Identification and quantification of gold engineered nanomaterials and impaired fluid transfer across the rat placenta via ex vivo perfusion.

Development and implementation of products incorporating nanoparticles are occurring at a rapid pace. These particles are widely utilized in domestic, occupational, and biomedical applications. Currently, it is unclear if pregnant women will be able to take advantage of the potential biomedical nanoproducts out of concerns associated with placental transfer and fetal interactions. We recently developed an ex vivo rat placental perfusion technique to allow for the evaluation of xenobiotic transfer and placental physiological perturbations. In this study, a segment of the uterine horn and associated placenta was isolated from pregnant (gestational day 20) Sprague-Dawley rats and placed into a modified pressure myography vessel chamber. The proximal and distal ends of the maternal uterine artery and the vessels of the umbilical cord were cannulated, secured, and perfused with physiological salt solution (PSS). The proximal uterine artery and umbilical artery were pressurized at 80 mmHg and 50 mmHg, respectively, to allow countercurrent flow through the placenta. After equilibration, a single 900 µL bolus dose of 20 nm gold engineered nanoparticles (Au-ENM) was introduced into the proximal maternal artery. Distal uterine and umbilical vein effluents were collected every 10 min for 180 min to measure placental fluid dynamics. The quantification of Au-ENM transfer was conducted via inductively coupled plasma mass spectrometry (ICP-MS). Overall, we were able to measure Au-ENM within uterine and umbilical effluent with 20 min of material infusion. This novel methodology may be widely incorporated into studies of pharmacology, toxicology, and placental physiology.

The effect of fibroblast growth factor 15 deficiency on the development of high fat diet induced non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a form of non-alcoholic fatty liver disease (NAFLD) characterized by steatosis, inflammation, and fibrosis often associated with metabolic syndrome. Fibroblast growth factor 15 (FGF15), an endocrine factor mainly produced in the distal part of small intestine, has emerged to be a critical factor in regulating bile acid homeostasis, energy metabolism, and liver regeneration. We hypothesized that FGF15 alters the development of each of the listed features of NASH. To test this hypothesis, four-week old male Fgf15-/- and their corresponding wild-type (WT) mice were fed either a high fat diet (HFD) or a control chow diet for six months. The results confirmed that HFD feeding for six months in WT mice recapitulated human NASH phenotype, including macrovesicular steatosis, inflammation, and fibrosis. Whereas FGF15 deficiency had no effect on the severity of liver steatosis or inflammation, it was associated with decreased liver fibrosis. Furthermore, FGF15 deficiency resulted in abnormal bile acid homeostasis, increased insulin resistance, increased HFD-induced serum triglycerides, decreased inductions of hepatic cholesterol content by HFD, and altered gene expression of lipid metabolic enzymes. These data suggest that FGF15 improves lipid homeostasis and reduces bile acid synthesis, but promotes fibrosis during the development of NASH.

Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.

During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.

Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils.

Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

Impact of missense mutations on biosynthesis of myeloperoxidase.

We have examined the biosynthesis of normal and mutant forms of myeloperoxidase (MPO) in order to gain insights into the critical features of normal biogenesis of MPO. The expression of wild-type and mutant forms of MPO in a stably transfected cell line devoid of endogenous MPO as well as in established human promyelocytic cell lines has allowed understanding of several features of MPO biosynthesis. It is clear that heme insertion into apoproMPO is necessary for proper folding, egress from the endoplasmic reticulum (ER), and eventual entry into the maturation pathway. In addition, molecular chaperones calreticulin and calnexin interact with normal MPO precursors in a sequential and regulated fashion. Studies of naturally occurring mutants, specifically missense mutations underlying inherited MPO deficiency, and mutations in putatively important residues in MPO have highlighted special features of the ER quality control system in the context of MPO biosynthesis. With identification of additional genotypes of MPO deficiency and the recent solution of MPO crystal structure at 1.8 A, this approach provides a powerful technique to assess structure-function relationships in MPO that are likely applicable to other members of the family of animal peroxidases.

A novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation.

Myeloperoxidase (MPO) deficiency is a common inherited disorder linked to increased susceptibility to infection and malignancy. We identified a novel missense mutation in the MPO gene at codon 173 whereby tyrosine is replaced with cysteine (Y173C) that is associated with MPO deficiency and assessed its impact on MPO processing and targeting in transfectants expressing normal or mutant proteins. Although the precursor synthesized by cells expressing the Y173C mutation (MPOY173C) was glycosylated, associated with the molecular chaperones calreticulin and calnexin, and acquired heme, it was neither proteolytically processed to mature MPO subunits nor secreted. After prolonged association with calreticulin and calnexin in the endoplasmic reticulum, MPOY173C was degraded. Furthermore, the 20S proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl inhibited its degradation, suggesting that the proteasome mediates proteolysis of MPOY173C and, thus, participates in quality control in this novel form of hereditary MPO deficiency.

