Distribution of trace elements in normal and tumor-bearing mice using the multitracer technique

A multitracer solution obtained from the nuclear reaction of selenium with 25-MeV/nucleon ⁴⁰Ar ions was orally administered to normal and tumor-bearing Balb/c male mice. After 96 h, the mice were sacrificed and the elemental distribution was determined in various tissues, organs, and blood. The uptake of Na, Rb, Ga, Sc, V, Cr, Mn, Co, Fe, Zn, Y, Zr, Tc, Ru, Ag, and In in normal and, except for zinc, in tumor-bearing mice was simultaneously detected. Most elements were distributed in about the same manner in the skin and liver of animals in both groups. The distribution of Rb, Ga, V, Cr, Tc, and In showed little or no significant differences between the two study groups. The distribution of Na, Mn, Fe, Ag, Sc, and Co showed significant differences between normal and tumor-bearing mice. In the blood, spleen, and kidney of the normal mice, there was good absorption of Na, Mn, Fe, Ag, Co, and Zn. In the heart, these elements were well absorbed, except for Na and Mn.     

Immunohistochemical distribution of epimorphin in human and mouse tissues

A novel protein epimorphin has been identified as a mesenchymal signal factor. We reported previously ubiquitous expression of epimorphin in normal skin and a significant increased expression in diseased human skin. The present immunofluorescence study was conducted to determine systematically the distribution of epimorphin in adult human organs with an anti-epimorphin monoclonal antibody. Epimorphin was found to be widely distributed in all human organs examined. It was present in the connective tissue adjacent to or around various epithelial tissues, muscles and vessels. In particular, strong staining was present on the endomysium of muscles, the adventitia of blood vessels, along the sinusoidal lining of hepatocytes and connective tissue around epithelial cells, exocrine and endocrine glands. The results suggest that epimorphin may play a key role in maintaining normal tissue structure and interaction between mesenchymal tissue and epithelial tissue in vivo. ©; 1998 Chapman & Hall     

Levels of Endogenous lnterleukin-1, Interleukin-6, and Tumor Necrosis Factor in Congenic Mice Infected with Borrelia garinii

This study describes the levels of interleukin-1 alpha (IL-1α), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the sera and parenchymal organs of various congenic mouse strains infected with Borrelia garinii. A significant elevation of inflammatory cytokine levels was found in the organs of C3H/HeN (H-2^k) and B10.BR (H-2^k) mice but not in those of BALB/c mice (H-2^d). Focally produced cytokines can contribute to antimicrobial defense against these organisms. High levels of IL-1α were observed in the sera of C3H/HeN, B10.BR and B10 (H-2^b) mice infected with B. garinii and they were associated with the presence of spirochetes in the skin. Thus, susceptible mice demonstrated a stronger cytokine response than resistant mice. This study presents in vivo evidence that B. garinii infection affects the immunopathogenesis of Lyme disease.     

Levels of D-serine in the brain and peripheral organs of serine racemase (Srr) knock-out mice

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor, plays an important role in mammalian brain neurotransmission, via the NMDA receptor. d-Serine is synthesized from l-serine by the pyridoxal-5′ phosphate-dependent enzyme serine racemase (SRR), and d-serine is metabolized by d-amino acid oxidase (DAAO). In this study, we measured levels of the neurotransmission related amino acids, d-serine, l-serine, glycine, glutamine and glutamate in the frontal cortex, hippocampus, striatum and cerebellum as well as in peripheral tissues of blood, heart, pancreas, spleen, liver, kidney, testis, epididymis, heart, lung, muscle and eyeball, in wild-type (WT) and Srr-knockout (Srr-KO) mice. Levels of d-serine in the frontal cortex, hippocampus, and striatum of Srr-KO mice were significantly lower than in WT mice, while levels in the cerebellum stayed the same. In contrast, levels of l-serine, glycine, glutamine and glutamate remained the same in all tested brain regions. In vivo microdialysis using free-moving mice showed that extracellular levels of d-serine in the hippocampus of Srr-KO mice were significantly lower than in WT mice while the other amino acid levels remained the same between mice. In peripheral organs, levels of d-serine in the kidney, testis, and muscle of Srr-KO mice were significantly lower than in WT mice. Tissue levels of the other tested amino acids in peripheral organs were not altered. These results suggest that SRR is the major enzyme responsible for d-serine production in the mouse forebrain, and that other pathways of d-serine production may exist in the brain and peripheral organs.     

