Lactation stages modulate the hematological, serum biochemical, and endocrinological profiles and oxidative stress markers in crossbred cows under tropical humid island ecosystem of Andaman and Nicobar Islands.

The present study was designed to assess the interrelationship between hematological, serum biochemical, and endocrinological profiles and oxidative stress markers and lactational stages in crossbred (CB) dairy cows of Andaman and Nicobar Islands (ANI). Healthy (n = 6) CB cows (50-62.50% exotic inheritance; Holstein Friesian × Andaman local) of 4th parity with age of 7-9 years and body weight of 350-400 kg were selected from Cattle Breeding Farm, ICAR-Central Island Agricultural Research Institute (ICAR-CIARI), Port Blair, ANI. These experimental cows were synchronized with Ovsynch protocol, and parturition was planned to happen in the month of May-June. Lactation was allowed for 305 days. Hematological profiles, serum biochemical profiles, oxidative stress markers and endocrinological profiles were measured at a 15-day interval from day 07 to 305 of lactation (after 6 days of colostrum). The lactation period was divided into first (day 07 to 90), second (day 91 to 180), and third (day 181 to 305) stage of lactation. Average daily milk yield (L) did not vary among the stages of lactations; however, first (8.56 ± 1.26) and second (9.79 ± 0.87) stages had higher milk yield compared to third (7.93 ± 0.79) stage of lactation. Hematological profiles did not vary among the stages of lactation; however, these values were within the range of bovine species at lactation. Serum glucose, triglyceride, total cholesterol, total protein, globulin, and blood urea nitrogen (BUN) increased (P < 0.05) and albumin and creatinine decreased (P < 0.05) gradually as lactation stages advanced. Activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) and concentration of calcium, phosphorous, and magnesium were nearly similar among the stages of lactation. Similarly, triiodothyronine (T3), and thyroxine (T4) increased and prolactin and cortisol decreased (P < 0.05) gradually as stages of lactation advanced. Total antioxidant capacity (TAC) increased and malondialdehyde (MDA) decreased (P < 0.05) as lactation stages advanced. The results of the present study indicated that the lactating CB cows suffered nutritional stress (deficiency of protein, carbohydrate, lipids, and minerals), physiological stress (higher cortisol), oxidative stress (higher MDA and deficiency of total antioxidant capacity), and hormonal imbalance (higher prolactin and cortisol and deficiency of thyroid hormones) during the early stages of lactation. Thus, the first and second stages are more stressful events compared to the third stage of lactation in the CB cows in ANI. Therefore, regular monitoring of blood components and accordingly suitable feeding strategies with balanced nutrients and minerals, supplementation of suitable antioxidants, and appropriate management practices need to be implemented to mitigate these stresses and to prevent metabolic disorders with maximum milk production during different stages of lactation in CB cows under humid tropical island ecosystem of Andaman and Nicobar Islands.

[Killing effect of anti-MSLN-iCAR-NK cells derived from induced pluripotent stem cells on ovarian epithelial cancer cells].

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD244 were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.

Iron Loading Exaggerates the Inflammatory Response to the Toll-like Receptor 4 Ligand Lipopolysaccharide by Altering Mitochondrial Homeostasis.

BACKGROUND: Perioperative and critically ill patients are often exposed to iron (in the form of parenteral-iron administration or blood transfusion) and inflammatory stimuli, but the effects of iron loading on the inflammatory response are unclear. Recent data suggest that mitochondrial reactive oxygen species have an important role in the innate immune response and that increased mitochondrial reactive oxygen species production is a result of dysfunctional mitochondria. We tested the hypothesis that increased intracellular iron potentiates lipopolysaccharide-induced inflammation by increasing mitochondrial reactive oxygen species levels. METHODS: Murine macrophage cells were incubated with iron and then stimulated with lipopolysaccharide. C57BL/6 wild-type mice were intraperitoneally injected with iron and then with lipopolysaccharide. Markers of inflammation and mitochondrial superoxide production were examined. Mitochondrial homeostasis (the balance between mitochondrial biogenesis and destruction) was assessed, as were mitochondrial mass and the proportion of nonfunctional to total mitochondria. RESULTS: Iron loading of mice and cells potentiated the inflammatory response to lipopolysaccharide. Iron loading increased mitochondrial superoxide production. Treatment with MitoTEMPO, a mitochondria-specific antioxidant, blunted the proinflammatory effects of iron loading. Iron loading increased mitochondrial mass in cells treated with lipopolysaccharide and increased the proportion of nonfunctional mitochondria. Iron loading also altered mitochondrial homeostasis to favor increased production of mitochondria. CONCLUSIONS: Acute iron loading potentiates the inflammatory response to lipopolysaccharide, at least in part by disrupting mitochondrial homeostasis and increasing the production of mitochondrial superoxide. Improved understanding of iron homeostasis in the context of acute inflammation may yield innovative therapeutic approaches in perioperative and critically ill patients.

