Apotransferrin in Combination with Ciprofloxacin Slows Bacterial Replication, Prevents Resistance Amplification, and Increases Antimicrobial Regimen Effect.

There has been renewed interest in combining traditional small-molecule antimicrobial agents with nontraditional therapies to potentiate antimicrobial effects. Apotransferrin, which decreases iron availability to microbes, is one such approach. We conducted a 48-h one-compartment in vitro infection model to explore the impact of apotransferrin on the bactericidal activity of ciprofloxacin. The challenge panel included four Klebsiella pneumoniae isolates with ciprofloxacin MIC values ranging from 0.08 to 32 mg/liter. Each challenge isolate was subjected to an ineffective ciprofloxacin monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio] ranging from 0.19 to 96.6) with and without apotransferrin. As expected, the no-treatment and apotransferrin control arms showed unaltered prototypical logarithmic bacterial growth. We identified relationships between exposure and change in bacterial density for ciprofloxacin alone (R2 = 0.64) and ciprofloxacin in combination with apotransferrin (R2 = 0.84). Addition of apotransferrin to ciprofloxacin enabled a remarkable reduction in bacterial density across a wide range of ciprofloxacin exposures. For instance, at a ciprofloxacin AUC/MIC ratio of 20, ciprofloxacin monotherapy resulted in nearly 2 log10 CFU increase in bacterial density, while the combination of apotransferrin and ciprofloxacin resulted in 2 log10 CFU reduction in bacterial density. Furthermore, addition of apotransferrin significantly reduced the emergence of ciprofloxacin-resistant subpopulations compared to monotherapy. These data demonstrate that decreasing the rate of bacterial replication with apotransferrin in combination with antimicrobial therapy represents an opportunity to increase the magnitude of the bactericidal effect and to suppress the growth rate of drug-resistant subpopulations.

Adjunctive transferrin to reduce the emergence of antibiotic resistance in Gram-negative bacteria.

BACKGROUND: New strategies are needed to slow the emergence of antibiotic resistance among bacterial pathogens. In particular, society is experiencing a crisis of antibiotic-resistant infections caused by Gram-negative bacterial pathogens and novel therapeutics are desperately needed to combat such diseases. Acquisition of iron from the host is a nearly universal requirement for microbial pathogens-including Gram-negative bacteria-to cause infection. We have previously reported that apo-transferrin (lacking iron) can inhibit the growth of Staphylococcus aureus in culture and diminish emergence of resistance to rifampicin. OBJECTIVES: To define the potential of apo-transferrin to inhibit in vitro growth of Klebsiella pneumoniae and Acinetobacter baumannii, key Gram-negative pathogens, and to reduce emergence of resistance to antibiotics. METHODS: The efficacy of apo-transferrin alone or in combination with meropenem or ciprofloxacin against K. pneumoniae and A. baumannii clinical isolates was tested by MIC assay, time-kill assay and assays for the selection of resistant mutants. RESULTS: We confirmed that apo-transferrin had detectable MICs for all strains tested of both pathogens. Apo-transferrin mediated an additive antimicrobial effect for both antibiotics against multiple strains in time-kill assays. Finally, adding apo-transferrin to ciprofloxacin or meropenem reduced the emergence of resistant mutants during 20 day serial passaging of both species. CONCLUSIONS: These results suggest that apo-transferrin may have promise to suppress the emergence of antibiotic-resistant mutants when treating infections caused by Gram-negative bacteria.

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

Iron overload results in significant morbidity and mortality in ß-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in ß-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Construction of a genetically engineered chimeric apoprotein consisting of sequences derived from lidamycin and neocarzinostatin.

Neocarzinostatin (NCS) consists of an enediyne chromophore and an apoprotein (NCP). Lidamycin (LDM) is composed of another active enediyne chromophore (AE) and an acidic protein (LDP). Although the structures of NCP and LDP are very similar, LDM has been shown to have an increased tumor-suppressive activity than that of NCS. The aim of this study was to construct a chimeric protein (CMP) that consists of both the terminus residue of NCP and an LDP pocket-forming residue that can bind AE. This CMP will have a structure similar to NCS and an antitumor activity similar to LDM. The assembling efficiency of LDP, CMP, and NCP was 73.9, 1.5, and 1.1%, respectively. The cytotoxicity was consistent with their assembling efficiency of AE in proteins. When CMP-AE and NCP-AE were administered at equivalent AE doses of LDM, the inhibition rate of CMP-AE was the same as LDM and significantly higher than that of NCP-AE. Our study implied that the binding activity between LDP and AE was very specific. The terminus residue of LDP could affect the specifically binding activity. The pocket-forming residue could confer a protective function to the chromophore. Further investigation of its bioactivity might serve as a new drug design strategy and drug-delivery carrier in targeted cancer therapy.