Effect of albumin fusion on the biodistribution of interleukin-2

Purpose We investigated and compared the biodistribution of Albuleukin, a human serum albumin (HSA)-interleukin-2 (IL-2) fusion protein, with those of IL-2 and HSA. The objective was to determine whether Albuleukin distributes differently to normal organs and lymphoid tissues than IL-2 by virtue of its genetic fusion with HSA.Methods The chelating agent 2-(p-isothiocyanato-benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-A^II was selected for radiolabeling with ¹¹¹In, and conjugation with CHX-A^II did not alter bioactivities of IL-2 and Albuleukin on proliferation of CTLL-2 cells. The radiolabeled proteins were injected intravenously into mice, uptake in organs was measured, and whole-body autoradiography was performed.Results Striking differences in the biodistribution of IL-2 and Albuleukin were noted. ¹¹¹In-IL-2 cleared from blood rapidly, with less than 1% ID/g (percentage of injected dose per gram of tissue) at 20 min after injection. At this time, the kidneys showed more than 120% ID/g uptake, and these high levels persisted through 6 h. ¹¹¹In-Albuleukin, by contrast, showed significantly longer circulation (14% ID/g at 6 h), lower kidney uptake (<6% ID/g), and higher localization in liver, spleen, and lymph nodes (maximal uptake ~22% ID/g for all three organs). Uptake in liver, spleen, and lymph nodes appears to be mediated in part by the IL-2 component of Albuleukin because ¹¹¹In-HSA showed significantly lower accumulation in those tissues despite more prolonged circulation in blood.Conclusion These data support the hypothesis that Albuleukin targets tissues where lymphocytes reside to a much greater extent than does IL-2, and suggest that Albuleukin may exhibit improved efficacy and reduced toxicity in the treatment of solid tumors. Clinical trials underway will determine whether the improved targeting in the mice translates into a better therapeutic index in humans.     

Measurements of radon activity concentration in mouse tissues and organs

The purpose of this study is to investigate the biokinetics of inhaled radon, radon activity concentrations in mouse tissues and organs were determined after mice had been exposed to about 1 MBq/m³ of radon in air. Radon activity concentrations in mouse blood and in other tissues and organs were measured with a liquid scintillation counter and with a well-type HP Ge detector, respectively. Radon activity concentration in mouse blood was 0.410?±?0.016 Bq/g when saturated with 1 MBq/m³ of radon activity concentration in air. In addition, average partition coefficients obtained were 0.74?±?0.19 for liver, 0.46?±?0.13 for muscle, 9.09?±?0.49 for adipose tissue, and 0.22?±?0.04 for other organs. With these results, a value of 0.414 for the blood-to-air partition coefficient was calculated by means of our physiologically based pharmacokinetic model. The time variation of radon activity concentration in mouse blood during exposure to radon was also calculated. All results are compared in detail with those found in the literature.     

Metabolism of glycosaminoglycans in tissues of adjuvant arthritic rat

The metabolic changes in the connective tissue glycosaminoglycans were studied in tissues of adjuvant induced arthritic rats. Arthritic process was induced in rats with the inoculation of Freund's adjuvant containing heat killed Mycobacterium tuberculosis in paraffin oil. The connective tissue glycosaminoglycans were fractionated into sulfated and non-sulfated glycosaminoglycans by chemical and enzymatic methods. The biosynthesis of sulfated glycosaminoglycans was examined using radioactive labeled (³⁵S)-sulfate incorporation measurements into the sulfated glycosaminoglycans in tissues such as liver, kidney, spleen and skin of arthritic rats. The catabolism of glycosaminoglycans was studied by measuring the activity of various connective tissue degrading lysosomal glycohydrolases in tissues of experimental animals. In addition, the changes in the contents of total glycosaminoglycans, mono-sulfated, highly-sulfated and non-sulfated glycosaminoglycans were quantitatively assessed in diseased tissues. Alterations in the metabolism of connective tissue glycosaminoglycans were demonstrated in tissues of arthritic rats. The uptake of (³⁵S)-sulfate into the tissue was found to be increased in liver, kidney and spleen, while that of skin decreased during the process of arthritis. The total glycosaminoglycan content was significantly elevated in diseased tissues compared to normal. Similarly, mono-sulfated, highly-sulfated and non-sulfated glycosaminoglycans were found to be increased in arthritic tissues. In addition, the activity of various connective tissue degrading lysosomal glycohydrolases such as -glucuronidase, -N-acetylglucosaminidase, cathepsin B, cathepsin L and collagenolytic cathepsin was increased in tissues of arthritic rat. The results presented in this communication indicate that the characteristic alterations were induced in the metabolism of glycosaminoglycans by the dynamic process of adjuvant arthritis.     

