Determination of the elastase activity of the leukocyte thermostable alpha-glycoprotein from human blood plasma

The possibility of isolation of specific human granulocyte antigen--leukocyte thermostable alpha-glycoprotein (LTG) from plasma was shown. The isolation of preparations containing LTG was carried out on CNBr-activated sepharose with immobilized soluble fraction of pus. Using immunochemical analysis with standard antisera, the absence of plasma and leukocyte proteins and the presence of LTG in the preparations obtained were demonstrated. Pus may comprise a protein component capable of binding LTG. Elastase activity of the preparations obtained was established. The identity of LTG and granulocyte elastase is suggested.     

Immunochemical determination of the heterogeneity of leukocyte thermostable alpha-glycoprotein

A comparative immunochemical and physico-chemical study of leukocyte thermostable alpha-glycoprotein (LTG) has been performed in pus extract, hemolysate, leukolysate and blood plasma. Immunochemically determined LTG is heterogeneous in respect of molecular charge, sialic acid content and temperature stability. LTG in the blood plasma has a form of alpha-globulin, while in hemolysate it is a beta-globulin, with both forms present in pus. In leukolysate LTG possessing incomplete immunochemical identity with pus and plasma LTG was detected. It is suggested that LTG forms detected differ in their biological activity as well.     

Evidence that a plasmid from a hyperthermophilic archaebacterium is relaxed at physiological temperatures.

A plasmid of 3.45 kb (pGT5) was recently discovered in a strain of hyperthermophilic archaebacterium which was isolated from samples collected in a deep-sea hydrothermal vent. This strain (GE5) grows within a temperature range of 68 to 101.5 degrees C, and we show here that it contains a strong ATP-dependent reverse gyrase activity (positive DNA supercoiling). By comparison with eubacterial plasmids of known superhelical densities, we estimated the superhelical density of the archaebacterial plasmid pGT5 to be -0.026 at 25 degrees C. The equation which relates the change of the rotation angle of the DNA double helix with temperature was validated at 95 degrees C, the optimal growth temperature of the GE5 strain. Considering these new data, the superhelical density of plasmid pGT5 was calculated to be -0.006 at the physiological temperature of 95 degrees C, which is close to the relaxed state. This finding shows that the DNA topology of a plasmid isolated from a hyperthermophilic archaebacterium containing reverse gyrase activity is strikingly different from that of typical eubacterial plasmids.     

Lamotrigine induced selective changes in 5-HT(1A) receptor mediated response in rat brain.

A new anticonvulsant drug lamotrigine (LTG) has recently been reported to be effective in treating patients with bipolar affective disorder, depression and schizoaffective disorder, suggesting that it is a mood stabilizer. However, the mechanism of action underlying its efficacy in mood disorders is not understood. This study examined the in vivo effect of LTG on 5-HT(1A) receptor-mediated adenylyl cyclase (AC) response in regions of rat brain, as this pathway has been implicated in the therapeutic action of various classes of mood stabilizers. The density of 5-HT(1A) receptors was measured by radioligand binding assay using [(3)H]8-OH-DPAT (0.05-0.8nM) in frontal cortex and hippocampus of rats treated orally with LTG (5mg/kg) for 7 days. AC activity was assayed using [(3)H]ATP. The oral administration of LTG significantly decreased the density of cortical (50%, P<0.001) but not hippocampal 5-HT(1A) receptors, without significant change in the affinity of [(3)H]8-OH-DPAT to 5-HT(1A) receptor in these regions. There was no significant alteration in basal or forskolin-stimulated AC activity in either of regions. However, a significant decrease (P<0.01) in the inhibition of forskolin-stimulated AC activity by 8-OH-DPAT was observed only in cortical membranes of LTG treated rats when compared to control. These results suggest that one mode of action of LTG may be by the downregulation of cortical 5-HT(1A) receptor-mediated AC response.     

