Abnormal lymphatic phenotype in a CRISPR mouse model of the human lymphedema-causing Connexin47 R260C point mutation.

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.

Non-contrast MR lymphography of rare lymphatic abnormalities.

Detailed imaging of the lymphatic system often requires direct injection of contrast into lymph nodes which can be technically challenging, time consuming, and produce painful stimuli. We sought to describe the use of non-contrast MR lymphography (NCMRL) for normal controls and patients with a variety of rare disorders associated with lymphatic pathologies. Two control subjects and five affected patients (decompensated Fontan circulation, central conducting lymphatic abnormality, familial lymphedema and two with Gorham-Stout disease) were studied. NCMRL images were segmented in a semiautomated fashion and the major lymphatic channels and thoracic duct (TD) highlighted for identification. Adequate imaging was available for both controls and 4/5 affected patients; the youngest patient could not be imaged given patient motion. For the two controls, the TD was seen in the expected anatomic location. For the decompensated Fontan patient, there were numerous tortuous lymphatic channels, predominantly in the upper chest and neck. For the familial lymphedema patient, a TD was not identified; instead, peripheral lymphatic collaterals near the lateral chest walls. For the first Gorham- Stout patient, the TD was enlarged with large intrathoracic lymph collections. For the second Gorham-Stout patient, there were bilateral TD with lymph collections in vertebral bodies. Using NCMRL, we were able to image normal and abnormal lymphatic systems. An important learning point is the potential need for sedation for younger patients due to long image acquisition times and fine resolution of the structures of interest.

Whole-body lymphangioscintigraphy and SPECT/CT in children with lymphatic complications after surgery for complex congenital heart disease.

The number of patients surviving repair of complex congenital heart disease (CCHD) has increased due to improved surgical techniques, post operative management and outpatient care. Likewise, this growing patient population has demonstrated an increasing number and complexity of complications involving the lymphatic system. To evaluate the peripheral and central lymphatic system, whole-body lymphangioscintigraphy (LAS) is considered as the initial imaging evaluation of choice. To date, very few publications exist on the value of lymphatic imaging techniques in infants and small children with lymphatic complications following surgery for congenital heart disease. A retrospective review of medical records from 2008 to 2018 was performed for pediatric patients referred for lymphatic complications after CCHD surgery at an academic medical center. LAS and SPECT/CT was performed using intradermal bipedal injections of Tc 99m labeled filtered sulfur colloid, and in some patients also bilateral hand injections, followed by dynamic imaging and whole- body planar imaging typically up to 180 minutes post injection. Clinical decision making and outcomes were recorded. LAS and SPECT/CT were performed without complication in pediatric patients with prior surgery for CCHD. LAS successfully localized various lymphatic abnormalities such as lymphatic obstruction, reflux, and leaks, which were further delineated by SPECT/CT. LAS findings directed further evaluation with more definitive studies, management and prognosis. Five of the ten patients had follow up outcome data - 2 years and up to 10 years. LAS and SPECT/CT are safe and effective techniques for the initial evaluation of lymphatic abnormalities in pediatric patients with CCHD. LAS, particularly with further 3D localization by SPECT/CT, provides functional imaging of peripheral and central lymphatic flow and thus provides guidance for medical therapy, non operative interventional management, and surgical therapy for these diverse, debilitating, and often life threatening disorders.

Early feeding and dietary lipids affect broiler tissue fatty acids, vitamin E status, and cyclooxygenase-2 protein expression upon lipopolysaccharide challenge.