Changes in fatty acid composition of thymus cells, liver, blood plasma, and muscle tissue in mice with solid Ehrlich carcinoma

The fatty acid composition of thymus cells, liver, blood plasma, muscle tissue, and tumor focus has been studied in mice with solid Ehrlich carcinoma. The tumor growth in the mice was associated with an increase in the total content of monounsaturated fatty acids in all organs and tissues studied and with a decrease in the total amount of polyunsaturated fatty acids in all tissues except blood plasma. The tumor tissue was characterized by increased levels of monounsaturated fatty acids in comparison with their levels in organs and tissues of intact animals. In the thymus of tumor-bearing mice, the contents of myristic and palmitic saturated fatty acids, which are associated with activation of the T-cell immunity, were increased. The most expressed and considerable changes in the fatty acid composition during tumor growth were observed in the muscle tissue of the animals. A possible role of changes in the fatty acid composition in the investigated organs and tissues of tumor-bearing mice in the organism’s response to tumor growth is discussed.     

In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals

The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67⁺ cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4–6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34⁺α6⁺ and Lrig-1⁺ bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.     

Substantial Differences between Organ and Muscle Specific Tracer Incorporation Rates in a Lactating Dairy Cow

We aimed to produce intrinsically L-[1-¹³C]phenylalanine labeled milk and beef for subsequent use in human nutrition research. The collection of the various organ tissues after slaughter allowed for us to gain insight into the dynamics of tissue protein turnover in vivo in a lactating dairy cow. One lactating dairy cow received a constant infusion of L-[1-¹³C]phenylalanine (450 µmol/min) for 96 h. Plasma and milk were collected prior to, during, and after the stable isotope infusion. Twenty-four hours after cessation of the infusion the cow was slaughtered. The meat and samples of the various organ tissues (liver, heart, lung, udder, kidney, rumen, small intestine, and colon) were collected and stored. Approximately 210 kg of intrinsically labeled beef (bone and fat free) with an average L-[1-¹³C]phenylalanine enrichment of 1.8±0.1 mole percent excess (MPE) was obtained. The various organ tissues differed substantially in L-[1-¹³C]phenylalanine enrichments in the tissue protein bound pool, the highest enrichment levels were achieved in the kidney (11.7 MPE) and the lowest enrichment levels in the skeletal muscle tissue protein of the cow (between 1.5–2.4 MPE). The estimated protein synthesis rates of the various organ tissues should be regarded as underestimates, particularly for the organs with the higher turnover rates and high secretory activity, due to the lengthened (96 h) measurement period necessary for the production of the intrinsically labeled beef. Our data demonstrates that there are relatively small differences in L-[1-¹³C]phenylalanine enrichments between the various meat cuts, but substantial higher enrichment values are observed in the various organ tissues. We conclude that protein turnover rates of various organs are much higher when compared to skeletal muscle protein turnover rates in large lactating ruminants.     

Stable isotope resolved metabolomics of lung cancer in a SCID mouse model

We have determined the time course of [U-¹³C]-glucose utilization and transformations in SCID mice via bolus injection of the tracer in the tail vein. Incorporation of ¹³C into metabolites extracted from mouse blood plasma and several tissues (lung, heart, brain, liver, kidney, and skeletal muscle) were profiled by NMR and GC–MS, which helped ascertain optimal sampling times for different target tissues. We found that the time for overall optimal ¹³C incorporation into tissue was 15–20 min but with substantial differences in ¹³C labeling patterns of various organs that reflected their specific metabolism. Using this stable isotope resolved metabolomics (SIRM) approach, we have compared the ¹³C metabolite profile of the lungs in the same mouse with or without an orthotopic lung tumor xenograft established from human PC14PE6 lung adenocarcinoma cells. The ¹³C metabolite profile shows considerable differences in [U-¹³C]-glucose transformations between the two lung tissues, demonstrating the feasibility of applying SIRM to investigate metabolic networks of human cancer xenograft in the mouse model.     