The carbohydrate at FcgammaRIIIa Asn-162. An element required for high affinity binding to non-fucosylated IgG glycoforms

FcgammaRIIIa plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. Non-fucosylated bisected IgGs bind this receptor with increased affinity and trigger FcgammaRIII-mediated effector functions more efficiently than native, fucosylated antibodies. In this study the contribution of the carbohydrates of both binding partners to the strength of the complex was analyzed. Glycoengineering of the antibody increased affinity for two polymorphic forms of soluble human FcgammaRIIIa (by up to 50-fold) but did not affect binding to the inhibitory FcgammaRIIb receptor. While the absence of carbohydrate at FcgammaRIIIa's Asn-162 increased affinity for native IgG, presumably due to the removal of steric hindrance caused by the bulky sugars, it unexpectedly reduced affinity for glycoengineered (GE) antibodies by over one order of magnitude, bringing the affinity down to the same level as for native IgG. We conclude that the high affinity between GE antibodies and FcgammaRIII is mediated by productive interactions formed between the receptor carbohydrate attached at Asn-162 and regions of the Fc that are only accessible when it is nonfucosylated. As FcgammaRIIIa and FcgammaRIIIb are the only human Fcgamma receptors glycosylated at this position, the proposed interactions explain the observed selective affinity increase of GE antibodies for only these receptors. Furthermore, we predict from our structural model that only one of the two Fc-fucose residues needs to be absent for increased binding affinity toward FcgammaRIII. This information can be exploited for the design of new antibodies with altered Fc receptor binding affinity and enhanced therapeutic potential.     

Insulin-induced redistribution of the insulin-like growth factor II/mannose 6-phosphate receptor in intact rat liver

The ability of acute insulin treatment to elicit a redistribution of the liver insulin-like growth factor-II/ mannose 6-phosphate (IGF-II/M6P) receptor has been studied in rats, using cell fractionation. Injection of insulin (0.4-50 microg) led to a time- and dose-dependent decrease in IGF-II binding activity in Golgi-endosomal (GE) fractions, along with an increase in activity in the plasma membrane (PM) fraction; only receptor number was affected. Quantitative subfractionation of the microsomal fraction on sucrose density gradients showed that IGF-II binding activity distributed similarly to galactosyltransferase (a Golgi marker), at slightly higher densities than in vivo internalized (125)I-insulin, and at lower densities than 5' nucleotidase and alkaline phosphodiesterase (two plasma membrane markers). Insulin treatment led to a slight time-dependent and reversible shift of IGF-II binding activity toward higher densities. Subfractionation of the GE fraction on Percoll gradients showed that IGF-II binding activity was broadly distributed, with about 60% at low densities coinciding with galactosyltransferase and early internalized (125)I-insulin and with 40% at high densities in the region of late internalized (125)I-insulin. Insulin treatment caused a time-dependent and reversible shift of the distribution of IGF-II binding activity toward low densities. On SDS-PAGE, the size of the affinity-labeled IGF-II/M6P receptor was comparable in GE and PM fractions (about 255 kDa), but on Western blots receptor size was slightly lower in the latter (245 kDa) than in the former (255 kDa). Insulin treatment did not affect the size, but modified the abundance of the IGF-II/M6P receptor in a manner similar to that of IGF-II binding. In vivo chloroquine treatment fully suppressed the changes in IGF-II binding activity in liver GE and PM fractions observed in insulin-treated rats. We conclude that insulin elicits a time-dependent and reversible redistribution of liver IGF-II receptors from Golgi elements and endosomes to the plasma membrane, presumably via early endosomes.     

In vivo anti-diabetic activity of derivatives of isoliquiritigenin and liquiritigenin

Isoliquiritigenin (ISL), a chalcone and liquiritigenin (LTG), a flavonoid found in licorice roots and several other plants. ISL displays antioxidant, anti-inflammatory, antitumor and hepatoprotective activities whereas LTG is an estrogenic compound, acts as an agonist selective for the β-subtype of the oestrogen receptor. Both the phenolics were isolated from the rhizomes of Glycyrrhiza glabra. Five derivatives from ISL and four derivatives from LTG were synthesized. All the compounds were established by extensive spectroscopic analyses and screened through oral glucose tolerance test to gain preliminary information regarding the antihyperglycemic effect in normal Swiss albino male mice. ISL (1), ISL derivatives 3, 4, 5, 7 and LTG derivatives 9 and 10 showed significant blood glucose lowering effect. The structure–activity relationship indicated that the presence of ether and ester groups in ISL and LTG analogues are important for exhibiting the activity. Compounds 1, 4 and 10 were selected for in vivo antidiabetic activity and found to be potential candidates for treatment of diabetes. It is the first report on antidiabetic activity of ISL derivative 4 and LTG derivative 10.     

TEM-1 beta-lactamase as a scaffold for protein recognition and assay

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.     

HIV-1 Receptor Binding Site-Directed Antibodies Using a VH1-2 Gene Segment Orthologue Are Activated by Env Trimer Immunization

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01.     