HER2 and HLA-A*02 dual CAR-T cells utilize LOH in a NOT logic gate to address on-target off-tumor toxicity.

BACKGROUND: One of the major challenges in chimeric antigen receptor (CAR)-T cell therapy for solid tumors is the potential for on-target off-tumor toxicity due to the expression of CAR tumor antigens in essential tissues and organs. Here, we describe a dual CAR NOT gate incorporating an inhibitory CAR (iCAR) recognizing HLA-A*02 ("A2") that enables effective treatment with a potent HER2 activating CAR (aCAR) in the context of A2 loss of heterozygosity (LOH). METHODS: A CAR-T cell screen was conducted to identify inhibitory domains derived from natural immune receptors (iDomains) to be used in a NOT gate, to kill A2- HER2+ lung cancer cell lines but spare A2+ HER2+ lung cancer cell-lines with high specificity. The extensive analysis of lead candidates included T-cell activation and killing, assays of reversibility and durability in sequential challenges, target cell specificity in mixed 3D spheroids and 2D cultures, and the characterization of CAR expression level and cell-trafficking. RESULTS: A leukocyte immunoglobulin-like receptor B1 (LIR1) iDomain iCAR was identified as most effective in regulating the cytotoxicity of a second generation HER2 aCAR. Target transfer experiments demonstrated that the 'on' and 'off' cell state of the LIR1 NOT gate CAR-T cell is both durable and reversible. Protection required iCAR signaling and was associated with reduced aCAR and iCAR surface expression. iCAR regulation was sufficient to generate high target specificity in a 3D adjacent spheroid assay designed to model the interface between clonal A2 LOH foci and normal tissue. However, we observed significant bystander killing of A2+ cells in admix culture through aCAR dependent and independent mechanisms. LIR1 NOT gate CAR-T cells conferred protection against H1703-A2+ tumors and high efficacy against H1703-A2- tumors in-vivo. We observed that the iCAR is inactive in A2+ donors due to cis-binding, but Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout of HLA-A fully restored iCAR activity. CONCLUSIONS: We have preclinically validated an iCAR NOT gate technology broadly applicable for targeting HER2 expression in the context of A2 LOH. This approach is designed to prevent off tumor toxicity while allowing highly potent antitumor activity.

Intraperitoneal injection of iron dextran induces peripheral iron overload and mild neurodegeneration in the nigrostriatal system in C57BL/6 mice.

AIMS: Elevated iron levels in the affected areas of brain are linked to several neurodegenerative diseases including Parkinson's disease (PD). This study investigated the influence of peripheral iron overload in peripheral tissues, as well as its entry into the brain regions on lysosomal functions. The survival of dopaminergic neurons in the nigrostriatal system and motor coordination were also investigated. MAIN METHODS: An intraperitoneal injection of iron dextran (FeDx) mouse model was established. Western blot was used to detect iron deposition and lysosomal functions in the liver, spleen, hippocampal (HC), striatum (STR), substantia nigra (SN) and olfactory bulb (OB). Iron in serum and cerebrospinal fluid (CSF) was determined by an iron assay kit. Immunofluorescence and immunohistochemical staining were applied to detect dopaminergic neurons and fibers. Motor behavior was evaluated by gait analysis. KEY FINDINGS: Iron was deposited consistently in the liver and spleen, and serum iron was elevated. While iron deposition occurred late in the HC, STR and SN, without apparently affecting CSF iron levels. Although cathepsin B (CTSB), cathepsin D (CTSD), glucocerebrosidase (GCase) and lysosome integrated membrane protein 2 (LIMP-2) protein levels were dramatically up-regulated in the liver and spleen, they were almost unchanged in the brain regions. However, CTSB was up-regulated in acute iron-overloaded OB and primary cultured astrocytes. The number of dopaminergic neurons in the SN remained unchanged, and mice did not exhibit significant motor incoordination. SIGNIFICANCE: Intraperitoneal injection of FeDx in mice induces largely peripheral iron overload while not necessarily sufficient to cause severe disruption of the nigrostriatal system.