Dietary Selenium Influences Calcium Release and Activation of MLCK in Uterine Smooth Muscle of Rats

We sought to elucidate the effects of different concentrations of dietary selenium on calcium ion release, MLCK levels, and muscle contraction in the uterine smooth muscle of rats. The selenium (Se) content of blood and of uterine smooth muscle tissues was detected by fluorescence spectrophotometry. Ca²⁺ content was measured by atomic absorption spectroscopy. Calmodulin (CaM) and MLCK RNA and protein levels were analyzed by quantitative real-time polymerase chain reaction and Western blot, respectively. Dietary Se intake increased the Se levels in the blood and in uterine smooth muscle tissues and increased the Ca²⁺ concentration in uterine smooth muscle tissues. The addition of Se also promoted CaM expression and enhanced MLCK activation in uterine smooth muscle tissues. In conclusion, Ca²⁺, CaM, and MLCK were regulated by Se in uterine smooth muscle; Se plays a major role in regulating smooth muscle contraction in the uterus.     

Newly discovered activities for calcitriol (1,25-dihydroxyvitamin D3): implications for future pharmacological use

Recent studies have yielded new insights into the critical importance of adequate vitamin D₃ intake and metabolism. Investigations of the actions of 1,25-dihydroxyvitamin D₃ (calcitriol) on novel target tissues has revealed that this hormone has functions other than its recognized action in regulating blood calcium and phosphate levels. Reports have characterized calcitriol receptors and activities in organs and tissues as diverse as pancreas, skeletal and heart muscle, blood cells, brain, skin, pituitary, parathyroid, kidney, bone and intestine. These studies suggest functions for calcitriol as varied as the regulation of insulin and prolactin secretion, muscle contractility, immune cell metabolism, melanin synthesis and differentiation of blood cells. This information may ultimately help us to understand the etiologies of several kinds of organ dysfunction and lead to the development of tissue-specific agents for new therapies.     

Alterations in Oral [1-14C] 18:1n-9 Distribution in Lean Wild-Type and Genetically Obese (ob/ob) Mice

Obesity may result from altered fatty acid (FA) disposal. Altered FA distribution in obese individuals is poorly understood. Lean wild-type C57BL/6J and obese C57BL/6J^ob/ob mice received an oral dose of [1-¹⁴C]18:1n-9 (oleic acid), and the radioactivity in tissues was evaluated at various time points. The ¹⁴C concentration decreased rapidly in gastrointestinal tract but gradually increased and peaked at 96 h in adipose tissue, muscle and skin in lean mice. The ¹⁴C concentration was constant in adipose tissue and muscle of obese mice from 4h to 168h. ¹⁴C-label content in adipose tissue was significantly affected by genotype, whereas muscle ¹⁴C-label content was affected by genotype, time and the interaction between genotype and time. There was higher total ¹⁴C retention (47.7%) in obese mice than in lean mice (9.0%) at 168 h (P<0.05). The ¹⁴C concentrations in the soleus and gastrocnemius muscle were higher in obese mice than in lean mice (P<0.05). Perirenal adipose tissue contained the highest ¹⁴C content in lean mice, whereas subcutaneous adipose tissue (SAT) had the highest ¹⁴C content and accounted for the largest proportion of total radioactivity among fat depots in obese mice. More lipid radioactivity was recovered as TAG in SAT from obese mice than from lean mice (P<0.05). Gene expression suggested acyl CoA binding protein and fatty acid binding protein are important for FA distribution in adipose tissue and muscle. The FA distribution in major tissues was altered in ob/ob mice, perhaps contributing to obesity. Understanding the disparity in FA disposal between lean and obese mice may reveal novel targets for the treatment and prevention of obesity.     