New antibiotic that acts specifically on the GTP-bound form of elongation factor Tu.

The new thiazolyl peptide antibiotic GE2270 A, isolated from Planobispora rosea strain ATCC 53773, is shown to inhibit bacterial protein biosynthesis in vitro by affecting specifically the GTP-bound form of elongation factor Tu (EF-Tu). The 'off' rate of EF-Tu.GTP is slowed down 400-fold, locking GTP on EF-Tu, whereas EF-Tu.GDP is unaffected. Therefore, on the EF-Tu.guanine nucleotide interaction, GE2270 A mimicks the effect of aa-tRNA. In line with this, the binding of aa-tRNA to EF-Tu.GTP is hindered by the antibiotic, as shown by the absence of a stable ternary complex and the inhibition of the enzymatic binding of aa-tRNA to the ribosome. This blocks the elongation cycle. GE2270 A does not essentially modify the intrinsic GTPase activity of EF-Tu, but impairs the stimulation by ribosomes of this reaction. The negative effect of GE2270 A on the EF-Tu.GTP interaction with aa-tRNA bears similarities with that of the structurally unrelated pulvomycin, whereas marked differences were found by comparing the effects of these two antibiotics on EF-Tu.GDP. This work emphasizes the varieties of the transitional conformations which tune the EF-Tu interaction with GTP and GDP.     

Formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection.

Five hybridomas that secrete monoclonal antibodies which neutralize the infectivity of lactate dehydrogenase-elevating virus (LDV) were isolated from BALB/c mice primed with Formalin-inactivated LDV. Competition analyses indicated that all five neutralizing monoclonal antibodies recognize contiguous, if not identical, epitopes on the envelope glycoprotein of LDV (VP-3) which are not recognized by nonneutralizing VP-3-specific monoclonal antibodies isolated from the same fusion. Despite the presence of neutralizing activity, polyclonal anti-LDV antibodies obtained from persistently infected mice did not compete for binding to LDV with four of the five neutralizing monoclonal antibodies tested. The results indicate that the envelope glycoprotein of LDV possesses a major neutralizing epitope which is poorly recognized, if at all, by mice during a natural infection but is rendered immunogenic by Formalin inactivation of the virus. The epitope was also not immunogenic in a rabbit, since its polyclonal LDV-neutralizing antibodies did not inhibit binding of the mouse monoclonal antibodies to LDV. Passive immunization with the neutralizing monoclonal antibodies did not protect mice from LDV infection and did not alter the course of infection. Neutralizing monoclonal antibodies have been used to select a neutralization escape variant by a novel combination of in vitro and in vivo isolation.     

Purification and characterization of two basic beta-1,3-glucanases induced in Colletotrichum lindemuthianum-infected bean seedlings.

Two beta-1,3-glucanases which are rapidly induced in the incompatible interaction between bean (cv. Processor) and Colletotrichum lindemuthianum race beta were purified to homogeneity. Characterization of the two enzymes, GE1 and GE2, showed that they both had a basic isolectric point and a similar molecular weight (36,500 for GE1 and 36,000 for GE2), but differed in their pH optimum, thermal stability, and specific activity. GE2 was present in higher amounts but was shown to be less active than GE1 against laminarin and fungal cell walls isolated from race beta of the fungus. Both enzymes were specific for beta-1,3 linkages and showed a strict endolytic mode of action. Further characterization of GE2 was achieved by amino acid sequence analysis of tryptic peptides; the degree of homology shared with other basic beta-1,3-glucanases depended on the plant source. A time-course study showed that GE1 and GE2 were increased during infection. They were also induced by fungal elicitors, thereby indicating that they originate from the host.     

Transient expression of CYP1A1 in rat epithelial cells cultured in suspension

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.     

Development and characterization of monoclonal antibodies against a mucin-type glycoprotein.