Iron therapy mitigates chronic kidney disease progression by regulating intracellular iron status of kidney macrophages.

Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-ß expression and suppressed TGF-ß-driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.

Reduced expression of ferroportin-1 mediates hyporesponsiveness of suckling rats to stimuli that reduce iron absorption.

BACKGROUND & AIMS: Suckling mammals absorb high levels of iron to support their rapid growth. In adults, iron absorption is controlled by systemic signals that alter expression of the iron-regulatory hormone hepcidin. We investigated whether hepcidin and absorption respond appropriately to systemic stimuli during suckling. METHODS: In Sprague-Dawley rats, iron levels increased following administration of iron dextran, and inflammation was induced with lipopolysaccharide. Gene expression was measured by quantitative reverse-transcription polymerase chain reaction; protein levels were measured by immunoblot analyses. Iron absorption was determined based on retention of an oral dose of 59Fe. RESULTS: Iron absorption was high during suckling and reduced to adult levels upon weaning. In response to iron dextran or lipopolysaccharide, iron absorption in adults decreased substantially, but, in suckling animals, the changes were minimal. Despite this, expression of hepcidin messenger RNA was strongly induced by each agent, before and after weaning. The hyporesponsiveness of iron absorption to increased levels of hepcidin during suckling correlated with reduced or absent duodenal expression of ferroportin 1 (Fpn1), normally a hepcidin target. Fpn1 expression was robust in adults. Predominance of the Fpn1A splice variant, which is under iron-dependent translational control, accounts for the low level of Fpn1 in the iron-deficient intestine of suckling rats. CONCLUSIONS: Iron absorption during suckling is largely refractory to changes in expression of the systemic iron regulator hepcidin, and this in turn reflects limited expression of Fpn1 protein in the small intestine. Iron absorption is therefore not always controlled by hepcidin.

icaR and icaT are Ancient Chromosome Genes Encoding Substrates of the Type III Secretion Apparatus in Shigella flexneri.

Shigella is an Escherichia coli pathovar that colonizes the cytosol of mucosal cells in the human large intestine. To do this, Shigella uses a Type III Secretion Apparatus (T3SA) to translocate several proteins into host cells. The T3SA and its substrates are encoded by genes of the virulence plasmid pINV or by chromosomal genes derived thereof. We recently discovered two chromosomal genes, which seem unrelated to pINV, although they are activated by MxiE and IpgC similarly to some of the canonical substrates of the T3SA. Here, we showed that the production of the corresponding proteins depended on the conservation of a MxiE box in their cognate promoters. Furthermore, both proteins were secreted by the T3SA in a chaperone-independent manner through the recognition of their respective amino-terminal secretion signal. Based on these observations, we named these new genes icaR and icaT, which stand for invasion chromosome antigen with homology for a transcriptional regulator and a transposase, respectively. icaR and icaT have orthologs in commensal and pathogenic E. coli strains belonging mainly to phylogroups A, B1, D and E. Finally, we demonstrated that icaR and icaT orthologs could be activated by the coproduction of IpgC and MxiE in strains MG1655 K-12 (phylogroup A) and O157:H7 ATCC 43888 (phylogroup E). In contrast, the coproduction of EivF and YgeG, which are homologs of MxiE and IpgC in the E. coli T3SS 2 (ETT2), failed to activate icaR and icaT. IMPORTANCEicaR and icaT are the latest members of the MxiE regulon discovered in the chromosome. The proteins IcaR and IcaT, albeit produced in small amounts, are nonetheless secreted by the T3SA comparably to canonical substrates. The high occurrence of icaR and icaT in phylogroups A, B1, D, and E coupled with their widespread absence in their B2 counterparts agree with the consensus E. coli phylogeny. The widespread conservation of the MxiE box among icaR and icaT orthologs supports the notion that both genes had already undergone coevolution with transcriptional activators ipgC and mxiE- harbored in pINV or a relative- in the last common ancestor of Shigella and of E. coli from phylogroups A, B1, D, and E. The possibility that icaR and icaT may contribute to Shigella pathogenesis cannot be excluded, although some of their characteristics suggest they are fossil genes.

