A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium

Background

Pulmonary neuroendocrine cells (PNEC) are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy.

Methods

Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table ​(Table1)1) undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5), sensory nerves (calcitonin gene related peptide, CGRP), and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP). The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop.

Table 1

Patient Demographic Data

A Clinical Study of Isoniazid Inactivation

The rate of inactivation of isoniazid (INH) in a host organism varies widely, probably because of genetic factors. A simple chemical test was used to determine INH levels in 24 tuberculous patients three and six hours after oral administration of the drug. Results are expressed in terms of half-life values of free INH in the body. Seven of the 24 patients inactivated INH rapidly (half-life average: 64 minutes); the remaining 17 metabolized INH at a slower rate (half-life average: 186 minutes). The range of individual half-life values was 30 to 305 minutes. A provisional half-life limit of 110 minutes was used to define “fast inactivators”; 110-160 minutes, “mo$\tilde{d}$erate inactivators”; and over 160 minutes, “slow inactivators”. Although INH inactivation may not be directly related to therapeutic failure, the security margin of the treatment may be diminished in those patients who inactivate INH rapidly.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Augmented analgesic effects of L-tryptophan combined with low current transcranial electrostimulation

The analgesic effects of low current transcranial electrostimulation are both naloxone and pCPA-reversible, suggesting that they may be mediated in part by endogenous opioid and serotonergic activity. The present experiments indicate that pretreatment with the serotonin precursor L-tryptophan results in an increased analgesic effect of electrostimulation as measured by the 50 degrees C wet tail flick test in the rat. Rats receiving both L-tryptophan and electrostimulation displayed significantly more analgesia than rats receiving electrostimulation and injection vehicle alone, rats receiving drug and sham stimulation or rats receiving vehicle and sham stimulation.     

Growth rate, size, and sex ratio of last-laid, last-hatched offspring in the tree swallow Tachycineta bicolor

In tree swallows Tachycineta bicolor, last‐laid eggs typically hatch one to two days after the other eggs in the clutch hatch, putting last‐hatched offspring at a disadvantage when competing for food delivered by parents. We studied the biology of last‐laid, last‐hatched tree swallow offspring over two years in a Wyoming, USA, population. Our first objective was to compare the growth of last‐hatched offspring to that of their earlier‐hatched nestmates. One previous study had suggested that last‐hatched, competitively disadvantaged offspring grow feathers faster than senior nestmates, even at the expense of other aspects of growth. This may allow last‐hatched offspring to fledge with senior nestmates and avoid abandonment by parents. A second objective was to determine the sex of nestlings from last‐laid eggs. If last‐laid eggs typically produce undersized, weak adults that are poor competitors for resources, and if the fitness costs of being undersized/weak are more severe for males than for females, then selection may favour having offspring from last‐laid eggs to be female. In this study, last‐laid eggs hatched in 63 of 66 (94%) nests and hatched last in 93% of cases. At hatching, offspring from last‐laid eggs weighed, on average, 63% as much as their three heaviest nestmates (range: 26–107%). Offspring from last‐laid eggs fledged from 71% of the nests that produced at least one fledgling and apparently starved to death in remaining nests. Last‐hatched offspring who were presumably at a substantial competitive disadvantage (those whose mass at hatching was no more than about 75% of the mean mass of their three heaviest nestmates), gained mass more slowly than their senior nestmates but they eventually attained the same peak mass before fledging. Last‐hatched offspring grew primary feathers more slowly than their senior nestmates although the difference in growth rate was slight (0.2 mm/d) and only marginally significant. As a group, offspring from last‐laid eggs did not differ from offspring from all other eggs in either maximum mass attained before fledging or tarsus length at fledging. This is atypical for species with asynchronous hatching and is possibly the result of another unusual trait: the tendency of parent tree swallows to distribute food equally among young within broods. The sex ratio of offspring from last‐laid eggs did not deviate from 1:1 (22 males, 21 females). Given that last‐hatched eggs do not routinely produce undersized/weak individuals in our study population, there should be little selection on parent females to bias the sex ratio of last‐laid offspring towards females.     

Genetic association between nest building and brown adipose tissue thermogenesis in female house mice

Summary Mice selectively bred for either high or low levels of thermoregulatory nest building were cold-acclimated (5°C) for 3 weeks without nesting material; then body weight and food intake were measured. The mice selected for low nest building (Lows) of both sexes showed lower feed efficiencies than the high nest-building mice (Highs), although their body weights were not significantly different (Table 1). This adds to a large body of evidence which suggests that nest building and feed efficiency were influenced by a common mechanism (Lacy et al. 1978; Sulzbach and Lynch 1984; Lunch et al. 1981; Lynch and Roberts 1984).Brown adipose tissue mitochondrial GDP binding and cytochrome c oxidase activity were measured in the above mice. In females, the Lows had 100% higher levels of total GDP binding than the Highs, while no difference between the lines was seen in males (Fig. 2). Thus in the High females, lower energy expenditure through brown fat thermogenesis may account for their greater feed efficiency. In males, the genetic differences in feed efficiency must be due to differences in either thermogenesis in tissues other than brown fat, or mechanisms which reduce heat loss.Abbreviations Highs Mice from lines selectively bred for high levels of nest-building;Lows mice from the low nest-building selected lines     

Differential effects of n-3 fatty acid deficiency on phospholipid molecular species composition in the rat hippocampus

In this study, we have examined the effects of n-3 fatty acid deficient diets on the phospholipids (PL) molecular species composition in the hippocampus. Female rats were raised for two generations on diets containing linoleic acid (18:2n-6), with or without supplementation of alpha-linolenic acid (18:3n-3) or 18:3n-3 plus docosahexaenoic acid (22:6n-3). At 84 days of age, the hippocampal phospholipids were analyzed by reversed phase HPLC-electrospray ionization mass spectrometry. Depleting n-3 fatty acids from the diet led to a reduction of 22:6n-3 molecular species in phosphatidylcholine (PC), phosphatidylethanolamine (PE), PE-plasmalogens (PLE), and phosphatidylserine (PS) by 70-80%. In general, 22:6n-3 was replaced with 22:5n-6 but the replacement at the molecular species level did not always occur in a reciprocal manner, especially in PC and PLE. In PC, the 16:0,22:6n-3 species was replaced by 16:0,22:5n-6 and 18:0,22:5n-6. In PLE, substantial increases of both 22:5n-6 and 22:4n-6 species compensated for the decreases in 22:6n-3 species in n-3 fatty acid deficient groups. While the total PL content was not affected by n-3 deficiency, the relative distribution of PS decreased by 28% with a concomitant increase in PC.The observed decrease of 22:6n-3 species along with PS reduction may represent key biochemical changes underlying losses in brain-hippocampal function associated with n-3 deficiency.