Density of Parasites in Various Organs and the Relation to Numbers of Trypomastigotes in the Blood during Acute Infections of Trypanosoma cruzi in Mice*

Quantitative methods were used to (a) determine the density of Trypanosoma cruzi in organs of CF₁ mice following intraperitoneal inoculation of 50,000 trypomastigotes of a Brazil strain of T. cruzi and (b) study the relation of the numbers of these intracellular stages to the numbers of trypomastigotes in the blood. Tissue stages (predominantly amastigotes) in heart, skeletal muscle (triceps), diaphragm, cerebrum, cerebellum, and musculature of stomach, duodenum, esophagus, jejunum, cecum, and rectum increased in numbers during the 1st 3 weeks of infection, reached maximum density 21–28 days after inoculation and subsequently declined in numbers until mice were histologically negative for intracellular parasites by 30–40 days. The density of tissue stages in the urinary bladder, uterine body, and ileum was similar with the exception that maximum numbers of parasites were observed slightly earlier at 15 days. The greatest density of intracellular stages was seen in heart, urinary bladder, diaphragm, and triceps muscle where mean counts of 44.6–60.0 × 10⁶ parasites/cc of muscle were recorded while maximum density of parasites in the uterine body, cerebrum, stomach, cerebellum, duodenum, esophagus, jejunum, ileum, cecum, and rectum was 13.0 × 10⁶/cc of muscle or less. Amastigotes were not observed in sections of lymph node, thymus, salivary glands, liver, spleen, or kidney and only a single pseudocyst containing 5 amastigotes was seen in lung. With the exception of the brain and lung, intracellular parasites were located exclusively in the musculature. Trypomastigotes in the blood increased during the 1st 3 weeks of infection, reached maximum numbers 21–28 days after initiation of infection, and subsequently decreased until by 30–40 days parasites were observed only rarely in the blood of a few animals. Thus generally close correlation was noted between the numbers of intracellular stages of T. cruzi in the organs and trypomastigotes in the blood throughout acute Chagas’ disease in mice as evidenced by the concomitant increase in numbers of both stages, the coincidence of days of maximum parasite levels, and the simultaneous decline in numbers of both stages. The mean number of parasites/pseudocyst section varied in the organs studied. Of the 15 positive organs studied, the pseudocyst sections in skeletal muscle contained the highest mean number of parasites (64.3 parasites/pseudocyst section) and those pseudocyst sections seen in the musculature of the small intestine contained the lowest mean number (5.5–6.8 parasites/pseudocyst section respectively in ileum and jejunum). Serial sections of skeletal muscle, heart, urinary bladder, and stomach revealed the largest pseudocysts in skeletal muscle while those in the musculature of the urinary bladder were the smallest.     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

A multicopy c-Myc transgene as a nuclear label: overgrowth of Myctg50 cells in allophenic mice.

To trace cell lineages and the origin and fate of cells in transplantation and embryo chimeras, a DNA/DNA in situ hybridization cell labelling system was developed, based on a 50-copy murine c-myc transgene on mouse chromosome 8. Elevated levels of cMyc mRNA were found in Myc*tg50 (Myc^tg50/0 and Myc^tg50/Myc^tg50) transgenic tissues, but adult transgenic NMRI mice were anatomically and histologically indistinguishable from control NMRI mice and did not develop tumours on a wild-type or nude (nu / nu) background. The hybridization label detected transgenic nuclei with an efficiency of ～80%. In muscle grafts, this transgene label was successfully applied to trace donor cells in a labelled host and to study the invasion of a graft by host cells. When the cMyc hybridization was used in allophenic mice of the control|acNMRI-Myc^tg50/? (nu /+ or +/+) type, an up to a three-fold excess of MYC*tg50 positive over control nuclei was found in all organs examined (ventricle, skeletal muscle, liver, small intestine). This overgrowth of MYC*tg50 cells is probably due to transgene expression. Four out of seven (C57BL/6×BALB/c) or (C57BL/6×NMRI)|acMYC*tg50 allophenic mice displayed anatomical abnormalities, e.g. an enlarged thymus and a tumour in the groin region. As these abnormalities were only observed in allophenic mice, they might be due to the imbalance of growth potential between MYC*tg50 transgenic and normal cells in the same individual.