Permeability of the ovarian follicle of Aedes aegypti mosquitoes.

The passage of tracers of various molecular weights into resting and vitellogenic ovarian follicles of Aedes aegypti mosquitoes was studied ultrastructurally. The outermost layer of the follicular sheath (the basement lamina) is a coarse mechanical filter. It is freely permeable to particles with molecular weights ranging from 12,000 to 500,000 (i.e. cytochrome c, peroxidase, hemoglobin, catalase, ferritin, immunoglobulin (IgG)-peroxidase, iron dextran and Thorotrast) that have dimensions less than 110 A. Molecules as large as carbon (300-500 A) are totally excluded. Whereas proteins and polysaccharide tracers permeate the basement lamina with apparent ease, certain inert particles (e.g. Thorotrast, Fellows-Testager Div., Fellows Mfg. Co., Inc., Detroit, Mich.) penetrate more slowly. With respect to the tracers tested, resting follicles are as permeable as vitellogenic follicles. The follicle epithelium of resting or vitellogenic follicles is penetrated by narrow intercellular channels. Our observations suggest that these spaces are lined with mucopolysaccharide material. After permeating the basement lamina, exogenous tracers fill these channels, while the bulk of material accumulates in the perioocytic space. Within 3 hr after imbibing blood, the pinocytotic mechanism of the oocyte is greatly augmented. Pinocytosis is not selective with regard to material in the perioocytic space, since double tracer studies show that exogenous compounds are not separated, but are incorporated into the same pinocytotic vesicle. During later stages of vitellogenesis, 36-48 hr after the blood-meal, the pinocytotic mechanism of the oocyte is diminished. Simultaneously, the intercellular channels become occluded by desmosomes, and the vitelline membrane plaques separate the oocyte and follicle epithelium.

Effects of colloidal iron overload on renal and hepatic siderosis and the femur in male rats.

Genetic hemochromatosis is an iron overload disorder, and osteopenic and osteoporotic. Femoral neck bone mineral density (BMD) appears to fall with rising hepatic iron concentrations. A critical role for iron in mediating tissue injury is played via hydroxyl radical formation in nephrotoxicity. We investigated the effects of a colloidal iron overload on renal function, organ siderosis, and femoral bone in male rats. Iron overload reduced body growth, and increased the weights of the liver and spleen. Marked deposition of iron was noted in liver and kidney. Activities of lactate dehydrogenase and alkaline phosphatase were decreased, and the concentrations of blood urea nitrogen and creatinine were increased with the reduction in plasma calcium and inorganic phosphorus levels, i.e. functions of the liver and kidney might be affected by reactive oxygen species such as the superoxide radical, H2O2, and the hydroxyl radical produced by overloaded iron. Damage to the proximal tubular epithelial cells of the kidney and a loss of connectivity of cancellous bone in the epiphysis and of trabecular bone in the metaphysis of the distal femur were observed in iron-overloaded rats with a reduction of femoral bone mineral density, i.e. reabsorption of calcium from the proximal tubular epithelial cells of the kidney might be affected and urinary discharge of calcium might be elevated. It was suggested that iron overload gave rise to osteoporosis combined with renal dysfunction and liver iron overload syndrome.

Peripheral iron dextran induced degeneration of dopaminergic neurons in rat substantia nigra.