The preparation of greater than 30 different hybridomas, all secreting IgM class antibodies against epiglycanin, a glycoprotein at the surface of the mouse mammary carcinoma cell line TA3-Ha, is described. The specificities of 10 of the antibodies, with affinity constants in the range of 10(8)-10(10) l/mol were compared in an enzyme competitive binding assay. The affinity of epiglycanin was strongly reduced for all antibodies tested by incubation with periodate (10 mM, 4 degrees C) and was reduced for most of the antibodies by endo-alpha-N-acetyl- D-galactosaminidase. This suggested that carbohydrate, and specifically the Gal beta (1----3)GalNAc disaccharide, formed an integral part of the epitopes of most of the antibodies. The isolated disaccharide, however, exhibited 250,000 times less inhibitory activity in the competitive binding assay than epiglycanin. The binding capacity of epiglycanin was also reduced by incubation with trypsin or pronase, suggesting a high molecular weight dependency for binding. Incubation with sialidase increased its affinity for the antibodies. The binding of the antibodies to epiglycanin was strongly inhibited by peanut agglutinin, and to a lesser extent by lectins from Triticum vulgaris, Ricinus communis, Pisum sativum and Phaseolus vulgaris. None of the antibodies bound to any of eight different gangliosides immobilized on HPTLC plates. Mono- (Fab) and divalent [F(ab')2] fragments of the antibodies possessed very low affinity for epiglycanin. The results demonstrated that the specificities of the antibodies are related, but distinguishable, and they suggest that this epiglycanin-IgM model may be useful for studies on the general principles of the interaction between IgM antibodies and mucin-type glycoproteins.     

Effects of human anti-IL-1 alpha autoantibodies on receptor binding and biological activities of IL-1.

Immunoglobulin G (IgG) antibodies to interleukin 1 alpha (IL-1 alpha) are frequently found in the sera of healthy human individuals. The effects of these autoantibodies on receptor binding and biological activities of human IL-1 were tested. Using the murine T-lymphocyte line NOB-1, human thyrocytes and human foreskin fibroblasts, the antibodies competitively inhibited the biological activity of human recombinant IL-1 alpha (rIL-1 alpha). The degree of inhibition correlated with 125I-rIL-1 alpha binding to IgG in different immunoglobulin preparations and in individual sera. These antibodies also neutralized the IL-1 activity of isolated membrane fragments and lysates of human blood monocytes activated by lipopolysaccharide. In contrast, the supernatant IL-1 activity was not affected. Stronger inhibition of biological activity and cell binding of 125I-rIL-1 alpha was obtained with NOB-1 cells than with human thyrocytes. The antibodies failed to interfere with the biological activity of rIL-1 beta. It is concluded that IgG autoantibodies of IL-1 alpha in the sera of healthy humans selectively inhibit the biological activity of the soluble and membrane-associated forms of IL-1 alpha in vitro, and that the degree of biological inhibition afforded by these antibodies depends upon the target cell.     

IgG autoantibody activity in normal mouse serum is controlled by IgM

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.     

Trivalent Combination Vaccine Induces Broad Heterologous Immune Responses to Norovirus and Rotavirus in Mice

Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs) and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.     

Granulocytic ehrlichiosis and tick infestation in mountain lions in California

Forty-seven mountain lions (Puma concolor) collected year-round in 1996 to 1998 from the Sierra Nevada foothills, the northern coast ranges, and in Monterey County (California, USA) were examined for infestation with Ixodes pacificus and Dermacentor variabilis ticks. Ticks were found predominantly in winter and spring. The seroprevalence of granulocytic ehrlichiae (GE) antibodies (Ehrlichia equi or the agent of human granulocytic ehrlichiosis) was 17% and the PCR-prevalence of DNA characteristic of GE in blood was 16%. There were eight polymerase chain reaction (PCR)-positive but seronegative mountain lions, one that was PCR-positive and seropositive, and eight that were PCR-negative and seropositive. Nineteen percent of engorged tick pools from mountain lions were PCR-positive. Because mountain lions inhabit tick-infested habitat and are frequently bitten by I. pacificus, surveillance for GE antibodies and DNA in mountain lions and other vertebrate hosts may be useful as indicators for geographical regions in which humans are at risk of GE infection.     

Seminal plasma of brown trout,Salmo trutta fario (L.) contains a factor able to retain iron at acid pH,typical feature of lactoferrin

Blood and seminal plasma of brown trout Salmo trutta fario were analyzed for their iron binding potential adopting two different methods. Seminal plasma showed an iron binding capacity that was retained even if samples were exposed at acid pH, similarly to mammalian lactoferrin that binds ferric iron also at acid pH. This suggests that the iron binding capacity is determined by a factor having a lactoferrin-like activity. Moreover, trout seminal plasma proteins were also analyzed in their pattern by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. When seminal plasma was subjected to immunoblotting using goat anti-bovine lactoferrin antibodies as a probe, only a single band having an apparent molecular weight of around 80 kDa was specifically detected, showing that this protein has homology with bovine lactoferrin.