Effects of murine tumor class I major histocompatibility complex expression on antitumor activity of tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TILs) are T cells that can be grown from enzyme-digested murine or human tumors. When adoptively transferred to tumor-bearing hosts concurrent with the administration of recombinant interleukin-2 (rIL-2), TILs can mediate significant regression of tumor. To examine whether expression of class I major histocompatibility complex on tumor cells influenced the generation and antitumor activity of TILs, we used clones of murine B16BL6 melanoma either transfected with or lacking the class I gene Kb to generate TILs at a high dose (1,000 U/mL) or at a low dose (20 U/mL) of human rIL-2. TILs grew from both tumors in high-dose rIL-2, but they grew from the class I-expressing tumor only in low-dose rIL-2. TILs from the class I-deficient tumor did not lyse any target tested in vitro, nor did they demonstrate any therapeutic effect in vivo on established tumors that lacked or expressed class I. In contrast, TILs from the class I-expressing tumor specifically lysed the tumor of origin in vitro and caused it to regress in vivo. Further, these TILs demonstrated activity in vitro against the non-class I-expressing melanoma treated with the combination of murine recombinant interferon gamma and human recombinant tumor necrosis factor alpha; in vivo, when administered with recombinant interferon gamma and recombinant tumor necrosis factor alpha, TILs from the class I-expressing tumor mediated regression of non-class I-expressing pulmonary metastases, presumably by augmenting class I expression.     

Iodothyronine 5'-deiodinase in brown adipose tissue: thiol activation and propylthiouracil inhibition

Brown adipose tissue (BAT) of hypothyroid rats contains a low Km (type II) iodothyronine 5'-deiodinase (I-5'D) that has been characterized as being insensitive to inhibition by propylthiouracil (PTU), based mainly on observations with homogenates prepared in a medium containing 10 mM dithiothreitol (DTT) and enzymatic assays in the presence of 20 mM DTT in vitro. In the studies reported herein, BAT homogenates from hypothyroid rats prepared in a DTT-free medium were found to contain I-5'D activity at 20 mM DTT, comparable to that in homogenates prepared in a DTT-containing medium, and were activated by submillimolar concentrations of DTT with an EC50 of approximately 0.5 mM. Almost all of the homogenate activities could be accounted for in microsomal preparations. The activity was substantially inhibited by 1 mM PTU. The PTU inhibition was progressively alleviated with increasing concentrations of added DTT and was not seen at DTT concentrations higher than 10 mM. At 250 microM DTT, the Km and maximum velocity values for rT3 and T4 were 2.9 and 1 nM and 70 and 200 fmol/mg protein X h, respectively, with a Ki for PTU of approximately 200 microM. On administration of PTU in vivo (2 mg/100 g BW; 1 h before killing) and subsequent assay at 250 microM DTT, the I-5'D in the homogenates was about 50% inhibited, and the microsomes showed a state of persistent inhibition, with activity levels about 70% of the control value. The data show that BAT type II I-5'D can be substantially activated at submillimolar concentrations of DTT, and this activation is sensitive to inhibition by PTU administered both in vitro and in vivo.     

Induction of heme oxygenase in the small intestinal epithelium: a response to oral cadmium exposure

The effects of oral cadmium administration on heme oxygenase activity and cytochrome P-450-dependent drug metabolism in intestinal epithelium were examined in male Sprague-Dawley rats. Cadmium chloride was administered via drinking water (0, 5 or 50 ppm cadmium) for 5 or 30 days, and heme oxygenase, 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and cytochrome P-450 were measured in the liver and in the epithelium of the proximal region of the small intestine. Cadmium exposure produced a marked, dose-related induction of intestinal heme oxygenase (up to 300% of control levels) in the small intestine at both time points examined. Concomitant decreases in intestinal ECOD (70%) and EROD (65%) activities were also observed, with a 65% decline in cytochrome P-450 levels at 30 days as compared with controls. Oral cadmium exposure, however, did not affect heme catabolism or cytochrome P-450 function in the liver, even at the highest concentration (50 ppm) administered, although cadmium levels accumulated in a dose-related manner in the liver as well as in the small intestine. Systemic absorption of cadmium was limited, as reflected by the relatively low accumulation of cadmium in the liver at 5 days (approximately 20 micrograms/g), as compared with the levels present in small intestine at this time ponit (approximately 100 micrograms/g). These findings emphasize the sensitivity of cytochrome P-450-dependent drug metabolism in small intestinal epithelium to orally ingested cadmium, and highlight the vulnerability of this tissue to low-dose exposure to this metal.     

Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene.

The human salivary amylase genes are associated with two inserted elements, a gamma-actin-processed pseudogene and an endogenous retroviral-like element. To test the contribution of these inserted elements to tissue specificity, 25 lines of transgenic mice carrying 10 amylase constructs were established. A 1-kb fragment of AMY1C (-1003 to +2) was found to be sufficient for parotid-specific expression of a human growth hormone reporter gene. The 1-kb fragment is entirely derived from inserted sequences. Deletion from -1003 to -826 resulted in reduced levels of transgene expression and loss of tissue specificity. The fragment -1003 to -327 was sufficient to transfer parotid specificity to the thymidine kinase promoter. The data demonstrate that the functional tissue-specific promoter of human AMY1C is derived from inserted sequences and that parotid expression can be conferred by sequences derived solely from the retrovirus. A role for retrotransposition in the evolution of gene regulation is indicated by these and other recent observations.     

Stimulus generalization of fear responses: effects of auditory cortex lesions in a computational model and in rats

The conditioning of fear responses to a simple acoustic stimulus (pure tone) paired with footshock can be mediated by the transmission of auditory information to the lateral nucleus of the amygdala from either the auditory thalamus or the auditory cortex. We examined the processing capacity of the thalamo-amygdala pathway by making lesions of the auditory cortex and testing the extent to which conditioned fear responses generalized to tones other than the one paired with footshock. Two studies were performed, one in an anatomically constrained computational model of the fear conditioning network and the other in rats. Stimulus generalization was unaffected in both. These findings support the validity of the model as an approach to studying the neural basis of conditioned fear learning, and in addition suggest that the thalamo-amygdala pathway, possibly by the use of population coding, is capable of performing at least crude stimulus discriminations.      

Visual acuity in an Australian Aboriginal population

Background: Australia is a developed country, However; Aboriginal Australians have rates of blindness comparable to Third World countries. There have been well-funded eye health programs for 15 years in Central Australia. This paper examines if there has been an improvement in visual disability of one traditional group of Aboriginal Australians. Methods: Results from an eye health survey of the Anangu Pitjantjatjara of South Australia in 1990 are presented. These data are compared with results for ‘blindness’ and ‘poor vision’ from a national survey undertaken in 1976. The two surveys were comparable in design, both were cross-sectional population-based prevalence surveys. Prevalence rates were adjusted for the size of the source population. Results: Young rural Aboriginal Australians have good visual acuity. Low vision and blindness (WHO definitions) occur in 19.6% and 10.4% of 60+ year olds, respectively. Women were more likely than men to be blind or have low vision (OR= 1.93; 1.06-3.58). There was a decline in ‘poor vision’ between surveys (OR=2.86; 1.86-4.75) but not in ‘blindness’. Conclusion: Although there has been a reduction in the prevalence of visual disability in rural Aboriginal Australians, improvements in the provision of eye care for the elderly need to occur.     

Universal linkage system: an improved method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) has become a powerful technique for studying gains and losses of DNA sequences in solid tumors. Importantly, DNA derived from archival tumor tissue is also applicable in CGH analysis. However, DNA isolated from routinely processed, formalin-fixed, paraffin-embedded tissue is often degraded, with the bulk of DNA showing fragment sizes of only 400-750 bp. Enzymatic labeling of archival DNA by standard nick translation (NT) decreases DNA size even further, until it becomes too small for CGH (<300 bp). This study presents application in CGH of a commercially available, non-enzymatic labeling method, called Universal Linkage System (ULS), that leaves the DNA fragment size intact. To compare the effect of chemical labeling of archival DNA by ULS vs. enzymatic by NT on the quality of CGH, DNA derived from 16 tumors was labeled by both ULS and NT. In those cases (n = 8), in which the bulk of DNA had a fragment size of 400-1,000 bp, CGH was successful with ULS-labeled probes, but not with NT-labeled probes. In the DNA samples (n = 6) with a fragment size > 1 kb, the intensity of CGH signals was comparable for both ULS- and NT-labeled probes, but CGH with ULS-labeled samples showed a high, speckled, background, which seriously hampered image analysis. In the remaining two cases, which had evenly distributed DNA fragment sizes (range 250-5,000 bp), CGH was successful with both labeling methods. Using DNA fragment size < 1 kb as a selection criterion for ULS labeling, we were able to obtain good quality CGH of a large panel (n = 77) of a variety of archival solid tumors. We conclude that ULS is an excellent labeling method for performing CGH on small-fragment-sized DNA.