Inhibition of TNF-alpha produced by Kupffer cells protects against the nonspecific liver toxicity of immunotoxin anti-Tac(Fv)-PE38, LMB-2

LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas: exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-alpha by these cells. Following LMB-2 administration to mice, the levels of TNF-alpha in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-alpha in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-alpha produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.     

The Red-Eared Slider Turtle: An Animal Model for the Study of Low Doses and Mixtures

Current risk assessment techniques for almost all chemicalsinvolve establishing a threshold dose, or the dose below whichno adverse effects are seen. But threshold doses may not applyin situations where the chemical mimics the actions of an endogenouscompound—such as a steroid hormone—that is importantto development. In such cases, any exposure may exceed the threshold.Recent studies with the red-eared slider turtle have shown thatexogenous estradiol—even when applied to eggshell in dosesas low 0.4 ng—will affect sex development during embryogenesis.Considering that only 0.2% of the estradiol applied to the eggshellends up in the embryo, it becomes apparent that even very lowdosages of steroid hormones or their mimics can have profoundbiological effects. We tested this idea using eight compoundsidentified in the yolk of alligator eggs from Lake Apopka, Fla.Five of the compounds—the PCB mixture Aroclor 1242,trans-Nonachlor,cis-Nonachlor,p,p'-DDE, and chlordane—altered sex ratio outcomes whenapplied to eggshells during development. Aroclor 1242 producedthe most powerful effects, shifting the ratio of females almosttwofold, while chlordane had the greatest effect when combinedwith estradiol. Administration of all eight compounds togetheralso increased the ratio of females to males. However, comparingthe single-compound exposures at the same dosages indicate thatthese compounds behave differently in combination than theydo singly, emphasizing the need for further studies using chemicalmixtures reflecting proportions found in nature. The effectof chlordane and Aroclor 1242 on aromatase activity levels duringembryogenesis in the brain and adrenal-kidney-gonad (AKG) complexwas also examined. Chlordane, a suspected anti-androgen in thisspecies, did not affect aromatase activity in either the brainor the AKG. However, Aroclor 1242 significantly altered aromataseactivity levels in the red-eared slider turtle brain—butnot in the AKG—during a crucial developmental period.After this crucial period, Aroclor 1242 caused an increase inaromatase activity in the AKG of embryos just prior to hatch.Additionally, hatchling males treated during embryogenesis withAroclor 1242 and chlordane exhibited significantly lower testosteronelevels than controls in response to follicle-stimulating hormoneadministration, while chlordane-treated females had significantlylower progesterone, testosterone, and 5-dihydrotestosteronelevels relative to controls. These results are similar to thosefound in juvenile alligators from Lake Apopka. Males treatedwith Aroclor 1242 and trans-Nonachlor displayed an elevatedestradiol response to FSH administration vs. control males.Taken together, these results suggest that EDCs exert effectsduring embryonic development that extend beyond birth. Theyalso suggest that the alterations in sex steroid hormone levelsobserved in animals from contaminated areas may result fromEDC-induced alterations in the neuroendocrine axis controllinggonadal sex steroid hormone production.     

Expression, transport properties, and chromosomal location of organic anion transporter subtype 3

The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na(+)-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant approximately 30 microM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.     

Ultrastructural immunocytochemical localization of clathrin in cultured fibroblasts

Using an affinity-purified antibody prepared against the major coat protein of brain-coated vesicles, clathrin, we have localized this protein by ultrastructural immunocytochemistry in Swiss 3T3 cultured fibroblasts. Fixation, permeabilization, and labeling were performed using the EGS and ferritin bridge labeling techniques. Localization of clathrin was detected on the coated regions of both the plasma membrane and the GERL apparatus. Almost no clathrin was found in the cytosol or in association with any other organelles. A very low concentration of labeling was occasionally seen randomly distributed on the inner surface of the plasma membrane and reticular membranous structures near the plasma membrane. The significance of these results in the role of the clathrin-coated regions in receptor-mediated endocytosis and Golgi function are discussed.     

Agronomic evaluation of prospective mnew crop species II. The AmericanLimnanthes

Limnanthes is a potential seed oil source of long chain fatty acids. Agronomic evaluations in Alaska, California, Maryland, and Oregon indicate that theL. alba group has the best crop potential because of superior seed retention and erect growth habit. Experimental seed yields of 1700 kg/ha or more indicate that economically feasible yields on a commercial scale will be attainable, as continued study reveals optimum cultural practices and utilizes variability present in the various accessions. For the best seed yields,Limnanthes must be handled as a winter annual in California, Maryland, and Oregon but as a spring-seeded annual in Alaska.Limnanthes plants mature rapidly during hot weather; thus the timing of harvest is critical for maximization of seed yields. Further studies on seeding rates, weed control, and harvest methods are suggested.     

Above‐ and belowground linkages in Sphagnum peatland: climate warming affects plant‐microbial interactions

Peatlands contain approximately one third of all soil organic carbon (SOC). Warming can alter above‐ and belowground linkages that regulate soil organic carbon dynamics and C‐balance in peatlands. Here we examine the multiyear impact of in situ experimental warming on the microbial food web, vegetation, and their feedbacks with soil chemistry. We provide evidence of both positive and negative impacts of warming on specific microbial functional groups, leading to destabilization of the microbial food web. We observed a strong reduction (70%) in the biomass of top‐predators (testate amoebae) in warmed plots. Such a loss caused a shortening of microbial food chains, which in turn stimulated microbial activity, leading to slight increases in levels of nutrients and labile C in water. We further show that warming altered the regulatory role of Sphagnum‐polyphenols on microbial community structure with a potential inhibition of top predators. In addition, warming caused a decrease in Sphagnum cover and an increase in vascular plant cover. Using structural equation modelling, we show that changes in the microbial food web affected the relationships between plants, soil water chemistry, and microbial communities. These results suggest that warming will destabilize C and nutrient recycling of peatlands via changes in above‐ and belowground linkages, and therefore, the microbial food web associated with mosses will feedback positively to global warming by destabilizing the carbon cycle. This study confirms that microbial food webs thus constitute a key element in the functioning of peatland ecosystems. Their study can help understand how mosses, as ecosystem engineers, tightly regulate biogeochemical cycling and climate feedback in peatlands     

Interactions between immunity,proliferation and molecular subtype in breast cancer prognosis

Background

Gene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified.

Results

To investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes.

Conclusions

These findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications.     

Low-pH Rescue of Acid-Sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-Regulated Arginine Decarboxylase System

For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the P_araBAD or P_rhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter P_rhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter P_araBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.