vitro evidence that urine composition affects the fraction of active furosemide in the nephrotic syndrome

Diuretic resistance to furosemide in the nephrotic syndrome (NS) may result from binding of drug to filtered albumin within the renal tubule. In buffer solutions intended to partially mimic the luminal environment of the distal nephron during the NS, we examined several chemical properties to determine their effect on furosemide-albumin binding equilibria. Dissociation constants were obtained by measuring furosemide's quenching of human serum albumin's intrinsic tryptophan fluorescence over ranges of pH, ionic strength (IS) and osmolarity. Neither pH nor osmolarity significantly affected binding; however, incremental increases in IS between 0.0 and 1.0 produced increases in Kd from 0.65 +/- 0.05 to 34.38 +/- 1.72 microM, resulting in a 5- and 28-fold increase in the unbound furosemide fraction when the furosemide-albumin concentrations were 3.0:5.0 and 10.0:45.0 microM, respectively. Our results indicate that human serum albumin contains one high affinity binding site for furosemide that is sensitive to IS. Because of changes in the concentrations of reactants as well as IS that can occur in nephron segments distal to furosemide's site of action, we conclude that the amount of unbound (i.e., pharmacologically active) drug in voided urine will not necessarily correspond to the amount at the active site. To clinically assess the pharmacodynamic consequence of protein binding in the NS, changes in the concentration of the reactants and IS in the distal nephron must be minimized so that the unbound furosemide measured in voided urine will accurately reflect the amount at the drug's active site.     

Apoptosis: understanding the new molecular pathway.

Advances in science have increased the knowledge of how cells die in the body (apoptosis). A basic understanding of this process can improve nurses' ability to review new scientific literature and enable them to provide safer bedside care.     

Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes.     

Immunisation with phage displaying peptides representing single epitopes of the glycoprotein G can give rise to partial protective immunity to HSV-2

Filamentous phage displaying peptides representing single epitopes of the glycoprotein G of HSV-2 (gG2) were used as immunogens via the subcutaneous route in Balb/c mice without additional adjuvant. The phage were isolated from a random phage peptide display library and contain 15-mer peptide inserts that mimic epitopes of gG2. In each case, an antibody response to gG2 was generated that was dependent on the dose of phage administered and on the presence of the peptide insert. Phage displaying epitopes of gG2, which map to amino acids 551-570, were the most immunogenic; interestingly, this region of gG2 is frequently recognised by patients infected with HSV-2. The data also provide interesting information as regards choice of peptide mimics for use as immunogens because, surprisingly, the most antigenic of the individual clones was the least immunogenic. In two of the experiments, mice immunised with phage displaying a single epitope of gG2 were protected against challenge with a lethal dose of whole HSV-2. This suggests a possible role for phage-displayed peptides in inducing protective immunity against pathogens and provides a model system for investigating the underlying mechanisms.     

Loss of CD8 and TCR binding to Class I MHC ligands following T cell activation

The capacity of T cells to bind peptide/MHC ligands changes with T cell development and differentiation. Here we study changes in peptide/MHC multimer binding following T cell activation. Surprisingly, T cell activation caused a marked reduction in specific peptide/MHC Class I multimer binding, which was distinct from transient TCR down-regulation, and was especially dramatic for engagement with low-affinity peptide/MHC ligands. Direct CD8-Class I interactions were also profoundly and rapidly impaired following T cell stimulation, even though surface CD8alpha and CD8beta levels were unchanged after activation, suggesting that decreased CD8 co-receptor binding contributes to this effect. Finally, we show that enzymatic desialylation restores much of the multimer binding on activated T cells, suggesting that altered glycosylation may inhibit TCR/CD8 binding to peptide/MHC ligands. These radical changes in activated T cells' ability to perceive peptide/MHC ligands may contribute to selective outgrowth of clones with high affinity for the stimulatory ligand.