Sleep-wake cycle and melatonin rhythms in adolescents and young adults with mood disorders: comparison of unipolar and bipolar phenotypes.

This study evaluated the potential of circadian measures as early markers of mood disorders subtypes. Patients with bipolar disorders had significantly lower levels and later onset of melatonin secretion than those with unipolar depression. Furthermore, abnormal phase angles between sleep, melatonin and temperature were found in several patients.

Circadian profiles in young people during the early stages of affective disorder.

Although disturbances of the circadian system are strongly linked to affective disorders, no known studies have examined melatonin profiles in young people in early stages of illness. In this study, 44 patients with an affective disorder underwent clinical and neuropsychological assessments. They were then rated by a psychiatrist according to a clinical staging model and were categorized as having an 'attenuated syndrome' or an 'established disorder'. During the evening, salivary melatonin was sampled under dim light conditions over an 8-h interval and for each patient, the time of melatonin onset, total area under the curve and phase angle (difference between time of melatonin onset and time of habitual sleep onset) were computed. Results showed that there was no difference in the timing of melatonin onset across illness stages. However, area under the curve analyses showed that those patients with 'established disorders' had markedly reduced levels of melatonin secretion, and shorter phase angles, relative to those with 'attenuated syndromes'. These lower levels, in turn, were related to lower subjective sleepiness, and poorer performance on neuropsychological tests of verbal memory. Overall, these results suggest that for patients with established illness, dysfunction of the circadian system relates clearly to functional features and markers of underlying neurobiological change. Although the interpretation of these results would be greatly enhanced by control data, this work has important implications for the early delivery of chronobiological interventions in young people with affective disorders.

Angiogenesis monitored by perfusion with a space-filling microbead suspension.

Numerous laboratories are focusing efforts on delivering gene products to induce or prevent the development of new blood vessels in adults, with the hope of rescuing ischemic tissues, circumventing cardiac bypass surgery, or inhibiting tumor growth. Current approaches to the assessment of vascular continuity involve the introduction of either dyes or fluorescent microspheres to track blood flow. However, dyes and dextrans are subject to leakage when vessels are hyperpermeable, a situation that may occur in studies of tumor vasculature and during efforts to stimulate therapeutic angiogenesis. Furthermore, the microspheres that are used for flow studies do not allow a comprehensive visual analysis of vascular continuity. Here we report a method for the visual assessment of microvascular continuity in mouse muscle under circumstances in which vessels are leaky. The approach involves perfusion of the vasculature with fluorescent beads that are much smaller than those used for flow studies. The suspension behaves like a fluid and completely fills the vessels, yet the beads do not leak from VEGF-permeablized capillaries and remain localized in histological sections. Use of beads with the proper fluorescence emission wavelengths allows immunofluorescent colocalization with vessel-specific markers. We compare this improved method with other methods for tracking vascular continuity involving dextrans and larger beads. This approach should aid in the dynamic study of tumor angiogenesis and the evaluation of efforts to deliver angiogenic factors.

Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1.

NK cells are able to lyse a variety of virally infected and neoplastic cells in an MHC-unrestricted manner. The cell-surface protein NKR-P1 is thought to play a key role in this process. NKR-P1, initially identified in rat IL-2 activated NK cells, is encoded in the mouse by at least three similar, but not identical, genes. We previously reported the isolation and characterization of three different NKR-P1 cDNA, termed cDNA 2, 34, and 40, from IL-2 activated mouse NK cells. This report describes the structure of the gene encoding NKR-P1 cDNA 2, the smallest of these three cDNA. Gene 2 is composed of six exons spanning approximately 14 kb of genomic DNA. The first exon encodes the N-terminal intracellular domain, and exons 4, 5, and 6 contain the sequences coding for the CRD. This organization is similar to that of other genes that encode C-type animal lectins. The expression of the NKR-P1 genes in A-LAK cells from 13 mouse strains was examined by Northern blot analysis. NKR-P1 expression appears to coincide with that of the NK1.1 Ag. This observation further supports the hypothesis that the NK1.1 Ag is encoded by one of the NKR-P1 genes. Nucleotide sequence analysis of the promoter region of the three NKR-P1 genes in BALB/c and C57BL/6 mice suggests that differences in the level of expression probably do not result from alterations in the upstream regions of these genes, but may be caused by the expression of strain-specific transacting factors.

Transport functions in a bioartificial kidney under uremic conditions.