Iron accumulation is considered to be involved in the pathogenesis of Parkinson's disease. To demonstrate the relationship between peripheral iron overload and dopaminergic neuron loss in rat substantia nigra (SN), in the present study we used fast cyclic voltammetry, tyrosine hydroxylase (TH) immunohistochemistry, Perls' iron staining, and high performance liquid chromatography-electrochemical detection to study the degeneration of dopaminergic neurons and increased iron content in the SN of iron dextran overloaded animals. The findings showed that peripheral iron dextran overload increased the iron staining positive cells and reduced the number of TH-immunoreactive neurons in the SN. As a result, dopamine release and content, as well as its metabolites contents were decreased in caudate putamen. Even more dramatic changes were found in chronic overload group. These results suggest that peripheral iron dextran can increase the iron level in the SN, where excessive iron causes the degeneration of dopaminergic neurons. The chronic iron overload may be more destructive to dopaminergic neurons than the acute iron overload.

Magnetic characterisation of rat muscle tissues after subcutaneous iron dextran injection.

Ex vivo freeze-dried rat muscle tissues, collected at different times t after a single dose of subcutaneously injected iron dextran, have been magnetically characterised. The AC susceptibility of the tissues shows an overall superparamagnetic behaviour and the dependence on t of, especially, the out-of-phase component is remarkably systematic despite the fact that each tissue originates in a different rat individual. The experiments show that the akaganéite (beta-FeOOH) nanoparticles contained in the injected drug are progressively degraded in the living tissue and, at times of the order of 1 month and for all the analysed rat individuals, converge to a magnetically well-defined species with much narrower magnetic activation energy distribution than iron dextran. Thorough transmission electron microscopy experiments of the same tissues indicate the presence of oxyhydroxide particles, whose size decreases for increasing t in agreement with the interpretation of the magnetic susceptibility. The conclusions drawn from the magnetic study do well correspond to the properties of the whole tissue since no biochemical extraction work has been done. The AC susceptibility appears to be a valuable and complementary tool in pharmacological studies of iron-containing drugs.

Bioinorganic transformations of liver iron deposits observed by tissue magnetic characterisation in a rat model.

The magnetic properties and the ultrastructure, with special emphasis on the nanometric range, of liver tissues in an iron overload rat model have been investigated. The tissues of the animals, sacrificed at different times after a single iron dextran injection, have been characterised by magnetic AC susceptibility measurements together with transmission electron microscopy (TEM) and selected area electron diffraction (SAED) as helping techniques. It has been observed that few days after the iron administration the liver contains at least two iron species: (i) akaganéite nanoparticles, coming from iron dextran and (ii) ferrihydrite nanoparticles corresponding to ferritin. The magnetic susceptibility of the tissues depends not only on the elemental iron content but also on its distribution among chemical species, and varies in a remarkable regular manner as a function of the elapsed time since the iron administration. The results are of relevance with respect to non-invasive techniques for liver iron determination, directly or indirectly based on the magnetic susceptibility of the tissues, as biomagnetic liver susceptometry (BLS) and magnetic resonance (MRI) image treatment.

Interaction of intravenously injected liposomes with mouse liver mitochondria. A fluorescence and electron microscopy study.

Megamitochondria, resulting from cuprizone feeding of Swiss ICR mice, were fluorescent in hepatocytes after the intravenous injection to mice of a liposome-encapsulated acridine orange-DNA complex (AO-DNA). Flow cytofluorimetric analysis of isolated megamitochondria showed that the proportion of liposome-encapsulated AO-DNA which localized in megamitochondria increased from 0.02% of the dose injected per liver cell at 3 min after injection to an average of 0.34% at 1 h after injection. Megamitochondria showed negligible fluorescence by fluorescence activated cell sorting (FACS) analysis when free AO-DNA was intravenously injected. Transmission electron micrographs of mouse liver tissue after intravenous injection of liposomes encapsulating iron dextran showed an association of the liposomes with megamitochondria which appeared identical to liposome association with normal mitochondria. These results support and extend our earlier observation that a fraction of the liposomes injected intravenously into mice associate with mitochondria in the liver, and possibly deliver their aqueous contents there.

Wheat germ agglutinin is selectively transported to multivesicular bodies.

Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.

Parenteral iron formulations: a comparative toxicologic analysis and mechanisms of cell injury.