Newly hatched chicks are often subjected to delayed access to feed and water because of shipment distances and hatchery practices, which may reduce growth and development of the immune system. The current study investigated the effects of early vs. late access to feed and dietary lipids (n-3 vs. n-6) on lipopolysaccharide (LPS)-induced alterations in tissue fatty acids, vitamin E status, and cyclooxygenase-2 (COX-2) protein expression. The chicks (n = 16/group) were fed a high or low n-3 diet within 5 to 5 h 30 min (early) or after 48 h (late) of hatching. Feeding high n-3 diets increased eicosapentaenoic acid (EPA, 20:5 n-3), docosapentaenoic acid (22:5 n-3), and docosahexaenoic acid (DHA, 22:6 n-3) in the liver, spleen, and plasma (P < 0.05). Feeding low n-3 diets increased arachidonic acid in the liver and plasma (P < 0.05). Early access to feed led to increases in liver oleic acid and reduction in arachidonic acid as compared with late-fed birds (P < 0.05). No effect of time of feeding on fatty acids in the spleen was observed. Early feeding led to significant increases in linoleic and arachidonic acids in the plasma (P < 0.05). Stearic acid was higher in the plasma of low n-3 early-fed as opposed to low n-3 late-fed birds (P < 0.05). The LPS challenge led to an increase in liver total fat content (P < 0.05). The total fat content in the spleen and plasma were not affected by LPS injection (P > 0.05). The LPS-injected birds had decreases in oleic acid in the liver and plasma as compared with saline-injected birds (P < 0.05). Stearic acid increased upon LPS injection in the spleen and plasma (P < 0.05). Liver vitamin E content was significantly higher in saline-injected birds from the early high n-3 group compared with all treatment groups, except for the late low n-3 saline-injected birds (P < 0.05). Plasma vitamin E was highest in the early low n-3 LPS-injected birds compared with all other treatment groups (P < 0.05). The COX2:actin ratio in the early high n-3 LPS-injected birds was higher than that of the saline-injected birds of the same treatment (P < 0.05). However, no difference in COX-2 expression was observed between LPS- or saline-injected fed early low n-3, late high n-3, or late low n-3 diets (P > 0.05). No effect of diet, time of feeding, or LPS challenge on plasma isoprostanes was observed (P > 0.05). These results suggest that dietary and management strategies directed at modulating tissue polyunsaturated fatty acid status may offer the promise of modulating lipid metabolism and COX-2 expression in commercial poultry.

Reporter gene transactivation by human p53 is inhibited in thioredoxin reductase null yeast by a mechanism associated with thioredoxin oxidation and independent of changes in the redox state of glutathione.

Reporter gene transactivation by human p53 is compromised in S. cerevisiae lacking the TRR1 gene encoding thioredoxin reductase. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Deltatrr1 yeast. The Deltatrr1 mutation affected the redox state of both molecules. About 34% of thioredoxin was in the disulfide form in wild-type yeast and increased to 70% in Deltatrr1 yeast. About 18% of glutathione was in the GSSG form in wild-type yeast and increased to 32% in Deltatrr1 yeast. The Deltatrr1 mutation also resulted in a 2.9-fold increase in total glutathione per mg extract protein. Highcopy expression of the GLR1 gene encoding glutathione reductase in Deltatrr1 yeast restored the GSSG:GSH ratio to wild-type levels, but did not restore p53 activity. Also, p53 activity was shown to be unaffected by a Deltaglr1 mutation, even though the mutation was known to result in glutathione oxidation. In summary, the results show that, although glutathione becomes more oxidized in Deltatrr1 cells, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. The results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment.

Measurement of glutathione and glutathione disulfide.

Measurements of glutathione should include quantification of both the reduced and oxidized forms. HPLC-based assays for glutathione and other cellular thiols and disulfides utilize a variety of detection methods, including ultraviolet, fluorescence, and electrochemical methods. This unit provides rapid, sensitive, highly versatile and reproducible HPLC methods for separating and quantifying sulfur-containing amino acids and related derivatives from a variety of biological samples.

The role of the redox protein thioredoxin in cell growth and cancer.