Human serum transferrin fibrils: nanomineralisation in bacteria and destruction of red blood cells.

Fibrils formed by human serum transferrin [(1-3 µM) apo-Tf, partially iron-saturated (Fe0.6 -Tf) and holo-Tf (Fe2 -Tf) forms], from dilute bicarbonate solutions, were deposited on formvar surfaces and studied by electron microscopy. We observed that possible bacterial contamination appears to give rise to long, pea-pod-like (PPL) structures for Fe2 -Tf, attributable to the formation of polyhydroxybutyrate (PHB) storage granules, under the nutrient-limiting conditions used. These PPL structures contained periodic nanomineralisation sites susceptible to uranyl stain. Extended incubation of transferrin solutions (about four days) gave rise to extensive transferrin fibril structures. Optical microscopy and AFM studies showed that red blood cells (RBCs) readily adhere to these fibrils. Moreover, the fibrils appear to penetrate RBC membranes and to induce rapid cell destruction (within about 5 h). It is speculated that in situations in vivo where transferrin fibrils can form, such interactions might have adverse physiological consequences, and further studies could aid the understanding of related pathological events.

Toxic side-effects of diaspirin cross-linked human hemoglobin are attenuated by the apohemoglobin-haptoglobin complex.

Alpha-alpha diaspirin-crosslinked human hemoglobin (DCLHb or ααHb) was a promising early generation red blood cell (RBC) substitute. The DCLHb was developed through a collaborative effort between the United States Army and Baxter Healthcare. The core design feature underlying its development was chemical stabilization of the tetrameric structure of hemoglobin (Hb) to prevent Hb intravascular dimerization and extravasation. DCLHb was developed to resuscitate warfighters on the battlefield, who suffered from life-threatening blood loss. However, extensive research revealed toxic side effects associated with the use of DCLHb that contributed to high mortality rates in clinical trials. This study explores whether scavenging Hb and heme via the apohemoglobin-haptoglobin (apoHb-Hp) complex can reduce DCLHb associated toxicity. Awake Golden Syrian hamsters were equipped with a window chamber model to characterize the microcirculation. Each group was first infused with either Lactated Ringer's or apoHb-Hp followed by a hypovolemic infusion of 10% of the animal's blood volume of DCLHb. Our results indicated that animals pretreated with apoHb-Hb exhibited improved microhemodynamics vs the group pretreated with Lactated Ringer's. While systemic acute inflammation was observed regardless of the treatment group, apoHb-Hp pretreatment lessened those effects with a marked reduction in IL-6 levels in the heart and kidneys compared to the control group. Taken together, this study demonstrated that utilizing a Hb and heme scavenger protein complex significantly reduces the microvasculature effects of ααHb, paving the way for improved HBOC formulations. Future apoHb-Hp dose optimization studies may identify a dose that can completely neutralize DCLHb toxicity.

The Notch signaling pathway: its role in focal CNS demyelination and apotransferrin-induced remyelination.

Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin-induced remyelination in a focal lysolecithin-induced demyelination model in rats. Notch was found activated in Nestin-expressing neural progenitor cells and in NG2-expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin-demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ-secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up-regulation of the myelin-associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non-canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin-induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin-mediated oligodendroglial maturation. Evidence of the participation of Notch signaling in the demyelination/remyelination process will help further understand demyelinating disorders such as Multiple Sclerosis and the use of aTf should be taken into consideration as a possible therapeutic intervention.

Lactoferrin inhibits neutrophil apoptosis via blockade of proximal apoptotic signaling events.

Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.

Bovine lactoferrin: involvement of metal saturation and carbohydrates in the inhibition of influenza virus infection.