BACKGROUND: Multiple parenteral iron (Fe) formulations exist for administration to patients with end-stage renal disease. Although there are concerns regarding their potential toxicities, no direct in vitro comparisons of these agents exist. Thus, the present study contrasted pro-oxidant and cytotoxic potentials of four available Fe preparations: Fe dextran (Fe dext), Fe sucrose (Fe sucr), Fe gluconate (Fe gluc), and Fe oligosaccharide (Fe OS). METHODS: Differing dosages (0.06 to 1 mg/mL) of each compound were added to either (1) isolated mouse proximal tubule segments, (2) renal cortical homogenates, or (3) cultured human proximal tubule (HK-2) cells (0.5- to 72-hour incubations). Oxidant injury (malondialdehyde generation) and lethal cell injury (percentage of lactate dehydrogenase release; tetrazolium dye uptake) were assessed. Effects of selected antioxidants (glutathione [GSH], catalase, dimethylthiourea (DMTU), and sodium benzoate also were assessed. RESULTS: Each test agent induced massive and similar degrees of lipid peroxidation. Nevertheless, marked differences in cell death resulted (Fe sucr >> Fe gluc > Fe dext approximately Fe OS). This relative toxicity profile also was observed in cultured aortic endothelial cells. Catalase, DMTU, and sodium benzoate conferred no protection. However, GSH and its constituent amino acid glycine blocked Fe sucr-mediated cell death. The latter was mediated by mitochondrial blockade, causing free radical generation and a severe adenosine triphosphate depletion state. CONCLUSIONS: (1) parenteral Fes are highly potent pro-oxidants and capable of inducing tubular and endothelial cell death, (2) markedly different toxicity profiles exist among these agents, and (3) GSH can exert protective effects. However, the latter stems from GSH's glycine content, rather than from a direct antioxidant effect.

Increased transfer of iron to the fetus after total dose infusion of iron dextran during pregnancy.

After total dose infusion of iron dextran to 14 pregnant women the saturation of transferrin and the concentration of ferritin were increased in the cord blood at birth, compared with untreated cases. These changes suggest that the fetal iron stores may be increased by such treatment.

Impaired transport of iron-dextran complex in myelinated central fibers.

The labeling of retinal ganglion cells by axonal transport of an iron-dextran complex (injected into the superior colliculus and the lateral geniculate nucleus) was compared to the previously described labeling of the dopaminergic neurons of the substantia nigra (after striatal injection), to test the validity of the method in myelinated and unmyelinated CNS pathways. It was shown that the retrograde labeling in the visual pathway was impaired, consecutive to the penetration of iron-dextran into the myelin sheaths and a subsequent myelin alteration along the optic nerve.

Uptake and retrograde axonal transport of various exogenous macromolecules in normal and crushed hypoglossal nerves.

Macromolecular tracers were injected into the tongue or around a crush in mouse hypoglossal nerves. At various times thereafter, the tracers were histochemically localized on the basis of peroxidase activity. The distribution of reaction product was then examined using light microscopy in order to study the influence of molecular charge and size on uptake and retrograde axonal transport from the periphery or from the crushed axon. Of various proteins with peroxidase activity, horseradish peroxidase and cytochrome-c showed the greatest penetration into axons proximal to the crush. Following injection into the tongue, intra-axonal cytochrome-c was detectable in some of the peripheral branches but not any of the other proteins. Retrograde transport to the nerve cell bodies was demonstrated for horseradish peroxidase and cytochrome-c, both from the tongue and from the axonal crush but not for microperoxidase, myoglobin, hemoglobin, lactoperoxidase and catalase. The number of neuronal cell bodies having detectable reaction product was higher for peroxidase-injected than for cytochrome-c-injected animals. Ferritin and iron-dextran (Imferon) also accumulated in hypoglossal neurons, but this could be detected only after repeated injections into the tongue. Uptake and retrograde transport from the tongue or from the crush occurred both for anionic and for cationic horseradish peroxidase. This is interpreted as evidence against absolute specificity in the uptake and transport of macromolecules on the basis of electrical charge.

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury.

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125 mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20 microM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20 microM) diminished ethanol-induced alkane production 0-40%, inhibition of aldehyde oxidase with menadione (20 microM) diminished alkane production 36-75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100 mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50 microM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.