The thioredoxins are ubiquitous proteins containing a conserved -Trp-Cys-Gly-Pro-Cys-Lys- redox catalytic site. Mammalian thioredoxin family members include thioredoxin-1 (Trx1), mitochondrial thioredoxin-2 (Trx2), and a larger thioredoxin-like protein, p32TrxL. Thioredoxin is reduced by NADPH and thioredoxin reductase and, in turn reduces oxidized cysteine groups on proteins. When thioredoxin levels are elevated there is increased cell growth and resistance to the normal mechanism of programmed cell death. An increase in thioredoxin levels seen in many human primary cancers compared to normal tissue appears to contribute to increased cancer cell growth and resistance to chemotherapy. Mechanisms by which thioredoxin increases cell growth include an increased supply of reducing equivalents for DNA synthesis, activation of transcription factors that regulate cell growth, and an increase in the sensitivity of cells to other cytokines and growth factors. The mechanisms for the inhibition of apoptosis by thioredoxin are just now being elucidated. Because of its role in stimulating cancer cell growth and as an inhibitor of apoptosis, thioredoxin offers a target for the development of drugs to treat and prevent cancer.

Thioredoxin reductase.

The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases.

Biliary secretion of alpha-tocopherol and the role of the mdr2 P-glycoprotein in rats and mice.

The mechanism by which alpha-tocopherol (alpha-T) is secreted into the bile is not known; however, we have previously demonstrated that treatment with piperonyl butoxide (PIP, 1 g/kg) results in increased biliary output of both alpha-T and phosphatidylcholine within 3 h of ip injection in rats and that the biliary output of both substances was prevented by chemicals that disrupt microtubules (Toxicol. Appl. Pharmacol. 139, 411-417 (1996)). The P-glycoprotein (Pgp) encoded by the mdr2 gene has been shown to transport phosphatidylcholine into the bile; therefore, in the current study, we utilized the Pgp inhibitor verapamil to investigate the possible involvement of Pgps in the biliary secretion of alpha-T. When rats were iv injected with verapamil (4 mg/kg) 10 min prior to PIP treatment, verapamil prevented the PIP-induced increases in biliary alpha-T and phosphatidylcholine output and resulted in biliary alpha-T outputs that were significantly less than controls. Also, we determined that the biliary alpha-T levels in mdr2 knockout mice were 25% of those in wildtype mice; furthermore, mdr2 liver, lung, and kidney levels of alpha-T and glutathione differed from those of wildtype. To investigate the fate of biliary alpha-T, we injected 14C-labeled alpha-T into the bile duct cannulae of rats and determined that approximately 60% of the radioactivity was reabsorbed within 1 h. Our results indicate that alpha-T undergoes enterohepatic circulation and that the biliary secretion of alpha-T, basally and following chemical treatment, is dependent on the presence of a functioning mdr2 Pgp in rats and mice.

Colchicine and vinblastine prevent the piperonyl butoxide-induced increase in rat biliary output of alpha-tocopherol.

alpha-Tocopherol, a lipid-soluble chain-breaking antioxidant, is important in the protection of biologic membranes and is transported in the body in association with lipids. It has been estimated that the liver contains up to 29% of the alpha-tocopherol stores of the body. Piperonyl butoxide (PIP) is a widely used synthetic methylenedioxyphenyl insecticide synergist. Bile was collected for 1 hr prior to, and 5 hr following, i.p. injection of 1 g/kg PIP. Within 3 hr of PIP administration, biliary alpha-tocopherol output increased to 132% of pretreatment measurements. Hepatic alpha-tocopherol levels of PIP-treated rats decreased to 26% below those of controls at the end of the bile collection period. Biliary bile acid and phospholipid output also increased significantly following PIP treatment. In a second set of experiments, rats were pretreated with either colchicine or one of two doses of vinblastine; both colchicine and the higher dose of vinblastine prevented the PIP-induced decrease in hepatic alpha-tocopherol and the increase in biliary output of alpha-tocopherol, bile acids, and phospholipids. These results suggest that (i) PIP-induced biliary secretion of alpha-tocopherol is associated with altered biliary phospholipid output, (ii) a significant portion of hepatic alpha-tocopherol may function as a highly mobilizable source of antioxidant for possible rapid enterohepatic distribution, and (iii) microtubules may be involved in this process.