Influenza is a highly contagious, acute respiratory illness, which represents one of the main plagues worldwide. Even though some antiviral drugs are available, the alarming increase of virus strains resistant to them highlights the need to find new antiviral compounds. As we have recently demonstrated that bovine lactoferrin (bLf) prevents influenza virus-induced apoptosis, in the present wor,k we have attempted to investigate in depth the mechanism of the anti-influenza virus effect of this protein. To this aim, experiments have been carried out whereby different forms of bLf were added to the cells during different phases of viral infection. Results obtained showed that bLf was able to prevent influenza virus cytopathic effects when incubated with the cells after the adsorption step, independently from ion saturation or carbohydrate content. Moreover, the influence of iron saturations or sialic acid/carbohydrates removal on bLf activity on the early phases of infection has been observed. Our results provide further insights on the antiviral activity of bLf and suggest novel strategies for treatment of influenza virus infection.

Antitumor efficacy of the scFv-based fusion protein and its enediyne-energized analogue directed against epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR), overexpressed in many epithelial tumors, plays important roles in the formation and the development of tumors, and thus it is regarded as a promising target for cancer therapy. Single-chain variable fragment (scFv), an engineered antibody fragment, is generally used for constructing antibody-targeted drugs, owing to its low immunogenicity and high penetration capability into solid tumors. A fusion protein ER(Fv-LDP), consisting of an anti-EGFR scFv and the apoprotein (LDP) of lidamycin (LDM), was prepared and then assembled with the active chomophore [active enediyne (AE)] of LDM to generate enediyne-energized analogue ER(Fv-LDP-AE). The fusion protein ER(Fv-LDP) bound specifically to EGFR-overexpressing cancer cells and internalized into the cytoplasm through receptor-mediated endocytosis. ER(Fv-LDP) possessed cytotoxicity against carcinoma cell lines, which was hundreds of times more potent than the separate moiety of ER(Fv) and LDP. The enediyne-energized fusion protein ER(Fv-LDP-AE) also showed stronger cytotoxicity to target-relevant cancer cells than LDM in vitro. In human epidermoid carcinoma A431 xenografts, ER(Fv-LDP) presented higher antitumor efficacy than that of ER(Fv), LDP, and their mixture, with tumor growth inhibition rates of 63.6, 46.7, 48.5, and 49.9%, respectively. The enediyne-energized fusion protein ER(Fv-LDP-AE) at a dose of 0.4 mg/kg inhibited tumor growth by 89.2%, while no significant body weight loss was seen in treated animals. The results show that an anti-EGFR scFv-based fusion protein and its enediyne-energized analogue can be prepared by DNA recombination and molecular reconstitution. Both ER(Fv-LDP) and ER(Fv-LDP-AE) are effective against EGFR-overexpressing cancer xenograft in athymic mice. An integrated technical platform for scFv-based enediyne-energized fusion proteins has been established.

Human apo-lactoferrin as a physiological mimetic of hypoxia stabilizes hypoxia-inducible factor-1 alpha.

Apo-form of human lactoferrin (LF) is a potent iron chelator, this feature being similar to the iron-binding properties of a synthetic chelator desferoxamine (DFO). The latter stabilizes the principal adaptive transcriptional hypoxia-inducible factor-1 alpha (HIF-1α). Since DFO is known as a pharmacological mimetic of hypoxia it was decided to test whether apo-LF is able to perform as such. Mice either injected intraperitoneally or given per os apo-LF displayed HIF-1α in liver, lungs, heart, brain, spleen and kidneys, as judged by results of Western blotting. Similar administration of iron-saturated LF (75 mg/kg) did not bring forth such effect. Synthesis of erythropoietin and ceruloplasmin became increased in the first case, which is explained by the respective genes being targets for HIF-1α. Apo-LF, but not Fe-LF, injected intraperitoneally to hypoxia low-resistant mice 24 h before animals were subjected to normobaric hypoxia with hypercapnia caused a significant increase of life-time by 40 %. The results obtained show that, like DFO, apo-LF performs as a normoxic mimetic of hypoxia, capable of stabilizing HIF-1α. Protective features of LF and DFO and their pharmacological properties involving HIF-1α are discussed.

Iron sensitizes keratinocytes and fibroblasts to UVA-mediated matrix metalloproteinase-1 through TNF-α and ERK activation.