Coordinated participation of calreticulin and calnexin in the biosynthesis of myeloperoxidase.

Myeloperoxidase (MPO) is a neutrophil lysosomal hemeprotein essential for optimal oxygen-dependent microbicidal activity. We have demonstrated previously that calreticulin, a luminal endoplasmic reticulum protein, functions as a molecular chaperone during myeloperoxidase biosynthesis, associating reversibly with the heme-free precursor apopro-MPO. Because the membrane-bound endoplasmic reticulum protein calnexin is structurally and functionally related to calreticulin, we assessed the role of calnexin in myeloperoxidase biosynthesis. Like calreticulin, calnexin coprecipitated exclusively with glycosylated MPO precursors and with apopro-MPO but, in contrast to calreticulin, also with the enzymatically active, heme-containing precursor pro-MPO. To determine if calnexin participated in heme insertion into MPO, we compared the kinetics of chaperone association with MPO precursors using stable transfectants expressing cDNA encoding wild type MPO or mutated forms that do not acquire heme. Transfectants expressing mutant cDNA had prolonged association of MPO-related precursors with calreticulin and especially with calnexin. These studies demonstrate that 1) both calreticulin and calnexin associated with glycosylated apopro-MPO; 2) only calnexin associated selectively with the enzymatically active, heme-containing precursor pro-MPO; and 3) mutants unable to incorporate heme had prolonged association with calnexin. These findings represent the first evidence of a specialized role for calnexin in facilitating protein maturation in the endoplasmic reticulum of myeloid cells.

Function and morphology of platelets produced for transfusion by intermittent-flow centrifugation plateletpheresis or combined platelet-leukapheresis.

The effects on platelet function of intermittent-flow centrifugation pheresis were measured employing platelets collected from ten donors by combined platelet-leukapheresis with hydroxyethyl starch (LP) and from ten by plateletpheresis (PP) by similar techniques except without starch. Greater numbers of platelets were produced by LP than by PP. Aggregation of platelets collected by both LP and PP was normal (did not differ from prepheresis baseline) to collagen and to 10(-5)M adenosine diphosphate (ADP). Slight impairment to aggregation with 2 X 10(-6)M ADP and 5.5 X 10(-5)M epinephrine occurred with both techniques. These abnormalities, however, were significantly less severe in platelets collected by LP. Platelet morphology by electron microscopy was nearly normal, although glycogen granules were absent in LP platelets. Thus, hydroxyethyl starch, at doses currently used during a single LP, does not enhance abnormalities of platelet aggregation over those expected to result from PP alone. Actually, LP platelets function better in vitro than those collected by PP. Large numbers of platelets can be harvested by LP, and their use as a component that is comparable to PP platelets may mean improved efficiency and reduced costs for pheresis centers.

Platelet function in donors undergoing intermittent-flow centrifugation plateletpheresis or leukapheresis.

Platelet dysfunction and prolonged bleeding have been seen in man when glucose polymer solutions, such as dextran, are used to volume replacement. Hydroxyethyl starch (HES) is a glucose polymer slightly different from dextran, which is used as a sedimenting agent in leukapheresis procedures. This controlled study was performed to evaluate the effect of HES on platelet function in centrifugation leukapheresis donors. Plateletpheresis using the same machines and techniques but without HES was the control. Platelet function was assessed by bleeding time, platelet count, adhesiveness to glass beads and aggregation to collagen, epinephrine and several concentrations of ADP, before and after the pheresis procedures. Except for a decrease in platelet count after both procedures, no other major changes in platelet function were seen. After one exposure in previously unpheresed donors, HES does not induce platelet dysfunction.

Gram-negative sepsis treated with neutrophils collected exclusively by intermittent flow centrifugation leukapheresis.

The effectiveness of neutrophils prepared for transfusion by intermittent flow centrifugation leukapheresis (IFCL) as treatment for serious bacterial infections in neutropenic patients has not been documented in the literature. Their value, in fact, has been questioned. We report that IFCL neutrophils are similar to cells prepared by other techniques in the ability to support neutropenic patients with culture-proven, gram-negative sepsis unresponsive to antibiotics.

Combined platelet-leukapheresis: a technique for preparing separate platelet and granulocyte-platelet units from single donors.

One donor can provide separate platelet and granulocyte-platelet units that have the potential to fulfill the needs of two recipients. Combined platelet and leukapheresis were performed in the presence of hyddroxyethyl starch and citrate employing the Haemonetics Model 30 Blood Separator. Platelets and granulocytes were separated subsequently by centrifugation and two individual units were prepared for transfusion. The platelet units contained a mean of 4.4 x 10(11) platelets/unit and were nearly devoid of leukocytes. The combined granulocyte-platelet units contained a mean of 7.38 x 10(9) neutrophils/unit and 3.06 x 10(11) platelets/unit. Thus two separate units containing sufficient numbers of cells for transfusion can be obtained from pheresis of one donor.