A bioartifical kidney, a confluent monolayer of renal tubular cells attached to a permselective synthetic membrane, may some day substitute for the failing renal function. In such a device, the cells would have to exhibit transport properties when exposed to uremic plasma. To test the feasibility of a bioartificial kidney, LLC-PK1 cells derived from the proximal tubule were grown to confluence on microporous, cellulose nitrate membranes and transepithelial transport of inulin, glucose and tetraethylammonium (TEA) was investigated with standard tissue culture medium and with the hemofiltrate obtained from a uremic patient. Inulin, which is neither reabsorbed nor secreted by renal epithelial cells, did not diffuse through the LLC-PK1 monolayer in either tissue culture fluid or hemofiltrate. Glucose was preferentially reabsorbed and TEA was preferentially secreted in both media. In hemofiltrate, LLC-PK1 cells reabsorbed glucose at the same rate as in tissue culture fluid but secreted TEA at a significantly higher rate. This difference was related to the pH of the fluids since it was abolished by correction to the same pH value. For both solutes, the passive transepithelial leakage was significantly lower in hemofiltrate. We conclude that LLC-PK1 cells retain their reabsorbtive and secretory transport characteristics under uremic conditions.

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells.

The growth and differentiation of an established renal epithelial cell line, LLC-PK1, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK1 monolayers grown on ECM-coated membranes displayed the highest transepithelial D-glucose transport (333 +/- 22 ng.cm-2.min-1) whereas cells plated on collagen-coated membranes displayed the lowest (94 +/- 23 ng.cm-2.min-1). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.

Renal epithelial-cell-controlled solute transport across permeable membranes as the foundation for a bioartificial kidney.

Despite the success of current blood purification techniques in allowing the survival of individuals with acute and chronic renal failure, the quality of life of people affected by end-stage renal disease remains unsatisfactory. Part of the reason is due to the nonphysiologic manner in which current blood purification techniques achieve homeostasis. Attempts to improve mechanical substitution of renal function by coupling the transport capability of living cells with conventional hemofiltration devices constitute the first step toward the development of an implantable bioartificial kidney.

Cellular control of membrane permeability. Implications for a bioartificial renal tubule.

A hemofiltrate processor, which consists of a cultured monolayer of renal epithelial cells on a permeable membrane, displayed transport functions that mimicked the renal tubule. Two cell lines, LLC-PK1 (a proximal tubule-like cell) and MDCK (a distal tubule-like cell), were cultured on microporous cellulose nitrate membranes and studied in a diffusion chamber. Dual-label tracer experiments revealed that inulin, glucose, and para-aminohippurate (PAH) moved across unseeded membranes with similar rates in either direction. Solute concentrations in both compartments reached equilibrium in less than 12 hours. MDCK cell-covered membranes were impermeable to all three solutes, as expected from a distal tubule-like cell. LLC-PK1 cell-covered membranes were impermeable to inulin and PAH, but demonstrated a net apical to basal glucose flux of 150 to 350 ng/cm2/min, depending on the conditions. This led within 72 hours to a zero glucose concentration in the compartment on the apical side of the cells, versus 4.6 mg/ml on the basal side. This glucose transport function was inhibited by 0.5 mmol/L phlorizin. Highest glucose flux was obtained using extracellular matrix as the cell attachment substrate on the membrane, suggesting the highest degree of cell differentiation. Based on estimates of the surface area of the normal kidney, the glucose reabsorption capacity of the LLC-PK1 cell line was about 20% that of the normal tubule. The absence of PAH secretion may have been due to the heterogeneity of cells derived from the proximal tubule. Different cell types, culture conditions, and attachment substrates may enhance the transport capacity of the bioartificial renal tubule.

The bioartificial kidney: progress towards an ultrafiltration device with renal epithelial cells processing.

The combination of an ultrafiltration device with an exchanger whose semipermeable hollow fibres are covered with renal epithelial cells is proposed as a design for a bioartificial kidney. We first demonstrated that continuous ultrafiltration can be maintained for relatively long periods in the absence of anticoagulation. As a second step, we report here the feasibility of attaching and growing two lines of kidney epithelial cells (MDCK and LLC-PK1) on two different semipermeable materials, an acrylic copolymer and a polysulphone. Cells seeded on acrylic copolymer hollow fibres reach confluence within three weeks. Depending on the chemical and/or physical properties of the polymer, the cells show distinct differentiated morphology, which may influence their ability to perform specialized tasks.