Oestrogen deficiency is regarded as the main causative factor in postmenopausal skin ageing and photoageing. While women after menopause experience low levels of oestrogen because of cease of ovarian function, they are also exposed to high levels of iron as a result of cessation of menstruation. In this study, we investigated whether this increase in iron presents a risk to the postmenopausal skin. Because of the lack of appropriate animal models to closely mimic the low oestrogen and high iron conditions, we tested the hypothesis in a high iron and low oestrogen culture model. Here, we showed that primary human dermal fibroblasts exposed to iron did not affect the baseline levels of matrix metalloproteinase-1 (MMP-1) activity. However, the iron-exposed fibroblasts were sensitized to UVA exposure, which resulted in a synergistic increase in MMP-1. UVA activated the three members of MAPK family: ERKs, p38, and JNKs. Additional activation of ERKs by iron contributed to the synergistic increases. Primary normal human epidermal keratinocytes (NHEK) did not respond to iron or UVA exposure as measured by MMP-1, but produced tumor necrosis factor-alpha (TNF-α) in the media, which then stimulated MMP-1 in fibroblasts. Our results indicate that iron and UVA increase MMP-1 activity in dermal fibroblasts not only directly through ERK activation but also by an indirect paracrine loop through TNF-α released by NHEK. We conclude that in addition to oestrogen deficiency, increased iron as a result of menopause could be a novel risk factor by sensitizing postmenopausal skin to solar irradiation.

CYP3A4-Mediated oxygenation versus dehydrogenation of raloxifene.

Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, (18)O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3'-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O(2)-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.

Fyn kinase is involved in oligodendroglial cell differentiation induced by apotransferrin.

Mechanisms that regulate oligodendroglial cell (OLGc) differentiation are the focus of intensive research in the field of cellular and molecular neurobiology. We have previously shown that the addition of apotransferrin (aTf) to primary OLGc cultures accelerates their differentiation and induces an increase in the expression of different components of the myelin cytoskeleton (CSK) such as actin, tubulin, and some of the microtubule-associated proteins, particularly the stable tubulin only peptide (STOP). Fyn protein-tyrosine kinase (Fyn kinase), a member of the Src family, participates in signalling pathways that regulate OLGs/myelin cytoskeletal reorganization. It is essential for myelin development in the central nervous system (CNS), and its absence results in hypomyelination. In the present study, we used both primary cell and N19 cell line cultures to investigate further the mechanisms of action involved in the accelerated differentiation of OLGcs induced by aTf. In particular, we were interested in studying the participation of Fyn kinase in the different pathways involved in the reorganization of the OLGc/myelin cytoskeleton. In agreement with results already published, we found that in OLGcs, Fyn kinase is associated with Tau and tubulin. Using a dominant-negative of Tau in which the Fyn-Tau-microtubules (MTs) interaction is blocked, we found that aTf was unable to induce OLGc morphological differentiation. It was also observed that aTf decreases the activated RhoA content in coincidence with a redistribution of actin immunoreactivity. These results give support to our hypothesis that Fyn kinase plays a key role in the differentiation process of OLGcs promoted by aTf.

Attenuation of massive cytokine response to the staphylococcal enterotoxin B superantigen by the innate immunomodulatory protein lactoferrin.

Staphylococcal enterotoxin B (SEB) is a pyrogenic exotoxin and a potent superantigen which causes massive T cell activation and cytokine secretion, leading to profound immunosuppression and morbidity. The inhibition of SEB-induced responses is thus considered a goal in the management of certain types of staphylococcal infections. Lactoferrin (LF) is a multi-functional glycoprotein with both bacteriostatic and bactericidal activities. In addition, LF is known to have potent immunomodulatory properties. Given the anti-microbial and anti-inflammatory properties of this protein, we hypothesized that LF can modulate T cell responses to SEB. Here, we report that bovine LF (bLF) was indeed able to attenuate SEB-induced proliferation, interleukin-2 production and CD25 expression by human leucocyte antigen (HLA)-DR4 transgenic mouse T cells. This inhibition was not due to bLF's iron-binding capacity, and could be mimicked by the bLF-derived peptide lactoferricin. Cytokine secretion by an engineered SEB-responsive human Jurkat T cell line and by peripheral blood mononuclear cells from healthy donors was also inhibited by bLF. These findings reveal a previously unrecognized property of LF in modulation of SEB-triggered immune activation and suggest a therapeutic potential for this naturally occurring protein during toxic shock syndrome.

A bactericidal effect for human lactoferrin.

Streptococcus mutans and Vibrio cholerae, but not Escherichia coli, were killed by incubation with purified human apolactoferrin. Concentrations of lactoferrin below that necessary for total inhibition resulted in a marked reduction in viable colony-forming units. This bactericidal effect was contingent upon the metal-chelating properties of the lactoferrin molecule.

Cytotoxicity of apo bovine α-lactalbumin complexed with La3+ on cancer cells supported by its high resolution crystal structure.

Cancer remains one of the biggest threats to human society. There are massive demands for compounds to selectively kill cancerous cells. Earlier studies have shown that bovine α -lactalbumin made lethal to tumor cells (BAMLET) becomes cytotoxic against cancer cells in complex with oleic acid {Hoque, M. et. al., PLoSOne 8, e68390 (2013)}. In our study, we obtained bovine α-lactalbumin complexed with lanthanum ion (La3+-B-α-LA) and determined its high resolution crystal structure. The natural calcium binding site of bovine α-lactalbumin is replaced by lanthanum. The La3+ complex formation by B-α-apo-LA was also supported by various biophysical methods. Interestingly, our complex, La3+-B-α-LA exhibits much greater anticancer activity against breast cancer cells as compared to the reported BAMLET-oleic acid complex. This study shows that La3+-B-α-LA complex is preferentially more toxic to MCF-7 cells as compared to KB (oral cancer) and HeLa (cervical) cells, while almost non-toxic to the healthy cells that we studied. Our data indicates that the cytotoxicity of La3+-B-α-LA against cancer cells is through apoptotic path way. The higher anticancer activity of La3+-B-α-LA is attributable to the requisite structural changes induced in the protein by La3+ binding as supported by the crystal structure of the complex.

IsdA protects Staphylococcus aureus against the bactericidal protease activity of apolactoferrin.

An important facet of the Staphylococcus aureus host-pathogen interaction is the ability of the invading bacterium to evade host innate defenses, particularly the cocktail of host antimicrobial peptides. In this work, we showed that IsdA, a surface protein of S. aureus which is required for nasal colonization, binds to lactoferrin, the most abundant antistaphylococcal polypeptide in human nasal secretions. The presence of IsdA on the surface of S. aureus confers resistance to killing by lactoferrin. In addition, the bactericidal activity of lactoferrin was inhibited by addition of phenylmethylsulfonyl fluoride, implicating the serine protease activity of lactoferrin in the killing of S. aureus. Recombinant IsdA was a competitive inhibitor of lactoferrin protease activity. Reciprocally, antibody reactive to IsdA enhanced killing of S. aureus. Thus, IsdA can protect S. aureus against lactoferrin and acts as a protease inhibitor.

Effect of transferrin on hypomyelination induced by iron deficiency.

We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.

Use of human surfactant low molecular weight apoproteins in the reconstitution of surfactant biologic activity.

Two low molecular weight (LMW) apoproteins were isolated from human pulmonary surfactant. SDS polyacrylamide gel analysis showed one protein (SP 18) to have an apparent molecular weight of 18,000 when unreduced and 9,000 D after reduction. The second protein (SP 9) migrated at approximately 9,000 D in the presence or absence of reducing agents. Both proteins contain a high number of hydrophobic amino acids. The NH2-terminal sequence of SP 18 was determined to be: NH2-phe-pro-ile-pro-leu-pro-tyr-. A cDNA clone isolated from a human adult lung cDNA library contained a long open reading frame encoding at an internal position the human SP 18 amino-terminal sequence. Mixtures of phospholipids (PL) and SP 9 and SP 18 were assessed for their capacity to reduce surface tensions on a pulsating bubble surfactometer. The addition of 1% apoprotein resulted in a reduction of surface tension after 15 s from 42.9 dyn/cm for PL alone to 16.7 and 6.3 dyn/cm for preparations containing SP 9 and SP 18, respectively. In vivo assessment of reconstituted surfactant activity was performed in fetal rabbits. Reconstituted surfactant consisting of PL + 0.5% SP 18 instilled intratracheally at delivery resulted in a marked increase in lung compliance, while the incorporation of 0.5% SP 9 yielded a moderate increase. These data show the ability to produce biologically active surfactant by the addition of isolated LMW apoproteins to defined PL.